In vitro characterisation of koala Chlamydia pneumoniae: morphology, inclusion development and doubling time.

Chlamydia pneumoniae is a common human and animal pathogen associated with upper and lower respiratory tract infections. Of the animal C. pneumoniae isolates, the koala nasal isolate (LPCoLN) is by far the best genetically characterised. This current study was designed to characterise the morphology and developmental events for the LPCoLN isolate, and our results showed several striking in vitro growth differences when compared to the human isolate, AR39. The LPCoLN inclusion size and morphology was distinct from AR39, and a much faster doubling time (3.4-4.9h versus 5.9-8.7h doubling time) was observed when grown in HEp-2 cell monolayers. Confocal and electron microscopy of LPCoLN confirmed large (9-30 microm in diameter) inclusions, that were heterogeneously shaped, compared to the small (5-9 microm in diameter), uniformly shaped inclusions of AR39. The morphology of the LPCoLN elementary body was round, and had a narrow or nonexistent periplasmic space, compared to the 'pear-shaped' morphology of AR39 EBs. While both isolates showed evidence of inclusion fusion, the level of fusion was much higher for LPCoLN (100%) compared to AR39 (30-40%). Our findings have provided new insights and identified key differences in the in vitro doubling time, size and morphology of an animal C. pneumoniae isolate.

Chlamydia trachomatis responds to heat shock, penicillin induced persistence, and IFN-gamma persistence by altering levels of the extracytoplasmic stress response protease HtrA.

BACKGROUND: Chlamydia trachomatis, an obligate intracellular human pathogen, is the most prevalent bacterial sexually transmitted infection worldwide and a leading cause of preventable blindness. HtrA is a virulence and stress response periplasmic serine protease and molecular chaperone found in many bacteria. Recombinant purified C. trachomatis HtrA has been previously shown to have both activities. This investigation examined the physiological role of Chlamydia trachomatis HtrA. RESULTS: The Chlamydia trachomatis htrA gene complemented the lethal high temperature phenotype of Escherichia coli htrA- (>42 degrees C). HtrA levels were detected to increase by western blot and immunofluorescence during Chlamydia heat shock experiments. Confocal laser scanning microscopy revealed a likely periplasmic localisation of HtrA. During penicillin induced persistence of Chlamydia trachomatis, HtrA levels (as a ratio of LPS) were initially less than control acute cultures (20 h post infection) but increased to more than acute cultures at 44 h post infection. This was unlike IFN-gamma persistence where lower levels of HtrA were observed, suggesting Chlamydia trachomatis IFN-gamma persistence does not involve a broad stress response. CONCLUSION: The heterologous heat shock protection for Escherichia coli, and increased HtrA during cell wall disruption via penicillin and heat shock, indicates an important role for HtrA during high protein stress conditions for Chlamydia trachomatis.

The cross-species prediction of bacterial promoters using a support vector machine.

Due to degeneracy of the observed binding sites, the in silico prediction of bacterial sigma(70)-like promoters remains a challenging problem. A large number of sigma(70)-like promoters has been biologically identified in only two species, Escherichia coli and Bacillus subtilis. In this paper we investigate the issues that arise when searching for promoters in other species using an ensemble of SVM classifiers trained on E. coli promoters. DNA sequences are represented using a tagged mismatch string kernel. The major benefit of our approach is that it does not require a prior definition of the typical -35 and -10 hexamers. This gives the SVM classifiers the freedom to discover other features relevant to the prediction of promoters. We use our approach to predict sigma(A) promoters in B. subtilis and sigma(66) promoters in Chlamydia trachomatis. We extended the analysis to identify specific regulatory features of gene sets in C. trachomatis having different expression profiles. We found a strong -35 hexamer and TGN/-10 associated with a set of early expressed genes. Our analysis highlights the advantage of using TSS-PREDICT as a starting point for predicting promoters in species where few are known.

The temperature activated HtrA protease from pathogen Chlamydia trachomatis acts as both a chaperone and protease at 37 degrees C.

Characterization of the protease, HtrA, from pathogen Chlamydia trachomatis is presented. The purified recombinant protein was a serine endoprotease, specific for unfolded proteins, and temperature activated above 34 degrees C. Chaperone activity was observed, although this appeared target-dependent. Inactive protease (S247A) was able to chaperone insulin B-chain, irrespective of temperature, but at 30 degrees C only HtrA and not S247A displayed significant chaperone activity for alpha-lactalbumin. These data demonstrate that chaperone activity may involve functional protease domain and that C. trachomatis HtrA functions as both a chaperone and protease at 37 degrees C. These properties are consistent with the developmental cycle of this obligate intracellular bacterium.

