Article Mycobacteria trehalose dimycolate interactions with host Mincle remodel blood-brain barrier junctions for brain invasion Graphical abstract Highlights • Macrophages release mycobacteria into the blood, where they bind to brain endothelial cells • Mycobacteria attach to the endothelium using trehalose dimycolate (TDM) • TDM binds its host receptor, Mincle, triggering cell junction opening • Mycobacteria cross the blood-brain barrier using a paracellular mechanism Authors Megan I. Hayes, Sumedha Ravishankar, Jonathan K. Shanahan, Adam J. Fountain, Lalita Ramakrishnan, Cressida A. Madigan Correspondence lalitar@mrc-lmb.cam.ac.uk (L.R.), cmadigan@ucsd.edu (C.A.M.) In brief Using a zebrafish model of brain infection, Hayes et al. find that mycobacteria infect the brain not within macrophages, but as extracellular bacteria that attach to endothelial cells of the blood-brain barrier. Using a glycolipid, mycobacteria trigger the opening of gaps in endothelial cell junctions, allowing transit into the brain. Hayes et al., 2025, Cell Reports 44, 116661 December 23, 2025 © 2025 The Authors. Published by Elsevier Inc. https://doi.org/10.1016/j.celrep.2025.116661 ll mailto:lalitar@mrc-lmb.cam.ac.uk mailto:cmadigan@ucsd.edu https://doi.org/10.1016/j.celrep.2025.116661 http://crossmark.crossref.org/dialog/?doi=10.1016/j.celrep.2025.116661&domain=pdf Article Mycobacteria trehalose dimycolate interactions with host Mincle remodel blood-brain barrier junctions for brain invasion Megan I. Hayes, 1,4 Sumedha Ravishankar, 1,4 Jonathan K. Shanahan, 2 Adam J. Fountain, 3 Lalita Ramakrishnan, 2,3, * and Cressida A. Madigan 1,5, * 1 School of Biological Sciences, University of California, San Diego, La Jolla, CA 92037, USA 2 Molecular Immunity Unit, Cambridge Institute of Therapeutic Immunology and Infectious Diseases, Department of Medicine, University of Cambridge, Cambridge CB2 0QH, UK 3 Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, UK 4 These authors contributed equally 5 Lead contact *Correspondence: lalitar@mrc-lmb.cam.ac.uk (L.R.), cmadigan@ucsd.edu (C.A.M.) https://doi.org/10.1016/j.celrep.2025.116661 SUMMARY Tuberculous meningitis is unique among bacterial meningitides because it occurs in two temporally sepa- rated steps: mycobacteria first invade the brain, then form infected macrophage aggregates called Rich foci, which later erode the meninges. Here, using transparent zebrafish larvae, we detail the first step—brain invasion. We find that whereas elsewhere in the body mycobacteria disseminate within phagocytes, only extracellular mycobacteria reach the brain microvasculature. There, they adhere to the microvascular endo- thelium and grow into microcolonies. These microcolonies induce endothelial tight junction reorganization, creating transient gaps through which bacteria enter the brain and infect microglia to initiate Rich foci. This reorganization is induced by mycobacterial surface glycolipid trehalose dimycolate interacting with its receptor, Mincle. Strikingly, the pathogens Mycobacterium tuberculosis and Mycobacterium marinum and the saprophyte Mycobacterium smegmatis can all invade the brain via this pathway. Thus, M. tubercu- losis initiates meningitis, the deadliest form of tuberculosis, using an ancestral determinant important for environmental fitness. INTRODUCTION Meningitis, one of the most serious bacterial diseases, carries a high mortality despite antimicrobial treatment. 1,2 Tuberculous meningitis is no exception; almost uniformly fatal without antitu- bercular chemotherapy, mortality remains 20%–40% with treat- ment, increasing to >80% for drug-resistant tuberculosis (TB). 3–5 Survivors often suffer lifelong neurological deficits, and children are disproportionately affected, compounding the burden placed by this devastating disease. 3,4 Meningitis occurs when organisms enter the circulation and invade the meninges through the endothelial cells of blood-brain barrier (BBB) vessels. 1,2,6 To protect the brain from toxins and pathogens, the BBB downregulates transcytosis and increases expression of specialized tight junction proteins that deter traversal between cells. 7,8 Therefore, few bacteria regularly cause meningitis. 2 Most are extracellular commensals that can breach mucosal barriers, enter the circulation, and traverse the BBB to cause acute meningeal infection. 1 In human brain micro- vascular endothelial cells (HBMECs), these bacteria are inferred to cross the BBB by transcytosis or lysis. 2 Neisseria meningitidis is exceptional in that it breaches endothelial cell tight junctions to traverse paracellularly. 2,9,10 While epidemiological studies sug- gest that M. tuberculosis also invades the brain through the cir- culation, 4,11–13 meningitis does not occur directly upon menin- geal seeding. 1,11,12,14 Histopathological analyses of brains from fatal tuberculous meningitis cases suggest a distinct pathophys- iology with disease occurring in two stages. First, M. tuberculosis invades the brain or meninges and forms an immune aggregate or granuloma called the Rich focus. Months later, meningitis oc- curs as the granuloma matures and becomes necrotic. 11,12,14,15 Identifying the mechanism by which mycobacteria invade the brain has been stymied by the lack of animal models, where these steps can be visualized. 4,16 The zebrafish larva’s optical transparency and genetic amenability enable real-time delinea- tion of the earliest events of mycobacterial pathogenesis. 17–21 It has also been used to examine M. marinum’s traversal of the BBB and the blood-retinal barrier 22,23 and group B Streptococ- cus’s and Cryptococcus neoformans’s traversal of the BBB. 24–26 We performed live imaging studies in zebrafish larvae to exper- imentally corroborate human autopsy studies showing that mycobacterial entry from blood vessels into the brain produces the Rich focus. We then dissected the entry process and found that extracellular bacteria first attach to brain microvascular Cell Reports 44, 116661, December 23, 2025 © 2025 The Authors. Published by Elsevier Inc. 1 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ll OPEN ACCESS http://creativecommons.org/licenses/by/4.0/ mailto:lalitar@mrc-lmb.cam.ac.uk mailto:cmadigan@ucsd.edu https://doi.org/10.1016/j.celrep.2025.116661 http://crossmark.crossref.org/dialog/?doi=10.1016/j.celrep.2025.116661&domain=pdf http://creativecommons.org/licenses/by/4.0/ Figure 1. Extracellular M. marinum invades the brain and then infects microglia to form nascent Rich foci (A) Schematic drawing of a 3 dpf zebrafish larva showing the caudal vein infection site (injection needle) and the brain region where imaging was performed (dashed box). (B) Image of brain region of the same larva at 3 dpi (left) and 5 dpi (right), infected with ∼120 CFU fluorescent M. marinum (Mm) showing microcolonies (ar- rowheads). Boxes indicate a microcolony that has enlarged between 3 and 5 dpi. Scale bars, 100 μm. (C) Proportion of larvae with brain infection after inoculation with ∼20 or ∼120 CFU Mm. Horizontal bars, means; s; Fisher’s exact test. Representative of 3 independent experiments. (D) Volume of fluorescent Mm microcolonies in the brain at 3 and 5 dpi in larvae infected with ∼120 CFU. Horizontal bars, means; Student’s t test. Representative of 3 independent experiments. (E) Representative confocal images of 3 dpi larvae with green-fluorescent blood vessels infected with blue-fluorescent Mm showing microcolonies inside, partially outside, and completely outside brain blood vessels. Scale bars, 10 μm. (F) Quantification of images from experiment in (E). Fisher’s exact test, ***p < 0.001, ****p < 0.0001, and ns, not significant. Representative of 3 independent experiments. (G) Representative confocal image of a 3 dpi transgenic larva with green-fluorescent blood vessels and red-fluorescent myeloid cells (arrows), including microglia (yellow arrow), infected with blue-fluorescent Mm (arrowhead). The right panel shows a 3D rendered version of the image in the left panel. Scale bars, 10 μm. (H) Representative images of green-fluorescent brain microvasculature in 2 dpi wild-type and myeloid-deficient (PU.1 morphant) larvae infected with red-fluo- rescent Mm (arrowheads). Scale bars, 10 μm. (I) Mm burden per larva quantified by fluorescent pixel counts from experiment in (H). Horizontal bars, means; ns, not significant; Student’s t test. Representative of 3 independent experiments. (legend continued on next page) 2 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS endothelial cells using the serine threonine kinase PknD, reported to promote M. tuberculosis-mediated actin polymerization and invasion of HBMECs. 27,28 However, our detailed in vivo analyses revealed that PknD’s role in brain invasion is limited to promoting attachment to the endothelium. We went on to discover that the attached bacteria then invade the brain by an unexpected mechanism of paracellular transit: transient tight junction remod- eling, mediated by their surface glycolipid trehalose 6,6 ′ -dimyco- late (TDM). Our findings explain the peculiarities of tuberculous meningitis, particularly that it occurs in two steps, with the inter- vening period between them being subclinical. RESULTS Circulating extracellular mycobacteria invade the brain to establish Rich foci To confirm that M. marinum invades the brain of zebrafish larvae via a hematogenous route, we intravenously injected ∼20 or ∼120 colony-forming units (CFUs) into larvae at 3 days post- fertilization (dpf) and imaged their brains serially some days later (Figure 1A). Most observations were made at 3 days post-infec- tion (dpi) (6 dpf), when the zebrafish BBB has matured by sup- pressing transcytosis and increasing tight junction integrity. 29 Discrete foci of living bacteria (or microcolonies) appeared in the brain by 3 dpi (Figure 1B, left panel). The frequency of brain dissemination correlated with inoculum: inocula of 20 and 120 bacteria produced brain dissemination in 42% and 96% of ani- mals, respectively (Figure 1C). After a single intravenous injec- tion, new microcolonies comprising approximately 8–12 bacte- ria appeared over time and expanded by 5 dpi (Figure 1B, compare left and right insets, 3 and 5 dpi, respectively). Thus, both continual dissemination from the blood and in situ growth contributed to increased total bacterial volume in the brain (Figure 1D). To determine if the bacteria had invaded the brain tissue from the microvasculature, transgenic larvae with green-fluorescent vascular endothelial cells (kdrl:GFP) 30 were infected with blue- fluorescent M. marinum and imaged sequentially over several days. At 1 dpi, M. marinum microcolonies were exclusively within brain blood vessels and began traversing the vessel wall by 3 dpi, before entering the brain at 5 dpi (Figures 1E and 1F). Together, these experiments confirmed that circulating M. mar- inum invades the brain. As M. tuberculosis resides predominantly in myeloid cells that can disseminate it throughout the body, it has been assumed that mycobacteria enter the brain within myeloid cells. 2,4,31 How- ever, mice lacking myeloid cells still develop mycobacterial men- ingitis, 32 and extracellular M. tuberculosis can invade and cross HBMECs, 28 although a predominant role for myeloid cells, which are absent in this in vitro system, could not be evaluated. In ze- brafish larvae, as in humans, myeloid cells play a major role in M. marinum tissue dissemination from the circulation into distal body sites. 20,33,34 One study reported that M. marinum used myeloid cells to traverse the BBB, and only when phagocytes were depleted were free bacteria reported to traverse via pre- sumed transcytosis. 22 To test if phagocytes are involved in mycobacterial brain inva- sion, we infected mpeg1:dsRed;kdrl:GFP larvae 35 that have red-fluorescent myeloid cells and green-fluorescent blood ves- sels. In contrast to the prior study, 22 few M. marinum microcol- onies in the brain microvasculature were within the myeloid cells in any of the five animals examined at 4 dpi (Figure 1G). Myeloid cells were observed only outside the blood vessels and within the brain tissue, 33 many displaying the ramified morphology typical of microglia, brain-resident macrophages (Figure 1G, yellow arrow). No myeloid cells were observed within the brain blood vessels of uninfected animals either; in three animals examined, all 111 mpeg1-positive myeloid cells observed were extravascular microglia. This is consistent with mammals that restrict cellular traffic into the brain, likely to reduce the risk of dysregulated inflammation in this vulnerable organ. 7,36 To further confirm that myeloid cells were dispensable, we depleted animals of myeloid cells by morpholino knockdown of the myeloid transcription factor spi1b (also called pu.1). 34 At 2 dpi, when the overall bacterial burdens throughout the larvae in wild-type and pu.1 morphant animals were equivalent, there were significantly more microcolonies in the pu.1 mor- phant brains (Figures 1H–1J). Thus, these data suggest that myeloid cells deter rather than promote mycobacterial brain in- vasion, likely because monocytes are restricted from the brain vasculature, thereby keeping mycobacteria out of the brain. Our observation that brain dissemination occurs predomi- nantly via extracellular mycobacteria might seem at odds with findings that infecting bacteria are rapidly taken up by macro- phages and reside within them in the early stages of infec- tion. 34,37 However, dead macrophages can release extracellular mycobacteria into circulation that could enter the brain blood vessels. 37 If so, mycobacteria invading the brain would have been macrophage-resident before becoming extracellular. To test this, we used Mi marinum expressing a macrophage-acti- vated promoter (map49) fused to GFP, in addition to constitutive red fluorescence (Figures S1A and S1B). 38 Macrophage-resi- dent mycobacteria continue to express GFP for some days after becoming extracellular, with the signal diluting due to replica- tion. We found that the majority of brain microcolonies (61.5%) retained some GFP (Figures S1C and S1D), consistent with the (J) Mm volume in the brains of larvae in (I). Horizontal bars, means; Student’s t test. Representative of 3 independent experiments. (K) Proportion of Mm microcolonies in the brain blood vessels or brain tissue of 3 dpi larvae infected with Mm; Fisher’s exact test. Representative of 3 independent experiments. (L–O) Representative images of interactions between blue-fluorescent Mm (arrowheads), green-fluorescent blood vessels, and red-fluorescent myeloid cells (arrows) in a 3 dpi larva infected with Mm. The right panels show the 3D rendered images of the left panels. Scale bars, 10 μm. Extracellular Mm crossing a vessel with microglia close by (L). Crossing Mm is being taken up by a myeloid cell (M and N). In the larva from (B), Mm that have already crossed completely are associated with aggregating myeloid cells (O). (P and Q) A 4 dpi larva with a red-fluorescent granuloma (Rich focus) (circled) forming near point of exit of blue-fluorescent Mm from green-fluorescent blood vessel. 3D rendering of Rich focus with transparent myeloid cells (right) to show infected (arrowhead) and uninfected cells (arrow). Scale bars, 10 μm (P). 