Final Report Summary - CHLTRT3SE (Analysis of the cellular function of type III secretion effectors of Chlamydia trachomatis) Chlamydia trachomatis are obligate intracellular bacterial pathogens that cause ocular and genital infections, which are a significant public health concern worldwide. A better understanding of C. trachomatis basic biology is important to eventually develop new ways to combat infections caused by these bacteria. Although genetic manipulation of C. trachomatis is still very difficult, it is known that this bacterial species uses a type III secretion system (T3SS) to inject host cells with possibly > 100 effector proteins. These effectors manipulate host cell processes to enable e. g. bacterial invasion, acquisition of nutrients, inhibition of apoptosis, or interference with immune signalling.The overall scientific objective of this project was to increase our fundamental understanding of mechanisms underlying C. trachomatis virulence. For this, specifically we aimed to (i) identify novel C. trachomatis type III secretion (T3S) effectors and (ii) further understand the secretion and function of known effectors.To screen for novel T3S effectors of C. trachomatis, we used Yersinia enterocolitica as a genetically tractable surrogate bacterial species assembling a T3S apparatus, which has been shown to recognise heterologous T3S substrates. We first confirmed that known T3S substrates (IncA and IncC effectors) of C. trachomatis possess a T3S signal within their first 20 amino acids, which is recognised by the Y. enterocolitica T3SS as efficiently as a Yersinia endogenous T3S substrate. For this, we constructed translational fusions comprising the first 20 amino acids of the IncA or IncC effectors and a reporter protein (the mature form of TEM-1 beta-lactamase), expressed under the control of the Y. enterocolitica yopE effector gene promoter (PyopE). As negative control, we used a fusion between the first 20 amino acids of a C. trachomatis ribosomal protein and TEM-1 (RplJ20-TEM-1). Then, we selected 48 genes that may encode uncharacterised effectors (e. g. no detectable amino acid identity to other proteins or with a particular putative biochemical activity) from the genome of C. trachomatis strain L2/434/Bu. T3S assays performed with T3S-competent Y. enterocolitica or with an isogenic T3S-deficient strain individually expressing the TEM-1 fusions revealed a statistically significant higher level of secretion of 22 fusion proteins relative to RplJ20-TEM-1. Because T3S substrates have evolved to be transported within a narrow secretion tube (inner diameter of about 25 ?), we tested if these 22 C. trachomatis proteins with an N-terminal T3S signal were also secreted as full-length proteins after PyopE-dependent expression in Y. enterocolitica. Thirteen proteins showed a statistically significant higher level of secretion than full-length C. trachomatis RplJ. We also tested secretion of 11 out of these 13 proteins when expressed from an arabinose-inducible promoter in Y. enterocolitica, which revealed T3S-dependent secretion of 7 proteins. In summary, we have identified 22 novel putative C. trachomatis T3S effectors, seven of which were also secreted as full-length proteins by Y. enterocolitica in different experimental conditions. Therefore, these 7 proteins are very likely novel effectors of C. trachomatis. We have raised rabbit polyclonal antibodies against 5 of these proteins and we are currently analysing their subcellular localisation in C. trachomatis infected cells.To study the function of known effectors, we focused on the C. trachomatis Inc proteins, an important group of potential chlamydial virulence factors. They share a unique hydrophobic motif thought to target them to the membrane of the large vacuole where Chlamydia resides intracellularly. We aimed to understand if Inc proteins could play a role in the different types of infections caused by distinct C. trachomatis serovars: infections by serovars A-C are usually restricted to the conjunctival epithelium; infections by serovars D-K are restricted to genital epithelial cells; infections by serovars L1 L3 are sexually transmitted but with dissemination to lymph nodes, resulting in lymphogranuloma venereum (LGV). In spite of these differences, the genomes of ocular, genital, and LGV strains exhibit a high degree of synteny and more than 98 % of identity at the DNA level. We analysed if differences in the nucleotide (nt) and amino acid (aa) sequence of 48 C. trachomatis inc genes and corresponding Inc proteins, or differences in the expression of incs, correlate to distinct tropism and disease outcomes. We identified 19 incs with a mean genetic distance among C. trachomatis reference strains from all serovars of at least 0. 5 % nt and 1. 0 % aa differences (ranging from 3. 0-0. 6 % nt and 5. 1-1. 0 % aa differences). This was largely due to differences in the sequences between distinct disease groups (ocular, genital, or LGV) and not to differences within groups. In phylogenetic reconstructions of nt or aa sequences of these 19 polymorphic incs, six of them showed separate clustering of the three disease groups, and the other 13 showed separate clustering of strains from at least one disease group. Accordingly, 14 incs showed a ratio of nonsynonymous to synonymous substitutions of > 1 for all strains, which was also observed in analyses between disease groups, indicating an evolutionary trend towards protein modification. We used real-time quantitative PCR to determine the expression levels of the 48 incs during the infectious cycle of reference and clinical C. trachomatis strains representing the three disease groups. We identified 10 incs with notable differences in gene expression between serovars, which in most cases was due to higher expression levels in LGV strains. Overall, we identified nucleotide and expression differences between incs that may relate to distinct evolutionary pressures and could contribute to the specificity of host-pathogen interactions underlying infections with different C. trachomatis serovars. Future work will focus at studying the molecular and cellular function of the Incs identified during this work.To study the secretion of known C. trachomatis effectors, we screened for T3S chaperones encoded in the genome of strain L2/434/Bu. Often, secretion of T3S substrates involves characteristic chaperones (with a low molecular weight, an acidic pI, and which form dimers, and do not bind or hydrolyze ATP). Although the number of known chlamydial T3S effectors has been increasing, little is known about their possible cognate chaperones. To identify new T3S chaperones of C. trachomatis, we first selected 20 proteins with a predicted low molecular weight and pI. These proteins were subsequently analyzed on their ability to oligomerise, by using a bacterial two-hybrid system (BACTH). The 7 proteins that self-associated were then systematically tested by BACTH for interactions with a collection of 40 known and candidate C. trachomatis T3S substrates. The protein-protein interactions found were subsequently validated by co-immunoprecipitation experiments using Y. enterocolitica as heterologous bacteria. We identified two T3S chaperones: one that interacted with three different chlamydial T3S effectors and one that interacted with a candidate effector.In summary, we have identified novel C. trachomatis T3S effectors and chaperones, and we have increased our current understanding on the function of C. trachomatis Inc proteins. We are performing further studies on these subjects and this may eventually lead to new ideas to fight infectious diseases.Furthermore, the project aimed at helping the re-integration of the researcher holding the grant in the Portuguese science and higher education system after benefiting from an Intra-European Marie Curie Fellowship, to work in the United Kingdom. From this social perspective, the re-integration grant was pivotal in helping the researcher to establish his own laboratory and research lines (http://www.itqb. unl. pt/labs/infection-biology) and eventually led the researcher to obtain an Assistant Professorship in a major Portuguese public university. Therefore, the grant had an important impact on the European health research and higher education system.