Silencing or permanent activation: host-cell responses in models of persistent Chlamydia pneumoniae infection

Chlamydia pneumoniae causes respiratory infections. In chronic diseases associated with Chlamydia, such as arteriosclerosis, C. pneumoniae is present in a persistent form, which might participate in pathogenesis of chronic inflammatory disease. To elucidate how these intracellular bacteria modulate host-cells during persistence, we compared the expression pattern of a range of host genes after short (24 h) and long (up to 7 days) times of chlamydia infection in HeLa-cells. One day post infection, in three cell-culture models of persistence, namely treatment with penicillin or IFN-gamma, or iron-depletion, infection induced the genes of CTGF, IL-6, IL-8, IL-11, LIF, EGR-1 and ETV4 in a similar fashion. However, after a longer time, two modes of host-cell reaction emerged that were dependent on the persistence model used. After IFN-gamma and penicillin treatment chlamydia-induced host-cell gene expression was inhibited, while it stayed upregulated in iron-depletion. Human monocytes/macrophages, in which persistence naturally occurs, were additionally investigated: for several genes, UV-inactivated and viable chlamydia caused long-lasting upregulation. Thus, this study reveals (i) the ability of C. pneumoniae to participate in two putative pathomechanisms of persistence, silencing and permanent activation, which might represent different in vivo situations and (ii) a strong dependence on the mode of persistence induction.     

Chlamydia trachomatis persistence: an update

Chlamydial persistence is a reversible state generated during conditions deleterious to growth. In persistence, Chlamydia trachomatis remains viable but atypical, with an enlarged, aberrant form and quiescent metabolism. It favours chronic chlamydiosis, leading to serious sequelae. Although the mechanism of persistence formation is still unknown, more reliable molecular approaches tend to confirm that its occurs in vivo, even lasting 3 years. One approach consists of identifying unprocessed rRNA found only in viable Chlamydia, when infection is not apparent. Another approach, referring to the fact that immunity is type-specific, consists of showing by genotyping that multiple recurrences are due to the same genovar. At the molecular level, persistence is characterized by increased expression of ct755, one of the three heat shock protein (hsp60)-coding genes. In addition, chromosomal replication occurs continuously, and cell division is blocked possibly due to the repression of genes such as ftsW and amiA. At the immunological level, persistence reveals the failure of host-defence mechanisms because of reduced or suppressed pro-inflammatory or cytotoxic responses.     

Chlamydia pneumoniae inhibits activated human T lymphocyte proliferation by the induction of apoptotic and pyroptotic pathways

Chlamydia pneumoniae is an omnipresent obligate intracellular bacterial pathogen that infects numerous host species. C. pneumoniae infections of humans are a common cause of community acquired pneumonia but have also been linked to chronic diseases such as atherosclerosis, Alzheimer's disease, and asthma. Persistent infection and immune avoidance are believed to play important roles in the pathophysiology of C. pneumoniae disease. We found that C. pneumoniae organisms inhibited activated but not nonactivated human T cell proliferation. Inhibition of proliferation was pathogen specific, heat sensitive, and multiplicity of infection dependent and required chlamydial entry but not de novo protein synthesis. Activated CD4(+) and CD8(+) T cells were equally sensitive to C. pneumoniae antiproliferative effectors. The C. pneumoniae antiproliferative effect was linked to T cell death associated with caspase 1, 8, 9, and IL-1β production, indicating that both apoptotic and pyroptotic cellular death pathways were activated after pathogen-T cell interactions. Collectively, these findings are consistent with the conclusion that C. pneumoniae could induce a local T cell immunosuppression and inflammatory response revealing a possible host-pathogen scenario that would support both persistence and inflammation.     

Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis

The ability of a high frequency (10(-2)) of Escherichia coli to survive prolonged exposure to penicillin antibiotics, called high persistence, is associated with mutations in the hipA gene. The hip operon is located in the chromosomal terminus near dif and consists of two genes, hipA and hipB. The wild-type hipA gene encodes a toxin, whereas hipB encodes a DNA-binding protein that autoregulates expression of the hip operon and binds to HipA to nullify its toxic effects. We have characterized the hipA7 allele, which confers high persistence, and established that HipA7 is non-toxic, contains two mutations (G22S and D291A) and that both mutations are required for the full range of phenotypes associated with hip mutants. Furthermore, expression of hipA7 in the absence of hipB is sufficient to establish the high persistent phenotype, indicating that hipB is not required. There is a strong correlation between the frequency of persister cells generated by hipA7 strains and cell density, with hipA7 strains generating a 20-fold higher frequency of persisters as cultures approach stationary phase. It is also demonstrated that relA knock-outs diminish the high persistent phenotype in hipA7 mutants and that relA spoT knock-outs eliminate high persistence altogether, suggesting that hipA7 facilitates the establishment of the persister state by inducing (p)ppGpp synthesis. Consistent with this proposal, ectopic expression of relA' from a plasmid was shown to increase the number of persistent cells produced by hipA7 relA double mutants by 100-fold or more. A model is presented that postulates that hipA7 increases the basal level of (p)ppGpp synthesis, allowing a significantly greater percentage of cells in a population to assume a persistent, antibiotic-insensitive state by potentiating a rapid transition to a dormant state upon application of stress.     