3D rendering of the front (left) and back (right) of a Rich focus with opaque myeloid cells to show extracellular Mm. Scale bars, 10 μm (Q). Cell Reports 44, 116661, December 23, 2025 3 Article ll OPEN ACCESS release of extracellular mycobacteria by dead macrophages. This mechanism could reconcile our findings with the prior report surmising that brain infection is predominantly mediated by macrophages. 22 As we and others observe, bacteria escaping from circulating monocytes may enter the brain extracellularly before infecting microglia. 27,32 Therefore, our findings suggest that because myeloid cells are largely excluded from the brain vasculature even during systemic infection, extracellular myco- bacteria released from macrophages are responsible for brain invasion. Mycobacteria infect microglia to initiate Rich foci We next sought to confirm human autopsy studies suggesting that M. tuberculosis invasion of the brain or meninges initially re- sults in the formation of granulomas termed Rich foci. 11,14,15 Because M. tuberculosis can infect microglia in culture, it has been inferred that it infects these cells in vivo to initiate the Rich focus. 4,39 Our finding that extracellular mycobacteria are responsible for brain invasion suggests that, after crossing the BBB, brain-resident microglia are infected first. At 3 dpi, several extracellular mycobacteria that had invaded the brain were within the microglia (Figure 1K). As mycobacteria traversed brain blood vessels, we observed microglia migrating toward the my- cobacteria and phagocytosing them, irrespective of whether the bacteria were still partially within the blood vessel (Figure 1L) or had just entered the brain parenchyma (Figures 1M and 1N). Initially, the foci were composed entirely of microglia, resem- bling brain invasion by cryptococcus. 40 Within days, infected and uninfected myeloid cells were recruited to form aggregates (Figures 1O–1Q and S2A–S2J), as expected from previous work. By 3 dpi, monocytes were recruited to aggregates, which we observed using intravenous injection of Hoechst, a nuclear dye that differentiates monocytes (Hoechst-positive) from microglia (Hoechst-negative), both of which were present in the aggre- gates 33 (Figure S2K). Thus, extracellular mycobacteria invading the brain are rapidly phagocytosed by microglia, initiating brain granulomas resembling Rich foci. Mycobacterial attachment and growth on the microvascular endothelium are associated with F-actin rearrangements but not endothelial transcytosis or damage Having established that extracellular mycobacteria are respon- sible for brain invasion, we sought to understand the route that these bacteria use to cross the BBB. Using time-lapse confocal microscopy, we observed that circulating M. marinum occa- sionally became attached to the brain endothelium (Figure 2A). Serial monitoring of individual microcolonies sug- gested that mycobacteria cross the BBB where they initially attached (Figure 2B). Furthermore, the attached microcolonies Figure 2. M. marinum microcolonies grow and recruit endothelial cell F-actin (A and B) Representative confocal images of the same larva brain with green-fluorescent blood vessels, infected with blue-fluorescent M. marinum (Mm). Insets highlight a microcolony inside the vessel at 2 dpi (A), crossing at 3 dpi (B). Scale bars, 10 μm. (C) Volume of attached and crossing Mm microcolonies at 1 dpi. Horizontal bars, means. Representative of 2 independent experiments. (D and E) Representative confocal images of uninfected (D) and infected (E) vessels from flk:GAL4;UAS:LifeAct-GFP larvae at 3 dpi with red-fluorescent Mm. Arrowhead, green-fluorescent F-actin accumulated around the Mm microcolony. Scale bars, 10 μm. (F) Quantification of F-actin in the infected vessel compared to the contralateral uninfected vessel from the same animal at 3 dpi. Horizontal bars, means; paired Student’s t test. Representative of 2 independent experiments. (G and H) Representative confocal images (xy axis) with optical cross sections (yz axis) of green-fluorescent vessels of 3 dpi larvae infected with blue-fluorescent Mm. Mm microcolony in blood vessel lumen, as indicated by the lack of co-localization of blue and green fluorescence. Scale bars, 10 μm (G). Mm inside the blood vessel endothelial cell, indicated by co-localization of blue and green fluorescence. Scale bars, 10 μm (H). (I) Proportion of vessels containing Mm in the lumen that are not crossing (gray), crossing (black), or inside of an endothelial cell (white) at 3 dpi. Representative of 4 independent experiments. 4 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS grew in situ before crossing. For instance, the microcolony shown in Figures 2A and 2B had grown 8.3-fold between 2 dpi, when it was not yet crossing, and 3 dpi, when it was crossing. Overall, the microcolonies in the process of crossing were larger than those that were attached but not crossing (Figure 2C). Thus, to enter the brain, circulating mycobacteria adhere to endothelial cells, replicate in situ to form microcolo- nies, and cross the BBB at the attachment site. This could sug- gest that the in situ growth on the vessel wall facilitates brain in- vasion or that increased microcolony size facilitates crossing, irrespective of the in situ growth. M. tuberculosis adherence to HBMECs was found to trigger actin rearrangements, leading the authors to infer that brain in- vasion occurs via transcytosis through the endothelial cells’ en- docytic route. 27,28 We confirmed that F-actin rearrangements occurred in vivo, using transgenic larvae expressing the GFP- tagged F-actin biosensor, LifeAct, in endothelial cells (flk:GA- L4;UAS:Lifeact-GFP). 41 We observed that brain blood vessels had increased LifeAct-GFP where M. marinum contacted the endothelial cells, compared to blood vessels without bacteria (Figures 2D–2F). However, a few endothelial cells were infected (in 2/39 vessels where they had attached) (Figures 2G–2I), and none of the bacteria crossed the BBB (Figure 2H). Only attached bacteria that had not been internalized crossed the BBB (19 instances), suggesting that transcytosis is not a predominant mechanism of mycobacterial brain invasion (Figure 2I). This apparent discrepancy with the HBMEC findings 27,28 is readily explained by recent studies, showing that the downregu- lation of transcytosis does not occur in vitro but is integral to BBB function in vivo, including in larval zebrafish, and is mediated by pericyte interactions with endothelial cells, which would be ab- sent in HBMECs. 6,29,42 Having ruled out transcytosis, we wanted to determine whether mycobacteria invade the brain using lysis, similar to more common meningeal pathogens, such as Streptococcus pneumoniae and group B Streptococcus. 2,26,43,44 Pathogenic mycobacteria have a membranolytic protein, ESAT-6, secreted by the ESX-1 secretion system, which can lyse cells in a con- tact-dependent manner and is required for full virulence in mac- rophages. 45,46 A previous study found that BBB crossing in larvae with macrophages requires ESX-1, suggesting endothelial cell damage. 22 In contrast, we found that in macrophage- depleted larvae, ESX-1 was dispensable for brain invasion (Figures S3A–S3E). Indeed, wild-type M. marinum did not result in endothelial cell staining by propidium iodide (PI), a cell- impermeant nuclear dye, in any of the 13 crossing events observed across 8 larvae, ruling out endothelial cell damage (Figure S3F). This is in stark contrast to group B Streptococcus, in which PI staining reveals extensive cell lysis. 44 We cannot explain the differences between our ESX-1 findings and those re- ported previously, in that we did not find reduced ESX-1 mutant bacteria in the brain or damage to endothelial cells, which is the proposed mechanism by which ESX-1 promotes BBB crossing. Importantly, M. tuberculosis does not lyse HBMECs, consistent with our findings that mycobacteria do not lyse endothelial cells to cross the BBB. 28 Given their barrier function, BBB endothelial cell membranes may be more resistant to bacterial cytolysins, making them able to resist ESAT-6 and other mycobacterial cy- tolysins, which might have weaker lysis activity than those of streptococci. Mycobacteria dynamically remodel endothelial cell tight junctions to create transient gaps through which they invade the brain paracellularly Having ruled out transcytosis and endothelial cell damage, we investigated whether mycobacteria might cross the BBB para- cellularly, through endothelial cell junctions. Consistent with a paracellular route, we observed small openings—‘‘gaps’’ (mean diameter 3.5 μm)—in the endothelial cell membrane where M. marinum was attached, but not in uninfected vessels (Figures 3A and 3B). Gaps were invariably associated with BBB crossing, as bacteria protruded through the gaps in the vessel (Figures 3A and 3C). We confirmed that these gaps allowed circulating materials to transit the BBB by imaging the brain immediately after injecting 0.02 μm fluorescent beads into the caudal vein of infected and uninfected animals. In infected ani- mals, the beads escaped into the brain, whereas they remained within the vessels of uninfected animals (Figures 3D and 3E). Moreover, the beads entered the brain only in regions where mi- crocolonies were attached (Figures 3D and 3E). Thus, microcol- ony attachment appears to trigger localized gaps in the micro- vascular endothelium through which mycobacteria invade the brain. To investigate the presence of paracellular gaps in more detail, we used transmission electron microscopy (TEM). The TEM of the brain confirmed that most of a crossing microcolony was attached to endothelial cells of the vessel lumen (Figures 3F– 3I), with two bacteria crossing the BBB (Figure 3I, white aster- isks) through an otherwise intact cell junction (Figure 3I, green cells). This confirmed that mycobacteria enter the brain paracell- ularly between intact endothelial cells. BBB function is maintained by multiple interconnected junc- tional complexes that promote adhesion of adjacent endothelial cells, 47 including tight and adherens junctions. 7 To cross the BBB paracellularly, these junctional complexes must undergo remodeling. We first investigated tight junctions, the most apical of these complexes, 47 which also prevent pathogens from crossing the gut epithelium. 48 To test if microcolony attachment alters tight junctions, we used an antibody to zonula occludens-1 (ZO-1), a cytosolic protein that regulates tight junctions. 7 Vessels without microcolonies showed localization of ZO-1 to the cell border, creating a single clean seam defining the tight junction (Figure 4A). In contrast, ZO-1 was dramatically reorganized into ring-like structures that surrounded the gaps where mycobacte- ria protruded into the brain (Figures 4B and 4C; Video S1). We were struck by the presence of vessel gaps, given the impor- tance of BBB integrity. Long-term time-lapse imaging resolved this apparent discrepancy, revealing that the gaps were tran- sient, closing minutes after forming. During 7 h of video moni- toring with images captured every 5 min, 27 gaps formed and closed (Figures 4D–4F; Video S2). The majority of the gaps (63%) closed within 5 min of forming and 33.3% within 30 min, with a single outlier (3.7%) remaining open for 55 min (Figure 4F). Open gaps allowed individual mycobacteria or microcolonies to protrude into the brain (Figures 4D and 4E; Video S2). Tight Cell Reports 44, 116661, December 23, 2025 5 Article ll OPEN ACCESS junction and adherens junction reorganization go hand in hand, owing to their close proximity and interconnectedness. To confirm that attached microcolonies reorganize adherens junc- tions, we imaged α-catenin, which stabilizes the junction and binds F-actin. 49 Using larvae expressing α-catenin-GFP in endo- thelial cells (flk1:α-catenin-eGFP), 41 we found that vessels with microcolonies showed a 7.5-fold increase in GFP puncta, sug- gesting active reorganization of α-catenin (Figures 4G and 4H). Vessels without a microcolony showed a mostly uniform distribu- tion of sparse α-catenin throughout the cell (Figure 4G). In sum, attached M. marinum microcolonies dynamically remodel both tight and adherens junctions, creating transient tight junction gaps in endothelial cells, through which some bacteria enter the brain. Mycobacterial PknD promotes brain invasion by promoting F-actin polymerization and attachment but does not cause tight junction remodeling We next wanted to understand the mycobacterial factors that are important for endothelial attachment and tight junction A D E F G IH CB Figure 3. M. marinum crosses through permeable gaps in brain blood vessels (A) Representative confocal image (top) and 3D rendering (bottom) of an infected brain blood vessel near an uninfected vessel, from a 3 dpi larva with red- fluorescent blood vessels (pseudo-colored green) infected with blue-fluorescent M. marinum (Mm). The dashed circle indicates the border of the gap underneath the microcolony in the vessel. Scale bars, 10 μm. (B) Maximum diameter of vessel gaps formed by Mm microcolonies from larvae in (A). Horizontal bar, mean. Representative of 4 independent experiments. (C) Proportion of attached or crossing Mm microcolonies associated with blood vessel gaps. Fisher’s exact test. Representative of 2 independent experiments. (D) Representative confocal images of uninfected (top) or infected (bottom) green-fluorescent brain blood vessels from 3 dpi larvae infected with blue-fluorescent Mm and injected intravenously with far red-fluorescent 0.02 μm latex beads (pseudo-colored magenta) just prior to imaging. Scale bars, 10 μm. The inset shows beads leaking from the infected vessel (arrowhead). Scale bar, 5 μm. (E) Proportion of vessels with retained beads or those that escaped into the brain in uninfected and infected vessels from the experiment in (D). Fisher’s exact test. Representative of 2 independent experiments. (F and G) Brain confocal images of a 3 dpi larva with green-fluorescent vessels infected with red-fluorescent Mm, which were used to identify a crossing mi- crocolony for transmission electron microscopy (TEM) in (H and I). The dotted box in (F), magnified in (G), indicates the area imaged by TEM. (H and I) TEM showing the microcolony (pseudo-colored red) primarily in the vessel lumen (pseudo-colored light green) with endothelial cells (pseudo-colored dark green). Scale bar, 10 μm (H). Two Mm bacilli (white asterisks) crossing between two endothelial cells (pseudo-colored green). Arrowhead, Mm that has fully crossed into the brain. Scale bar, 1 μm (I). 