SUMMARY Chlamydia trachomatis is a leading cause of genital and ocular infections for which no vaccine exists. Upon entry into host cells, C. trachomatis resides within a membrane bound compartment—the inclusion--and secretes inclusion membrane proteins (Incs) that are thought to modulate the host-bacterium interface. To expand our understanding of Inc function(s), we subjected putative C. trachomatis Incs to affinity purification-mass spectroscopy (AP-MS). We identified Inc-human interactions for 38/58 Incs with enrichment in host processes consistent with Chlamydia’s intracellular lifecycle. There is significant overlap between Inc targets and viral proteins, suggesting common pathogenic mechanisms among obligate intracellular microbes. IncE binds to sorting nexins (SNXs) 5/6, components of the retromer, resulting in SNX5/6 relocalization to the inclusion membrane and enhanced inclusion membrane tubulation. Depletion of retromer components enhances progeny production, revealing that retromer restricts Chlamydia infection. This study demonstrates the value of proteomics in unveiling host-pathogen interactions in genetically challenging microbes.

Antibiotic resistant bacteria are an emerging global health threat. New antimicrobials are urgently needed. The injectisome type III secretion system (T3SS), required by dozens of Gram-negative bacteria for virulence but largely absent from non-pathogenic bacteria, is an attractive antimicrobial target. We previously identified synthetic cyclic peptomers, inspired by the natural product phepropeptin D, that inhibit protein secretion through the Yersinia Ysc and Pseudomonas aeruginosa Psc T3SSs, but do not inhibit bacterial growth. Here we describe identification of an isomer, 4EpDN, that is two-fold more potent (IC50 of 4 μM) than its parental compound. Furthermore, 4EpDN inhibited the Yersinia Ysa and the Salmonella SPI-1 T3SSs, suggesting that this cyclic peptomer has broad efficacy against evolutionarily distant injectisome T3SSs. Indeed, 4EpDN strongly inhibited intracellular growth of Chlamydia trachomatis in HeLa cells, which requires the T3SS. 4EpDN did not inhibit the unrelated Twin arginine translocation (Tat) system, nor did it impact T3SS gene transcription. Moreover, although the injectisome and flagellar T3SSs are evolutionarily and structurally related, the 4EpDN cyclic peptomer did not inhibit secretion of substrates through the Salmonella flagellar T3SS, indicating that cyclic peptomers broadly but specifically target the injestisome T3SS. 4EpDN reduced the number of T3SS needles detected on the surface of Y. pseudotuberculosis as detected by microscopy. Collectively, these data suggest that cyclic peptomers specifically inhibit the injectisome T3SS from a variety of Gram-negative bacteria, possibly by preventing complete T3SS assembly.

Chlamydia trachomatis is an obligate intracellular pathogen that replicates within a specialized membrane-bound compartment, called the inclusion. Chlamydia species express a unique class of effectors, Incs, which are translocated from the bacteria by a Type III secretion system and are inserted into the inclusion membrane where they modulate the host-bacterium interface. C. trachomatis repositions specific host organelles during infection to acquire nutrients and evade host cell surveillance, however the bacterial and host proteins controlling these processes are largely unknown. Here, we identify an interaction between the host dynactin complex and the C. trachomatis Inc CT192 (CTL0444), hereafter named Dre1 for Dynactin Recruiting Effector 1. We show that dynactin is recruited to the inclusion in a Dre1-dependent manner and that loss of Dre1 diminishes the recruitment of specific host organelles, including the centrosome, mitotic spindle, and Golgi apparatus to the inclusion. Inactivation of Dre1 results in decreased C. trachomatis fitness in cell-based assays and in a mouse model of infection. By targeting particular functions of the versatile host dynactin complex, Dre1 facilitates re-arrangement of certain organelles around the growing inclusion. Our work highlights how C. trachomatis employs a single effector to evoke specific, large-scale changes in host cell organization that establish an intracellular replicative niche without globally inhibiting host cellular function.

Antibiotic resistant bacteria are an emerging global health threat. New antimicrobials are urgently needed. The injectisome type III secretion system (T3SS), required by dozens of Gram-negative bacteria for virulence but largely absent from non-pathogenic bacteria, is an attractive antimicrobial target. We previously identified synthetic cyclic peptomers, inspired by the natural product phepropeptin D, that inhibit protein secretion through the Yersinia Ysc and Pseudomonas aeruginosa Psc T3SSs, but do not inhibit bacterial growth. Here we describe identification of an isomer, 4EpDN, that is two-fold more potent (IC50 4 mM) than its parental compound. Furthermore, 4EpDN inhibited the Yersinia Ysa and the Salmonella SPI-1 T3SSs, suggesting that this cyclic peptomer has broad efficacy against evolutionarily distant injectisome T3SSs. Indeed, 4EpDN strongly inhibited intracellular growth of Chlamydia trachomatis in HeLa cells, which requires the T3SS. 4EpDN did not inhibit the unrelated Twin arginine translocation (Tat) system, nor did it impact T3SS gene transcription. Moreover, although the injectisome and flagellar T3SSs are evolutionarily and structurally related, the 4EpDN cyclic peptomer did not inhibit secretion of substrates through the Salmonella flagellar T3SS, indicating that cyclic peptomers broadly but specifically target the injestisome T3SS. 4EpDN reduced the number of T3SS basal bodies detected on the surface of Y. enterocolitica, as visualized using a fluorescent derivative of YscD, an inner membrane ring with low homology to flagellar protein FliG. Collectively, these data suggest that cyclic peptomers specifically inhibit the injectisome T3SS from a variety of Gram-negative bacteria, possibly by preventing complete T3SS assembly.