ABSTRACT
Chlamydia trachomatis is an obligate intracellular bacterium, which undergoes a biphasic developmental cycle inside a vacuole termed the inclusion. Chlamydia-specific effector proteins embedded into the inclusion membrane, the Inc proteins, facilitate inclusion interaction with cellular organelles. A subset of Inc proteins engages with specific host factors at the endoplasmic reticulum (ER)-inclusion membrane contact site (MCS), which is a discrete point of contact between the inclusion membrane and the endoplasmic reticulum (ER). Here, we report that the C. trachomatis Inc protein CTL0402/IncSCt is a novel component of the ER-inclusion MCS that specifically interacts with and recruits STIM1, a previously identified host component of the ER-inclusion MCS with an unassigned interacting partner at the inclusion membrane. In comparison, the Chlamydia muridarum IncS homologue (TC0424/IncSCm) does not interact with or recruit STIM1 to the inclusion, indicating species specificity. To further investigate IncS function and overcome the recently reported early developmental defect of the incS mutant, we achieved temporal complementation by expressing IncS exclusively during the early stages of the developmental cycle. Additionally, we used allelic exchange to replace the incSCt open reading frame with incSCm in the C. trachomatis chromosome. Inclusions harboring either of these strains progressed through the developmental cycle but were STIM1 negative and displayed increased inclusion lysis 48 h postinfection. Expression of incSCt in trans complemented these phenotypes. Altogether, our results indicate that IncS is necessary and sufficient to recruit STIM1 to C. trachomatis inclusion and that IncS plays an early developmental role conserved in C. trachomatis and C. muridarum and a late role in inclusion stability specific to C. trachomatis. IMPORTANCE Obligate intracellular pathogens strictly rely on the host for replication. Specialized pathogen-encoded effector proteins play a central role in sophisticated mechanisms of host cell manipulation. In Chlamydia, a subset of these effector proteins, the inclusion membrane proteins, are embedded in the membrane of the vacuole in which the bacteria replicate. Chlamydia encodes 50 to 100 putative Inc proteins. Many are conserved among species, including the human and mouse pathogens Chlamydia trachomatis and Chlamydia muridarum, respectively. However, whether the function(s) of Inc proteins is indeed conserved among species is poorly understood. Here, we characterized the function of the Inc protein IncS conserved in C. trachomatis and C. muridarum. Our work reveals that a single effector protein can play multiple functions at various stages of the developmental cycle. However, these functions are not necessarily conserved across species, suggesting a complex evolutionary path among Chlamydia species.
Subject(s)
Chlamydia muridarum , Chlamydia trachomatis , Humans , Animals , Mice , Chlamydia trachomatis/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Chlamydia muridarum/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Endoplasmic Reticulum/metabolismABSTRACT
We have previously shown that circRNAs in host cells are involved in the process of Chlamydia trachomatis infection. In this study we aimed to identify significantly altered circRNAs/lncRNAs/mRNAs in Chlamydia muridarum infected cells and investigate their biological functions in the interaction between Chlamydia muridarum and host cells. For this purpose, circRNA, lncRNA and mRNA expression profiles were screened and identified in HeLa cells with or without Chlamydia muridarum infection by microarray. Bioinformatics analyses including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) analysis were then carried out and the circRNA-miRNA ceRNA network was constructed. The differentially expressed circRNAs and lncRNAs were selected for validation by RT-qPCR. The results shown that a total of 834 circRNAs, 2149 lncRNAs and 1283 mRNAs were found to be differentially expressed. Enrichment analysis of GO and KEGG showed that the dysregulated genes involved nuclear-transcribed mRNA catabolic process, protein binding, RNA catabolic process and translation, the MAPK signaling pathway, apoptosis, Toll-like receptor signaling pathway, cAMP signaling pathway and Notch signaling pathway may play important roles in Chlamydia infection. Our study provides a systematic outlook on the potential function of non-coding RNAs in the molecular basis of Chlamydia infection.