6 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS A D E F G H CB Figure 4. M. marinum reorganizes endothelial cell junctions to cross paracellularly (A and B) Representative confocal images of uninfected (A) and infected vessels (B) from 3 dpi larvae with green-fluorescent vessels infected with red-fluorescent M. marinum (Mm), fixed and stained with anti-ZO-1 antibody (pseudo-colored magenta). Bottom panels: 3D rendered versions of the top panels. In (B), 3D renderings show a gap (dashed circle), ringed by ZO-1 under Mm microcolony. Yellow indicates parts of the microcolony that have exited the vasculature and entered the brain. Scale bars, 10 μm. (C) Proportion of gaps associated with a complete, incomplete, or no ZO-1 ring for Mm microcolonies that are crossing blood vessels. Representative of 2 independent experiments. (D) 3D rendered images of red-fluorescent brain blood vessel (pseudo-colored green) in a 4 dpi larva infected with blue-fluorescent Mm, which is protruding from a gap, taken from Video S2. Scale bars, 10 μm. Boxed area is magnified in the bottom panel. Scale bars, 5 μm. (E) Sequential 3D rendered images from Video S2, showing gaps (outlined) forming and resealing near an Mm microcolony in a brain blood vessel. Time (t), h:min after the start of time-lapse video recording. Scale bars, 10 μm. (F) Histogram of frequency of duration of all gaps forming and closing in Video S2. The x axis details the 5-min time windows (e.g., 5: 0–5 min, 30: 26–30 min). (G) Representative confocal images of uninfected (top) and infected vessels (bottom) from a 3 dpi larva with green-fluorescent α-catenin, infected with blue- fluorescent Mm. Arrowheads indicate punctate α-catenin signal. Scale bars, 10 μm. (H) Total α-catenin puncta in uninfected and infected vessels. Horizontal bars, means; paired Student’s t test. Cell Reports 44, 116661, December 23, 2025 7 Article ll OPEN ACCESS A D E F G I J K L H M O P N CB Figure 5. PknD contributes to M. marinum attachment, but not crossing through vessel gaps (A) Representative confocal images of the green-fluorescent brain vasculature in 1 dpi larvae infected with ∼1000 CFU red-fluorescent wild-type (WT) M. marinum (Mm) (left), or ∼3000 CFU red-fluorescent pknD::Tn Mm (right). Scale bars, 10 μm. (B) Total WT and pknD::Tn Mm microcolonies in the brain vasculature (attached and crossing). Horizontal bars, means; Student’s t test. (C) WT or pknD::Tn Mm burden per larva at 1 dpi quantified by fluorescent pixel counts (FPC) from the experiment in (A). Horizontal bars, means; Student’s t test; ns, not significant. (D) Total WT and pknD::Tn:pknD Mm microcolonies in the brain vasculature (attached and crossing). Horizontal bars, means; Student’s t test; ns, not significant. (E) WT and pknD::Tn:pknD Mm burden per larva at 1 dpi quantified by FPC. Horizontal bars, means; ns, not significant; Student’s t test. (F) Representative confocal images of red-fluorescent pknD::Tn Mm-infected vessels from flk:GAL4;UAS:LifeAct-GFP larvae at 1 dpi. Scale bar, 10 μm. (legend continued on next page) 8 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS remodeling. The M. tuberculosis serine/threonine kinase PknD was previously identified in a transposon mutant screen, which found that a PknD mutant had reduced dissemination to the brain in guinea pigs. 27 In HBMECs, the M. tuberculosis PknD mutant displayed decreased invasion, and the authors inferred that transcytosis was occurring. 27 Given our findings that M. marinum predominantly invades paracellularly, we hy- pothesized that, in vivo, PknD must promote F-actin rear- rangements, which increase attachment to endothelial cells, tight junction remodeling, and gap formation. Compared to wild-type, we found fewer M. marinum PknD mutant bacteria attached to the brain microvasculature (Figures 5A–5C) and the absence of F-actin rearrangements (Figures 5F–5H), despite similar overall bacterial burdens. Complementing the PknD mutant with M. marinum PknD (Figures 5D and 5E, 5H, and 5J) rescued these defects, confirming that M. mari- num PknD promotes brain invasion in zebrafish, similar to M. tuberculosis in guinea pigs. 27 In addition to promoting attachment, if PknD-mediated F-actin rearrangements also caused cell junction remodeling to create gaps, then the PknD mutant should exhibit a crossing defect over and above its attachment defect. However, the PknD mutant, once attached, did not have a crossing defect. In fact, it crossed more often than the wild type (62% vs. 47%) (Figure 5I). Complemented PknD microcolonies crossed at a similar ratio to wild type, showing that this increased crossing was specifically caused by the absence of PknD (Figure 5J). Thus, PknD promotes brain invasion by increasing attachment but not crossing, which it slightly deters. Furthermore, the PknD mutant, complement, and wild type crossed through ZO-1- ringed gaps of similar sizes (Figures 5K–5O). However, the dy- namics of gap formation differed, with PknD mutant gaps remain- ing open much longer than wild type (Figure 5P, mean 45.8 min compared to 11.5 min), perhaps due to the lack of F-actin accu- mulation (compare Figures 2E and 5K). This suggests that F-actin accumulation is associated with gaps closing more quickly for the wild type and less quickly for the PknD mutant, allowing bacteria to cross more easily. Our findings are consistent with the appre- ciation that tight junction permeability depends on optimal inter- action with F-actin, as both too weak and too strong associations diminish tight junction integrity. 50 Thus, the F-actin rearrange- ments that aid attachment paradoxically counteract the tight junction reorganization required for crossing. In sum, PknD promotes brain invasion by inducing F-actin re- arrangements that help attach mycobacteria to endothelial cells. Importantly, our findings show that attachment and junctional remodeling are mediated by distinct mycobacterial functions. Cell surface determinants shared with nonpathogenic mycobacteria remodel junctions Our findings suggested a model where remodeling is promoted by a surface-exposed or secreted factor from mycobacteria attached to the endothelium. To determine the importance of these factors, we used M. marinum killed by γ-irradiation, which are structurally intact but incapable of active protein secretion. Given that killed bacteria are rapidly destroyed by macrophages, we used macrophage-depleted larvae infected with similar numbers of live or γ-irradiated M. marinum and found equivalent brain infection (Figures S4A–S4C). We found that small clumps of γ-irradiated M. marinum attached to the microvasculature, crossed as efficiently as live microcolonies (Figure S4D), and were of a similar size (mean volumes, live 181 μm 3 and γ-irradiated 151 μm 3 ). Like live M. marinum, crossing clumps were larger than attached clumps (mean volumes, crossing 386 μm 3 and attached 64 μm 3 ), supporting the hypothesis that microcolony size increases crossing (Figure S4E). Similar to live bacteria, attachment was associated with F-actin recruitment (Figures S4F and S4G) followed by crossing through ZO-1-ringed gaps (mean diameter 4.8 μm) (Figures S4H–S4K). Thus, myco- bacterial surface determinants, rather than active protein secre- tion, cause tight junction remodeling. While both pathogenic and environmental mycobacterial spe- cies share conserved, complex cell walls, pathogenic species have some specific surface-associated determinants. Phthio- cerol dimycoceroserate (PDIM) was an attractive candidate as it perturbs epithelial cell membranes. 51,52 However, when we tested Mycobacterium smegmatis, the prototypical nonpatho- genic, environmental mycobacterium that lacks PDIM, 37 we found that it behaved identically to M. marinum, with equivalent brain bacterial burden and similar numbers of microcolonies at- taching to and crossing the BBB (Figures S4L–S4O). The attach- ment was associated with F-actin rearrangements (Figures S4P and S4Q), and crossing invariably occurred through gaps sur- rounded by ZO-1 (Figures S4R and S4S). These findings led to the conclusion that junctional remodeling is induced by one or (G and H) Quantification of F-actin in WT and pknD::Tn Mm infected vessels (G), or uninfected and pknD::Tn:pknD Mm. Horizontal bars, means; Student’s t test. Representative of 2 independent experiments. (I) Proportion of attached or crossing WT and pknD::Tn Mm microcolonies (excluding those in the brain) in blood vessels from larvae in (A); Fischer’s exact test. Representative of 2 independent experiments. (J) Proportion of attached or crossing WT and pknD::Tn:pknD Mm microcolonies (excluding microcolonies in the brain) in blood vessels. Fischer’s exact test; ns, not significant. (K) Representative confocal image (top) and 3D rendering (bottom) of a 2 dpi larva with green-fluorescent blood vessels infected with red-fluorescent pknD::Tn Mm. Dashed circle indicates the border of the gap underlying the microcolony in the vessel. Scale bar, 10 μm. (L and M) Maximum diameter of vessel gaps formed underneath Mm microcolonies infected with WT or pknD::Tn Mm (L), or pknD::Tn:pknD Mm (M). Horizontal bars, means; ns, not significant; Student’s t test. Representative of 2 independent experiments. (N) Proportion of gaps associated with a complete, incomplete, or no ZO-1 ring for pknD::Tn Mm microcolonies that are crossing blood vessels. (O) 3D rendered, representative confocal image from a 1 dpi larva with green-fluorescent vessels infected with red-fluorescent pknD::Tn Mm, then fixed and stained with anti-ZO-1 antibody (pseudo-colored magenta). Dashed circle, gap ringed by ZO-1 under pknD::Tn Mm microcolony. Yellow indicates parts of the microcolony that have exited the vasculature and entered the brain. Scale bars, 10 μm. (P) Length of time (min) that gaps remain open in the green-fluorescent vessels of a larva infected with WT or pknD::Tn Mm. Error bars, means; Student’s t test. Cell Reports 44, 116661, December 23, 2025 9 Article ll OPEN ACCESS A D E F G I J K L H M O P Q R S T U V N CB Figure 6. TDM recognition induces junctional remodeling (A) Representative confocal images of green-fluorescent brain vasculature in 2 dpi larvae infected with ∼1000 CFU wild-type (WT) M. marinum (Mm) (left), or ∼1,500 CFU pcaA::Tn Mm (right). Scale bars, 10 μm. (B) Total WT and pcaA::Tn Mm microcolonies in the brain vasculature (attached and crossing microcolonies). Horizontal bars, means; Student’s t test. Repre- sentative of 2 independent experiments. (legend continued on next page) 10 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS more surface factors shared between pathogenic (M. tubercu- losis and M. marinum) and saprophytic (M. smegmatis) mycobacteria. Cyclopropanated TDM promotes invasion by increasing attachment and remodeling junctions Another strong candidate was TDM, an abundant outer cell wall glycolipid that is present in M. tuberculosis, M. marinum, and M. smegmatis. 53–55 Cyclopropanated TDM is important for the characteristic cording morphology of mycobacteria, which has been recently linked to the ability of M. tuberculosis to penetrate between alveolar epithelial cells in vitro. 53,56 Independent of cording, cyclopropanated TDM engages several eukaryotic signaling pathways, 56–59 such as VEGF-mediated angiogenesis in zebrafish. 54 We tested an M. marinum transposon mutant in PcaA, the cyclopropane synthase that cis-cyclopropanates TDM at the proximal position. 53,54 Despite equivalent overall bacterial burden to wild type, the PcaA mutant had reduced brain dissemination (Figures 6A–6C), which was reversed by complementation (Figures 6D and 6E). Similar to the PknD mutant, attachment by the PcaA mutant was not associated with F-actin rearrangements, indicating an attachment defect (Figure 6F). However, in contrast to the PknD mutant, the PcaA mutant exhibited a crossing defect over and above its attachment defect. Fewer microcolonies crossed the BBB (Figure 6G), which was reversed by complementation (Figure 6H). Thus, while PknD promotes attachment, PcaA promotes both attachment and crossing. Strikingly, only 33% of PcaA mutant microcolonies crossed through obvious gaps, compared to nearly all in wild type and the PknD mutant (Figures 6I and 6J). While most PcaA mutant microcolonies protruded into the brain without an obvious gap (Figure 6K, compare top and bottom panels), they were still likely crossing between junctions rather than by transcytosis, as the bacteria emerging from the vessel had no endothelial membrane (green fluorescence) surrounding them (Figure 6K, ar- rowheads). Furthermore, as with wild-type bacteria, only a minor- ity (5%) of PcaA mutant microcolonies were found inside endo- thelial cells, none of which were crossing (Figure 6L). Finally, PcaA mutants crossing through gaps often had absent or incom- plete ZO-1 rings, which were complete in only 17% of instances, compared to 60% for wild type or complement (Figures 6M–6Q). PcaA mutant microcolonies did not have the corded growth phenotype seen in wild-type bacteria (Figures S5A–S5C). To clarify if the attachment and crossing defects of the PcaA mutant were due to TDM or its defect in cording, we tested brain inva- sion by Erp mutant M. marinum, which has functional TDM but deficient cording. 60 The Erp mutant attached to the brain micro- vasculature and accumulated F-actin as frequently as wild type in equivalently infected larvae (Figures S5D–S5F). The majority of these microcolonies crossed from the vessel lumen through gaps without infecting endothelial cells (Figures S5G–S5I). Like wild type, the majority of gaps were associated with a ZO-1 ring (Figures S5J and S5K). These findings suggest that cording does not contribute to attachment, F-actin rearrangements, or remodeling of ZO-1-ringed gaps. In sum, cyclopropanated TDM is necessary for brain micro- vascular endothelial F-actin rearrangements that increase myco- bacterial attachment as well as promote tight junction remodel- ing manifested by openings in the endothelium surrounded by ZO-1. In its absence, individual members of attached microcol- onies can still cross by squeezing between the junctions. How- ever, the overall reduction in brain invasion in the PcaA mutant suggests that this process is not as efficient as when the junc- tions can be remodeled to create gaps. (C) WT or pcaA::Tn Mm burden per larva at 2 dpi quantified by fluorescent pixel counts (FPC) from the experiment in (A). Horizontal bars, means; ns, not sig- nificant; Student’s t test. Representative of 2 independent experiments. (D) Total WT and pcaA::Tn::pcaA Mm microcolonies in the brain vasculature (attached and crossing microcolonies). Horizontal bars, means; Student’s t test. (E) WT or pcaA::Tn::pcaA Mm burden per larva at 2 dpi quantified by FPC. Horizontal bars, means. (F) Representative confocal images of red-fluorescent pcaA::Tn Mm-infected vessels from flk:GAL4;UAS:LifeAct-GFP larvae at 2 dpi. Scale bars, 10 μm. (G) Proportion of attached or crossing WT and pcaA::Tn Mm microcolonies (excluding microcolonies in the brain) in brain blood vessels from larvae in (A). Fisher’s exact test. Representative of 2 independent experiments. (H) Proportion of attached or crossing WT and pcaA::Tn::pcaA Mm microcolonies (excluding microcolonies in the brain) in blood vessels. Fisher’s exact test; ns, not significant. (I) Proportion of vessels with or without gaps in pcaA::Tn Mm infected vessels. Representative of 2 independent experiments. (J) Proportion of vessels associated with gaps in pcaA::Tn:pcaA Mm infected vessels. (K) 3D rendered, representative confocal image of 3 dpi larvae with green-fluorescent blood vessels, infected with red-fluorescent pcaA::Tn Mm. Top, pcaA::Tn Mm microcolony crossing blood vessel without apparent gap (arrowheads). Bottom, pcaA::Tn Mm microcolony crossing through a gap. Scale bars, 10 μm. (L) Proportion of vessels containing pcaA::Tn Mm that are attached, crossing, or inside of an endothelial cell. (M) Proportion of gaps associated with a complete, incomplete, or no ZO-1 ring for pcaA::Tn Mm microcolonies that are crossing blood vessels. (N) Proportion of WT or pcaA::Tn Mm associated gaps associated with a complete or incomplete/no ZO-1 ring. WT data from Figure 4C; Fisher’s exact test. (O) Proportion of gaps associated with a complete, incomplete, or no ZO-1 ring for pcaA::Tn::pcaA Mm microcolonies that are crossing blood vessels. (P and Q) 3D rendered, representative confocal images from a 3 dpi larva with green-fluorescent vessels infected with red-fluorescent pcaA::Tn Mm, fixed and stained with anti-ZO-1 antibody (pseudo-colored magenta). pcaA::Tn Mm microcolony without associated gap or ZO-1 ring (P). pcaA::Tn Mm microcolony with associated gap, partially ringed by ZO-1 (Q). Yellow indicates parts of the microcolony that have exited the vasculature and entered the brain. Scale bars, 10 μm. (R) Total Mm microcolonies in the brain vasculature (attached and crossing microcolonies) in WT (scramble) and Mincle crispant larvae. Horizontal bars, means; Student’s t test. Representative of 2 independent experiments. (S) WT or pcaA::Tn Mm burden per larva at 2 dpi quantified by FPC from experiment in (R). Horizontal bars, means; ns, not significant; Student’s t test. Representative of 2 independent experiments. (T) Proportion of attached or crossing Mm microcolonies (excluding microcolonies in the brain) in blood vessels from WT or Mincle crispant larvae. Fisher’s exact test; ns, not significant. Representative of 2 independent experiments. (U) Proportion of vessels associated with blood vessel gaps in Mm-infected vessels from Mincle crispant larvae. Representative of 2 independent experiments. (V) Proportion of gaps associated with a complete, incomplete, or no ZO-1 ring for microcolonies that are crossing blood vessels in Mincle crispant larvae. Cell Reports 44, 116661, December 23, 2025 11 Article ll OPEN ACCESS TDM is a potent immunostimulatory glycolipid that is recog- nized by the C-type lectin, Mincle, expressed on the surface of myeloid cells. 