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  • Periodic Report Summary 1 - GENCHLADEATH (Bacterial host cell death modulators – a genetic approach to identify anti-apoptotic factors of Chlamydia trachomatis and to explore their role during infection)
FP7

GenChlaDeath Report Summary

Project ID: 626116
Funded under: FP7-PEOPLE
Country: France

Periodic Report Summary 1 - 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 Objectives

The induction of host cell death is a cell-autonomous defense mechanism against intracellular pathogens. It can deprive the pathogen from its replicative niche and can influence disease progression and adaptive immune responses (Labbé and Saleh, 2008). Successful bacterial and viral pathogens have thus evolved sophisticated strategies to modulate host cell viability (Lamkanfi and Dixit, 2010). Bacteria of the genus Chlamydia are particularly dependent on their host cell due to their obligate intracellular life-style and their peculiar biphasic developmental cycle that needs to be complete for infectious particles to be generated (Ward, 1988). It is thus not surprising that Chlamydia spp. actively protect infected host cells from apoptosis (Sharma and Rudel, 2009).The Chlamydia virulence factors that mediate this protection are unknown and studies aiming at their identification were hampered by the recalcitrance of Chlamydia spp. to molecular genetic analysis. The Chlamydia research landscape changed dramatically as a result of recent advances, such as the generation of large collections of mutant strains (Kari et al., 2011; Kokes et al., 2015; Nguyen and Valdivia, 2012), the introduction of a plasmid-based transformation system (Wang et al., 2011) and the development of techniques for targeted gene disruption or allelic replacement in Chlamydia trachomatis (Johnson and Fisher, 2013; Mueller et al., 2016).

The MC-IOF project GenChlaDeath exploits these new genetic tools to: (1) isolate Chlamydia mutants that have an altered ability to maintain host cell viability, (2) identify the bacterial virulence factors whose deficiency cause the observed phenotypes, (3) determine the significance of Chlamydia-mediated host cell death modulation on bacterial replication and pathogenesis, and (4) define the molecular basis of the cell-autonomous induction of defensive cell death and the mechanism of bacteria-mediated interference with such processes.

Project Progress & Results

The project GenChlaDeath is carried out in an interdisciplinary collaboration between the lab of Dr. Valdivia (Duke University, USA), a pioneer in the emerging field of molecular genetic analysis of Chlamydia, and the lab or Dr. Kroemer (INSERM U1138, France), an expert in cell death research. The first period, in which Dr. Sixt conducted research at Duke University, was recently concluded.

At the beginning of this period Dr. Sixt developed a screening protocol that subsequently enabled her to screen a pre-existing library of C. trachomatis mutants (Kokes et al., 2015), for strains that have either a decreased ability to block experimentally-induced apoptosis or that display spontaneously enhanced cytotoxicity towards host cells. One strain, which spontaneously induced both apoptotic and necrotic cell death in a variety of cell lines, was selected for an in-depth characterization. This particular strain was chosen due to the strength of the observed phenotype.

In the next phase of the project Dr. Sixt applied a combination of molecular genetic techniques, including whole genome sequencing, analysis of Chlamydia recombinants, plasmid-based complementation, and targeted insertional gene disruption, to identify the mutation that caused increased cytotoxicity in infected cells. Enhanced induction of host cell death by the Chlamydia mutant was linked to a mutation that disrupted a gene coding for a secreted chlamydial effector protein. The phenotype could be complemented in the mutant strain by plasmid-driven expression of the protein. Moreover, targeted disruption of the gene encoding the effector in wild-type Chlamydia recapitulated the phenotype of the mutant.

Finally, Dr. Sixt applied cell culture experiments and a murine transcervical infection model to investigate the importance of the secreted effector protein for Chlamydia replication and pathogenesis. These studies revealed that the mutant was defective in its ability to form infections progeny and was cleared faster from the murine genital tract.

A molecular dissection of the host cell death pathways induced during infection with the Chlamydia mutant and a characterization of the mode of action of the chlamydial effector protein are ongoing and will be the major focus of research during the second period of the project which will be conducted at the European host institute.

The results of the project were summarized in a research article which is currently under revision for publication in a peer-reviewed journal (Sixt et al.). The expertise gained during her stay at Duke University moreover enabled Dr. Sixt to make major contributions to a review article that summarizes current technology and future perspectives of molecular genetics analysis of Chlamydia (Sixt and Valdivia, 2016).