Chlamydia pneumoniae infection results in generalized bone loss in mice

Osteoporosis is associated with a general bone loss. Whether infections could contribute to osteoporosis is not known. Chlamydia pneumoniae causes chronic infections and produces potentially bone resorptive cytokines. The effect of C. pneumoniae infection was investigated in vivo in 10-week old mice (c57BL/6) and in vitro in the human osteoblast-like cell line hFOB 1.19 (hFOB). Bone mineral density (BMD) was measured before and 16 days after infection. C. pneumoniae-infected mice had decreased (p<0.05) total and subcortical BMD at the distal femur and proximal tibia compared with controls, but no body-weight gain differences. IL-6 (56 vs. 39pg/mL, p=0.02) and IL-1beta (11 vs. 0pg/mL, p=0.003) levels in sera, and CD3(+) T-cells (p=0.04) were higher in infected mice compared with controls. In vitro, hFOB infected with C. pneumoniae was associated with increased IL-6 (p=0.01) and RANKL (p<0.05) mRNA expression; additionally, IL-6 secretion increased in a dose-dependent manner (p<0.05). In summary, mice infected with C. pneumoniae had generalized bone loss associated with increased IL-6 and IL-1. In addition, C. pneumoniae established an infection in an osteoblast cell line in vitro with similar cytokine profiles as those in vivo, supporting a causal linkage.     

Analysis of synonymous codon usage bias in Chlamydia

Chlamydiae are obligate intracellular bacterial pathogens that cause ocular and sexuallytransmitted diseases,and are associated with cardiovascular diseases.The analysis of codon usage mayimprove our understanding of the evolution and pathogenesis of Chlamydia and allow reengineering of targetgenes to improve their expression for gene therapy.Here,we analyzed the codon usage of C.muridarum,C.trachomatis(here indicating biovar trachoma and LGV),C.pneumoniae,and C.psittaci using the codonusage database and the CUSP(Create a codon usage table)program of EMBOSS(The European MolecularBiology Open Software Suite).The results show that the four genomes have similar codon usage patterns,with a strong bias towards the codons with A and T at the third codon position.Compared with Homosapiens,the four chlamydial species show discordant seven or eight preferred codons.The ENC(effectivenumber of codons used in a gene)-plot reveals that the genetic heterogeneity in Chlamydia is constrained bythe G+C content,while translational selection and gene length exert relatively weaker influences.Moreover,mutational pressure appears to be the major determinant of the codon usage variation among the chlamydialgenes.In addition,we compared the codon preferences of C.trachomatis with those of E.coli,yeast,adenovirus and Homo sapiens.There are 23 codons showing distinct usage differences between C.trachomatisand E.coli,24 between C.trachomatis and adenovirus,21 between C.trachomatis and Homo sapiens,butonly six codons between C.trachomatis and yeast.Therefore,the yeast system may be more suitable for theexpression of chlamydial genes.Finally,we compared the codon preferences of C.trachomatis with those ofsix eukaryotes,eight prokaryotes and 23 viruses.There is a strong positive correlation between the differ-ences in coding GC content and the variations in codon bias(r=0.905,P<0,001).We conclude that thevariation of codon bias between C.trachomatis and other organisms is much less influenced by phylogeneticlineage and primarily determined by the extent of disparities in GC content.     

Chlamydia pneumoniae infected macrophages exhibit enhanced plasma membrane fluidity and show increased adherence to endothelial cells

Chlamydia pneumoniae, an intracellular prokaryote, is known to have requirement for some lipids which it is incapable of synthesizing, and these lipids have important fluidizing roles in plasma membrane. We decided to examine if the trafficking of these lipids to C. pneumoniae alters the physicochemical properties of macrophage plasma membrane, affects the expression of genes and proteins of enzymes associated with metabolism of some of these lipids and assess if Ca2+ signaling usually induced in macrophages infected with C. pneumoniae modulates the genes of these selected enzymes. Chlamydia pneumoniae induced the depletion of macrophage membrane cholesterol, phosphatidylinositol and cardiolipin but caused an increase in phosphotidylcholine resulting in a relative increase in total phospholipids. There was increased membrane fluidity, enhanced macrophage fragility and heightened adherence of macrophages to endothelial cells despite the application of inhibitor of adhesion molecules. Also, there was impairment of macrophage 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene and protein expression independent of Ca2+ signaling, while phospholipase C gene and protein were up-regulated in a manner minimally dependent on Ca2+ signaling. The implications of these findings are that macrophages infected with C. pneumoniae have altered membrane physicochemical characteristics which may render them atherogenic.     

Enzymology and molecular biology of prokaryotic sulfite oxidation

The intracellular bacterial pathogen Chlamydia pneumoniae causes respiratory tract infection and has been associated with atherosclerosis and coronary artery disease. Since atherosclerosis is a progressive disease and is considered to be a chronic inflammation of the artery vessel wall, the interaction of C. pneumoniae with cells of the vasculature that can result in a local inflammatory response is of paramount importance. In this essay we review the pathophysiology of atherosclerosis in the context of C. pneumoniae infection and present an integrated model that includes the involvement of C. pneumoniae in all stages of atherogenesis including initiation, inflammation, fibrous plaque formation, plaque rupture and thrombosis. We hypothesize that acute and persistent infection of professional immune cells (T-cells, monocytes and macrophages) and non-immune cells (endothelial cells and smooth muscle cells) contributes to a sustained inflammatory response mediated by extensive cellular 'crosstalk' and numerous cytokines/chemokines. This cascade of inflammatory mediators may contribute to cellular dysfunction and tissue remodelling of the arterial intima. An improved understanding of the precise mechanism(s) of C. pneumoniae involvement in atherogenesis may help resolve the question of causality however, at the present time, we interpret the data as favoring a contributory rather than a causal role. Future research directed at the discovery of chlamydial virulence factors necessary for intracellular survival and subsequent alterations in host cell gene expression including signalling pathways may be important for the design of future clinical trials.