Immunobiology of monocytes and macrophages during Chlamydia trachomatis infection

Infections caused by the intracellular bacterium Chlamydia trachomatis are a global health burden affecting more than 100 million people annually causing damaging long-lasting infections. In this review, we will present and discuss important aspects of the interaction between C. trachomatis and monocytes/macrophages.     

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia -containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia -containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis .     

Developmental expression of non-coding RNAs in Chlamydia trachomatis during normal and persistent growth

Chlamydia trachomatis is an obligate intracellular bacterium that exhibits a unique biphasic developmental cycle that can be disrupted by growth in the presence of IFN-γ and β-lactams, giving rise to an abnormal growth state termed persistence. Here we have examined the expression of a family of non-coding RNAs (ncRNAs) that are differentially expressed during the developmental cycle and the induction of persistence and reactivation. ncRNAs were initially identified using an intergenic tiling microarray and were confirmed by northern blotting. ncRNAs were mapped, characterized and compared with the previously described chlamydial ncRNAs. The 5'- and 3'-ends of the ncRNAs were determined using an RNA circularization procedure. Promoter predictions indicated that all ncRNAs were expressed from σ(66) promoters and eight ncRNAs contained non-templated 3'-poly-A or poly-AG additions. Expression of ncRNAs was studied by northern blotting during (i) the normal developmental cycle, (ii) IFN-γ-induced persistence and (iii) carbenicillin-induced persistence. Differential temporal expression during the developmental cycle was seen for all ncRNAs and distinct differences in expression were seen during IFN-γ and carbenicillin-induced persistence and reactivation. A heterologous co-expression system was used to demonstrate that one of the identified ncRNAs regulated the expression of FtsI by inducing degradation of ftsI mRNA.     

Immunology of Chlamydia infection: implications for a Chlamydia trachomatis vaccine

Sexually transmitted Chlamydia trachomatis infections are a serious public-health problem. With more than 90 million new cases occurring annually, C. trachomatis is the most common cause of bacterial sexually transmitted disease worldwide. Recent progress in elucidating the immunobiology of Chlamydia muridarum infection of mice has helped to guide the interpretation of immunological findings in studies of human C. trachomatis infection and has led to the development of a common model of immunity. In this review, we describe our current understanding of the immune response to infection with Chlamydia spp. and how this information is improving the prospects for development of a vaccine against infection with C. trachomatis.     

CFTR is required for cellular entry and internalization of Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular Gram‐negative pathogen affecting over 600 million people worldwide with 92 million new cases occurring globally each year. C. trachomatis enter the cells and replicate to infect different tissues/organs, giving rise to a spectrum of pathological conditions; however, the exact mechanism or receptor(s) for their entry is not well understood. Here we report that CFTR (cystic fibrosis transmembrane conductance regulator), an apical epithelial anion channel, is required for cellular entry and internalization of C. trachomatis. Human epithelial cell lines expressing functional CFTR internalized more C. trachomatis than the cells expressing mutant Δ508 CFTR. The in vitro cellular uptake of C. trachomatis can be blocked by CFTR inhibitors or antibody, and the in vivo cellular uptake of C. trachomatis in CFTR mutant (CFTR^?/?) mice was significantly less compared with that in the wild‐type. Direct interaction between CFTR and C. trachomatis LPS (lipopolysaccharide) is demonstrated by their immune‐co‐localization and co‐immunoprecipitation. Despite an increase in CFTR expression observed upon C. trachomatis LPS challenge, a reduction in its ion channel activity is observed, consistent with the notion that CFTR functions as a receptor for cellular entry and internationization of C. trachomatis, with compromised ion‐channel function. These findings, for the first time, demonstrate that CFTR functions as a cell‐surface receptor for epithelial cell entry, and internalization of C. trachomatis and these findings may lead to the development of new treatment strategies to curtail the spread of chlamydial infections.     

NF-kappa B activation is not required for Chlamydia trachomatis inhibition of host epithelial cell apoptosis

Chlamydia trachomatis, an obligate intracellular bacterial species, is known to inhibit host cell apoptosis. However, the chlamydial antiapoptotic mechanism is still not clear. Because NF-kappaB activation is antiapoptotic, we tested the potential role of NF-kappaB activation in chlamydial antiapoptotic activity in the current study. First, no obvious NF-kappaB activation was detected in the chlamydia-infected cells when these cells were resistant to apoptosis induced via either the intrinsic or extrinsic apoptosis pathways. Second, inhibition of NF-kappaB activation with pharmacologic reagents failed to block the chlamydial antiapoptotic activity. Finally, NF-kappaB p65 gene deletion did not prevent chlamydia from inhibiting host cell apoptosis. These observations together have demonstrated that NF-kappaB activation is not required for the chlamydial antiapoptotic activity.     

Expression of Chlamydia trachomatis inclusion membrane protein genes IncB and IncC in Escherichia coli

In this study, we have cloned the Chlamydia trachomatis genes incB and incC into the expression plasmid vectors from pET series for the subsequent isolation of recombinant proteins. As a result, we have obtained the first full-length recombinant C. trachomatis proteins IncB and IncC, which can be used for following antibody production and for study of their protein-protein interaction.     

Modular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis

Abstract Peptide synthetases are large multienzyme complexes that catalyze the non-ribosomal synthesis of a structurally diverse family of bioactive peptides. They possess a multidomain structure and employ the thiotemplate mechanism to activate, modify and link together by amide or ester bonds the constituent amino acids of the peptide product. The domains, which represent the functional building units of peptide synthetases, appear to act as independent enzymes whose specific linkage order forms the protein-template that defines the sequence of the incorporated amino acids. Two types of domains have been characterized in peptide synthetases of bacterial and fungal origin: type I comprises about 600 amino acids and contains at least two modules involved in substrate recognition, adenylation and thioester formation, whereas type II domains carry in addition an insertion of about 430 amino acids that may function as a N-methyltransferase module. The role of other genes associated with bacterial opérons encoding peptide synthetases is also discussed.     

