HtrA is known to be an important stress response protease for many bacteria and has been shown to be critical for virulence in many bacteria, including intracellular pathogens Salmonella enterica and Legionella pneumophila [29, 30]. There is considerable evidence from both microarray and proteomic studies that HtrA is expressed in Chlamydia.
In the absence of a genetic manipulation system, a complementation approach was used to test the physiological function of C. trachomatis HtrA in a heterologous host (E. coli). E. coli HtrA protein (EcHtrA) and C. trachomatis HtrA protein (CtHtrA) are known to have differences in substrate specificity for their protease activities, although both have temperature activated protease activity, and are specific for unfolded proteins [4, 8]. The findings reported here show that the C. trachomatis htrA was able to protect E. coli htrA
- against its lethal high temperature phenotype. This suggests that the ability to chaperone and degrade unfolded proteins, regardless of specificity for residues at the site of peptide bond cleavage, is sufficient to protect against the damage caused by heat stress. Minor differences observed in complementation by echtrA and cthtrA (Fig. 2) could be attributed to; differences in substrate specificities of the enzymes, potential differences in the 'switch' to chaperone activity at higher temperatures as E. coli has been previously reported to act only as a chaperone at temperatures below 30°C (Speiss et al., 1999) although more recent studies suggest EcHtrA may have chaperone activity at higher temperatures (Skorko-Glonek et al., 2007), or finally due to different copy number requirements for the two genes. However, regardless of the minor differences in complementation by growth curve, it is clear that Chlamydia htrA can protect E. coli htrA
- against the detrimental affects of heat stress. This data provides direct evidence for in vivo physiological functionality of C. trachomatis HtrA as a molecular chaperone and/or protease to protect against protein stress induced by high temperatures. The lack of a genetic manipulation system for Chlamydia limits the ability to collect in vivo physiological evidence, however we feel the use of this heterologous system has provided strong evidence that Chlamydia HtrA protects against protein stress in vivo.
Protein levels were examined in C. trachomatis L2 cultures under acute and heat stressed conditions to examine the role of the HtrA. Heat stress is an ideal laboratory model to induce protein level stress and is highly relevant for many bacteria, but it is important to note that C. trachomatis would more commonly encounter other forms of protein damaging stress such as, immune related oxidative and nitrosative stress, temperature flux in the genital tract, rapid pH changes during early development of the inclusion vacuole, and possibly osmotic flux. The increased level of HtrA during heat stress was observed both by western blot and immunofluorescence (relative to MOMP and LPS). The use of the major outer membrane protein (MOMP) as a comparative protein for molecular studies in Chlamydia is widely reported. MOMP levels have been reported to decrease during C. trachomatis persistence and stress conditions [15, 18]. However, as different fixatives were required for the HtrA and MOMP antibodies used during this study, LPS was used in conjunction with HtrA immunofluorescence. The heat stress model we tested was consistent with previous studies on C. pneumoniae and C. trachomatis with both morphological similarity when examined by TEM and similar decreased levels of MOMP and LPS [14, 16, 17]. Furthermore, we tested under conditions for which it is known a high proportion of the C. trachomatis cells remain viable. That is, after 3 hrs heat shock at 42°C once heat stress is removed, C. trachomatis was able to complete the developmental cycle, and form infectious EBs . This suggests that CtHtrA is important during heat stress and could be one of the key factors for protection of cellular envelope proteins sufficiently to allow restoration of normal growth once heat stress is removed. The protection is likely to be mediated by both the protease and chaperone functions we previously reported during in vitro investigations of HtrA . The data presented here clearly indicates a cell envelope or extracytoplasmic localisation of HtrA which seems likely to be periplasmic as indicated by in silico predictions. Further experimental data would be needed to confirm the exact location of HtrA (periplasmic, cytoplasmic membrane, outer membrane), however, regardless of the exact location it is clear that HtrA's protein maintenance function would still serve much of the extracytoplasmic proteome.
Two well established persistence models were used during this investigation to test for the potential significance of HtrA for persistent infections. The penicillin induced persistence model affects the development of Chlamydia by binding to three penicillin binding proteins halting binary fission and preventing later phases of the chlamydial developmental cycle (formation of EBs)[17, 32]. This affect of penicillin on Chlamydia is somewhat paradoxical as there is no unequivocal evidence for the presence of peptidoglycan, although it is predicted that peptidoglycan synthesis functions in RBs during chlamydial cell division (reviewed ). Thus, in the presence of penicillin at 20 h PI, the RBs will have no peptidoglycan (PG) or components thereof, and less MOMP and outer membrane cysteine rich proteins such as OmcB [18, 19] which normally contribute to the rigidity of the outer membrane by extensive disulfide crosslinking. The decreased outer membrane disulfide crosslinking and peptidoglycan likely results in reduced extracytoplasmic integrity, reduced protection from osmotic and redox stress for extracytoplasmic proteins due to these reduced physical and chemical barriers. Osmotic and redox stress is known to affect protein integrity . Thus, HtrA may be present at increased levels during penicillin persistence to protect against this possible additional protein stress. These findings suggest that HtrA is less important for the initial adaptation to penicillin but rather functions during longer term persistence presumably via maintenance of extracytoplasmic proteins possibly required for ongoing viability or for the capability to return to normal development once the selective pressure is removed.
HtrA protein levels were reduced at both 20 h PI and 44 h PI during IFN-γ persistence, possibly indicating that extracytoplasmic protein stress is less important during this form of persistence until at least 44 h PI. IFN-γ persistence for Chlamydia is effectively a form of amino acid deprivation stress, which results in a lack of binary fission and prevention of RB development to EBs. The amino acid deprivation is not likely to result in direct stress on existing extracytoplasmic proteins, which could explain the reduced levels of HtrA under these conditions. The considerable reduction in HtrA during IFN-γ persistence, when compared to acute, heat stress and penicillin data, suggests that IFN-γ persistence doesn't involve chlamydial extracytoplasmic stress response, unlike previous suggestions for C. pneumoniae that IFN-γ persistence is a stress response .