Subject(s)
Chlamydia Infections , Chlamydia muridarum , RNA, Long Noncoding , Chlamydia Infections/genetics , Chlamydia muridarum/genetics , Chlamydia muridarum/metabolism , Computational Biology , Gene Regulatory Networks , HeLa Cells , Humans , RNA, Circular/genetics , RNA, Long Noncoding/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Chlamydia muridarum actively grows in murine mucosae and is a representative model of human chlamydial genital tract disease. In contrast, C. trachomatis infections in mice are limited and rarely cause disease. The factors that contribute to these differences in host adaptation and specificity remain elusive. Overall genomic similarity leads to challenges in the understanding of these significant differences in tropism. A region of major genetic divergence termed the plasticity zone (PZ) has been hypothesized to contribute to the host specificity. To evaluate this hypothesis, lateral gene transfer was used to generate multiple hetero-genomic strains that are predominately C. trachomatis but have replaced regions of the PZ with those from C. muridarum. In vitro analysis of these chimeras revealed C. trachomatis-like growth as well as poor mouse infection capabilities. Growth-independent cytotoxicity phenotypes have been ascribed to three large putative cytotoxins (LCT) encoded in the C. muridarum PZ. However, analysis of PZ chimeras supported that gene products other than the LCTs are responsible for cytopathic and cytotoxic phenotypes. Growth analysis of associated chimeras also led to the discovery of an inclusion protein, CTL0402 (CT147), and homolog TC0424, which was critical for the integrity of the inclusion and preventing apoptosis.
Subject(s)
Chlamydia Infections/microbiology , Chlamydia muridarum/genetics , Chlamydia trachomatis/genetics , Gene Transfer, Horizontal , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chlamydia muridarum/metabolism , Chlamydia trachomatis/metabolism , Female , Genetic Variation , Humans , Mice, Inbred C57BLABSTRACT
The obligate intracellular pathogen Chlamydia trachomatis is the leading cause of noncongenital blindness and causative agent of the most common sexually transmitted infection of bacterial origin. With a reduced genome, C. trachomatis is dependent on its host for survival, in part due to a need for the host cell to compensate for incomplete bacterial metabolic pathways. However, relatively little is known regarding how C. trachomatis is able to hijack host cell metabolism. In this study, we show that two host glycolytic enzymes, aldolase A and pyruvate kinase, as well as lactate dehydrogenase, are enriched at the C. trachomatis inclusion membrane during infection. Inclusion localization was not species specific, since a similar phenotype was observed with C. muridarum Time course experiments showed that the number of positive inclusions increased throughout the developmental cycle. In addition, these host enzymes colocalized to the same inclusion, and their localization did not appear to be dependent on sustained bacterial protein synthesis or on intact host actin, vesicular trafficking, or microtubules. Depletion of the host glycolytic enzyme aldolase A resulted in decreased inclusion size and infectious progeny production, indicating a role for host glycolysis in bacterial growth. Finally, quantitative PCR analysis showed that expression of C. trachomatis glycolytic enzymes inversely correlated with host enzyme localization at the inclusion. We discuss potential mechanisms leading to inclusion localization of host glycolytic enzymes and how it could benefit the bacteria. Altogether, our findings provide further insight into the intricate relationship between host and bacterial metabolism during Chlamydia infection.
