Final Report Summary - GENCHLADEATH (Bacterial host cell death modulators – a genetic approach to identify anti-apoptotic factors of Chlamydia trachomatis and to explore their role during infection) Project ObjectivesThe induction of host cell death can serve as a cell-autonomous defense mechanism against intracellular pathogens. It can deprive a pathogen of its replicative niche and can influence disease progression and adaptive immune responses [1]. Successful pathogens have thus evolved sophisticated strategies to modulate host cell viability and to reduce the immunogenicity of host cell death [2,3]. Bacteria of the genus Chlamydia, which represent significant agents of sexually-transmitted disease, ocular infections, and respiratory disease in humans [4-6], are particularly dependent on their host cell due to their obligate intracellular lifestyle and their peculiar biphasic developmental cycle that needs to be complete for infectious particles to be generated [7]. It is thus not surprising that Chlamydia spp. actively protect infected host cells from apoptosis [8]. However, the experimental identification and characterization of virulence factors that mediate this protection was severely hindered by the recalcitrance of Chlamydia spp. to molecular genetic analysis. This situation changed dramatically as a result of recent methodological advances, such as the generation of large collections of mutant strains [9-11], the introduction of a plasmid-based transformation system [12] and the development of techniques for targeted gene disruption or allelic replacement in Chlamydia spp. [13,14].The MC-IOF project GenChlaDeath aimed to exploit these new genetic tools to enable (1) the isolation of Chlamydia mutants that have a reduced ability to counteract host cell death, (2) the identification of the bacterial virulence factors whose deficiency causes the observed phenotypes, (3) an assessment of the significance of Chlamydia-mediated host cell death modulation for bacterial replication and pathogenesis, and (4) the analysis of the mechanisms of the cell-autonomous induction of defensive cell death and of the bacterial countermeasures at the molecular level. To achieve these objectives, project GenChlaDeath was carried out by the postdoctoral researcher Dr. Barbara Sixt in the framework of an interdisciplinary international collaboration between the laboratory of Dr. Raphael Valdivia (Duke University, USA), a pioneer in the emerging field of molecular genetic analysis of Chlamydia, and the laboratory of Dr. Guido Kroemer (INSERM U1138, France), an expert in cell death research.Project ResultsAt the beginning of the outgoing phase of the project (conducted at Duke University), Dr. Sixt received extensive training in the application of molecular genetic tools for the analysis of Chlamydia spp. She then developed a forward-genetic approach to screen a pre-existing library of Chlamydia trachomatis mutants [11] for strains that had a decreased ability to block host cell death. The screen uncovered several mutant strains that induced premature cell death spontaneously, in line with the initial assumptions that (a) Chlamydia infection itself represents a pro-death signal, (b) host cells induce cell death as part of the cell-autonomous defense, and (c) Chlamydia spp. have evolved to block this response.One strain that spontaneously induced both apoptotic and necrotic cell death in a variety of cell lines was selected for an in-depth characterization. To identify the mutation that caused increased cytotoxicity in infected cells, Dr. Sixt applied a combination of various molecular genetic techniques, including the analysis of Chlamydia recombinants, plasmid-based complementation, and targeted insertional gene disruption approaches. This led to the identification of CpoS, a secreted chlamydial effector protein that is inserted into the membrane of the bacteria-containing vacuole (also known as inclusion), as the critical virulence factor that counteracts premature host cell death. Time lapse fluorescence microscopy of cells co-infected with CpoS-deficient bacteria together with CpoS-proficient strains that either form inclusions that can fuse with inclusions of CpoS-deficient bacteria or that form inclusions that remain separate, furthermore revealed that CpoS needs to be present at each Chlamydia inclusion inside an infected cells to prevent the induction of host cell death. Dr. Sixt furthermore discovered that CpoS-deficient bacteria caused an enhanced activation of the pathogen-associated molecular pattern receptor STING, leading to a strongly enhanced type I interferon (IFN) response at early time points of infection. These findings suggested that CpoS prevents the induction of cellular defense responses by hindering host cells from detecting the presence of the pathogen. Importantly, cell death induced by CpoS-deficient bacteria was also dependent on STING, but independent of the IFN response. Hence, these investigations uncovered a new role for STING as regulator of host cell death, which is independent from its known role as regulator of the type I IFN response. Apart from obtaining first mechanistic insights, Dr. Sixt also demonstrated the significance of CpoS as a virulence factor by showing that CpoS-deficient bacteria