Disulfide bonding within components of the Chlamydia type III secretion apparatus correlates with development

Chlamydia spp. exhibit a unique biphasic developmental cycle whereby infectious elementary bodies (EBs) invade host epithelial cells and differentiate into noninfectious, metabolically active reticulate bodies (RBs). EBs posses a unique outer envelope where rigidity is achieved by disulfide bonding among cysteine-rich envelope-associated proteins. Conversely, these disulfide bonds become reduced in RBs to accommodate vegetative growth, thereby linking the redox status of cysteine-rich envelope proteins with progression of the developmental cycle. We investigated the potential role of disulfide bonding within the chlamydial type III secretion system (T3SS), since activity of this system is also closely linked to development. We focused on structural components of the T3S apparatus that contain an unusually high number of cysteine residues compared to orthologs in other secretion systems. Nonreducing SDS-PAGE revealed that EB-localized apparatus proteins such as CdsF, CdsD, and CdsC form higher-order complexes mediated by disulfide bonding. The most dramatic alterations were detected for the needle protein CdsF. Significantly, disulfide bonding patterns shifted during differentiation of developmental forms and were completely reduced in RBs. Furthermore, at later time points during infection following RB to EB conversion, we found that CdsF is reoxidized into higher-order complexes. Overall, we conclude that the redox status of specific T3SS apparatus proteins is intimately linked to the developmental cycle and constitutes a newly appreciated aspect of functionally significant alterations within proteins of the chlamydial envelope.     

Proteomic analysis of the outer membrane of Protochlamydia amoebophila elementary bodies

Chlamydiae are obligate intracellular bacteria, comprising some of the most important bacterial pathogens of animals and humans. During their unique developmental cycle they have to attach to and enter their eukaryotic host cells, a process mediated by proteins in the chlamydial outer membrane. So far the only experimental data for chlamydial outer membrane proteins are available from members of the Chlamydiaceae, a family comprising exclusively human and animal pathogens. To get further insights into the evolution of the protein composition of the chlamydial outer membrane and into host-dependent differences, we performed an extensive experimental analysis of outer membrane fractions of Protochlamydia amoebophila elementary bodies, which constitute the infectious form of this non-pathogenic member of the Chlamydiae that thrives as a symbiont in Acanthamoeba spp. We used 1-D and 2-DE in combination with MALDI-TOF, MALDI-TOF/TOF and nanoLC-ESI-MS/MS, and compared our experimental results with a previously published in silico analysis of chlamydial outer membrane proteins. This resulted in the identification of 38 proteins supported by both studies and therefore very likely to be located in the P. amoebophila outer membrane. The obtained experimental data provide the first comprehensive overview of outer membrane proteins of a chlamydial organism outside the Chlamydiaceae. They reveal both fundamental differences and convergent evolution between pathogenic and symbiotic chlamydiae.     

Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle

The obligate intracellular bacterium Chlamydia trachomatis possesses a biphasic developmental cycle that is manifested by differentiation of infectious, metabolically inert elementary bodies (EBs) to larger, metabolically active reticulate bodies (RBs). The cycle is completed by asynchronous differentiation of dividing RBs back to a population of dormant EBs that can initiate further rounds of infection upon lysis of the host cell. Chlamydiae express a type III secretion system (T3SS) that is presumably employed to establish and maintain the permissive intracellular niche by secretion of anti-host proteins. We hypothesize that T3SS activity is essential for chlamydial development and pathogenesis. However, the lack of a genetic system has confounded efforts to establish any role of the T3SS. We therefore employed the small molecule Yersinia T3SS inhibitor N'-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, designated compound 1 (C1), to examine the interdependence of the chlamydial T3SS and development. C1 treatment inhibited C. trachomatis but not T4SS-expressing Coxiella burnetii development in a dose-dependent manner. Although chlamydiae remained viable and metabolically active, they failed to divide significantly and RB to EB differentiation was inhibited. These effects occurred in the absence of host cell cytotoxicity and were reversible by washing out C1. We further demonstrate that secretion of T3S substrates is perturbed in C1-treated chlamydial cultures. We have therefore provided evidence that C1 can inhibit C. trachomatis development and T3SS activity and present a model in which progression of the C. trachomatis developmental cycle requires a fully functional T3SS.     