Subject(s)
Chlamydia Infections/metabolism , Chlamydia trachomatis/metabolism , Fructose-Bisphosphate Aldolase/metabolism , Glycolysis , Host Microbial Interactions , Inclusion Bodies/metabolism , L-Lactate Dehydrogenase/metabolism , Pyruvate Kinase/metabolism , Actins/metabolism , Bacterial Outer Membrane/enzymology , Bacterial Outer Membrane/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chlamydia Infections/enzymology , Chlamydia Infections/genetics , Chlamydia muridarum/metabolism , Chlamydia trachomatis/enzymology , Chlamydia trachomatis/growth & development , Chlamydia trachomatis/pathogenicity , Fructose-Bisphosphate Aldolase/genetics , HeLa Cells , Humans , Inclusion Bodies/enzymology , Inclusion Bodies/microbiology , L-Lactate Dehydrogenase/genetics , Microtubules/metabolism , Protein Biosynthesis/drug effects , Pyruvate Kinase/geneticsABSTRACT
Functional genetic analysis of Chlamydia has been a challenge due to the historical genetic intractability of Chlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for Chlamydia muridarum, a mouse-adapted Chlamydia species that is widely used in Chlamydia infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing C. muridarum transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions in glgB, pmpI, pmpA, and pmpD were investigated further for in vitro and in vivo phenotypes, including growth, inclusion morphology, and attachment to host cells. The glgB mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 pmp mutants, pmpI was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum The generation of a complete library of C. muridarum mutants will ultimately enable comprehensive identification of the functional genetic requirements for Chlamydia infection in vivoIMPORTANCE Historical issues with genetic manipulation of Chlamydia have prevented rigorous functional genetic characterization of the â¼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adapted Chlamydia species that is widely used for in vivo investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized in vitro phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.
Subject(s)
Bacterial Proteins/genetics , Chlamydia muridarum/genetics , Chromosomes, Bacterial/chemistry , DNA Transposable Elements , Mutagenesis , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Base Sequence , Chlamydia Infections/microbiology , Chlamydia muridarum/drug effects , Chlamydia muridarum/metabolism , Chloramphenicol/pharmacology , Chromosomes, Bacterial/metabolism , Clone Cells , Gene Library , Mice , Mutation , Open Reading Frames , Plasmids/chemistry , Plasmids/metabolism , Whole Genome SequencingABSTRACT
Lateral gene transfer (LGT) among Chlamydia trachomatis strains is common, in both isolates generated in the laboratory and those examined directly from patients. In contrast, there are very few examples of recent acquisition of DNA by any Chlamydia spp. from any other species. Interspecies LGT in this system was analyzed using crosses of tetracycline (Tc)-resistant C. trachomatis L2/434 and chloramphenicol (Cam)-resistant C. muridarum VR-123. Parental C. muridarum strains were created using a plasmid-based Himar transposition system, which led to integration of the Camr marker randomly across the chromosome. Fragments encompassing 79% of the C. muridarum chromosome were introduced into a C. trachomatis background, with the total coverage contained on 142 independent recombinant clones. Genome sequence analysis of progeny strains identified candidate recombination hot spots, a property not consistent with in vitroC. trachomatis × C. trachomatis (intraspecies) crosses. In both interspecies and intraspecies crosses, there were examples of duplications, mosaic recombination endpoints, and recombined sequences that were not linked to the selection marker. Quantitative analysis of the distribution and constitution of inserted sequences indicated that there are different constraints on interspecies LGT than on intraspecies crosses. These constraints may help explain why there is so little evidence of interspecies genetic exchange in this system, which is in contrast to very widespread intraspecies exchange in C. trachomatisIMPORTANCE Genome sequence analysis has demonstrated that there is widespread lateral gene transfer among strains within the species C. trachomatis and with other closely related Chlamydia species in laboratory experiments. This is in contrast to the complete absence of foreign DNA in the genomes of sequenced clinical C. trachomatis strains. There is no understanding of any mechanisms of genetic transfer in this important group of pathogens. In this report, we demonstrate that interspecies genetic exchange can occur but that the nature of the fragments exchanged is different than those observed in intraspecies crosses. We also generated a large hybrid strain library that can be exploited to examine important aspects of chlamydial disease.