61 Mice deficient for Mincle have significantly diminished macrophage activation and fail to form TDM-induced lung granulomas. 57 Mincle is also expressed on human and mouse brain endothelial cells. 62,63 Therefore, we surmised that TDM recognition by Mincle expressed by endothelial cells could be involved in mycobacterial brain invasion. To test this, we generated G0 Mincle crispants. The intravenous injection of M. marinum produced an infection phenotype similar to PcaA mutant infection in wild-type larvae, even with comparable over- all bacterial burdens. Specifically, we observed reduced dissem- ination to the brain (Figures 6R and 6S), impaired gap-associated crossing of the brain microvasculature (compare Figures 3C; 6T, 6U, and S6A), and absent or incomplete ZO-1 rings surrounding the gaps that formed (Figures 6V, S6B, and S6C). Thus, TDM recognition by Mincle interactions facilitates gap formation and crossing of the brain microvasculature. M. tuberculosis invades the brain via attachment and junctional remodeling, with conserved roles for PknD and PcaA To see if M. tuberculosis also invades the brain paracellularly, we used a fluorescently labeled double leucine and pantothenic acid auxotrophic strain of M. tuberculosis, mc 2 6206, which can be safely handled in our biosafety level 2 microscopy suite. 64 In- jection of M. tuberculosis mc 2 6206 intravenously into macro- phage-depleted larvae showed that it appeared in the brain microvasculature within days (Figure 7A). Similar to M. marinum, attachment of pre-existing clumps was associated with F-actin recruitment (Figure 7B). Neither internalization nor transcytosis was observed for 23 attached clumps, of which 12 were in the process of crossing. Instead, as with M. marinum, crossing occurred through ZO-1-ringed gaps of a similar size (mean diameter 6.6 μm) and morphology to those with M. marinum (Figures 7C and 7D). Next, to verify if M. tuberculosis uses PknD and PcaA for attachment and crossing, we tested the corresponding M. tuber- culosis mutants. Like their M. marinum counterparts, the M. tuberculosis PknD and PcaA mutants both had reduced attach- ment to the brain microvasculature and failed to mediate F-actin rearrangements (Figures 7E–7J). Consistent with M. marinum, the M. tuberculosis PknD mutant only had an attachment defect, whereas the PcaA mutant had both an attachment and crossing defect. Among attached clumps, the M. tuberculosis PknD mutant crossed slightly better, similar to M. marinum; in contrast, the attached PcaA clumps crossed less frequently (Figures 7K and 7L). Consistent with these phenotypes, the PknD mutant crossed through ZO-1-ringed gaps (Figures 7M and 7N), while few PcaA mutant crossings were associated with gaps (2/23) (Figure 7O). Thus, M. tuberculosis invades the brain using the same paracellular mechanism identified for M. marinum. M. smegmatis PcaA is required for junctional remodeling The finding that a cell surface lipid shared between M. marinum and M. smegmatis had brought us to the discovery that cyclo- propanated TDM disrupts tight junctions to enable mycobacte- rial brain invasion. This suggests that M. smegmatis also uses PcaA to cyclopropanate TDM and mediate junctional reorgani- zation. The M. smegmatis PcaA homolog (MSMEG_1351) has been shown to cis-cyclopropanate α-mycolic acids and restore both α-mycolic acid cis-cyclopropanation and cording in an M. bovis BCG PcaA mutant. 55 To test this, we created an M. smeg- matis PcaA mutant. M. smegmatis does not exhibit as strong cording as M. marinum and M. tuberculosis; the M. smegmatis PcaA mutant nevertheless showed the expected reduction in cording (Figure S5C). Like the M. marinum and M. tuberculosis PcaA mutant strains, the M. smegmatis PcaA mutant had reduced numbers in the brain microvasculature, decreased inva- sion, and attached colonies without F-actin rearrangements (Figures S7A–S7D). Crossing was reduced and was not associ- ated with tight junction openings (Figures S7E–S7F). Rather, the bacteria often crossed through imperceptible gaps in the tight junctions that did not have ZO-1 rings (Figures S7G–S7I). Thus, PcaA can mediate brain microvasculature traversal in both path- ogens and saprophytes. DISCUSSION The use of time-lapse microscopy in the transparent zebrafish larva has provided sequential, granular details of the very first and most elusive step of TB meningitis, how mycobacteria invade the brain. Our work brings into question the long-stand- ing dogma of the ‘‘Trojan horse’’ model that macrophages carry mycobacteria into the brain vasculature and into the brain. Instead, free mycobacteria enter the brain microvasculature where they attach to endothelial membranes, by inducing endo- thelial cell F-actin rearrangements and then invade by inducing dynamic junctional reorganization that results in transient gaps through which the bacteria enter the brain. The two mycobacte- rial mutants—PknD and PcaA—enable separation of the distinct roles of the F-actin rearrangements and junctional reorganiza- tion in the invasion process. TDM is required for both attachment and crossing through its receptor Mincle, which is associated with F-actin rearrangements and tight junction reorganization. In contrast, PknD is required only for F-actin rearrangements and attachment, but is dispensable for the reorganization of tight junctions. Those PknD mutant microcolonies that do manage to attach in the absence of F-actin rearrangements can remodel junctions and cross through the ensuing gaps. Thus, mycobac- teria mediate tight junction reorganization independently of actin cytoskeleton rearrangements, whereas these are linked under homeostatic conditions. 65 Indeed, our findings with the PknD mutant show that F-actin cytoskeletal rearrangements can impede crossing. Although we saw a dramatic rearrangement of ZO-1, we cannot exclude that TDM’s direct interactions are with other or additional proteins, either from among the tight junction complex that in turn causes ZO-1 reorganization or from other linked junctional complexes, such as the adherens junctions. 65 Another unresolved issue is how cyclopropanated TDM reorga- nizes junctions. One possibility is that it mediates the cording morphology that causes physical disruption of junctions, as has been recently proposed. 56 A second is that it acts as a signaling molecule on one or more tight junction proteins, which 12 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS A B C D E F G H I J K L M N O Figure 7. M. tuberculosis crosses the blood-brain barrier through ZO-1 rings (A) Representative confocal image of the green-fluorescent brain vasculature in a 3 dpi larvas infected with ∼100 CFU red-fluorescent mc 2 6206 M. tuberculosis (Mtb). White arrowheads, Mtb in blood vessels; orange arrowheads, Mtb in brain parenchyma. Scale bar, 10 μm. (B) Representative confocal image of infected vessels from a flk:GAL4;UAS:LifeAct-GFP larva infected at 3 dpi with ∼300 CFU red-fluorescent Mtb, showing green-fluorescent F-actin accumulation around the micro-clump. Scale bar, 10 μm. (C) 3D rendered, representative confocal image of a 3 dpi larva with green-fluorescent vessels infected with ∼300 CFU red-fluorescent Mtb, fixed, and stained with anti-ZO-1 antibody (pseudo-colored magenta). Dashed circle, gap ringed by ZO-1 under Mtb micro-clump. Yellow indicates parts of the micro-clump that have exited the vasculature and entered the brain. Scale bar, 10 μm. (D) Proportion of gaps associated with a complete, incomplete, or no ZO-1 ring for Mtb micro-clumps that are crossing blood vessels. (E) Total WT and ΔpknD Mtb micro-clumps in the brain vasculature (attached and crossing micro-clumps). Horizontal bars, means; Student’s t test. (F) WT or ΔpknD Mtb burden per larva at 3 dpi quantified by fluorescent pixel counts (FPC) infected with ∼300 CFU. Horizontal bars, means; Student’s t test; ns, not significant. (G) Total WT and ΔpcaA Mtb micro-clumps in the brain vasculature (attached and crossing). Horizontal bars, means; Student’s t test. (H) WT or ΔpcaA Mtb burden per larva at 3 dpi quantified by FPC infected with ∼300 CFU. Horizontal bars, means; Student’s t test; ns, not significant. (I) Representative confocal image of a red-fluorescent ΔpknD Mtb-infected vessel from flk:GAL4;UAS:LifeAct-GFP larvae at 4 dpi. Scale bar, 10 μm. (J) Representative confocal image of a red-fluorescent ΔpcaA Mtb-infected vessel from flk:GAL4;UAS:LifeAct-GFP larvae at 4 dpi. Scale bar, 10 μm. (K and L) Proportion of attached or crossing WT and ΔpknD Mtb (K) or WT and ΔpcaA Mtb (L) micro-clumps (excluding micro-clumps in the brain) in brain blood vessels. Fischer’s exact test. (M) Proportion of infected vessels associated with gaps in ΔpknD Mtb-infected larvae. (N) Proportion of gaps associated with a complete, incomplete, or no ZO-1 ring for ΔpcaA Mtb clumps that are crossing blood vessels. (O) Proportion of infected vessels associated with gaps in ΔpcaA Mtb-infected larvae. Cell Reports 44, 116661, December 23, 2025 13 Article ll OPEN ACCESS would be consistent with its role in signaling in a variety of eu- karyotic processes. 56–59 Cording and signaling could work in concert, with the corded morphology apposing bacterial micro- colonies and the endothelium, to optimize signaling. Our find- ings that γ-irradiated M. marinum, Δerp M. marinum, and M. smegmatis have less corded morphology than wild-type M. marinum and M. tuberculosis, which cross through ZO-1-ringed membrane disruptions, suggest that cyclopropanated TDM- mediated signaling contributes to tight junction reorganization, over and above contributing to cording. In previous work using HBMECs to study the role of PknD, the authors, upon observing PknD-mediated actin polymeriza- tion, attachment, and internalization, reasonably surmised that PknD promotes bacterial transcytosis. 27 Subsequently pub- lished findings explain the discrepancy with our findings— transcytosis is greatly downregulated in vivo through interac- tions of the BBB endothelium with pericytes in the brain, including in the zebrafish larvae. 8,29,66,67 PknD has been pro- posed to bind endothelial cell laminin α2 through its sensor domain, 27 but it can also phosphorylate proteins involved in cell wall transport through its kinase domain. 68 The association of laminin α2 with the basolateral rather than luminal surface of blood vessels rules out that it uses this interaction for tight bind- ing. Rather, PknD likely modifies the mycobacterial cell surface to promote attachment. That TDM, another cell surface modi- fier, is also necessary for F-actin rearrangements and tight attachment supports this model. Our finding that mycobacteria cross in vivo predominantly by paracellular transit is striking, as among other meningeal patho- gens, only N. meningitidis has a predominantly paracellular mechanism. 1,9 Similar to our observations for mycobacteria, N. meningitidis also adheres to the endothelium and forms mi- crocolonies. However, the mode of tight junction disruption ap- pears to be distinct for the two pathogens. The attached N. meningitidis microcolony recruits both cellular actin and multi- ple junctional proteins to it. 31,69 This sequestration of junctional proteins away from the junctions makes the junctions leaky. 31,69,70 Indeed, our finding that the PknD mutant remodels tight junctions without causing F-actin rearrangements demon- strates that mycobacteria have a distinct mechanism. Another unique feature of mycobacteria’s paracellular transit is that it occurs through dynamic junctional remodeling, creating only transient gaps that seal quickly. This finding explains how TB meningitis is a two-step event with mycobacterial invasion into the brain first causing granuloma formation and meningitis occurring only months later, if and when these granulomas erode into the meninges. The host can be asymptomatic in the inter- vening period. A mystery has been how mycobacteria could invade the brain in the first place without causing consequential BBB disruption that would be clinically apparent. Our finding that the disruptions are transient, allowing mycobacteria to invade while leaving the BBB intact, provides the answer. Furthermore, we find that the invading mycobacteria initiate Rich foci by attracting and infecting microglia. This again ad- dresses the two-step model, as to how tuberculous granulomas might form in the brain in the first place, without causing too much inflammation. It uses the already available macrophages in the brain. Like many important mycobacterial virulence factors, cyclo- propanated TDM is present in nonpathogenic mycobacteria, where it likely provides environmental protection by forming the bacteria into multicellular communities. 55 In vivo work has linked mycobacterial cording to increased mycobacterial growth by in- hibiting re-phagocytosis into macrophages, which can be growth restricting. 