CD1d degradation in Chlamydia trachomatis-infected epithelial cells is the result of both cellular and chlamydial proteasomal activity.

Chlamydia trachomatis is an obligate intracellular pathogen that can persist in the urogenital tract. Mechanisms by which C. trachomatis evades clearance by host innate immune responses are poorly described. CD1d is MHC-like, is expressed by epithelial cells, and can signal innate immune responses by NK and NKT cells. Here we demonstrate that C. trachomatis infection down-regulates surface-expressed CD1d in human penile urethral epithelial cells through proteasomal degradation. A chlamydial proteasome-like activity factor (CPAF) interacts with the CD1d heavy chain, and CPAF-associated CD1d heavy chain is then ubiquitinated and directed along two distinct proteolytic pathways. The degradation of immature glycosylated CD1d was blocked by the proteasome inhibitor lactacystin but not by MG132, indicating that degradation was not via the conventional proteasome. In contrast, the degradation of non-glycosylated CD1d was blocked by lactacystin and MG132, consistent with conventional cellular cytosolic degradation of N-linked glycoproteins. Immunofluorescent microscopy confirmed the interruption of CD1d trafficking to the cell surface, and the dislocation of CD1d heavy chains into both the cellular cytosol and the chlamydial inclusion along with cytosolic CPAF. C. trachomatis targeted CD1d toward two distinct proteolytic pathways. Decreased CD1d surface expression may help C. trachomatis evade detection by innate immune cells and may promote C. trachomatis persistence.

Selective promoter recognition by chlamydial sigma28 holoenzyme.

The sigma transcription factor confers the promoter recognition specificity of RNA polymerase (RNAP) in eubacteria. Chlamydia trachomatis has three known sigma factors, sigma(66), sigma(54), and sigma(28). We developed two methods to facilitate the characterization of promoter sequences recognized by C. trachomatis sigma(28) (sigma(28)(Ct)). One involved the arabinose-induced expression of plasmid-encoded sigma(28)(Ct) in a strain of Escherichia coli defective in the sigma(28) structural gene, fliA. The second was an analysis of transcription in vitro with a hybrid holoenzyme reconstituted with E. coli RNAP core and recombinant sigma(28)(Ct). These approaches were used to investigate the interactions of sigma(28)(Ct) with the sigma(28)(Ct)-dependent hctB promoter and selected E. coli sigma(28) (sigma(28)(Ec))-dependent promoters, in parallel, compared with the promoter recognition properties of sigma(28)(EC). Our results indicate that RNAP containing sigma(28)(Ct) has at least three characteristics: (i) it is capable of recognizing some but not all sigma(28)(EC)-dependent promoters; (ii) it can distinguish different promoter structures, preferentially activating promoters with upstream AT-rich sequences; and (iii) it possesses a greater flexibility than sigma(28)(EC) in recognizing variants with different spacing lengths separating the -35 and -10 elements of the core promoter.

Identification of surface-exposed components of MOMP of Chlamydia trachomatis serovar F.

The identification of surface-exposed components of the major outer membrane protein (MOMP) of Chlamydia is critical for modeling its three-dimensional structure, as well as for understanding the role of MOMP in the pathogenesis of Chlamydia-related diseases. MOMP contains four variable domains (VDs). In this study, VDII and VDIV of Chlamydia trachomatis serovar F were proven to be surface-located by immuno-dot blot assay using monoclonal antibodies (MAbs). Two proteases, trypsin and endoproteinase Glu-C, were applied to digest the intact elementary body of serovar F under native conditions to reveal the surface-located amino acids. The resulting peptides were separated by SDS-PAGE and probed with MAbs against these VDs. N-terminal amino acid sequencing revealed: (1) The Glu-C cleavage sites were located within VDI (at Glu61) and VDIII (at Glu225); (2) the trypsin cleavage sites were found at Lys79 in VDI and at Lys224 in VDIII. The tryptic peptides were then isolated by HPLC and analyzed with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer and a quadrupole-orthogonal-TOF mass spectrometer coupled with a capillary liquid chromatograph. Masses and fragmentation patterns that correlated to the peptides cleaved from VDI and VDIII regions, and C-terminal peptides Ser333-Arg358 and Ser333-Lys350 were observed. This result demonstrated that these regions are surface-exposed. Data derived from comparison of nonreduced outer membrane complex proteolytic fragments with their reduced fractions revealed that Cys26, 29, 33, 116, 208, and 337 were involved in disulfide bonds, and Cys26 and 337, and 116 and 208 were paired. Based on these data, a new two-dimensional model is proposed.

