Introduction
Chlamydia spp. are obligate intracellular bacterial pathogens responsible for a wide range of diseases in humans and animals 1. Chlamydia trachomatis, the most prevalent human pathogenic Chlamydia species, is the causative agent of blinding trachoma, an ocular disease that is endemic in many developing countries 2. Moreover, with over 100 million annual cases, C. trachomatis is the most frequent bacterial agent of sexually transmitted diseases worldwide and as such a frequent cause of infertility and adverse pregnancy outcomes 3.
Within its host cell, C. trachomatis undergoes a biphasic developmental cycle consisting of two distinct developmental stages 4. The infectious stage, the elementary body (EB), invades a human host cell and subsequently resides within a membrane-bound intracellular compartment termed inclusion. The EB then differentiates into the replicative stage, the reticulate body (RB), which proliferates within the inclusion. RBs are non-infectious and fragile and therefore highly dependent on the integrity of their host cell. At late stages of the infection cycle, typically 2-3 days after invasion, RBs retro-differentiate into EBs, which are released from the host cell by host cell lysis or extrusion to infect neighboring cells 5.
Its unique lifestyle, its pronounced dependence on a human host cell, and its clinical significance make Chlamydia an excellent system for studies on cell-autonomous immunity and bacterial evasion mechanisms.
Molecular action of the inclusion membrane protein CpoS
Chlamydia spp. actively modulate host cellular functions by exploiting their type III secretion system to deliver effector proteins into the host cell cytosol 6. A special class of these effectors, the inclusion membrane proteins (Incs), are inserted into the membrane of the inclusion 7. In a forward genetic screen using a library of chemically mutagenized C. trachomatis strains 8, we recently identified a strain that failed to block host cell driven cellular defense responses 9. More precisely, infection with the mutant led to a strongly enhanced induction of the STING-mediated type I interferon response, as well as to premature host cell death at early to mid-stages of infection. The mutant had a reduced ability to form infectious EBs in cell culture and was cleared faster from the mouse genital tract in an in vivo genital tract infection model 9. Molecular genetic analysis of the mutant revealed that the causative mutation resulted in the loss of the inclusion membrane protein CpoS 9. Our current studies focus on CpoS’ molecular interactions with host cellular proteins with the aim to obtain a detailed understanding of their contributions to CpoS-dependent immune evasion.
Investigated by: Karsten Meier and Lana Jachmann (formerly: Lucía Pérez Jiménez)
Molecular composition of a novel defensive host cell death program
Host cell death is a cell-autonomous defense response that can restrict the growth of intracellular pathogens by removing their replicative niche 10. The discovery that many clinically important pathogens, including Chlamydia spp., have evolved strategies to actively suppress cell death in infected cells further highlights the significance of this defense mechanism 11. We exploit the availability of a CpoS-deficient C. trachomatis strain, which causes premature host cell death 9, to obtain a deeper understanding of the molecular machinery that induces cell death in response to infection. In this context, we analyze the involvement of known innate immune pathways and programmed cell death programs in the execution of host cell death during infection with the mutant. Moreover, we complement these studies with unbiased genome-wide genetic screening approaches to uncover novel regulators of cell death and defense.
Investigated by: Mohammed Rizwan Babu Sait (formerly: Samada Muraleedharan, Emma Jousseaume)
Identification of cellular defense pathways with hidden protective potential
While induction of premature host cell death can disrupt Chlamydia development and growth very effectively 9, human cells also have defense responses that act in different ways. For instance, cells may restrict pathogen growth via nutrient depletion or may destroy intracellular pathogens via delivery into autophagosomes or lysosomes 12. However, the protective potential of cell-autonomous defense programs often remains obscure, because clinically important pathogens are usually well adapted to their host and have evolved efficient countermeasures. To obtain a better understanding of the full potential of cell-autonomous immunity in restricting Chlamydia infection, we use a forward genetic screening approach to uncover defense pathways that could confer significant protection when relieved from pathogen-mediated suppression. Currently, we identify the cognate counteracting virulence factors, which will in the future enable in-depth analyses of their mode of action.
Investigated by: Karsten Meier and Gözde Türköz
Expanding the experimental toolkit for Chlamydia
Our ability to characterize Chlamydia virulence factors and immune evasion mechanisms has historically been limited by the genetic intractability of these bacteria. However, in spite of technical difficulties arising from the obligate intracellular and developmental lifestyles of Chlamydia spp., various genetic techniques have been developed in the past decade, in particular for the human pathogen C. trachomatis 13. These techniques now enable us to transform C. trachomatis with plasmids that mediate heterologous protein expression 14, 15 and to introduce targeted genetic modifications, such as gene disruptions and gene replacements 16, 17. Together with our collaborators from the University of Maryland (Baltimore, MD, USA) and Duke University (Durham, NC, USA), we recently demonstrated the versatility of the TargeTron system, the currently most widely used experimental strategy for targeted gene disruption in C. trachomatis 16, by disrupting for the first time virulence factors in a distinct Chlamydia species, the zoonotic pathogen C. caviae 18. Our current efforts focus on exploiting the available genetic tools for the development of new reporter systems to study Chlamydia-host interactions with a focus on cell-autonomous immunity and bacterial evasion mechanisms.
Investigated by: Lana Jachmann (formerly Anastasiia Chaban, Partha Mohanty, Samada Muraleedharan, Celia Llorente, Sepideh Farmand Azadeh)
A multi-strategy discovery approach to find new ways to treat Chlamydia
Infections with C. trachomatis are usually effectivly treated with azithromycin or doxycycline 19. However, treatment failures have been described 20, and these broad-spectrum antibiotics bring negative consequences for patient and society by disrupting the commensal microbiota 21 and by potentially facilitating resistance development in Chlamydia as well as in off-target bacterial populations 22. The availability of pathogen-specific drugs for the treatment of Chlamydia infections would thus benefit patient health and aid antibiotic stewardship. In this project, we use a combination of different experimental and computational techniques to discover and characterize novel compounds that inhibit the growth of C. trachomatis while leaving the microbiota intact. We envision that such specific antichlamydials could for instance act by boosting cellular defense programs (host-directed compounds) or by blocking the pathogenic countermeasures (anti-virulence compounds). However, other modes of action are conceivable as well.
Investigated by: Magnus Ölander, Karsten Meier, Daniel Rea, and Eduard Calpe (formerly Nelumika Panagoda, María Rayón Díaz, Lieke Mooij, Johanna Fredlund)
References
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