71 It is striking that M. tuberculosis invades the brain us- ing this virulence determinant shared with nonpathogens. From an evolutionary standpoint, this is not surprising since meningitis caused by any bacterium is an accidental dead end, providing no benefit to the bacterium in terms of transmission and thereby evolutionary survival. This is also the case for M. tuberculosis, where only the pulmonary form is transmissible. Thus, the bacte- rial factors that cause meningitis in all cases have evolved for other purposes; in the case of meningitis caused by commensal pathogens, these have been described as colonization factors that run amok. 37 Our work extends this paradigm to the obligate pathogen M. tuberculosis to show that determinants that clearly evolved for environmental survival are responsible for the dead- liest form of disease caused by humanity’s greatest killer. 20 Limitations of the study Our study demonstrates the mechanism by which mycobacteria breach the BBB in zebrafish larvae. While it has been demon- strated previously that M. marinum causes a TB-like infection in zebrafish, we recognize that mycobacterial pathogenesis in a mammalian system may present differently. However, there are several factors that support the translatability of zebrafish infection models to human or mammalian disease: (1) ∼70% of human genes have known zebrafish orthologs; (2) the innate im- mune responses are similar between humans and zebrafish; (3) granulomas in zebrafish resemble those found in human TB; (4) lta4h is a susceptibility locus for M. marinum disease in zebrafish, as it is for TB in humans, and (5) both PknD and PcaA are impor- tant for mycobacterial virulence in mammals, and PknD for brain infection. Nevertheless, future study is required to confirm if my- cobacteria use the Mincle-TDM pathway to disrupt endothelial junctions to cross into the brain in mammals. In addition to dissecting the mechanism of M. marinum brain in- vasion in zebrafish, we confirmed our findings with M. tubercu- losis, the causative agent of tuberculous meningitis in humans. For biosafety requirements, we utilized the double auxotrophic M. tuberculosis strain, mc 2 6206, instead of the more virulent parent strain, H37Rv. The mc 2 6206 and the H37Rv strains have been shown to behave similarly in several ways, displaying similar growth rates, both in vitro and within macrophages, and respond- ing similarly to anti-TB agents. However, mc 2 6206 has also been shown to display an increased stress response and can induce higher cytokine and chemokine responses in immune cells compared to H37Rv. Therefore, how the H37Rv M. tuberculosis strain behaves in this system remains to be seen. RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Cressida A. Madigan (cmadigan@ucsd.edu). 14 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS mailto:cmadigan@ucsd.edu Materials availability Zebrafish lines and bacterial strains generated in this study are available from the lead contact. Data and code availability • All data reported in this paper will be shared by the lead contact upon request. • This paper does not report original code. • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. ACKNOWLEDGMENTS We thank P. Edelstein for discussion and guidance throughout the project and advice and help with statistical analysis; N. Yamaguchi, R.K. Rao, B. Cormack, M. Troll, and J. Bö hning for discussion; P. Edelstein and N. Yamaguchi for critical review of the paper; the University of California, San Diego – Cellular and Molecular Medicine Electron Microscopy Core (UCSD-CMM-EM Core, RRID: SCR_022039) for sample preparation and technical assistance; M. Reitano and the University of California, San Diego aquatics facility staff, J. Cameron, and the University of Washington aquatics facility staff for zebrafish husbandry; and E. Chiang from the MRC Laboratory of Molecular Biology VisLab for designing and making the graphical abstract. This study was funded by the following grants: National Institutes of Health MERIT award R37 AI054503 (L.R.), National Institutes of Health Pioneer award (L.R.), National Institutes of Health R01AI054503 (L.R.), Wellcome Trust Principal Research Fellowship 223103/Z/21/Z and 223103/Z/21/Z (L.R.), National Institutes of Health grant F32 AI104240-02 (C.A.M.), Pew Biomedical Scholars Program grant 2021-A-17088 (C.A.M.), National Insti- tutes of Health Director’s New Innovator award 1DP2NS127277 (C.A.M.), Na- tional Institutes of Health Grant T32 Cell and Molecular Genetics Graduate Training Grant Fellowship 5T32GM007240-43 (S.R.), National Institutes of Health Grant T32 Cell and Molecular Genetics Graduate Training Grant Fellowship 5T32GM007240-42 (M.I.H.), and National Science Foundation Graduate Research Fellowship Program grant no. DGE-2038238 (M.I.H.). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. AUTHOR CONTRIBUTIONS Conceptualization, L.R. and C.A.M.; supervision, L.R. and C.A.M.; methodol- ogy, M.I.H., S.R., J.K.S., A.J.F., L.R., and C.A.M.; investigation, M.I.H., S.R., J.K.S., A.J.F., L.R., and C.A.M.; formal analysis, M.I.H., S.R., L.R., and C.A.M.; project administration, L.R. and C.A.M.; funding acquisition, M.I.H., S.R., L.R., and C.A.M.; visualization, M.I.H., S.R., C.A.M., and L.R.; writing – original draft, M.I.H., S.R., L.R., and C.A.M.; writing – review and editing, M.I.H., S.R., J.K.S., A.J.F., L.R., and C.A.M. DECLARATION OF INTERESTS The authors declare that they have no competing interests. STAR★METHODS Detailed methods are provided in the online version of this paper and include the following: • KEY RESOURCES TABLE • EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS ○ Zebrafish husbandry and infections • METHOD DETAILS ○ Bacterial strains ○ Plasmid construction ○ Monocyte depletion, fluorospheres, and stains ○ α-ZO-1 whole mount immunofluorescence ○ Zebrafish larva microscopy and image analysis • QUANTIFICATION AND STATISTICAL ANALYSIS SUPPLEMENTAL INFORMATION Supplemental information can be found online at https://doi.org/10.1016/j. celrep.2025.116661. Received: November 12, 2024 Revised: September 25, 2025 Accepted: November 13, 2025 REFERENCES 1. Le Guennec, L., Coureuil, M., Nassif, X., and Bourdoulous, S. (2020). Stra- tegies used by bacterial pathogens to cross the blood-brain barrier. Cell. Microbiol. 22, e13132. https://doi.org/10.1111/cmi.13132. 2. Coureuil, M., Lé cuyer, H., Bourdoulous, S., and Nassif, X. (2017). A journey into the brain: insight into how bacterial pathogens cross blood-brain bar- riers. Nat. Rev. Microbiol. 15, 149–159. https://doi.org/10.1038/nrmicro. 2016.178. 3. Thwaites, G.E., van Toorn, R., and Schoeman, J. (2013). Tuberculous men- ingitis: more questions, still too few answers. Lancet Neurol. 12, 999–1010. https://doi.org/10.1016/S1474-4422(13)70168-6. 4. Wilkinson, R.J., Rohlwink, U., Misra, U.K., van Crevel, R., Mai, N.T.H., Dooley, K.E., Caws, M., Figaji, A., Savic, R., Solomons, R., et al. (2017). Tuberculous meningitis. Nat. Rev. Neurol. 13, 581–598. https://doi.org/ 10.1038/nrneurol.2017.120. 5. Huynh, J., Donovan, J., Phu, N.H., Nghia, H.D.T., Thuong, N.T.T., and Thwaites, G.E. (2022). Tuberculous meningitis: progress and remaining questions. Lancet Neurol. 21, 450–464. https://doi.org/10.1016/s1474-44 22(21)00435-x. 6. Ayloo, S., and Gu, C. (2019). Transcytosis at the blood-brain barrier. Curr. Opin. Neurobiol. 57, 32–38. https://doi.org/10.1016/j.conb.2018.12.014. 7. Daneman, R., and Prat, A. (2015). The blood-brain barrier. Cold Spring Harb. Perspect. Biol. 7, a020412. https://doi.org/10.1101/cshperspect.a0 20412. 8. Langen, U.H., Ayloo, S., and Gu, C. (2019). Development and Cell Biology of the Blood-Brain Barrier. Annu. Rev. Cell Dev. Biol. 35, 591–613. https:// doi.org/10.1146/annurev-cellbio-100617-062608. 9. Coureuil, M., Join-Lambert, O., Lé cuyer, H., Bourdoulous, S., Marullo, S., and Nassif, X. (2012). Mechanism of meningeal invasion by Neisseria men- ingitidis. Virulence 3, 164–172. https://doi.org/10.4161/viru.18639. 10. Coureuil, M., Bourdoulous, S., Marullo, S., and Nassif, X. (2014). Invasive meningococcal disease: a disease of the endothelial cells. Trends Mol. Med. 20, 571–578. https://doi.org/10.1016/j.molmed.2014.08.002. 11. Donald, P.R., Schaaf, H.S., and Schoeman, J.F. (2005). Tuberculous men- ingitis and miliary tuberculosis: the Rich focus revisited. J. Infect. 50, 193–195. https://doi.org/10.1016/j.jinf.2004.02.010. 12. Rich, A.R., and McCordock, H.A. (1933). The Pathogenesis of Tuberculous Meningitis. Bull. Johns Hopkins Hosp. 52, 5–37. 13. Gothenburg, A.W. (1934). Some aspects of tuberculous meningitis and the possibility of its prevention. J. Pediatr. 5, 291–298. 14. Zaharie, S.D., Franken, D.J., van der Kuip, M., van Elsland, S., de Bakker, B.S., Hagoort, J., Roest, S.L., van Dam, C.S., Timmers, C., Solomons, R., et al. (2020). The immunological architecture of granulomatous inflamma- tion in central nervous system tuberculosis. Tuberculosis 125, 102016. https://doi.org/10.1016/j.tube.2020.102016. 15. Macgregor, A.R., and Green, C.A. (1937). Tuberculosis of the central ner- vous system, with special reference to tuberculous meningitis. J. Pathol. Bacteriol. 45, 613–645. https://doi.org/10.1002/path.1700450312. Cell Reports 44, 116661, December 23, 2025 15 Article ll OPEN ACCESS https://doi.org/10.1016/j.celrep.2025.116661 https://doi.org/10.1016/j.celrep.2025.116661 https://doi.org/10.1111/cmi.13132 https://doi.org/10.1038/nrmicro.2016.178 https://doi.org/10.1038/nrmicro.2016.178 https://doi.org/10.1016/S1474-4422(13)70168-6 https://doi.org/10.1038/nrneurol.2017.120 https://doi.org/10.1038/nrneurol.2017.120 https://doi.org/10.1016/s1474-4422(21)00435-x https://doi.org/10.1016/s1474-4422(21)00435-x https://doi.org/10.1016/j.conb.2018.12.014 https://doi.org/10.1101/cshperspect.a020412 https://doi.org/10.1101/cshperspect.a020412 https://doi.org/10.1146/annurev-cellbio-100617-062608 https://doi.org/10.1146/annurev-cellbio-100617-062608 https://doi.org/10.4161/viru.18639 https://doi.org/10.1016/j.molmed.2014.08.002 https://doi.org/10.1016/j.jinf.2004.02.010 http://refhub.elsevier.com/S2211-1247(25)01433-0/sref12 http://refhub.elsevier.com/S2211-1247(25)01433-0/sref12 http://refhub.elsevier.com/S2211-1247(25)01433-0/sref13 http://refhub.elsevier.com/S2211-1247(25)01433-0/sref13 https://doi.org/10.1016/j.tube.2020.102016 https://doi.org/10.1002/path.1700450312 16. Jain, S.K., Tobin, D.M., Tucker, E.W., Venketaraman, V., Ordonez, A.A., Jayashankar, L., Siddiqi, O.K., Hammoud, D.A., Prasadarao, N.V., Sandor, M., et al. (2018). Tuberculous meningitis: a roadmap for advancing basic and translational research. Nat. Immunol. 19, 521–525. https://doi.org/ 10.1038/s41590-018-0119-x. 17. Roca, F.J., Whitworth, L.J., Prag, H.A., Murphy, M.P., and Ramakrishnan, L. (2022). Tumor necrosis factor induces pathogenic mitochondrial ROS in tuberculosis through reverse electron transport. Science 376, eabh2841. https://doi.org/10.1126/science.abh2841. 18. Pagá n, A.J., Lee, L.J., Edwards-Hicks, J., Moens, C.B., Tobin, D.M., Busch- Nentwich, E.M., Pearce, E.L., and Ramakrishnan, L. (2022). mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity. Cell 185, 3720–3738.e3713. https://doi.org/10.1016/j.cell.2022.08.018. 19. Davis, J.M., Clay, H., Lewis, J.L., Ghori, N., Herbomel, P., and Ramak- rishnan, L. (2002). Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish em- bryos. Immunity 17, 693–702. https://doi.org/10.1016/s1074-7613(02)00 475-2. 20. Ramakrishnan, L. (2020). Mycobacterium tuberculosis pathogenicity viewed through the lens of molecular Koch’s postulates. Curr. Opin. Mi- crobiol. 54, 103–110. https://doi.org/10.1016/j.mib.2020.01.011. 21. Madigan, C.A., Cambier, C.J., Kelly-Scumpia, K.M., Scumpia, P.O., Cheng, T.Y., Zailaa, J., Bloom, B.R., Moody, D.B., Smale, S.T., Sagasti, A., et al. (2017). A Macrophage Response to Mycobacterium leprae Phenolic Glycolipid Initiates Nerve Damage in Leprosy. Cell 170, 973– 985.e10. https://doi.org/10.1016/j.cell.2017.07.030. 22. van Leeuwen, L.M., Boot, M., Kuijl, C., Picavet, D.I., van Stempvoort, G., van der Pol, S.M.A., de Vries, H.E., van der Wel, N.N., van der Kuip, M., van Furth, A.M., et al. (2018). Mycobacteria employ two different mecha- nisms to cross the blood-brain barrier. Cell. Microbiol. 20, e12858. https://doi.org/10.1111/cmi.12858. 23. Takaki, K., Ramakrishnan, L., and Basu, S. (2018). A zebrafish model for ocular tuberculosis. PLoS One 13, e0194982. https://doi.org/10.1371/ journal.pone.0194982. 24. Chalakova, Z.P., and Johnston, S.A. (2023). Zebrafish Larvae as an Exper- imental Model of Cryptococcal Meningitis. Methods Mol. Biol. 2667, 47–69. https://doi.org/10.1007/978-1-0716-3199-7_4. 25. Nielson, J.A., and Davis, J.M. (2023). Roles for Microglia in Cryptococcal Brain Dissemination in the Zebrafish Larva. Microbiol. Spectr. 11, e0431522. https://doi.org/10.1128/spectrum.04315-22. 26. Ravishankar, S., Tuohey, S.M., Ramos, N.O., Uchiyama, S., Hayes, M.I., Kang, K., Nizet, V., and Madigan, C.A. (2025). Group B Streptococci lyse endothelial cells to infect the brain in a zebrafish meningitis model. PLoS Biol. 23, e3003236. https://doi.org/10.1371/journal.pbio.3003236. 27. Be, N.A., Bishai, W.R., and Jain, S.K. (2012). Role of Mycobacterium tuberculosis pknD in the pathogenesis of central nervous system tubercu- losis. BMC Microbiol. 12, 7. https://doi.org/10.1186/1471-2180-12-7. 28. Jain, S.K., Paul-Satyaseela, M., Lamichhane, G., Kim, K.S., and Bishai, W.R. (2006). Mycobacterium tuberculosis invasion and traversal across an in vitro human blood-brain barrier as a pathogenic mechanism for cen- tral nervous system tuberculosis. J. Infect. Dis. 193, 1287–1295. https:// doi.org/10.1086/502631. 29. O’Brown, N.M., Megason, S.G., and Gu, C. (2019). Suppression of trans- cytosis regulates zebrafish blood-brain barrier function. eLife 8, e47326. https://doi.org/10.7554/eLife.47326. 30. Choi, J., Dong, L., Ahn, J., Dao, D., Hammerschmidt, M., and Chen, J.N. (2007). FoxH1 negatively modulates flk1 gene expression and vascular formation in zebrafish. Dev. Biol. 304, 735–744. https://doi.org/10.1016/ j.ydbio.2007.01.023. 31. Maissa, N., Covarelli, V., Janel, S., Durel, B., Simpson, N., Bernard, S.C., Pardo-Lopez, L., Bouzinba-Segard, H., Faure, C., Scott, M.G.H., et al. (2017). Strength of Neisseria meningitidis binding to endothelial cells re- quires highly-ordered CD147/beta2-adrenoceptor clusters assembled by alpha-actinin-4. Nat. Commun. 8, 15764. https://doi.org/10.1038/ncomms 15764. 32. Wu, H.S., Kolonoski, P., Chang, Y.Y., and Bermudez, L.E. (2000). Invasion of the brain and chronic central nervous system infection after systemic Mycobacterium avium complex infection in mice. Infect. Immun. 68, 2979–2984. https://doi.org/10.1128/IAI.68.5.2979-2984.2000. 33. Davis, J.M., and Ramakrishnan, L. (2009). The role of the granuloma in expansion and dissemination of early tuberculous infection. Cell 136, 37–49. https://doi.org/10.1016/j.cell.2008.11.014. 34. Clay, H., Davis, J.M., Beery, D., Huttenlocher, A., Lyons, S.E., and Ramak- rishnan, L. (2007). Dichotomous role of the macrophage in early Mycobac- terium marinum infection of the zebrafish. Cell Host Microbe 2, 29–39. https://doi.org/10.1016/j.chom.2007.06.004. 