Subject(s)
Chlamydia muridarum/genetics , Chlamydia trachomatis/genetics , Chromosomes, Bacterial/chemistry , Gene Expression Regulation, Bacterial , Gene Transfer, Horizontal , Recombination, Genetic , Anti-Bacterial Agents/pharmacology , Base Sequence , Chlamydia muridarum/drug effects , Chlamydia muridarum/metabolism , Chlamydia trachomatis/drug effects , Chlamydia trachomatis/metabolism , Chromosomes, Bacterial/metabolism , Crosses, Genetic , DNA Transposable Elements , Plasmids/chemistry , Plasmids/metabolism , Tetracycline/pharmacology , Tetracycline Resistance/geneticsABSTRACT
Chlamydia trachomatis infections are the most prevalent sexually transmitted infections with potentially debilitating sequelae, such as infertility. Mouse models are generally used for vaccine development, to study the immune response and histopathology associated with Chlamydia infection. An important question regarding murine models is the in vivo identification of murine host genes responsible for the elimination of the murine and human Chlamydia strains. RNA sequencing of the Chlamydia muridarum infected BALB/c lung transcriptome revealed that several genes with direct antichlamydial functions were induced at the tissue level, including the already described and novel members of the murine interferon-inducible GTPase family, the CXCL chemokines CXCL9, CXCL11, immunoresponsive gene 1, nitric oxide synthase-2 (iNOS), and lipocalin-2. Indoleamine 2,3-dioxygenase 1-2 (IDO1-2) previously described potent antichlamydial host enzymes were also highly expressed in the infected murine lungs. This finding was novel, since IDO was considered as a unique human antichlamydial defense gene. Besides a lower level of epithelial cell positivity, immunohistochemistry showed that IDO1-2 proteins were expressed prominently in macrophages. Detection of the tryptophan degradation product kynurenine and the impact of IDO inhibition on Chlamydia muridarum growth proved that the IDO1-2 proteins were functionally active. IDO1-2 activity also increased in Chlamydia muridarum infected C57BL/6 lung tissues, indicating that this phenomenon is not mouse strain specific. Our study shows that the murine antichlamydial response includes a variety of highly up-regulated defense genes in vivo. Among these genes the antichlamydial effectors IDO1-2 were identified. The potential impact of murine IDO1-2 expression on Chlamydia propagation needs further investigation.
Subject(s)
Chlamydia Infections/metabolism , Chlamydia muridarum/drug effects , Chlamydia muridarum/metabolism , Chlamydophila pneumoniae/drug effects , Chlamydophila pneumoniae/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/pharmacology , Lung/metabolism , Animals , Chemokine CXCL11/genetics , Chemokine CXCL11/metabolism , Chemokine CXCL9/genetics , Chemokine CXCL9/metabolism , Chemokines/genetics , Chemokines/metabolism , Chlamydia Infections/genetics , Chlamydia Infections/microbiology , Chlamydia Infections/pathology , Disease Models, Animal , Enzyme Inhibitors/metabolism , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Gene Expression , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/drug effects , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Kynurenine , Lipocalin-2/genetics , Lipocalin-2/metabolism , Lung/pathology , Macrophages/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , RNA, Messenger/metabolism , Transcriptome , Tryptophan/analogs & derivatives , Tryptophan/antagonists & inhibitors , Tryptophan/metabolismABSTRACT
Recent studies have affirmed that higher-order epistasis is ubiquitous and can have large effects on complex traits. Yet, we lack frameworks for understanding how epistatic interactions are influenced by central features of cell physiology. In this study, we assess how protein quality control machinery-a critical component of cell physiology-affects epistasis for different traits related to bacterial resistance to antibiotics. Specifically, we disentangle the interactions between different protein quality control genetic backgrounds and two sets of mutations: (i) SNPs associated with resistance to antibiotics in an essential bacterial enzyme (dihydrofolate reductase, or DHFR) and (ii) differing DHFR bacterial species-specific amino acid background sequences (Escherichia coli, Listeria grayi, and Chlamydia muridarum). In doing so, we improve on generic observations that epistasis is widespread by discussing how patterns of epistasis can be partly explained by specific interactions between mutations in an essential enzyme and genes associated with the proteostasis environment. These findings speak to the role of environmental and genotypic context in modulating higher-order epistasis, with direct implications for evolutionary theory, genetic modification technology, and efforts to manage antimicrobial resistance.