Chlamydia trachomatis sigma28 recognizes the fliC promoter of Escherichia coli and responds to heat shock in chlamydiae.

The rpsD gene of Chlamydia trachomatis encodes the alternative sigma factor sigma28, which bears strong homology to many bacterial sigma factors, including Escherichia coli sigma8 and Bacillus subtilis sigmaB and sigmaD. Recently, a sigma28 promoter was identified upstream of the late-cycle-expressed gene hctB, which encodes the Chlamydia-histone-like protein 2 (Yu & Tan, 2003). In this study it is shown that the product of chlamydial rpsD is an E. coli sigma28 homologue. It was found that recombinant chlamydial sigma8, in combination with E. coli core RNA polymerase, initiates transcription in vitro from the E. coli sigma28-dependent promoter of fliC. It was also demonstrated that the recombinant chlamydial sigma28 does not recognize major sigma factor sigma70-consensus-like sequences in vitro. In C. trachomatis-infected cells, two rpsD transcripts were detected with 5' ends located 18 (transcript I) and 54 bp (transcript II) upstream of the translational initiation codon at 16 and 30 h post-infection. When the temperature of cultures infected with C. trachomatis was shifted from 35 to 42 degrees C, the rpsD transcript I increased dramatically. The levels of chlamydial sigma28, relative to EF-Tu, were greater throughout the exponential growth phase of the reticulate body, but lower late in the developmental cycle. These data support the hypothesis that sigma28 plays a role in the regulatory network that allows chlamydiae to survive changes in its environment, enabling it to complete its unique developmental cycle.

Chlamydia pneumoniae accelerates coronary artery disease progression in transgenic hyperlipidemia-genetic hypertension rat model.

Chlamydia pneumoniae (Cpn) has been associated with human coronary artery disease but causal relevance as a risk factor has not been shown. Several rabbit and mouse model studies demonstrate exacerbation of aortic atherosclerosis by Cpn, however impact of Cpn on coronary artery disease (CAD) and survival outcomes has not been shown. To study this, we used specific pathogen-free, inbred, transgenic-CAD Dahl salt-sensitive (S) hypertensive (Tg53) rats and control inbred, non-transgenic Dahl S (nonTg) rats to analyze the effects of Cpn infection on macrophage foam cell formation, coronary artery disease progression, and effect on survival. Cpn infection induced acceleration of foam cell formation in hyperlipidemic Tg53 recruited peritoneal macrophages. This effect is hyperlipidemia-dependent. The transcription profile of Tg53-Cpn macrophage foam cells is different from control mock-inoculated (Tg53-spg) and heat-inactivated (Tg53-iCpn) macrophages (ANOVA P < 0.0001). Decreased survival was detected in Tg53-Cpn compared with control nonTg-Cpn and mock-infected Tg53-mouse pneumonitic rats (P = 0.009) and was associated with "culprit" coronary plaques and left atrial thrombi. These data demonstrate that in the presence of significant hyperlipidemia and hypertension, one-time Cpn infection at 5 mo of age (associated with early CAD stage) accelerates progression to overt-CAD in the Tg53 rat model. The data support the hypothesis that untreated Cpn infection is a causal risk factor for CAD progression most likely mediated by Cpn-induced accelerated macrophage foam cell formation.