Identification of Chlamydia pneumoniae proteins in the transition from reticulate to elementary body formation.

Chlamydia pneumoniae is an important human respiratory pathogen that is responsible for an estimated 10% of community-acquired pneumonia and 5% of bronchitis and sinusitis cases. We examined changes in global protein expression profiles associated with the redifferentiation of reticulate body (RB) to elementary body (EB) as C. pneumoniae cells progressed from 24 to 48 h postinfection in HEp2 cells. Proteins corresponding to those showing the greatest changes in abundance in the beginning of the RB to EB transition were then identified from purified EBs. Among the 300 spots recognized, 35 proteins that were expressed at sufficiently high levels were identified by mass spectrometry. We identified C. pneumoniae proteins that showed more than 2-fold increases in abundance in the early stages of RB to EB transition, including several associated with amino acid and cofactor biosynthesis (Ndk, TrxA, Adk, PyrH, and BirA), maintenance of cytoplasmic protein function (GroEL/ES, DnaK, DksA, GrpE, HtrA, ClpP, ClpB, and Map), modification of the bacterial cell surface (CrpA, OmpA, and OmcB), energy metabolism (Tal and Pyk), and the putative transcriptional regulator TctD. This study identified C. pneumoniae proteins involved in the process of redifferentiation into mature, infective EBs and indicates bacterial metabolic pathways that may be involved in this transition. The proteins involved in RB to EB transition are key to C. pneumoniae infection and are perhaps suitable targets for therapeutic intervention.

Protein expression profiles of Chlamydia pneumoniae in models of persistence versus those of heat shock stress response.

Chlamydia pneumoniae is an obligate intracellular pathogen that causes both acute and chronic human disease. Several in vitro models of chlamydial persistence have been established to mimic chlamydial persistence in vivo. We determined the expression patterns of 52 C. pneumoniae proteins, representing nine functional subgroups, from the gamma interferon (IFN-gamma) treatment (primarily tryptophan limitation) and iron limitation (IL) models of persistence compared to those following heat shock (HS) at 42 degrees C. Protein expression patterns of C. pneumoniae persistence indicates a strong stress component, as evidenced by the upregulation of proteins involved in protein folding, assembly, and modification. However, it is clearly more than just a stress response. In IFN persistence, but not IL or HS, amino acid and/or nucleotide biosynthesis proteins were found to be significantly upregulated. In contrast, proteins involved in the biosynthesis of cofactors, cellular processes, energy metabolism, transcription, and translation showed an increased in expression in only the IL model of persistence. These data represent the most extensive protein expression study of C. pneumoniae comparing the chlamydial heat shock stress response to two models of persistence and identifying the common and unique protein level responses during persistence.

Differential expression of genes encoding membrane proteins between acute and continuous Chlamydia pneumoniae infections.

Chlamydia pneumoniae is associated with several chronic human diseases, including chronic obstructive pulmonary disease and atherosclerotic cardiovascular disease. During chronic disease, organisms are believed to exist in a persistent phase that is not well understood at the genetic level. Long-term in vitro continuous infections are spontaneously persistent and are less susceptible than in vitro acute infections to treatment with antibiotics, and are therefore particularly relevant as an in vitro model of in vivo chronic disease. Real-time reverse transcriptase-PCR (r-t RT-PCR) was used to quantitate transcript copy numbers of 13 genes in continuous and acute infections with C. pneumoniae. The set of genes studied encodes proteins with known or predicted functions in the cell membrane, the inclusion membrane, cell division, metabolism, and immunopathology. Significant upregulation was seen for five genes (CPn0483, nlpD, ompA, pmp1 and porB) in continuous cultures. The genes omcB, pmp1, and porB, all of which encode membrane proteins, shared similar patterns of expression over both acute and continuous profiles. These results show that Chlamydia in the long-term continuous model of persistence have a unique transcription profile, adding to our knowledge of regulation of this important stage of chlamydial growth.

DNA structure and novel amino and carboxyl termini of the Chlamydia sigma 70 analogue modulate promoter recognition.

Genes from the eubacterium Chlamydia typically do not share promoter consensus sequences with those of Escherichia coli and are not expressed when cloned in E. coli; nevertheless, the major sigma-subunit identified from Chlamydia trachomatis has nearly identical amino acid sequence to E. coli sigma 70 in regions that contact DNA. Following expression of the chlamydial sigma-subunit gene in E. coli, expression was specifically initiated from chlamydial promoter regions. Selective recognition of chlamydial promoters by holoenzyme was dependent upon the structure of the promoter DNA coupled with novel amino- and carboxyl-terminal extensions of the chlamydial sigma-subunit.