Subject(s)
Drug Resistance, Bacterial/genetics , Epistasis, Genetic , Polymorphism, Single Nucleotide , Proteostasis , Tetrahydrofolate Dehydrogenase/genetics , Chlamydia muridarum/drug effects , Chlamydia muridarum/genetics , Chlamydia muridarum/metabolism , Drug Resistance, Bacterial/drug effects , Epistasis, Genetic/drug effects , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Association Studies , Genetic Pleiotropy , Listeria/drug effects , Listeria/genetics , Listeria/metabolism , MutationABSTRACT
Pgp3 consists of globular N- and C-terminal domains connected by a triple-helical coiled-coil middle domain. We demonstrated previously that Pgp3 is required for induction of hydrosalpinx by Chlamydia muridarum. We constructed C. muridarum transformants harboring deletion of the Pgp3 N-terminus (pgp3Δn), C-terminus (pgp3Δc), or middle domain (pgp3Δm). C3H/HeJ and CBA/J mice infected with pgp3Δn or pgp3Δm failed to induce hydrosalpinx in oviduct tissue. However, the pgp3Δc transformant induced mild hydrosalpinx in 20% of C3H/HeJ mice (severity score 0.2 ± 0.6) and in 40% of CBA/J mice (severity score 0.8 ± 1.3). The attenuated pathogenicity of the transformants harboring Pgp3 domain deletions was correlated with impaired in vitro growth and significantly reduced infectivity in the mouse lower genital tract. Moreover, the oviduct tissue of C3H/HeJ and CBA/J mice infected with the Pgp3-domain-deficient transformants displayed less inflammatory cell infiltration. Thus, the structural integrity of plasmid-encoded Pgp3 is essential for induction of hydrosalpinx by C. muridarum.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Chlamydia Infections/pathology , Chlamydia muridarum/metabolism , Chlamydia muridarum/pathogenicity , Fallopian Tube Diseases/pathology , Plasmids , Virulence Factors/metabolism , Animals , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Chlamydia muridarum/genetics , Disease Models, Animal , Female , Mice, Inbred C3H , Mice, Inbred CBA , Sequence Deletion , Virulence Factors/geneticsABSTRACT
The unfolded protein response (UPR) contributes to chlamydial pathogenesis, as a source of lipids and ATP during replication, and for establishing the initial anti-apoptotic state of host cell that ensures successful inclusion development. The molecular mechanism(s) of UPR induction by Chlamydia is unknown. Chlamydia use type III secretion system (T3SS) effector proteins (e.g, the Translocated Actin-Recruiting Phosphoprotein (Tarp) to stimulate host cell's cytoskeletal reorganization that facilitates invasion and inclusion development. We investigated the hypothesis that T3SS effector-mediated assembly of myosin-II complex produces activated non-muscle myosin heavy chain II (NMMHC-II), which then binds the UPR master regulator (BiP) and/or transducers to induce UPR. Our results revealed the interaction of the chlamydial effector proteins (CT228 and Tarp) with components of the myosin II complex and UPR regulator and transducer during infection. These interactions caused the activation and binding of NMMHC-II to BiP and IRE1α leading to UPR induction. In addition, specific inhibitors of myosin light chain kinase, Tarp oligomerization and myosin ATPase significantly reduced UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through T3SS effector-mediated activation of NMMHC-II components of the myosin complex to facilitate infectivity. The finding provides greater insights into chlamydial pathogenesis with the potential to identify therapeutic targets and formulations.