had a reduced potential to form infectious progeny in cultured cells and that the bacteria were cleared faster from the mouse genital tract in a murine transcervical infection model. The findings from investigations conducted during the outgoing phase of the project were recently published in the peer-reviewed journal Cell Host & Microbe [15].At the end of the outgoing phase of the project, Dr. Sixt used a proteomic approach to identify CpoS-interacting proteins, i.e. bacterial and host proteins that co-immunoprecipitated with FLAG-tagged CpoS expressed from a plasmid introduced into the CpoS-deficient strain. During the return phase of the project (conducted at INSERM), these interactions were validated by additional co-immunoprecipitation assays and by the analysis of the subcellular localization of potential CpoS interactors in cells infected with wild-type or CpoS-deficient bacteria. These studies identified a class of host proteins that were recruited to the Chlamydia inclusion in a strictly CpoS-dependent manner. A detailed investigation of the role of CpoS-mediated interactions in preventing the induction of cellular defense responses is still in progress. A second major focus of the investigations conducted during the return phase of the project was the analysis of the immunogenicity of premature host cell death induced by CpoS-deficient bacteria and of the cell death induced naturally by wild-type Chlamydia at the end of their infection cycle. These studies involved the use of various assays to assess the occurrence of cellular markers of immunogenic cell death and the induction of connected cellular stress pathways [3] during the time course of infection with wild-type or CpoS-deficient Chlamydia. It is expected that the results from the two investigations, the analysis of CpoS-mediated interactions and the assessment of the immunogenicity of Chlamydia-induced cell death, will be published separately in peer-reviewed journals.ImpactThe cell-autonomous immunity - which is the ability of almost every cell in the human body to detect invading microbes and to respond to the threat by inducing microbial growth-restricting, microbicidal, or host cell death programs - is the most ancient branch of immunity. It is tempting to assume that it could be most effective in fighting obligate intracellular pathogens and that new innovative therapeutic and preventive treatment regimens could be developed based on the idea to activate its effector mechanisms. However, the power of cell-intrinsic defense responses is poorly understood, because pathogen-mediated defense suppression conceals their action. This paucity of knowledge can only be overcome by molecular genetic approaches, which are however still rarely conducted in obligate intracellular bacteria, due to technical difficulties. Project GenChlaDeath successfully faced these challenges. The work represents one of the very first studies that exploited the full repertoire of Chlamydia genetics and as such helps to define a benchmark for research in the age of Chlamydia genetics. It provided first evidence for the protective power of cell-autonomously induced defensive host cell death against Chlamydia spp., identified a novel virulence factor of importance for bacterial replication and survival in the host, and identified a novel role of the host sensor STING in regulating cell death. The latter is also of high interest beyond the field of infection biology, given reported associations of STING hyperactivation with autoinflammatory disease and the suggested role of STING in promoting antitumor immunity [16]. Ongoing work will shed light onto mechanisms used by intracellular bacteria to suppress the immunogenicity of host cell death. Overall, findings from GenChlaDeath significantly advanced our understanding of the defensive power of host cell death against intracellular pathogens and will motivate further investigations to assess how it can be exploited for therapeutic purposes.ContactBarbara S. Sixt (email: barbara.s.sixt@gmail.com)Guido Kroemer (email: kroemer@orange.fr)Raphael H. Valdivia (email: raphael.valdivia@duke.edu)References1. Labbé K, Saleh M (2008) Cell Death Differ 15: 1339-1349.2. Lamkanfi M, Dixit VM (2010) Cell Host Microbe 8: 44-54.3. Galluzzi L, Buque A, Kepp O, Zitvogel L, Kroemer G (2017) Nat Rev Immunol 17: 97-111.4. Taylor HR, Burton MJ, Haddad D, West S, Wright H (2014) Lancet 384: 2142-2152.5. Mylonas I (2012) Arch Gynecol Obstet 285: 1271-1285.6. Burillo A, Bouza E (2010) Infect Dis Clin North Am 24: 61-71.7. Ward ME (1988) In: Micobiology of Chlamydia. Boca Raton FL: CRC Press. pp. 71-95.8. Sharma M, Rudel T (2009) FEMS Immunol Med Microbiol 55: 154-161.9. Kari L, Goheen MM, Randall LB, Taylor LD, et al. (2011) Proc Natl Acad Sci U S A 108: 7189-7193.10. Nguyen BD, Valdivia RH (2012). Proc Natl Acad Sci U S A 109: 1263-1268.11. Kokes M, Dunn JD, Granek JA, Nguyen BD, et al. (2015) Cell Host Microbe 17: 716-725.12. Wang Y, Kahane S, Cutcliffe LT, Skilton RJ, et al. (2011) PLoS Pathog 7: e1002258.13. Johnson CM, Fisher DJ (2013) PLoS One 8: e83989.14. Mueller KE, Wolf K, Fields KA (2016) MBio 7: e01817-01815.15. Sixt BS, Bastidas RJ, Finethy R, Baxter RM, et al. (2017) Cell Host Microbe 21: 113-121.16. Barber GN (2015) Nat Rev Immunol 15: 760-770.