Inclusion Biogenesis and Reactivation of Persistent Chlamydia trachomatis Requires Host Cell Sphingolipid Biosynthesis

Chlamydiae are obligate intracellular pathogens that must coordinate the acquisition of host cell-derived biosynthetic constituents essential for bacterial survival. Purified chlamydiae contain several lipids that are typically found in eukaryotes, implying the translocation of host cell lipids to the chlamydial vacuole. Acquisition and incorporation of sphingomyelin occurs subsequent to transport from Golgi-derived exocytic vesicles, with possible intermediate transport through endosomal multivesicular bodies. Eukaryotic host cell-derived sphingomyelin is essential for intracellular growth of Chlamydia trachomatis, but the precise role of this lipid in development has not been delineated. The present study identifies specific phenotypic effects on inclusion membrane biogenesis and stability consequent to conditions of sphingomyelin deficiency. Culturing infected cells in the presence of inhibitors of serine palmitoyltransferase, the first enzyme in the biosynthetic pathway of host cell sphingomyelin, resulted in loss of inclusion membrane integrity with subsequent disruption in normal chlamydial inclusion development. Surprisingly, this was accompanied by premature redifferentiation to and release of infectious elementary bodies. Homotypic fusion of inclusions was also disrupted under conditions of sphingolipid deficiency. In addition, host cell sphingomyelin synthesis was essential for inclusion membrane stability and expansion that is vital to reactivation of persistent chlamydial infection. The present study implicates both the Golgi apparatus and multivesicular bodies as key sources of host-derived lipids, with multivesicular bodies being essential for normal inclusion development and reactivation of persistent C. trachomatis infection.     

Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells

Chlamydiae are obligate intracellular bacteria which occupy a non-acidified vacuole (the inclusion) throughout their developmental cycle. Little is known about events leading to the establishment and maintenance of the chlamydial inclusion membrane. To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library of Chlamydia psittaci DNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin-killed purified chlamydiae. Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera. Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product. The ORF1 sequence was amplified and fused to the malE gene of Escherichia coli and antisera were raised against the resulting fusion protein. Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purified C. psittaci elementary bodies. Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion. Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designated incA, (inc lusion membrane protein A ) and its gene product, IncA. In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells. Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post-translational modification. This pattern was not observed in immunoblots of RBs or in the E. coli expressing IncA. Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected cells.     

Penicillin Induced Persistence in Chlamydia trachomatis: High Quality Time Lapse Video Analysis of the Developmental Cycle

Background

Chlamydia trachomatis is a major human pathogen with a unique obligate intracellular developmental cycle that takes place inside a modified cytoplasmic structure known as an inclusion. Following entry into a cell, the infectious elementary body (EB) differentiates into a non - infectious replicative form known as a reticulate body (RB). RBs divide by binary fission and at the end of the cycle they redifferentiate into EBs. Treatment of C.trachomatis with penicillin prevents maturation of RBs which survive and enlarge to become aberrant RBs within the inclusion in a non - infective persistent state. Persistently infected individuals may be a reservoir for chlamydial infection. The C.trachomatis genome encodes the enzymes for peptidoglycan (PG) biosynthesis but a PG sacculus has never been detected. This coupled to the action of penicillin is known as the chlamydial anomaly. We have applied video microscopy and quantitative DNA assays to the chlamydial developmental cycle to assess the effects of penicillin treatment and establish a framework for investigating penicillin induced chlamydial persistence.

Principal Findings

Addition of penicillin at the time of cell infection does not prevent uptake and the establishment of an inclusion. EB to RB transition occurs but bacterial cytokinesis is arrested by the second binary fission. RBs continue to enlarge but not divide in the presence of penicillin. The normal developmental cycle can be recovered by the removal of penicillin although the large, aberrant RBs do not revert to the normal smaller size but remain present to the completion of the developmental cycle. Chromosomal and plasmid DNA replication is unaffected by the addition of penicillin but the arrest of bacterial cytokinesis under these conditions results in RBs accumulating multiple copies of the genome.

Conclusions

We have applied video time lapse microscopy to the study of the chlamydial developmental cycle. Linked with accurate measures of genome replication this provides a defined framework to analyse the developmental cycle and to investigate and provide new insights into the effects of antibiotic treatments. Removal of penicillin allows recovery of the normal developmental cycle by 10–20 hrs and the process occurs by budding from aberrant RBs.     

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.     

Cyclic AMP inhibits developmental regulation of Chlamydia trachomatis.

The effect of cyclic AMP (cAMP) on the chlamydial growth cycle was studied with Chlamydia trachomatis-infected HeLa cells. At concentrations of 1 mM, cAMP had a profound effect on the chlamydial developmental cycle, resulting in small, immature inclusions. Immunoblot analysis revealed the absence of elementary body (EB)-specific antigens in the cAMP-treated cells. This effect was observed only if cAMP was added within the first 12 h of incubation and continued thereafter. Its withdrawal at any time from the medium led to the reappearance of fully mature, infectious organisms. Analogs or breakdown products of cAMP exerted no inhibitory effect on chlamydial development. Intracellular inclusions from the cAMP-treated cells were unable to infect fresh HeLa monolayers, in contrast to the completely infectious nontreated inclusions. Protein profiles of the cAMP-treated organisms (at any time point) resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis very closely resembled reticulate bodies (RB) and did not possess characteristic EB-binding proteins. Collectively, these observations suggest an inhibitory role for cAMP at the RB stage of intracellular development. We also identified a cAMP receptor protein which is associated with RB and not with EB, further supporting a role for this system in the developmental regulation of chlamydiae.     