35. Ellett, F., Pase, L., Hayman, J.W., Andrianopoulos, A., and Lieschke, G.J. (2011). mpeg1 promoter transgenes direct macrophage-lineage expres- sion in zebrafish. Blood 117, e49–e56. https://doi.org/10.1182/blood-20 10-10-314120. 36. Prinz, M., and Priller, J. (2017). The role of peripheral immune cells in the CNS in steady state and disease. Nat. Neurosci. 20, 136–144. https:// doi.org/10.1038/nn.4475. 37. Cambier, C.J., Falkow, S., and Ramakrishnan, L. (2014). Host evasion and exploitation schemes of Mycobacterium tuberculosis. Cell 159, 1497– 1509. https://doi.org/10.1016/j.cell.2014.11.024. 38. Cosma, C.L., Humbert, O., and Ramakrishnan, L. (2004). Superinfecting mycobacteria home to established tuberculous granulomas. Nat. Immu- nol. 5, 828–835. https://doi.org/10.1038/ni1091. 39. Peterson, P.K., Gekker, G., Hu, S., Sheng, W.S., Anderson, W.R., Ulevitch, R.J., Tobias, P.S., Gustafson, K.V., Molitor, T.W., and Chao, C.C. (1995). CD14 receptor-mediated uptake of nonopsonized Mycobacterium tuber- culosis by human microglia. Infect. Immun. 63, 1598–1602. https://doi. org/10.1128/iai.63.4.1598-1602.1995. 40. Nielson, J.A., Jezewski, A.J., Wellington, M., and Davis, J.M. (2024). Sur- vival in macrophages induces enhanced virulence in Cryptococcus. mSphere 9, e0050423. https://doi.org/10.1128/msphere.00504-23. 41. Helker, C.S.M., Schuermann, A., Karpanen, T., Zeuschner, D., Belting, H.G., Affolter, M., Schulte-Merker, S., and Herzog, W. (2013). The zebra- fish common cardinal veins develop by a novel mechanism: lumen en- sheathment. Development 140, 2776–2786. https://doi.org/10.1242/dev. 091876. 42. Ayloo, S., Lazo, C.G., Sun, S., Zhang, W., Cui, B., and Gu, C. (2022). Peri- cyte-to-endothelial cell signaling via vitronectin-integrin regulates blood- CNS barrier. Neuron 110, 1641–1655.e6. https://doi.org/10.1016/j.neuron. 2022.02.017. 43. Nizet, V., Kim, K.S., Stins, M., Jonas, M., Chi, E.Y., Nguyen, D., and Ru- bens, C.E. (1997). Invasion of brain microvascular endothelial cells by group B streptococci. Infect. Immun. 65, 5074–5081. https://doi.org/10. 1128/iai.65.12.5074-5081.1997. 44. Ravishankar, S.T.,S.M., Ramos, N.O., Uchiyama, S., Hayes, M.I., Nizet, V., and Madigan, C.A. (2024). Group B streptococci lyse endothelial cells to infect the brain in a zebrafish meningitis model. Preprint at bioRxiv. https://doi.org/10.1101/2024.10.01.616123. 45. Osman, M.M., Shanahan, J.K., Chu, F., Takaki, K.K., Pinckert, M.L., Pagá n, A.J., Brosch, R., Conrad, W.H., and Ramakrishnan, L. (2022). The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence. Proc. Natl. Acad. Sci. USA 119, e2122161119. https://doi.org/10.1073/ pnas.2122161119. 46. Conrad, W.H., Osman, M.M., Shanahan, J.K., Chu, F., Takaki, K.K., Ca- meron, J., Hopkinson-Woolley, D., Brosch, R., and Ramakrishnan, L. (2017). Mycobacterial ESX-1 secretion system mediates host cell lysis through bacterium contact-dependent gross membrane disruptions. Proc. Natl. Acad. Sci. USA 114, 1371–1376. https://doi.org/10.1073/ pnas.1620133114. 16 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS https://doi.org/10.1038/s41590-018-0119-x https://doi.org/10.1038/s41590-018-0119-x https://doi.org/10.1126/science.abh2841 https://doi.org/10.1016/j.cell.2022.08.018 https://doi.org/10.1016/s1074-7613(02)00475-2 https://doi.org/10.1016/s1074-7613(02)00475-2 https://doi.org/10.1016/j.mib.2020.01.011 https://doi.org/10.1016/j.cell.2017.07.030 https://doi.org/10.1111/cmi.12858 https://doi.org/10.1371/journal.pone.0194982 https://doi.org/10.1371/journal.pone.0194982 https://doi.org/10.1007/978-1-0716-3199-7_4 https://doi.org/10.1128/spectrum.04315-22 https://doi.org/10.1371/journal.pbio.3003236 https://doi.org/10.1186/1471-2180-12-7 https://doi.org/10.1086/502631 https://doi.org/10.1086/502631 https://doi.org/10.7554/eLife.47326 https://doi.org/10.1016/j.ydbio.2007.01.023 https://doi.org/10.1016/j.ydbio.2007.01.023 https://doi.org/10.1038/ncomms15764 https://doi.org/10.1038/ncomms15764 https://doi.org/10.1128/IAI.68.5.2979-2984.2000 https://doi.org/10.1016/j.cell.2008.11.014 https://doi.org/10.1016/j.chom.2007.06.004 https://doi.org/10.1182/blood-2010-10-314120 https://doi.org/10.1182/blood-2010-10-314120 https://doi.org/10.1038/nn.4475 https://doi.org/10.1038/nn.4475 https://doi.org/10.1016/j.cell.2014.11.024 https://doi.org/10.1038/ni1091 https://doi.org/10.1128/iai.63.4.1598-1602.1995 https://doi.org/10.1128/iai.63.4.1598-1602.1995 https://doi.org/10.1128/msphere.00504-23 https://doi.org/10.1242/dev.091876 https://doi.org/10.1242/dev.091876 https://doi.org/10.1016/j.neuron.2022.02.017 https://doi.org/10.1016/j.neuron.2022.02.017 https://doi.org/10.1128/iai.65.12.5074-5081.1997 https://doi.org/10.1128/iai.65.12.5074-5081.1997 https://doi.org/10.1101/2024.10.01.616123 https://doi.org/10.1073/pnas.2122161119 https://doi.org/10.1073/pnas.2122161119 https://doi.org/10.1073/pnas.1620133114 https://doi.org/10.1073/pnas.1620133114 47. Campbell, H.K., Maiers, J.L., and DeMali, K.A. (2017). Interplay between tight junctions & adherens junctions. Exp. Cell Res. 358, 39–44. https:// doi.org/10.1016/j.yexcr.2017.03.061. 48. Paradis, T., Begue, H., Basmaciyan, L., Dalle, F., and Bon, F. (2021). Tight Junctions as a Key for Pathogens Invasion in Intestinal Epithelial Cells. Int. J. Mol. Sci. 22, 2506. https://doi.org/10.3390/ijms22052506. 49. Kobielak, A., and Fuchs, E. (2004). Alpha-catenin: at the junction of inter- cellular adhesion and actin dynamics. Nat. Rev. Mol. Cell Biol. 5, 614–625. https://doi.org/10.1038/nrm1433. 50. Belardi, B., Hamkins-Indik, T., Harris, A.R., Kim, J., Xu, K., and Fletcher, D.A. (2020). A Weak Link with Actin Organizes Tight Junctions to Control Epithelial Permeability. Dev. Cell 54, 792–804.e7. https://doi.org/10.1016/ j.devcel.2020.07.022. 51. Augenstreich, J., Haanappel, E., Ferré , G., Czaplicki, G., Jolibois, F., De- stainville, N., Guilhot, C., Milon, A., Astarie-Dequeker, C., and Chavent, M. (2019). The conical shape of DIM lipids promotes Mycobacterium tuberculosis infection of macrophages. Proc. Natl. Acad. Sci. USA 116, 25649–25658. https://doi.org/10.1073/pnas.1910368116. 52. Cambier, C.J., Banik, S.M., Buonomo, J.A., and Bertozzi, C.R. (2020). Spreading of a mycobacterial cell-surface lipid into host epithelial mem- branes promotes infectivity. eLife 9, e60648. https://doi.org/10.7554/eL- ife.60648. 53. Glickman, M.S., Cox, J.S., and Jacobs, W.R., Jr. (2000). A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis. Mol. Cell 5, 717–727. https:// doi.org/10.1016/s1097-2765(00)80250-6. 54. Walton, E.M., Cronan, M.R., Cambier, C.J., Rossi, A., Marass, M., Foglia, M.D., Brewer, W.J., Poss, K.D., Stainier, D.Y.R., Bertozzi, C.R., and Tobin, D.M. (2018). Cyclopropane Modification of Trehalose Dimycolate Drives Granuloma Angiogenesis and Mycobacterial Growth through Vegf Signaling. Cell Host Microbe 24, 514–525.e6. https://doi.org/10.1016/j. chom.2018.09.004. 55. Alibaud, L., Alahari, A., Trivelli, X., Ojha, A.K., Hatfull, G.F., Guerardel, Y., and Kremer, L. (2010). Temperature-dependent regulation of mycolic acid cyclopropanation in saprophytic mycobacteria: role of the Mycobac- terium smegmatis 1351 gene (MSMEG_1351) in CIS-cyclopropanation of alpha-mycolates. J. Biol. Chem. 285, 21698–21707. https://doi.org/10. 1074/jbc.M110.125724. 56. Mishra, R., Hannebelle, M., Patil, V.P., Dubois, A., Garcia-Mouton, C., Kirsch, G.M., Jan, M., Sharma, K., Guex, N., Sordet-Dessimoz, J., et al. (2023). Me- chanopathology of biofilm-like Mycobacterium tuberculosis cords. Cell 186, 5135–5150.e28. https://doi.org/10.1016/j.cell.2023.09.016. 57. Ishikawa, E., Ishikawa, T., Morita, Y.S., Toyonaga, K., Yamada, H., Take- uchi, O., Kinoshita, T., Akira, S., Yoshikai, Y., and Yamasaki, S. (2009). Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle. J. Exp. Med. 206, 2879–2888. https://doi.org/ 10.1084/jem.20091750. 58. Rao, V., Fujiwara, N., Porcelli, S.A., and Glickman, M.S. (2005). Mycobac- terium tuberculosis controls host innate immune activation through cyclo- propane modification of a glycolipid effector molecule. J. Exp. Med. 201, 535–543. https://doi.org/10.1084/jem.20041668. 59. Bowdish, D.M.E., Sakamoto, K., Kim, M.J., Kroos, M., Mukhopadhyay, S., Leifer, C.A., Tryggvason, K., Gordon, S., and Russell, D.G. (2009). MARCO, TLR2, and CD14 are required for macrophage cytokine responses to mycobacterial trehalose dimycolate and Mycobacterium tuberculosis. PLoS Pathog. 5, e1000474. https://doi.org/10.1371/journal.ppat.1000474. 60. de Mendonç a-Lima, L., Picardeau, M., Raynaud, C., Rauzier, J., Goguet de la Salmoniè re, Y.O., Barker, L., Bigi, F., Cataldi, A., Gicquel, B., and Reyrat, J.M. (2001). Erp, an extracellular protein family specific to myco- bacteria. Microbiology (Read.) 147, 2315–2320. https://doi.org/10.1099/ 00221287-147-8-2315. 61. Lang, R. (2013). Recognition of the mycobacterial cord factor by Mincle: relevance for granuloma formation and resistance to tuberculosis. Front. Immunol. 4, 5. https://doi.org/10.3389/fimmu.2013.00005. 62. Kostarnoy, A.V., Gancheva, P.G., Kireev, I.I., Soloviev, A.I., Lepenies, B., Kulibin, A.Y., Malolina, E.A., Scheglovitova, O.N., Kondratev, A.V., Soko- lova, M.V., et al. (2022). A mechanism of self-lipid endocytosis mediated by the receptor Mincle. Proc. Natl. Acad. Sci. USA 119, e2120489119. https://doi.org/10.1073/pnas.2120489119. 63. Suzuki, Y., Nakano, Y., Mishiro, K., Takagi, T., Tsuruma, K., Nakamura, M., Yoshimura, S., Shimazawa, M., and Hara, H. (2013). Involvement of Mincle and Syk in the changes to innate immunity after ischemic stroke. Sci. Rep. 3, 3177. https://doi.org/10.1038/srep03177. 64. Vilcheze, C., Copeland, J., Keiser, T.L., Weisbrod, T., Washington, J., Jain, P., Malek, A., Weinrick, B., and Jacobs, W.R., Jr. (2018). Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacte- rium tuberculosis through Nutrient Auxotrophy. mBio 9, 10–1128. https:// doi.org/10.1128/mBio.00938-18. 65. Zhao, Z., Nelson, A.R., Betsholtz, C., and Zlokovic, B.V. (2015). Establish- ment and Dysfunction of the Blood-Brain Barrier. Cell 163, 1064–1078. https://doi.org/10.1016/j.cell.2015.10.067. 66. Daneman, R., Zhou, L., Kebede, A.A., and Barres, B.A. (2010). Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature 468, 562–566. https://doi.org/10.1038/nature09513. 67. Naik, P., and Cucullo, L. (2012). In vitro blood-brain barrier models: current and perspective technologies. J. Pharm. Sci. 101, 1337–1354. https://doi. org/10.1002/jps.23022. 68. Hatzios, S.K., Baer, C.E., Rustad, T.R., Siegrist, M.S., Pang, J.M., Ortega, C., Alber, T., Grundner, C., Sherman, D.R., and Bertozzi, C.R. (2013). Osmosensory signaling in Mycobacterium tuberculosis mediated by a eukaryotic-like Ser/Thr protein kinase. Proc. Natl. Acad. Sci. USA 110, E5069–E5077. https://doi.org/10.1073/pnas.1321205110. 69. Eugè ne, E., Hoffmann, I., Pujol, C., Couraud, P.O., Bourdoulous, S., and Nassif, X. (2002). Microvilli-like structures are associated with the inter- nalization of virulent capsulated Neisseria meningitidis into vascular endothelial cells. J. Cell Sci. 115, 1231–1241. https://doi.org/10.1242/jcs. 115.6.1231. 70. Coureuil, M., Mikaty, G., Miller, F., Lé cuyer, H., Bernard, C., Bourdoulous, S., Dumé nil, G., Mè ge, R.M., Weksler, B.B., Romero, I.A., et al. (2009). Meningococcal type IV pili recruit the polarity complex to cross the brain endothelium. Science 325, 83–87. https://doi.org/10.1126/science.117 3196. 71. Bernut, A., Herrmann, J.L., Kissa, K., Dubremetz, J.F., Gaillard, J.L., Lut- falla, G., and Kremer, L. (2014). Mycobacterium abscessus cording pre- vents phagocytosis and promotes abscess formation. Proc. Natl. Acad. Sci. USA 111, E943–E952. https://doi.org/10.1073/pnas.1321390111. 72. Takaki, K., Davis, J.M., Winglee, K., and Ramakrishnan, L. (2013). Evalu- ation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish. Nat. Protoc. 8, 1114–1124. https://doi.org/10. 1038/nprot.2013.068. 73. Roca, F.J., Whitworth, L.J., Redmond, S., Jones, A.A., and Ramakrishnan, L. (2019). TNF Induces Pathogenic Programmed Macrophage Necrosis in Tuberculosis through a Mitochondrial-Lysosomal-Endoplasmic Reticulum Circuit. Cell 178, 1344–1361.e11. https://doi.org/10.1016/j.cell.2019. 08.004. 74. Mizoguchi, T., Kawakami, K., and Itoh, M. (2016). Zebrafish lines express- ing UAS-driven red probes for monitoring cytoskeletal dynamics. Genesis 54, 483–489. https://doi.org/10.1002/dvg.22955. 75. Hogan, B.M., Bos, F.L., Bussmann, J., Witte, M., Chi, N.C., Duckers, H.J., and Schulte-Merker, S. (2009). Ccbe1 is required for embryonic lymphan- giogenesis and venous sprouting. Nat. Genet. 41, 396–398. https://doi. org/10.1038/ng.321. 76. Lawson, N.D., and Weinstein, B.M. (2002). In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318. https://doi.org/10.1006/dbio.2002.0711. 77. Liu, J., Fraser, S.D., Faloon, P.W., Rollins, E.L., Vom Berg, J., Starovic-Sub- ota, O., Laliberte, A.L., Chen, J.N., Serluca, F.C., and Childs, S.J. (2007). A Cell Reports 44, 116661, December 23, 2025 17 Article ll OPEN ACCESS https://doi.org/10.1016/j.yexcr.2017.03.061 https://doi.org/10.1016/j.yexcr.2017.03.061 https://doi.org/10.3390/ijms22052506 https://doi.org/10.1038/nrm1433 https://doi.org/10.1016/j.devcel.2020.07.022 https://doi.org/10.1016/j.devcel.2020.07.022 https://doi.org/10.1073/pnas.1910368116 https://doi.org/10.7554/eLife.60648 https://doi.org/10.7554/eLife.60648 https://doi.org/10.1016/s1097-2765(00)80250-6 https://doi.org/10.1016/s1097-2765(00)80250-6 https://doi.org/10.1016/j.chom.2018.09.004 https://doi.org/10.1016/j.chom.2018.09.004 https://doi.org/10.1074/jbc.M110.125724 https://doi.org/10.1074/jbc.M110.125724 https://doi.org/10.1016/j.cell.2023.09.016 https://doi.org/10.1084/jem.20091750 https://doi.org/10.1084/jem.20091750 https://doi.org/10.1084/jem.20041668 https://doi.org/10.1371/journal.ppat.1000474 https://doi.org/10.1099/00221287-147-8-2315 https://doi.org/10.1099/00221287-147-8-2315 https://doi.org/10.3389/fimmu.2013.00005 https://doi.org/10.1073/pnas.2120489119 https://doi.org/10.1038/srep03177 https://doi.org/10.1128/mBio.00938-18 https://doi.org/10.1128/mBio.00938-18 https://doi.org/10.1016/j.cell.2015.10.067 https://doi.org/10.1038/nature09513 https://doi.org/10.1002/jps.23022 https://doi.org/10.1002/jps.23022 https://doi.org/10.1073/pnas.1321205110 https://doi.org/10.1242/jcs.115.6.1231 https://doi.org/10.1242/jcs.115.6.1231 https://doi.org/10.1126/science.1173196 https://doi.org/10.1126/science.1173196 https://doi.org/10.1073/pnas.1321390111 https://doi.org/10.1038/nprot.2013.068 https://doi.org/10.1038/nprot.2013.068 https://doi.org/10.1016/j.cell.2019.08.004 https://doi.org/10.1016/j.cell.2019.08.004 https://doi.org/10.1002/dvg.22955 https://doi.org/10.1038/ng.321 https://doi.org/10.1038/ng.321 https://doi.org/10.1006/dbio.2002.0711 betaPix Pak2a signaling pathway regulates cerebral vascular stability in ze- brafish. Proc. Natl. Acad. Sci. USA 104, 13990–13995. https://doi.org/10. 1073/pnas.0700825104. 78. Wang, Y., Kaiser, M.S., Larson, J.D., Nasevicius, A., Clark, K.J., Wadman, S.A., Roberg-Perez, S.E., Ekker, S.C., Hackett, P.B., McGrail, M., and Ess- ner, J.J. (2010). Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis. Development 137, 3119–3128. https://doi. org/10.1242/dev.048785. 