Subject(s)
Chlamydia muridarum/pathogenicity , Chlamydia trachomatis/pathogenicity , Host Microbial Interactions/physiology , Unfolded Protein Response/physiology , Animals , Chlamydia Infections/etiology , Chlamydia Infections/metabolism , Chlamydia Infections/microbiology , Chlamydia muridarum/metabolism , Chlamydia trachomatis/metabolism , HeLa Cells , Humans , Inclusion Bodies/metabolism , Mice , Myosin Type II/metabolism , Type III Secretion Systems/metabolismABSTRACT
Chlamydial pathogenicity in the upper genital tract relies on chlamydial ascending from the lower genital tract. To monitor chlamydial ascension, we engineered a luciferase-expressing C. muridarum. In cells infected with the luciferase-expressing C. muridarum, luciferase gene expression and enzymatic activity (measured as bioluminescence intensity) correlated well along the infection course, suggesting that bioluminescence can be used for monitoring chlamydial replication. Following an intravaginal inoculation with the luciferase-expressing C. muridarum, 8 of 10 mice displayed bioluminescence signal in the lower with 4 also in the upper genital tracts on day 3 after infection. By day 7, all 10 mice developed bioluminescence signal in the upper genital tracts. The bioluminescence signal was maintained in the upper genital tract in 6 and 2 mice by days 14 and 21, respectively. The bioluminescence signal was no longer detectable in any of the mice by day 28. The whole body imaging approach also revealed an unexpected airway infection following the intravaginal inoculation. Although the concomitant airway infection was transient and did not significantly alter the genital tract infection time courses, caution should be taken during data interpretation. The above observations have demonstrated that C. muridarum can not only achieve rapid ascending infection in the genital tract but also cause airway infection following a genital tract inoculation. These findings have laid a foundation for further optimizing the C. muridarum intravaginal infection murine model for understanding chlamydial pathogenic mechanisms.
Subject(s)
Chlamydia Infections/pathology , Chlamydia muridarum , Molecular Imaging , Reproductive Tract Infections/pathology , Animals , Chlamydia muridarum/genetics , Chlamydia muridarum/metabolism , Female , HeLa Cells , Humans , Luciferases/biosynthesis , Mice , Mice, Inbred BALB C , Reproductive Tract Infections/microbiologyABSTRACT
Transformation of Chlamydia trachomatis should greatly advance the chlamydial research. However, significant progress has been hindered by the failure of C. trachomatis to induce clinically relevant pathology in animal models. Chlamydia muridarum, which naturally infects mice, can induce hydrosalpinx in mice, a tubal pathology also seen in women infected with C. trachomatis. We have developed a C. muridarum transformation system and confirmed Pgp1, -2, -6, and -8 as plasmid maintenance factors, Pgp3, -5, and -7 as dispensable for in vitro growth, and Pgp4 as a positive regulator of genes that are dependent on plasmid for expression. More importantly, we have discovered that Pgp5 can negatively regulate the same plasmid-dependent genes. Deletion of Pgp5 led to a significant increase in expression of the plasmid-dependent genes, suggesting that Pgp5 can suppress the expression of these genes. Replacement of pgp5 with a mCherry gene, or premature termination of pgp5 translation, also increased expression of the plasmid-dependent genes, indicating that Pgp5 protein but not its DNA sequence is required for the inhibitory effect. Replacing C. muridarum pgp5 with a C. trachomatis pgp5 still inhibited the plasmid-dependent gene expression, indicating that the negative regulation of plasmid-dependent genes is a common feature of all Pgp5 regardless of its origin. Nevertheless, C. muridarum Pgp5 is more potent than C. trachomatis Pgp5 in suppressing gene expression. Thus, we have uncovered a novel function of Pgp5 and developed a C. muridarum transformation system for further mapping chlamydial pathogenic and protective determinants in animal models.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Chlamydia muridarum/metabolism , Plasmids/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Animals , Chlamydia muridarum/genetics , Cloning, Molecular , Female , Gene Expression Regulation, Bacterial , HeLa Cells , Humans , Mice , Plasmids/genetics , Transformation, GeneticABSTRACT