Type III Secretion, Contact-dependent Model for the Intracellular Development of Chlamydia

The medically significant genus Chlamydia is a class of obligate intracellular bacterial pathogens that replicate within vacuoles in host eukaryotic cells termed inclusions. Chlamydia's developmental cycle involves two forms; an infectious extracellular form, known as an elementary body (EB), and a non-infectious form, known as the reticulate body (RB), that replicates inside the vacuoles of the host cells. The RB surface is covered in projections that are in intimate contact with the inclusion membrane. Late in the developmental cycle, these reticulate bodies differentiate into the elementary body form. In this paper, we present a hypothesis for the modulation of these developmental events involving the contact-dependent type III secretion (TTS) system. TTS surface projections mediate intimate contact between the RB and the inclusion membrane. Below a certain number of projections, detachment of the RB provides a signal for late differentiation of RB into EB. We use data and develop a mathematical model investigating this hypothesis. If the hypothesis proves to be accurate, then we have shown that increasing the number of inclusions per host cell will increase the number of infectious progeny EB until some optimal number of inclusions. For more inclusions than this optimum, the infectious yield is reduced because of spatial restrictions. We also predict that a reduction in the number of projections on the surface of the RB (and as early as possible during development) will significantly reduce the burst size of infectious EB particles. Many of the results predicted by the model can be tested experimentally and may lead to the identification of potential targets for drug design.     

Productive Chlamydia trachomatis lymphogranuloma venereum 434 infection in cells with augmented or inactivated autophagic activities

Autophagy, a eukaryotic cellular activity leading to the degradation of cellular components, serves as a defense mechanism against facultative intracellular bacteria as well as a growth niche for the obligate intracellular bacterium Coxiella burnetii . We here demonstrate that the obligate intracellular bacterial pathogen Chlamydia trachomatis lymphogranuloma venereum strongly induced autophagy in the middle of the chlamydial developmental cycle (24 h after infection), a time point with maximal level of chlamydial replication, but not during the early stages with low overall chlamydial metabolism (before 8 h). No autophagy induction was evident in cells exposed to heat- and UV-inactivated elementary bodies (EBs, the infectious form of Chlamydia ) or to inocula from which EBs had been removed before inoculation. Blocking chlamydial development with chloramphenicol also prevented autophagy induction in cells infected with infectious EBs. It appears that autophagy is activated primarily in response to the metabolic stress consequent to chlamydial replication. However, autophagy-defective ATG5^−/− cells supported chlamydial development as efficiently as autophagy-proficient ATG5^+/+ cells.     

Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis.

The disulfide bond cross-linked major outer membrane protein (MOMP) of the extracellular elementary bodies (EBs) of Chlamydia psittaci was reduced to its monomeric form within 1 h of entry of EBs into host cells by a process which was inhibited by chloramphenicol, while monomeric forms of three cross-linked cysteine-rich proteins could not be detected in Sarkosyl outer membrane complexes at any time in either extracellular or intracellular forms of C. psittaci. Synthesis and incorporation of the MOMP into outer membrane complexes were detected early in the infection cycle (12 h postinfection), while synthesis and incorporation of the cysteine-rich proteins were not observed until reticulate bodies had begun to reorganize into EBs at 20 to 22 h postinfection. By 46 h postinfection, the intracellular population of C. psittaci consisted mainly of EBs, the outer membrane complexes of which were replete with monomeric MOMP and cross-linked cysteine-rich proteins. Upon lysis of infected cells at 46 h, the MOMP was rapidly cross-linked, and infectious EBs were released. The status of the MOMP of intracellular Chlamydia trachomatis was similar to the status of the MOMP of C. psittaci in that the MOMP was largely uncross-linked at 24 and 48 h postinfection, but formed interpeptide disulfide bonds when it was exposed to an extracellular environment late in the developmental cycle. In contrast to C. psittaci, only a fraction of the cross-linked MOMP of infecting EBs of C. trachomatis was reduced by 4 h postinfection, and reduction of the MOMP was not inhibited by chloramphenicol. Exposure of extracellular EBs of C. trachomatis and C. psittaci to dithiothreitol reduced the MOMP but failed to stimulate metabolic activities normally associated with reticulate bodies.     

Disulfide-linked oligomers of the major outer membrane protein of chlamydiae

The major outer membrane protein of chlamydial elementary bodies was identified in dimer, trimer, and other multimeric forms. These natural multimers were stabilized by disulfide-mediated cross-linking. Such cross-linking of outer membrane proteins may play an important role in the formation and evolution of chlamydial cell wall structure.     

