Secreting proteins into host cell cytosol represents one of the most effective strategies for C. trachomatis organisms to constantly improve their intracellular living. Identifying chlamydia-secreted proteins will be essential for uncovering the mysteries of the chlamydial intracellular life and understanding the molecular mechanisms of C. trachomatis pathogenesis. In the current manuscript, we have presented convincing evidence that the hypothetical protein CT311 is secreted into host cell cytosol during C. trachomatis infection. First, both the anti-CT311 polyclonal antiserum and mAb detected CT311 both inside and outside of the chlamydial inclusions in C. trachomatis-infected cells. The intra-inclusion CT311 molecules were labeled in granules free of chlamydial organisms while the extra-inclusion CT311 molecules were in the host cell cytoplasm just like the chlamydia-secreted protease CPAF, demonstrating that at least a portion of CT311 was completely secreted into the host cell cytosol. Second, the detection of the endogenous CT311 was specific. The anti-CT311 antibody labeling observed under the fluorescence microscope was only removed by absorption with CT311 but not CPAF fusion proteins. Furthermore, both anti-CT311 and anti-CPAF antibodies only detected their corresponding endogenous proteins without cross-reacting with each other or any other antigens in the whole cell lysates of C. trachomatis-infected cells. Third, like CPAF, CT311 also contains a N-terminal signal sequence and the signal sequence is functional in directing a heterologous protein across the inner membrane, suggesting that a sec-dependent pathway may contribute to the secretion of both CT311 and CPAF into host cell cytosol. Finally, despite the many similarities between CT311 and CPAF, the secretion kinetics of CT311 seemed to be distinct from that of CPAF. Although both CT311 and CPAF proteins were first detected 12h after infection, secretion of CT311 into host cell cytosol was delayed until 24h while CPAF secretion was already very obvious by 18h after infection, indicating that CPAF secretion was rapid, which is consistent with what was reported previously (Gong S, 2011
; Heuer et al., 2003
; Hobolt-Pedersen et al., 2009
). The secretion of CPAF also seemed to be more complete since once the protein was detectable, most CPAF was detected outside of the inclusions while there were a lot of CT311 signals retained in the inclusion throughout the infection course. The distinct secretion kinetics suggests that the chlamydial organisms may use CT311 to accomplish a function that is different from what CPAF does.
The next question is how CT311 is exported into host cell cytosol during chlamydial infection. This is a common question faced by the entire chlamydia research fields. The observations that the CT311 N-terminal signal sequence directed the translocation of mature PhoA into E. coli periplasm and secretion of CT311 in Chlamydia-infected cells was inhibited by a signal peptidase I inhibitor strongly suggest that a sec-dependent pathway may play an important role in the secretion of CT311 into host cell cytosol. However, the sec-dependent pathway can only translocate its cargoes into the periplasmic region. How is the periplasmic CT311 further exported out of the chlamydial organisms and into host cell cytoplasm? The C. trachomatis organisms do express homologs of the general secretion proteins (Gsps) in the outer membrane, including CT570-572 for GspF, E & D respectively. However, these outer membrane secretion proteins export periplasmic proteins out of the organisms in free form. It is hard to imagine how soluble proteins once dumped into the inclusion lumen can further cross the inclusion membrane to enter host cell cytosol in a controlled/regulated manner. Since CT311-laden vesicule-like structures free of chlamydial organisms were detected in the lumen of chlamydial inclusions, we like to propose that the chlamydial organisms may use an outer membrane vesicular (OMV) budding mechanism to export the periplasmic cargoes into host cell cytosol. This hypothesis is also consistent with various previous observations that the chlamydial RB outer membrane was induced to undergo vesiculation (Matsumoto & Manire, 1970a
; Matsumoto & Manire, 1970b
) and chlamydial organism-free vesicles were detected both inside (Jorgensen & Valdivia, 2008
) and outside of inclusion membrane (Giles et al., 2006
). The vesiculized CT311 may further enter host cell cytosol by vesicle fusing with or passing through the inclusion membrane. Although OMVs have been recognized as an essential means for gram-negative bacteria to secrete virulence factors (Ellis & Kuehn, 2010
; Ellis et al., 2010
; Parker et al., 2010
; Unal et al., 2010
), the precise mechanisms on how OMVs are regulated remain unknown (Haurat et al., 2010
), which has led some scientists to question whether OMVs can really represent a specific mechanism for protein secretion. The C. trachomatis-infected cells may provide a unique model system for dissecting the molecular pathways of OMVs.
Regardless of how CT311 is secreted into host cell cytosol, a more important question is what role the secreted CT311 may play during C. trachomatis infection. CT311 is categorized as a Chlamydia-specific hypothetical protein with a total of 235 amino acids and a pI of 9.75 (http://stdgen.northwestern.edu
). CT311 is conserved among all chlamydial genomes sequenced so far, suggesting that it is essential for maintaining the chlamydial intracellular parasitism. However, little is known about how CT311 works. No putative conserved domains have been detected using bioinformatics programs (http://toolkit.tuebingen.mpg.de/hhpred
). Indeed, identifying the secretion pathway and determining the functionality of Chlamydia trachomatis-secreted proteins (CtSPs) have been the common challenges faced by modern chlamydial researchers. As more CtSPs are identified, more knowledge will be accumulated and more tools will be available for figuring out biological significance of CtSPs. While we continue our path to identify new CtSPs, we are also using approaches such as yeast-two hybrid and co-precipitation to gain in-depth knowledge on the already identified CtSPs.