We previously demonstrated that plasmid-deficient Chlamydia muridarum retains the ability to infect the murine genital tract but does not elicit oviduct pathology because it fails to activate Toll-like receptor 2 (TLR2). We derived a plasmid-cured derivative of the human genital isolate Chlamydia trachomatis D/UW-3/Cx, strain CTD153, which also fails to activate TLR2, indicating this virulence phenotype is associated with plasmid loss in both C. trachomatis and C. muridarum. As observed with plasmid-deficient C. muridarum, CTD153 displayed impaired accumulation of glycogen within inclusions. Transcriptional profiling of the plasmid-deficient strains by using custom microarrays identified a conserved group of chromosomal loci, the expression of which was similarly controlled in plasmid-deficient C. muridarum strains CM972 and CM3.1 and plasmid-deficient C. trachomatis CTD153. However, although expression of glycogen synthase, encoded by glgA, was greatly reduced in CTD153, it was unaltered in plasmid-deficient C. muridarum strains. Thus, additional plasmid-associated factors are required for glycogen accumulation by this chlamydial species. Furthermore, in C. trachomatis, glgA and other plasmid-responsive chromosomal loci (PRCLs) were transcriptionally responsive to glucose limitation, indicating that additional regulatory elements may be involved in the coordinated expression of these candidate virulence effectors. Glucose-limited C. trachomatis displayed reduced TLR2 stimulation in an in vitro assay. During human chlamydial infection, glucose limitation may decrease chlamydial virulence through its effects on plasmid-responsive chromosomal genes.
Subject(s)
Chlamydia Infections/genetics , Chlamydia muridarum/genetics , Chlamydia trachomatis/genetics , Gene Expression Regulation, Bacterial/genetics , Plasmids/genetics , Toll-Like Receptor 2/metabolism , Animals , Cell Line , Chlamydia Infections/metabolism , Chlamydia muridarum/metabolism , Chlamydia muridarum/pathogenicity , Chlamydia trachomatis/metabolism , Chlamydia trachomatis/pathogenicity , Chromosomes, Bacterial/genetics , Gene Expression , Genetic Loci , Glucose/metabolism , Glycogen/metabolism , Glycogen Synthase/biosynthesis , Glycogen Synthase/genetics , Humans , Inclusion Bodies/metabolism , Mice , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Virulence/geneticsABSTRACT
BACKGROUND: Chlamydiae are obligate intracellular bacteria comprising some of the most important bacterial pathogens of animals and humans. Although chlamydial outer membrane proteins play a key role for attachment to and entry into host cells, only few have been described so far. We developed a comprehensive, multiphasic in silico approach, including the calculation of clusters of orthologues, to predict outer membrane proteins using conservative criteria. We tested this approach using Escherichia coli (positive control) and Bacillus subtilis (negative control), and applied it to five chlamydial species; Chlamydia trachomatis, Chlamydia muridarum, Chlamydia (a.k.a. Chlamydophila) pneumoniae, Chlamydia (a.k.a. Chlamydophila) caviae, and Protochlamydia amoebophila. RESULTS: In total, 312 chlamydial outer membrane proteins and lipoproteins in 88 orthologous clusters were identified, including 238 proteins not previously recognized to be located in the outer membrane. Analysis of their taxonomic distribution revealed an evolutionary conservation among Chlamydiae, Verrucomicrobia, Lentisphaerae and Planctomycetes as well as lifestyle-dependent conservation of the chlamydial outer membrane protein composition. CONCLUSION: This analysis suggested a correlation between the outer membrane protein composition and the host range of chlamydiae and revealed a common set of outer membrane proteins shared by these intracellular bacteria. The collection of predicted chlamydial outer membrane proteins is available at the online database pCOMP http://www.microbial-ecology.net/pcomp and might provide future guidance in the quest for anti-chlamydial vaccines.