Expected Final Results & Impact

Chlamydia spp. are important pathogens of humans and livestock. C. trachomatis, for instance, is the causative agent of blinding trachoma, a major health concern in developing countries (Taylor et al., 2014), and a prevalent bacterial agent of sexually-transmitted disease that can cause severe complications including lymphogranuloma venereum, pelvic inflammatory disease and infertility (Mylonas, 2012). A deep understanding of Chlamydia-mediated modulation of host cell-autonomous defense programs will aid in the development of new means for the prevention and treatment of Chlamydia diseases. The project GenChlaDeath highlighted the importance of the ability of Chlamydia to actively counteract induction of cell death pathways in infected cells. It moreover identified one of the bacterial virulence factors that mediate this activity, highlighting a potential target for therapeutic intervention. Furthermore, ongoing investigations have high potential to provide new insights into the host cell signaling pathways that mediate induction of cell death in response to infectious agents.

Contact

Guido Kroemer (email: kroemer@orange.fr)
Raphael H. Valdivia (email: raphael.valdivia@duke.edu
Barbara S. Sixt (email: barbara.s.sixt@gmail.com)

References

Johnson, C.M., and Fisher, D.J. (2013). Site-specific, insertional inactivation of incA in Chlamydia trachomatis using a group II intron. PLoS One 8, e83989.
Kari, L., Goheen, M.M., Randall, L.B., Taylor, L.D., Carlson, J.H., Whitmire, W.M., Virok, D., Rajaram, K., Endresz, V., McClarty, G., et al. (2011). Generation of targeted Chlamydia trachomatis null mutants. Proc Natl Acad Sci U S A 108, 7189-7193.
Kokes, M., Dunn, J.D., Granek, J.A., Nguyen, B.D., Barker, J.R., Valdivia, R.H., and Bastidas, R.J. (2015). Integrating chemical mutagenesis and whole-genome sequencing as a platform for forward and reverse genetic analysis of Chlamydia. Cell Host Microbe 17, 716-725.
Labbé, K., and Saleh, M. (2008). Cell death in the host response to infection. Cell Death Differ 15, 1339-1349.
Lamkanfi, M., and Dixit, V.M. (2010). Manipulation of host cell death pathways during microbial infections. Cell Host Microbe 8, 44-54.
Mueller, K.E., Wolf, K., and Fields, K.A. (2016). Gene deletion by fluorescence-reported allelic exchange mutagenesis in Chlamydia trachomatis. MBio 7, e01817-01815.
Mylonas, I. (2012). Female genital Chlamydia trachomatis infection: where are we heading? Arch Gynecol Obstet 285, 1271-1285.
Nguyen, B.D., and Valdivia, R.H. (2012). Virulence determinants in the obligate intracellular pathogen Chlamydia trachomatis revealed by forward genetic approaches. Proc Natl Acad Sci U S A 109, 1263-1268.
Sharma, M., and Rudel, T. (2009). Apoptosis resistance in Chlamydia-infected cells: a fate worse than death? FEMS Immunol Med Microbiol 55, 154-161.
Sixt, B.S., Bastidas, R.J., Finethy, R., Baxter, R.M., Carpenter, V.K., Kroemer, G., Coers, J., and Valdivia, R.H. The Chlamydia trachomatis inclusion membrane protein CpoS counteracts cellular surveillance and suicide programs Cell Host Microbe in revision.
Sixt, B.S., and Valdivia, R.H. (2016). Molecular Genetic Analysis of Chlamydia Species. Annu Rev Microbiol 70, 179-198.
Taylor, H.R., Burton, M.J., Haddad, D., West, S., and Wright, H. (2014). Trachoma. Lancet 384, 2142-2152.
Wang, Y., Kahane, S., Cutcliffe, L.T., Skilton, R.J., Lambden, P.R., and Clarke, I.N. (2011). Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathog 7, e1002258.
Ward, M.E. (1988). The chlamydial developmental cycle. In Micobiology of Chlamydia, A.L. Barron, ed. (Boca Raton FL: CRC Press), pp. 71-95.

Contact

Isabelle VERDIER, (Coordinatrice Ressources Externes)
Tel.: +33 1 48073433
Fax: +33 1 48073426
E-mail

Subjects

Life Sciences
Record Number: 193155 / Last updated on: 2017-01-16
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