Small molecule inhibitors of type III secretion and their effect on chlamydia development

Chlamydiae are obligate intracellular pathogens that cause a variety of diseases with clinical and public health importance. Like many Gram-negative bacteria, Chlamydiae employ a type III secretion (T3S) system for invasion and establishment of a protected intracellular niche for successful replication and survival within host cells. Understanding the role of T3S and bacterial effector proteins in Chlamydia infection will provide new insights into chlamydial pathogenesis and is important to identify novel therapeutic targets for drug intervention. In this thesis we employed different small molecule inhibitors of T3S activity in Yersinia, named INPs, and analyzed their effect on Chlamydia development. In addition, we identified and characterized a new family of T3S effector proteins. In paper I, we assessed the effect of INP0400 on C. trachomatis development and invasion. INP0400 caused a dose and growth phase-dependent inhibition of RB multiplication at micromolar concentrations. When INP0400 was given at different stages during the infectious cycle, we observed a partial inhibition of Chlamydia entry, inhibition of translocation of IncG and IncA and a bacterial detachment from the inclusion membrane during the late stage of infection concomitant with an inhibition of RB to EB conversion causing a marked decrease in infectivity. Our data suggest that INPs impair progression through the infectious cycle suggesting that the T3S system is essential for Chlamydia pathogenesis. In paper II, we found that INP0010 displays a strong growth inhibitory effect on C. pneumoniae development, affects translocation of the C. pneumoniae effector proteins IncB and IncC and leads to down-regulation of T3S associated genes collectively suggesting that INP0010 impairs T3S activity in C. pneumoniae. In paper III, we further investigated the effect of INPs on Chlamydia invasion. We show that INPs impair Chlamydia development after entry into host cells because the efficiency of C. trachomatis L2 and C. caviae entry into epithelial cells was not altered in the presence of INPs. Moreover, entry appeared normally with recruitment of actin and the small GTPases Rac, Cdc42 and Arf6 to the bacterial entry site. Finally, in paper IV we set out to identify novel T3S effectors in Chlamydia. We found a family of chlamydial proteins, represented by a C-terminal domain of unknown function referred to as DUF582 that contains an amino-terminal T3S signal. C. trachomatis members of this family were expressed late during the infectious cycle and found to be secreted into the lumen of the inclusion and the cytoplasm of infected cells. LIST OF PUBLICATIONS This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Muschiol, S. , L. Bailey, A. Gylfe, C. Sundin, K. Hultenby, S. Bergstrom, M. Elofsson, H. Wolf-Watz, S. Normark, and B. Henriques-Normark. 2006. A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis. Proc Natl Acad Sci USA. 103:14566-14571. II Bailey, L. , A. Gylfe, C. Sundin, S. Muschiol, E. Elofsson, P. Nordstrom, B. Henriques-Normark, R. Lugert, A. Waldenstrom, H. Wolf-Watz, and S. Bergstrom. 2007. Small molecular inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle. FEBS Let. 581:587-595. III Muschiol, S. , S. Normark, B. Henriques-Normark, and A. Subtil. 2009. Small molecule inhibitors of the Yersinia type III secretion system impair the development of Chlamydia after entry into host cells. BMC Microbiol. 2009. 9:75. IV Muschiol, S. , G. Boncompain, P. Dehoux, S. Normark, B. HenriquesNormark, and A. Subtil. 2009. A new family of secreted proteins in Chlamydia. Manuscript.