79. Murphy, K.C., Papavinasasundaram, K., and Sassetti, C.M. (2015). Myco- bacterial recombineering. Methods Mol. Biol. 1285, 177–199. https://doi. org/10.1007/978-1-4939-2450-9_10. 80. Murphy, K.C., Nelson, S.J., Nambi, S., Papavinasasundaram, K., Baer, C.E., and Sassetti, C.M. (2018). ORBIT: a New Paradigm for Genetic Engi- neering of Mycobacterial Chromosomes. mBio 9, e01467-18. https://doi. org/10.1128/mBio.01467-18. 81. Cosma, C.L., Swaim, L.E., Volkman, H., Ramakrishnan, L., and Davis, J.M. (2006). Zebrafish and frog models of Mycobacterium marinum infection. Curr Protoc Microbiol 10, Unit 10B 12. https://doi.org/10.1002/0471729 256.mc10b02s3. 82. Garcia-Nafria, J., Watson, J.F., and Greger, I.H. (2016). IVA cloning: A sin- gle-tube universal cloning system exploiting bacterial In Vivo Assembly. Sci. Rep. 6, 27459. https://doi.org/10.1038/srep27459. 83. Tobin, D.M., Roca, F.J., Oh, S.F., McFarland, R., Vickery, T.W., Ray, J.P., Ko, D.C., Zou, Y., Bang, N.D., Chau, T.T.H., et al. (2012). Host genotype- specific therapies can optimize the inflammatory response to mycobacterial infections. Cell 148, 434–446. https://doi.org/10.1016/j.cell.2011.12.023. 84. van Rooijen, N., Sanders, A., and van den Berg, T.K. (1996). Apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine. J. Immunol. Methods 193, 93–99. https:// doi.org/10.1016/0022-1759(96)00056-7. 18 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS https://doi.org/10.1073/pnas.0700825104 https://doi.org/10.1073/pnas.0700825104 https://doi.org/10.1242/dev.048785 https://doi.org/10.1242/dev.048785 https://doi.org/10.1007/978-1-4939-2450-9_10 https://doi.org/10.1007/978-1-4939-2450-9_10 https://doi.org/10.1128/mBio.01467-18 https://doi.org/10.1128/mBio.01467-18 https://doi.org/10.1002/0471729256.mc10b02s3 https://doi.org/10.1002/0471729256.mc10b02s3 https://doi.org/10.1038/srep27459 https://doi.org/10.1016/j.cell.2011.12.023 https://doi.org/10.1016/0022-1759(96)00056-7 https://doi.org/10.1016/0022-1759(96)00056-7 STAR★METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-ZO1 Monoclonal (ZO1-1A12) ThermoFisher Cat# 339100; RRID: AB_2663169 Goat anti-Mouse AF647 Life Technologies Cat# A21237 Bacterial and virus strains Mycobacterium marinum M strain transformed with pTEC18 or pTEC27 Takaki et al., 2013 72 derivatives of ATCC #BAA-535 Δesx-1 M. marinum M strain transformed with pTEC18 or pTEC27 Conrad et al., 2017 46 N/A M. marinum M strain pknD::Tn6042 transformed with pTEC18 or pTEC27 This paper N/A M. marinum transformed with pDL4912 (msp12:dsRedII;map49:GFP) Cosma et al., 2004 38 N/A M. tuberculosis ΔleuDΔpanCD mc 2 6206 transformed with pTEC27 Roca et al., 2019 73 N/A M. marinum M strain pcaA::Tn20324 transformed with pTEC18 or pTEC27 This paper N/A Δerp M. marinum M strain transformed with pTEC27 Takaki et al. 2013 72 N/A M. marinum M strain pcaA::Tn20324:pcaA – pcaA::Tn20324 transformed with pmsp12:cerulean;hsp60:pcaA Walton et al. 2018. 54 N/A M. marinum M strain pknD::Tn6042:pknD pknD::Tn6042 transformed with pJKS226 This paper N/A M. tuberculosis ΔleuDΔpanCD mc 2 6206 ΔpknD transformed with pTEC31 This paper N/A M. tuberculosis ΔleuDΔpanCD mc 2 6206 ΔpcaA transformed with pTEC31 This paper N/A Mycobacterium smegmatis mc 2 155 transformed with pTEC27 This paper N/A M. smegmatis mc 2 155 ΔMSMEG_1351 transformed with pTEC27 This paper N/A Chemicals, peptides, and recombinant proteins Sodium chloride JT Baker Cat# 3628-F7 Potassium chloride Sigma Cat# P3911 Calcium chloride G-Biosciences Cat# RC-030 Magnesium sulfate heptahydrate MP Biomedicals Cat# 194833 Methylene blue chloride Millipore Sigma Cat# 284 1-phenyl-2-thiourea (PTU) Sigma-Aldrich Cat# 189235 Clodronate liposomes Liposoma Cat# C-005 Paraformaldehyde solution, 4% in PBS Fisher scientific Cat# AAJ19943K2 Tango buffer ThermoFisher Cat# BY5 Fluorospheres carboxylate-modified, 0.02μm crimson (625/645) Invitrogen Cat# F8782 Middlebrook Low Melt Point agarose IBI Scientific Cat# IB70051 Phenol Red Sigma -Aldrich Cat# P3532 Syncaine Syndel Cat# 886-86-2 Triton X-100 Electron Microscopy Sciences Cat# 22140 Propidium Iodide (1.0 mg/mL Solution in Water) Invitrogen Cat# P3566 Tween 20 Sigma Cat# P2287 (Continued on next page) Cell Reports 44, 116661, December 23, 2025 19 Article ll OPEN ACCESS Continued REAGENT or RESOURCE SOURCE IDENTIFIER Proteinase K Fisher Scientific Cat# BP1700 Methanol Fisher Scientific Cat# A452 Normal goat serum Fisher Scientific Cat# NC9660079 FITC-Dextran Invitrogen Cat# D1820 Hoechst Invitrogen Cat# H21486 Durcupan N/A 7H10 Middlebrook Agar Base HiMedia Cat# M199 7H9 Middlebrook Broth Base Sigma-Aldrich Cat# M0178 Hygromycin B (in PBS 50 mg/mL) ThermoFisher Cat# 10687010 Kanamycin monosulfate TCI Cat# K0047 Tween-80 Sigma Cat# P1754 Glycerol ThermoFisher Cat# Pl17904 Bovine serum albumin Sigma Cat# A9647 Glucose Sigma Cat# D9434-500g NaOH Sigma Cat# 221465 Oleic acid Sigma Cat# O1008 Albumin Sigma Cat# A9647 Catalase Sigma-Aldrich Cat# C1345-1G Dextrose Sigma-Aldrich Cat# D9434-500g L-leucine Milipore Cat# 4330-100GM Calcium pantothenate Sigma-Aldrich Cat# PHR1232 Anhydrotetracycline hydrochloride Acros Organics Cat# 13803-65-1 Glycine Sigma-Aldrich Cat# G8898-1KG Isovaleronitrile, 98% Sigma-Aldrich Cat# 308528-5G Experimental models: Organisms/strains Zebrafish: wildtype AB University of Washington University of California, Los Angeles ZFIN ID: ZDB-GENO-960809-7 Zebrafish: Tg(kdrl:GFP) Choi et al., 2007 30 ZFIN ID: ZDB-TGCONSTRCT-070529-1 Zebrafish: Tg(flk:GAL4;UAS:Life-Act-GFP) Mizoguchi et al., 2016 74 N/A Zebrafish: Tg(flt1:Tomato) Hogan et al., 2009 75 ZFIN ID: ZDB-TGCONSTRCT-110504-1 Zebrafish: Tg(fliE:GFP) Lawson and Weinstein, 2002 76 ZFIN ID: ZDB-TGCONSTRCT-070117-94 Zebrafish: Tg(flk:GFP) Liu et al., 2007 77 ZFIN ID: ZDB-TGCONSTRCT-070529-1 Zebrafish: Tg(fliE:Gal4; UAS:dsRed) Lawson and Weinstein, 2002 76 N/A Zebrafish: Tg(flk:alpha-catenin-GFP) Wang et al., 2010 78 N/A Zebrafish: Tg(mpeg1:dsRed) Ellett et al., 2011 35 ZFIN ID: ZDB-FISH-150901-6828 Zebrafish: Tg(flk:moesin-GFP) Wang et al., 2010 78 N/A Oligonucleotides pu.1 morpholino component 1, sequence: CCTCCATTCTGTACGGATGCAGCAT Clay et al., 2007 34 N/A pu.1 morpholino component 2, sequence: GGTCTTTCTCCTTACCATGCTCTCC Clay et al., 2007 34 N/A pknD::Tn M. marinum sequencing F primer sequence: TAGCGTGAATATGTAGGGTC This paper N/A pknD::Tn M. marinum sequencing R primer sequence: ATCTACACCGAGCTCACCAA This paper N/A mincle sequencing F primer sequence: GAATTTCTGCGCTAGCCTG This paper N/A (Continued on next page) 20 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS Continued REAGENT or RESOURCE SOURCE IDENTIFIER mincle sequencing R primer sequence: GCTATGCTCTTGAATAAGATGTGC This paper N/A MSMEG_1531del ultramer: GGGTTCGATGCTGCCCCCCGGTTTCTCCGCCGAGA GGTGCACGTCAGTGACCAAGCAGCCCGGAAAGCTA CAACCAGGTTTGTCTGGTCAACCACCGCGGTCTC AGTGGTGTACGGTACAAACCATCGACGTCAACCA GTTCACCCTGGCGAAGTAGCGGCTCTTCAGAT TCGCTATGCGGGACCTGCCCCGACACGATA This paper N/A JS285 (add lox66/71 to pKM464 F primer sequence): GGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGA TCATTACCGTTCGTATAGCATACATTATACGAAGTT ATCTCGAGTCTAGAGCATGCACTAGT This paper N/A JS286 (add lox66/71 to pKM464 R primer sequence): TACCGTTCGTATAATGTATGCTATACGAAGT TATAAGCTTATCGATGTCGACGTAGT This paper N/A JS360 (amplify pknD Mtb downstream R primer sequence): GGCTACGGTCTCAACGAGATATCACGCCCTGTACG This paper N/A JS358 (amplify vector for GGA F primer sequence): GGCTACGGTCTCTCAAAGGCGGTAATACGGTTATC This paper N/A JS359 (amplify vector for GGA R primer sequence): GGCTACGGTCTCATCGTGATACGCCTATTTTTATAG This paper N/A JS361 (amplify pknD Mtb downstream F primer sequence): GGCTACGGTCTCTCACTAGTTCTAGAGAACGACCG AGTGGTGAAAC This paper N/A JS362 (amplify hygR for GGA F primer sequence): GGCTACGGTCTCTAGTGGATCCATAACTTCG This paper N/A JS363 (amplify hygR for GGA R primer sequence): GGCTACGGTCTCATTCCGCAGGTAGGGTCGC TCGAGGGTACCGGCGCG This paper N/A JS364 (amplify pknD Mtb upstream R primer sequence): GGCTACGGTCTCAGGAACGGCATCGCTCACC This paper N/A JS365 (amplify pknD Mtb downstream F primer sequence): GGCTACGGTCTCTTTTGGATATCCACAGATCCAAGCCC This paper N/A JS372 (ΔpknD M. tuberculosis sequencing R primer sequence): GGTGGTGTTTCGTCCGCTTAC This paper N/A JS373 (ΔpknD M. tuberculosis sequencing F primer sequence): ATGGAGCAGTTCGTCTATGCCTAC This paper N/A JS444 (amplify pcaA Mtb downstream R primer sequence): GGCTACGGTCTCAACGAGATATCGGTGCCTTCGAAGCATTC This paper N/A JS445 (amplify pcaA Mtb downstream F primer sequence): GGCTACGGTCTCGCACTACCGACGTCGACCAGTTCACA This paper N/A JS446 (amplify pcaA Mtb upstream R primer sequence): GGCTACGGTCTCCGGAATCCAAAATGCGGCGTGAGCTG This paper N/A JS447 (amplify pcaA Mtb upstream F primer sequence): GGCTACGGTCTCTTTTGGATATCGACAAGATCGGTTACGACG This paper N/A JS448 (MSMEG_1351 deletion sequencing F primer sequence): ATCTTCTCCAGCAACAACCA This paper N/A JS449 (MSMEG_1351 deletion sequencing R primer sequence): ACCTTGGACAGCTTTTCGATG This paper N/A JS465 (ΔpcaA M. tuberculosis R primer sequence): AGACCCCACGATGGCTTACAC This paper N/A JS466 (ΔpcaA M. tuberculosis sequencing F primer sequence): GCACATCGCCAACAACCTTAT This paper N/A (Continued on next page) Cell Reports 44, 116661, December 23, 2025 21 Article ll OPEN ACCESS Continued REAGENT or RESOURCE SOURCE IDENTIFIER JS485 (linearise pMV261 F primer sequence): CATTGCGAAGTGATTCCTCC This paper N/A JS486 (linearise pMV261 R primer sequence): TAGCGTACGATCGACTGC This paper N/A JS487 (amplify pknD Mm F primer sequence): TCCCCGATCCGGAGGAATCACTTCGCAA TGGTGAGCGAGACCGGGCCG This paper N/A JS488 (amplify pknD Mm R primer sequence): ATTTGATGCCTGGCAGTCGATCGTACGCTA TCAGGATCCTTGGGCCAGTTTCAG This paper N/A JS493 (linearise pTEC27 F primer sequence): CACTTTCTGGCTGGATGATG This paper N/A JS494 (linearise pTEC27 R primer sequence): CCACGGTTGATGAGAGCT This paper N/A JS495 (amplify hsp60:pknD Mm F primer sequence): CACCTACAACAAAGCTCTCATCAACCGTGGAAA TCTAGACGGTGACCACAACG This paper N/A JS496 (amplify hsp60:pknD Mm R primer sequence): TGAATCGCCCCATCATCCAGCCAGAAAGT GTTGTTGGCTAGCTGATCACC This paper N/A Recombinant DNA pTEC18 (msp12:eBFP2) Takaki et al., 2013 72 Addgene #30177 pTEC27 (msp12:tdTomato;hygR) Takaki et al., 2013 72 Addgene #30182 pTEC31 (msp12:tdTomato;kanR) Conrad et al., 2017 46 N/A pNitET-SacB-Kan Murphy et al., 2015 79 Addgene #107692 pKM461 Murphy et al., 2018 80 Addgene #108320 pCre-SacB-Zeo Murphy et al., 2015 79 Addgene #107706 pJKS146 (pKM464 [Addgene #108322] modified to have lox66/71 sites flanking attB) This paper N/A pJKS181 (pKM342 [Addgene #71486] with inserts to delete pknD) This paper N/A pJKS217 (pKM342 [Addgene #71486] with inserts to delete pcaA) This paper N/A pJKS226 (msp12:tdTomato;hsp60:pknD Mm ) This paper N/A gRNAs Mincle gRNA 1: GGTGAAGAGAAGCGTAACTT This paper N/A Mincle gRNA 2: GAGATCTGTGACACTCAAAT This paper N/A Mincle gRNA 3: AGCCTAACATGGGTCTGCAG This paper N/A Software and algorithms Imaris Bitplane https://imaris.oxinst.com/ Adobe Illustrator Adobe https://www.adobe.com/ products/illustrator.html Prism GraphPad https://www.graphpad.com/ ImageJ ImageJ https://imagej.net/ FPC (ImageJ); macro for quantification of bacterial burden by fluorescence imaging Takaki et al., 2013 72 N/A Zen Black Zeiss N/A 22 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS https://imaris.oxinst.com/ https://www.adobe.com/products/illustrator.html https://www.adobe.com/products/illustrator.html https://www.graphpad.com/ https://imagej.net/ EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Zebrafish husbandry and infections Zebrafish husbandry and experiments were conducted in compliance with guidelines from the U.S. National Institutes of Health and approved by the University of California San Diego Institutional Animal Care and Use Committee and the Institutional Biosafety Committee of the University of California San Diego. Wildtype AB strain zebrafish or transgenics in the AB background were used, including Tg(kdrl:GFP), 30 Tg(fliE:GAL4;UAS:dsRed), 76 Tg(fliE:GFP), 76 Tg(flk:GFP), 77 Tg(flk:GAL4;UAS:Lifeact-GFP), 74 Tg(mpeg1:dsRed), 35 Tg(flt1:tomato), 75 Tg(flk:alpha-catenin-GFP), 78 and Tg(flk:moesin-GFP). 78 Larvae were anesthetized with 2.8% Syncaine (Syndel #886-86-2) prior to imaging or infection. Larvae of indeterminate sex were infected by injection of 10 nL into the caudal vein at 3 days post fertilization (dpf) using a capillary needle containing bacteria diluted in PBS + 2% phenol red (Sigma #P3532), as previously described. 72 Titered, single-cell suspensions were prepared for all M. marinum strains prior to infec- tion by passing cell pellets from mid-log phase cultures (OD 600 0.5 ± 0.1) repeatedly through a syringe to remove clumps, as described. 72 After caudal vein injections were done, the same needle was used to inject onto 7H10 (Sigma-Aldrich #M199) agar plates containing 50 μg/mL hygromycin B (Thermofisher #10687010) or 50 μg/mL kanamycin (TCI #K0047) in triplicate to determine colony forming units (CFUs) of the inoculum. When two different bacterial strains were compared for bacterial burden directly, several groups of larvae (n=20 or more) were infected with different inocula of each strain. On the day of the comparison, equiva- lently infected groups of larvae were determined by FPC, as described, 72 to assure the comparison was not biased by in vivo growth differences between the two strains. ∼100–500 CFUs of wildtype M. mainum were administered to the larvae for experiments un- less otherwise specified. After infection, larvae were housed at 28.5 ◦ C, in fish water containing ddH 2 O, 14.61 g/L sodium chloride (JT Baker #3628-F7), 0.63 g/L potassium chloride (Sigma-Aldrich #P3911), 1.83 g/L calcium chloride (G-Biosciences #RC-030), 1.99 g/L magnesium sulfate heptahydrate (MP Biomedicals #194833), methylene blue chloride (Millipore Sigma #284), and 0.003% 1-phenyl-2-thiourea (PTU, Sigma-Aldrich #189235) to prevent melanocyte development. To generate G0 Mincle crispants, guide RNAs were prepared by combining equimolar concentrations of Alt-R CRISPR-Cas9 tracrRNA (IDT #1072532) with mincle crRNA (IDT; sequences listed in the key resources table below) or Alt-R Negative Control crRNA (IDT #1072545) in nuclease- free Duplex Buffer (IDT #11-01-03-01) at 95 ◦ C for 5 min. Alt-R S.p. Cas9 Nuclease V3 (IDT #1081058) was diluted to a working con- centration of 0.5 μg/μL in Cas9 dilution buffer (20 mM HEPES; 150 mM KCI, pH 7.5) and then combined with the prepared gRNAs at equimolar concentrations and heated at 37 ◦ C for 10 min to create the ribonucleoprotein (RNP) complexes. Larvae of indeterminate sex were injected with either 5 nL of the mincle or negative control RNP complexes at the single-cell stage. After imaging, larvae were individually sacrificed to collect genomic DNA and the mincle gene was PCR-amplified and Sanger-sequenced to verify gene disruption. METHOD DETAILS Bacterial strains M. marinum M strain (ATCC #BAA-535), mutants strains (pknD::Tn6042, Δesx1, pcaA::Tn20324, Δerp), and complemented strains (pknD::Tn6042:pknD and pcaA::Tn20324:pcaA) expressing tdTomato, wasabi, cerulean, or eBFP2 under control of the msp12 pro- moter, 72,81 were grown in 50 μg/mL hygromycin B (ThermoFisher, #10687010) or 50 μg/mL kanamycin (TCI, #K0047) in liquid culture, consisting of 7H9 Middlebrook medium (Sigma-Aldrich, #M0178) supplemented with 2.5% oleic acid (Sigma, #O1008), 50% glucose, and 20% Tween-80 (Sigma, #P1754). 