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Ion channel modulation during Chlamydia trachomatis development https://doi.org/10.17863/CAM.124072 Weild, Rachel Hayward, Richard Summary The obligate intracellular bacterial pathogen Chlamydia trachomatis causes sexually transmitted and ophthalmic infections (trachoma) in humans. C. trachomatis resides within a specialised membrane-bound replicative compartment termed the inclusion and exhibits a biphasic lifecycle requiring interconversion between two bacterial forms, infectious non-replicative elementary bodies (EB) and non-infectious replicative reticulate bodies (RB). RB redifferentiate into EB via transitional intermediate bodies (IB). C. trachomatis exploits potassium ion (K+) flux to mediate differentiation, host sensing, and inter-bacterial communication. During the early stages of the infection cycle RB accumulate K+ from the host cell when in close contact with the luminal face of the inclusion membrane. Higher cytosolic [K+] relative to the inclusion lumen and cell cytosol, generates an electrochemical K+ gradient across the inclusion membrane. K+ acquisition is RB specific as during redifferentiation EB lose contact with the inclusion membrane and K+ dissipate. Perturbing this K+ gradient early during the infection cycle using the K+ ionophore nigericin or sulfonylurea inhibitor glibenclamide promotes bacterial persistence. While these observations revealed an essential role for K+ during infection, the focus was the early replicative phase of the cycle. Since mid- cycle and late-cycle inclusions contain a heterogenous mixture of RB, IB, and EB, this study aimed to examine the dynamics and role/s of K+ later during the infection cycle. Using the K+ specific probe APG-2 and live-cell fluorescence confocal microscopy it was demonstrated that as the inclusion expands, a sub- population of RB maintain high cytosolic [K+] at the inclusion periphery from 30-56hpi relative to the inclusion lumen and cell cytosol. When the K+ gradient was perturbed using ionophores, a time-sensitive mid-cycle treatment window enhanced a population of novel sub-compartmentalised ‘vesiculated’ inclusions in the late cycle, which contained bacteria and otherwise retained typical features of intact inclusions. Treatments with K+ targeting reagents and Golgi disrupters revealed vesiculated inclusion formation was driven primarily 4 by disruption of inclusion-associated Golgi fragments, and that a functional Golgi apparatus was required for K+ sequestration by RB. Treatment with K+ ionophores in the mid-cycle reduced bacterial infectivity 81% by the late-cycle, whereas bacterial number was reduced by 45%. Quantification of bacterial forms present within the inclusion following treatment revealed a 40% loss of the EB, and temporally regulated increases in RB, RB dividing by binary fission, and IB. These data demonstrated that infectivity loss primarily resulted from impaired EB infectivity rather than stalls in RB to EB redifferentiation. Together these data showed that K+ gradients are essential for normal progression through the infection cycle. Confocal fluorescence microscopy showed that C. trachomatis specifically recruits the host inwardly rectifying K+ channel Kir6 to the inclusion membrane from early in the infection cycle. Channel recruitment was most prevalent in stages of the C. trachomatis infection cycle when RB predominate, with Kir6 recruitment gradually diminishing in correspondence with reducing RB populations. Conversely the canonical regulatory subunit of Kir6, SUR1/2, was never recruited to the inclusion. Correspondingly, the positive modulator of the heteromeric channel PIP2 did not colocalise with Kir6 at the inclusion. Analysis of K+ flux following siRNA-mediated knockdown of Kir6 in infected cells revealed Kir6 was essential for RB acquisition of K+ at the inclusion host interface, identifying host ion channel Kir6 as a key mediator of K+ flux at the inclusion membrane. The work presented in this thesis revealed K+ as a key regulator of bacterial differentiation and infectivity during the mid and late infection cycle. Since K+ dependent regulatory mechanisms are conserved in multiple human pathogens these data highlight a potentially universal K+ dependent mechanism of infection cycle regulation, revealing that ion channel modulation might present an attractive therapeutic target. Furthermore, a host mammalian ion channel was shown to be recruited to a pathogen compartment for the first time. Given the absence of sequence homology to known ion channels in the chlamydial genome, these data support a model in which bacterial ion channels are not required for K+ uptake, rather host K+ channels are hijacked. As Kir6 is uncoupled from its canonical regulators further work should address 5 how C. trachomatis coopts the host channel from within the inclusion. These data highlight the complex roles of K+ during the infection cycle of this medically important intracellular bacterial pathogen. University of Cambridge 2025-08-20 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/393944 embargo 2026-12-16 https://www.repository.cam.ac.uk/bitstreams/9007b1ae-f4d3-4972-b8d9-5859428c9c03/download 7884dbe5a4e60309255debd8d812b9cd https://www.repository.cam.ac.uk/bitstreams/5cfa5e68-15e9-46ca-ad29-a179d3550154/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved Chlamydia Ion channel Potassium Intracellular bacteria