Examination of an inducible expression system for limiting iron availability during Chlamydia trachomatis infection

The obligate intracellular bacterium Chlamydia trachomatis requires iron in order to complete its developmental cycle. Addition of an iron-chelating drug, Desferal (deferoxamine mesylate), to infected cell culture causes Chlamydia to enter persistence. Here, we explore the ability of a stably-transfected cell line with inducible over-expression of the eukaryotic iron efflux protein ferroportin to starve C. trachomatis serovar E for iron. Ferroportin-induced iron removal is perhaps a more direct method of removing iron from the intracellular compartment versus exposure to an exogenous chemical chelator. Following induction, ferroportin-green fluorescent protein (Fpn-GFP) was detected in the plasma membrane, and cells expressing Fpn-GFP remained viable throughout the timescale required for Chlamydia to complete its developmental cycle. Following Fpn-GFP induction in infected cells, chlamydial infectivity remained unchanged, indicating chlamydiae were not in persistence. Ferritin levels indicate only a small decrease in cellular iron following Fpn-GFP expression relative to cultures exposed to Desferal. These data indicate that expression of Fpn-GFP in chlamydiae-infected cells is not capable of reducing iron below the threshold concentration needed to cause chlamydiae to enter persistence.     

The yeast MYO1 gene encoding a myosin-like protein required for cell division.

A yeast gene MYO1 that contains regions of substantial sequence homology with the nematode muscle myosin gene (unc54) has been isolated and sequenced. Although the disruption of MYO1 is not lethal, it leads to aberrant nuclear migration and cytokinesis. The 200-kd myosin heavy chain-like protein, the product of MYO1, cross-reacts with anti-nematode myosin heavy chain IgG and is present in wild-type strains but not in strains carrying the disrupted gene. Instead, a truncated polypeptide with a molecular mass of 120 kd can be detected in some myo1 mutants.     

DNA synthesis and nuclear division during formation of infection structures by bean rust uredospore germlings

Plectonema boryanum can grow in the dark with ribose, sucrose, mannitol, maltose, glucose, or fructose. Cell doubling times with 10 mM substrate are the following: 5 days with ribose, 6 days with sucrose or mannitol, 10 days with maltose, 12 days with glucose, and 13 days with fructose; with ribose plus 0.1% casamino acids it is 2.5 days. Dark-grown cells appear morphologically similar to light-grown cells. Cells grown in the dark for several years remain pigmented and resume photoautotrophic growth when placed in the light. Dim light (85 lux) increases the growth rate with ribose and with ribose plus casamino acids to nearlytwice that of the dark rate. In moderate light, growth takes place with ribose even in the presence of 1x10^-5 M DCMU.     

Lysosome repair enables host cell survival and bacterial persistence following Chlamydia trachomatis infection

Chlamydiae are obligate intracellular bacteria that replicate within the confines of a membrane-bound vacuole termed the inclusion. The final event in the infectious process is the disruption of the inclusion membrane and release of a multitude of infectious elementary bodies, each capable of eliciting a new infection. Strains of the trachoma biovar of Chlamydia trachomatis are released from the host cell without concomitant host cell death. In this study, analysis of events associated with chlamydial egress revealed that the integrity of the host cell plasma membrane was compromised prior to the inclusion membrane. This disruption was accompanied by the appearance of LAMP-1 at the infected cell surface, implicating lysosome repair of plasma membrane lesions in response to infection. Analysis of the effects of calcium chelators and actin stabilizing agents, indicated calcium-induced actin depolymerization as a requisite to lysosome-plasma membrane fusion and host cell survival. A consequence of this lysosome-mediated repair process, was the retention of residual bacteria within the surviving host cell, providing a unique mechanism for intracellular persistence of C. trachomatis.     

Nucleotide sequence of DNA encoding the major outer membrane protein of Chlamydia trachomatis serovar L3

DNA encoding the major outer membrane protein of Chlamydia trachomatis serovar L3 was sequenced following amplification by the polymerase chain reaction. A comparison with the deduced amino acid (aa) sequence of the C. trachomatis serovar L2 showed that the L3 had three extra aa and 55 aa substitutions.     

Primary and secondary immune responses of mucosal and peripheral lymphocytes during Chlamydia trachomatis infection

Chlamydia trachomatis infection is followed by the development of antigen-specific cell-mediated immunity, which is detectable as a positive lymphocyte proliferation response to the chlamydial major outer membrane protein (MOMP) antigen. To date, however, there have been no studies on the mucosal immune responses to chlamydial antigens. This study aimed to study the primary and secondary immune responses of cervical lymphocytes in response to the chlamydial antigen. Median proliferative responses were found to be significantly (P<0.05) higher in patients with chlamydial infections than in controls. The chlamydial MOMP induced significantly higher IL-6 and IL-10 and lower interferon-gamma (IFN-gamma) secretion in cervical lymphocytes of Chlamydia-positive women, resulting in a T helper 2 response. On stimulation of peripheral blood mononuclear cells (PBMC) obtained from Chlamydia-positive women with the chlamydial antigen, the median levels of IL-10, IL-12 and IFN-gamma were higher than in controls, but the differences were not significant. Our study suggests that the mucosal immune responses towards Chlamydia trachomatis are different from those of PBMCs and are more helpful in understanding the cytokine responses in the female genital tract during chlamydial infection.