72 Agar plates contained 7H10 Middlebrook agar (HiMedia, #M199), supplemented with oleic acid, albumin (Sigma, #A9647), dextrose, and Tween-80. 72 To transform M. marinum pknD::Tn6042 with pmsp12:eBFP2, the bacte- rial pellet from a 10 mL liquid culture of M. marinum (OD 600 0.8) was collected by centrifugation at 4000 x g for 10 min at 4 ◦ C. After washing in 10 mL ice-cold 10% glycerol, the bacterial pellet was resuspended in 1 mL ice-cold 10% glycerol, and centrifuged at 7300 x g, for 2 min, at 4 ◦ C. The bacterial pellet was resuspended in 100 μL of ice-cold 10% glycerol. 1 μL containing 100 ng of pmsp12:eBFP2 was added to 30 μL of the bacterial resuspension, which was electroporated (800 Ω, 25 μF, 2.5kV) in a 2 mm sterile cuvette. Cells were recovered in 900 μL 7H9+OADS+Tween-80 for 24 h at 33 ◦ C. Cells were plated on 7H10 Middlebrook agar plates containing hygromycin B. Colonies were isolated by verifying for the presence of hygromycin B resistance, blue fluorescence, and the presence of the transposon at pknD (via Sanger sequencing; pknD F primer sequence: TAGCGTGAATATGTAGGGTC; pknD R primer sequence: ATCTACACCGAGCTCACCAA). The unlabelled pknD::Tn6042 was complemented by transforming with pJKS226 and isolated by verifying the presence of hygromycin B resistance, presence of the pknD gene and red fluorescence. For zebrafish larvae infection, ∼1000 CFUs of Δesx1 M. marinum, ∼3000 CFUs of pknD::Tn M. marinum, or ∼1500 CFUs of pcaA::Tn M. marinum were injected. Higher CFUs of the pknd::Tn and pcaA::Tn mutant were administered to the larvae compared to wildtype to ensure infection matching in vivo. M. tuberculosis ΔleuDΔpanCD mc 2 6206 expressing tdTomato was grown at 37 ◦ C under hygromycin B selection in Middlebrook 7H9 medium (Sigma-Aldrich, #M0178) supplemented with oleic acid (Sigma, #O1008), albumin (Sigma, #A9647), dextrose (Sigma- Aldrich, #D9434-500g), Tween-80 (Sigma, #P1754), catalase (Sigma-Aldrich, #C1345-1G), 0.05 mg/mL L-leucine (Milipore, #4330- 100GM), and 0.024 mg/mL calcium pantothenate (Sigma-Aldrich, # PHR1232). ΔpcaA and ΔpknD strains were constructed by re- combineering as previously described. 79 Briefly, pNit-SacB-Kan transformed mc 2 6206 was induced with 1 μM isovaleronitrile at OD 600 = 0.8 for 8 h followed by addition of 0.2 M glycine and incubation at 37 ◦ C. Electrocompetent cells were prepared (at room Cell Reports 44, 116661, December 23, 2025 23 Article ll OPEN ACCESS temperature) and transformed with ∼1 μg PCR-purified DNA fragments from an EcoRV digest of pJKS181 (PknD) and pJKS217 (PcaA) at 37 ◦ C for 16 h. Transformants were electroporated (1000 Ω, 25 μF, 2.5kV) in a 2 mm sterile cuvette and cells recovered in 2 mL 7H9 + OADC + Tween-80 + 50 μg/mL L-leucine + 24 μg/mL D-pantothenic acid for 24 h at 37 ◦ C, then plated on supplemented 7H10 Middlebrook agar plates containing 50 μg/mL hygromycin B. After 4–6 weeks hygromycin resistant isolates were screened for correct insertion of the deletion cassette by sequencing PCR products spanning the junction between the integrated knockout construct and flanking genome regions. For zebrafish larvae infection, ∼300 CFUs of M. tuberculosis were injected. γ-irradiated M. marinum-td:Tomato was made by irradiating 5 uL single-cell aliquots with 2000 Gy γ-irradiation using a JL Shep- herd MK I Cesium-137 irradiator. γ-irradiated bacterial cells were confirmed non-viable by plating on 7H10 agar plates. For this reason, experiments involving γ-irradiated M. marinum do not have CFU counts listed. M. smegmatis strain mc 2 155 was grown in liquid medium containing 50 μg/ml hygromycin B (ThermoFisher, #10687010) in 7H9 Middlebrook medium (Sigma-Aldrich, t# M0178) supplemented with 2.5% oleic acid (Sigma, #O1008), 50% glucose, and 20% Tween-80 (Sigma, #P1754). 72 Agar plates were 7H10 Middlebrook agar (HiMedia, #M199) supplemented with 2.5% oleic acid (Sigma, #O1008), 50% glucose, and 20% Tween-80 (Sigma, #P1754). 72 The ΔpcaA (MSMEG_1351) strain was constructed by ORBIT as previously described. 80 Briefly, pKM461 transformed mc 2 155 was induced with 500 ng/mL ATc at OD 600 = 0.6 for 4 h and incubated at 37 ◦ C. Electrocompetent cells were prepared and transformed with 1 μg MSMEG_1351del ultramer and ∼300 ng pJKS146. Transformants were electroporated (1000 Ω, 25 μF, 2.5kV) in a 2 mm sterile cuvette and cells recovered in 2 mL 7H9 + OADS + Tween-80 overnight then plated on 7H10 Middlebrook agar plates containing 50 μg/mL hygromycin B. After 7 days hygrom- ycin resistant isolates were screened for insertion of the deletion cassette by PCR. The deletion cassette was excised by transforming deletion mutants with pCre-SacB-Zeo, isolating zeocin resistant colonies and screening for excision of the deletion cassette by PCR of the region followed by sequencing. M. smegmatis strains were grown in liquid culture (OD 600 = 0.8) and transformed as described above with pTEC27. 72,81 Transformants were isolated by verifying the presence of hygromycin B resistance and red fluorescence. For zebrafish larvae infection, ∼500 CFUs of M. smegmatis were injected. Plasmid construction For ORBIT, pJKS146 was constructed by Q5 mutagenesis of pKM464 with primers JS285 and JS286. For recombineering plasmids, pKM342 (Addgene #71486) was domesticated by Q5 mutagenesis to remove the BsaI sites. The hygromycin cassette was amplified with JS362 and JS363 and the vector backbone was amplified with JS358 and JS359. The upstream flanking region of pknD was amplified with JS364 and JS365. The downstream flanking region of pknD was amplified with JS360 and JS361. The vector, up- stream, downstream and hyg fragments were assembled by Golden Gate assembly to generate pJKS181. The upstream flanking region of pcaA was amplified with JS446 and JS447. The downstream flanking region of pcaA was amplified with JS444 and JS445. The vector, upstream, downstream and hyg fragments were assembled by Golden Gate assembly to generate pJKS217. For complementation, M. marinum pknD was amplified with JS487 and JS488 and cloned by IVA 82 into pMV261 (Novopro V012795) linearised with JS485 and JS486. hsp60:pknD was amplified from the resulting vector with JS495 and JS496 and cloned by IVA into pTEC27, linearised by JS493 and JS494, to generate pJKS226. Monocyte depletion, fluorospheres, and stains Macrophage depletion was accomplished by morpholinos or clodronate-loaded liposomes (Liposoma #C-005). pu.1 morpholinos were designed to the transcription initiation site (CCTCCATTCTGTACGGATGCAGCAT) and the exon 4–5 boundary (GGTCTTTCTCC TTACCATGCTCTCC) and combined to final concentrations of 0.375 mM and 0.025 mM, respectively. 34 Morpholinos were diluted in tango buffer (Thermo Scientific #BY5) containing 2% phenol red (Sigma-Aldrich #P3532) and injected into the yolk of 1–2 cell-stage embryos in 1 nL. 83 Chlodronate liposomes (LC) or PBS 84 were diluted 1:5 in PBS + 2% phenol red and injected in 10 nL into 2 dpf larvae via the caudal vein. LC depletion was done for all experiments involving M. tuberculosis, Δesx-1 M. marinum, pknD::Tn M. marinum, γ-irradiated M. marinum, M. smegmatis, and pcaA::Tn M. marinum and Mincle crispants. For experiments involving 0.02 μm fluorospheres (Invitrogen #F8782), the reagent was diluted 1:10 in PBS and injected into the caudal vein on the day of im- aging. To visualize vessels in larvae without transgenic fluorescent vessels, Alexa 647 Dextran (ThermoFisher, #D22914) or FITC- Dextran (Invitrogen, #D1820) was diluted 1:10 in PBS and injected into the caudal vein at the time of imaging. To visualize cell lysis, propidium iodide (Invitrogen #P3566), was diluted to 100 μg/mL in PBS and injected in 10 nL into the caudal vein on the day of im- aging. To label monocytes that were in circulation before entering the brain, Hoechst (Invitrogen #H21486) was diluted to 100 μg/mL in PBS and injected in 10 nL into the caudal vein every day prior to imaging. α-ZO-1 whole mount immunofluorescence For ZO-1 immunohistochemistry, larvae were fixed in 1 mL 4% paraformaldehyde solution (PFA) (Fischer scientific #AAJ19943K2) overnight at 4 ◦ C. Fixed embryos were washed in 0.1% Tween 20 (Sigma, #P2287) in PBS and washed once with 1 mL 100% meth- anol (Fisher Scientific #A452) before being stored in 1 mL fresh 100% methanol at -20 ◦ C overnight. Stored larvae were rehydrated through a series of methanol dilutions before washing in 1 mL 1% Triton X-100 (Electron Microscopy Sciences #22140) in PBS (PBSTx). Larvae were permeabilized in 1 mL 50 μg/mL proteinase K (Fisher Scientific, #BP1700) in PBSTx for 30 min at room tem- perature. Permeabilized larvae were then refixed in 1 mL 4% PFA for 20 min at room temperature, washed in 1 mL PBSTx, and blocked with 1 mL blocking solution made with 10% normal goat serum (Cell Signaling 5425S) and 1% bovine serum albumin (Sigma, 24 Cell Reports 44, 116661, December 23, 2025 Article ll OPEN ACCESS #A9647) in PBSTx for 5 h at room temperature. Larvae were then incubated with 1:50 anti-ZO1 monoclonal antibody (ZO1-1A12, ThermoFisher, #339100) overnight at room temperature. Larvae were washed in PBSTx, re-blocked in 1 mL 10% normal goat serum (Fisher Scientific, #NC9660079) in PBSTx for 1 h, and incubated in 1:400 goat anti-Mouse AF647 (Life Technologies, # A21237) in PBSTx overnight at room temperature. Larvae were washed again in 1 mL PBS before imaging. Zebrafish larva microscopy and image analysis For confocal imaging, larvae were embedded in 1.2% low melting-point agarose (IBI Scientific #IB70051). 72 A series of z stack im- ages with a 0.82–1 μm step size were generated through the brain using the Zeiss LSM 880 laser scanning microscope with an LD C-Apochromat 40× objective. Imaris (Bitplane Scientific Software) was used to measure fluorescence intensity and construct three- dimensional surface renderings. When comparing infected to uninfected vessels, threshold sizes and values were determined using the uninfected vessel and were then applied to the paired (usually contralateral) infected vessel in the same fish. When events were compared between larvae, identical confocal laser settings, software settings, and Imaris surface-rendering algorithms were used. For imaging blood vessels, transgenic animals with fluorescent blood vessels (Tg(kdrl:GFP), 30 Tg(fliE:GAL4;UAS:dsRed), 76 Tg(fliE:GFP), 76 Tg(flk:GFP), 77 Tg(flt1:tomato), 75 and Tg(flk:moesin-GFP) 78 ) were used, or Alexa 647 Dextran (ThermoFisher, #D22914) or FITC-Dextran (Invitrogen, #D1820) were injected intravenously. For imaging myeloid cells, transgenic animals with fluo- rescent myeloid cells (Tg(mpeg1:dsRed) 35 ) were used. For transmission electron microscopy, larvae were imaged by confocal microscopy in order to measure the distance from the top of the head to a region of interest containing a crossing microcolony. After larvae were rescued from 1.5% agarose used for confocal imaging, larvae were euthanized and fixed. Zebrafish larvae were incubated in a fixative solution (2% Paraformaldehyde + 2.5% Glutaraldehyde in 0.15 M Sodium cacodylate buffer pH 7.4) at room temperature for 30 min, then transferred to 4 ◦ C for 24 h. Samples were washed three times with 4 ◦ C 0.15 M Sodium cacodylate buffer pH 7.4 and post-fixed with 1% tetroxide osmium in 0.15 M So- dium cacodylate buffer pH 7.4 at 4 ◦ C. After 3 washes with cold double-distilled water, the fish were incubated in cold 2% uranyl ac- etate in double-distilled water for 2 h. Samples were then incubated in a series of 4 ◦ C solutions with increasing ethanol concentra- tions (50%, 70%, 90%, 3 times 100%) for 5 min each, then in room temperature 50% ethanol/50% acetone, and three times in 100% acetone. Next, the samples were infused with a mixture of 75% acetone/25% Durcupan (Sigma, #44610), then 50% acetone/50% Durcupan, and 25% acetone/75% Durcupan, for 2 h each. Then, the samples were incubated overnight at room temperature in 100% Durcupan and 3 times 2 h in 100% Durcupan. Finally, the samples were mounted with 100% Durcupan into an embedding mold, and oriented to be later sectioned along the frontal plane and cured at 60 ◦ C for 48 h. Sections were obtained using an ultramicrotome Leica UC6. To find the region of interest, every 10 μm, a 500 nm thick section was stained with toluidine blue and observed by light microscopy. Using the blood vessels as space markers and comparing their relative position in the 3D confocal volume, we decided when to start collecting 70 nm serial sections covering the area containing the bacterial microcolony. Transmission electron micrographs were acquired using a JEOL 1400 plus operated at 80KeV and equipped with a Gatan One- view camera. The quantity of bacilli per microcolony in vivo was determined by generating 3D renderings and acquiring the volumes of single bacilli and microcolonies with Imaris software. The volume of a microcolony was divided by the volume of a single bacillus to deter- mine how many bacilli were within a microcolony. QUANTIFICATION AND STATISTICAL ANALYSIS Most experiments were repeated multiple times to ensure reproducibility. The number of experimental replicates is indicated in the corresponding figure legend. If no number is listed, the experiment was conducted once. The following statistical analyses were per- formed using Prism 8 (GraphPad): Student’s and paired t test, Mann-Whitney U-test, and Fisher’s exact test. The statistical tests used for each figure can be found in the corresponding figure legend. The n values for larvae and microcolonies are given below each corresponding graph. Cell Reports 44, 116661, December 23, 2025 25 Article ll OPEN ACCESS CELREP116661_proof_v44i12.pdf Mycobacteria trehalose dimycolate interactions with host Mincle remodel blood-brain barrier junctions for brain invasion Introduction Results Circulating extracellular mycobacteria invade the brain to establish Rich foci Mycobacteria infect microglia to initiate Rich foci Mycobacterial attachment and growth on the microvascular endothelium are associated with F-actin rearrangements but not end ... Mycobacteria dynamically remodel endothelial cell tight junctions to create transient gaps through which they invade the br ... Mycobacterial PknD promotes brain invasion by promoting F-actin polymerization and attachment but does not cause tight junc ... Cell surface determinants shared with nonpathogenic mycobacteria remodel junctions Cyclopropanated TDM promotes invasion by increasing attachment and remodeling junctions M. tuberculosis invades the brain via attachment and junctional remodeling, with conserved roles for PknD and PcaA M. smegmatis PcaA is required for junctional remodeling Discussion Limitations of the study Resource availability Lead contact Materials availability Data and code availability Acknowledgments Author contributions Declaration of interests Supplemental information References STAR★Methods Key resources table Experimental model and study participant details Zebrafish husbandry and infections Method details Bacterial strains Plasmid construction Monocyte depletion, fluorospheres, and stains α-ZO-1 whole mount immunofluorescence Zebrafish larva microscopy and image analysis Quantification and statistical analysis