Effector proteins of chlamydiae

This review summarizes the recently published data on the molecular mechanisms of Chlamydiae-host cell interaction, first of all, on chlamydial effector proteins. Such proteins, along with type III transport system proteins, which transfer many effector proteins into the host cytoplasm, are attractive targets for drug therapy of chlamydial infections. The majority of the data concerns two species, Chlamydia trachomatis and Chlamydophila pneumoniae. The C. trachomatis protein TARP, which is presynthesized in elementary bodies, plays an essential role in the initial stages of infection. The pathogen proteins that are involved in the next stage, which is the intracellular inclusion traffic to the centrosome, are C. trachomatis CT229 and C. pneumoniae Cpn0585, which interact with cell Rab GTPases. In C. trachomatis, IncA plays a key role in the fusion of chlamydial inclusions, CT847 modulates the life cycle of the host cell, and LDA3 is essential for the acquisition of nutrients. The protease CPAF and the inclusion membrane proteins IncG and CADD are involved in suppressing apoptosis of infected cells. The proteases CPAF and CT441 and the deubiquitinating protein ChlaDub1 help the pathogen to evade the immune response.     

Apoptosis of host cells regulation by Chlamydia

In a study on the impact of chlamydial infection on host cell apoptosis, C. trachomatis were shown to protect host cell against staurosporin-induced apoptosis only at the middle stage of infection development (at 20 hours post infection), C. pneumoniae--at different stages of its growth cycle (from 2 to 7 day post infection). We found, that C. trachomatis elementary bodies fail to inhibit staurosporin-induced apoptotic stimuli. The clear antiapoptotic effect of cell lysate filtrate, infected with C. trachomatis, was demonstrated by cytometric analysis and luminescent microscopy. Our findings make it possible to use biochemical approach to identification of chlamydial antiapoptotic factors in future. Investigations directed at chlamydial antiapoptotic activities may aim to create the therapies of chronic chlamydial infection.     

Electron microscopic determination of the localization of the group-specific antigen of Halprowise (Chlamydiae) by the direct immunoperoxidase method]

The direct immunoperoxidase technique was applied to the study of the localization of a group-specific antigen of Halprowiae (Chlamydiae) in the causative agents of meningopneumonia (strain MP) and of paratrachoma (strain LB-I) at the individual stages of their developmental cycle in the L-cell (16, 24 and 48 hours after infection of the culture). The product of reaction to peroxidase pointing to the localization of the group-specific antigen was localized not only on the surface, but in the whole thickness of the cell wall of the initial bodies in the form of an even electron dense layer, 200--280 A in thickness. A weak positive reaction was also noted on the outer layer of the sytoplasmic membrane. The periplasmic space remained free of the reaction product. Localization of the reaction product in the intermediate and elementary bodies remained unchanged. At the initial stages of the developmental cycle (16 hours after the infection) the reaction) the reaction product was revealed not in the whole cell wall, butin some of its areas only. There were found no qualitative differences in the localization of the group-specific antigen in the Malprowiae strains under study.     

Ultrastructural analysis of chlamydial antigen-containing vesicles everting from the Chlamydia trachomatis inclusion

Several chlamydial antigens have been detected in the infected epithelial cell cytosol and on the host cell surface prior to their presumed natural release at the end of the 72-96 h developmental cycle. These extra-inclusion antigens are proposed to influence vital host cell functions, antigen trafficking and presentation and, ultimately, contribute to a prolonged inflammatory response. To begin to dissect the mechanisms for escape of these antigens from the chlamydial inclusion, which are enhanced on exposure to antibiotics, polarized endometrial epithelial cells (HEC-1B) were infected with Chlamydia trachomatis serovar E for 36 h or 48 h. Infected cells were then exposed to chemotactic human polymorphonuclear neutrophils not loaded or pre-loaded in vitro with the antibiotic azithromycin. Viewed by electron microscopy, the azithromycin-mediated killing of chlamydiae involved an increase in chlamydial outer membrane blebbing followed by the appearance of the blebs in larger vesicles (i) everting from but still associated with the inclusion as well as (ii) external to the inclusion. Evidence that the vesicles originated from the chlamydial inclusion membrane was shown by immuno-localization of inclusion membrane proteins A, F, and G on the vesicular membranes. Chlamydial heat shock protein 60 (chsp60) copies 2 and 3, but not copy 1, were released from RB and incorporated into the everted inclusion membrane vesicles and delivered to the infected cell surface. These data represent direct evidence for one mechanism of early antigen delivery, albeit membrane-bound, beyond the confines of the chlamydial inclusion.     

In vitro and in vivo functional activity of Chlamydia MurA,a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance

Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal DeltamurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.