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Chlamydia/metabolism , Computational Biology/methods , Evolution, Molecular , Lipoproteins/metabolism , Bacterial Outer Membrane Proteins/genetics , Chlamydia/classification , Chlamydia/genetics , Chlamydia/physiology , Chlamydia muridarum/classification , Chlamydia muridarum/genetics , Chlamydia muridarum/metabolism , Chlamydia muridarum/physiology , Chlamydia trachomatis/classification , Chlamydia trachomatis/genetics , Chlamydia trachomatis/metabolism , Chlamydia trachomatis/physiology , Lipoproteins/genetics , SoftwareABSTRACT
BACKGROUND: Immunity to chlamydia is thought to rely on interferon (IFN)-gamma-secreting T helper cells type 1 (Th1) with an additional effect of secreted antibodies. A need for Th1-polarizing adjuvants in experimental chlamydia vaccines has been demonstrated, and antigen conformation has also been reported as being important for raising protective immunity. METHODS: C57BL/6 mice vaccinated with native refolded Chlamydia muridarum major outer membrane protein (MOMP) adjuvanted with either Th1-promoting cationic adjuvant formulation 1 (CAF01) or T helper cells type 2-promoting aluminum hydroxide (alum) received a genital inoculation of 1.5 x 10(5) inclusion-forming units of C. muridarum. The role played by CD4(+) T cells in MOMP/CAF01-raised immunity was investigated by depleting CD4(+) T cells in vaccinated mice, and antigen conformation dependence was evaluated by vaccination with recombinant MOMP. RESULTS: Mice vaccinated with MOMP/alum displayed a strong anti-MOMP humoral response with high IgG1 titers, low levels of IFN-gamma and tumor necrosis factor (TNF)-alpha, and only a slight reduction in chlamydial load. Mice vaccinated with MOMP/CAF01 displayed high titers of IgG2b, IFN-gamma, and TNF-alpha and a profoundly reduced vaginal chlamydial load, compared with control mice. The protection was CD4(+) T cell dependent and was not dependent on MOMP conformation. CONCLUSION: CAF01 adjuvant facilitates a protective anti-MOMP CD4(+) T cell response independent of MOMP conformation.
Subject(s)
Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/immunology , Chlamydia Infections/prevention & control , Chlamydia muridarum/immunology , Th1 Cells/immunology , Animals , Antibodies, Bacterial/biosynthesis , Antibodies, Bacterial/blood , Bacterial Outer Membrane Proteins/administration & dosage , Bacterial Vaccines/administration & dosage , CD4-Positive T-Lymphocytes/immunology , Chlamydia Infections/immunology , Chlamydia muridarum/metabolism , Cytokines/biosynthesis , Female , Immunoglobulin G/biosynthesis , Immunoglobulin G/blood , Liposomes , Mice , Mice, Inbred C57BL , Salpingitis/pathology , Vagina/microbiologyABSTRACT
Epithelial cells play an important role in host defense as sentinels for invading microbial pathogens. Chlamydia trachomatis is an intracellular bacterial pathogen that replicates in reproductive tract epithelium. Epithelial cells lining the reproductive tract likely play a key role in triggering inflammation and adaptive immunity during Chlamydia infections. For this report a murine oviduct epithelial cell line was derived in order to determine how epithelial cells influence innate and adaptive immune responses during Chlamydia infections. As expected, oviduct epithelial cells infected by Chlamydia muridarum produced a broad spectrum of chemokines, including CXCL16, and regulators of the acute-phase response, including interleukin-1alpha (IL-1alpha), IL-6, and tumor necrosis factor alpha. In addition, infected epithelial cells expressed cytokines that augment gamma interferon (IFN) production, including IFN-alpha/beta and IL-12-p70. To my knowledge this is the first report of a non-myeloid/lymphoid cell type making IL-12-p70 in response to an infection. Equally interesting, infected epithelial cells significantly upregulated transforming growth factor alpha precursor expression, suggesting a mechanism by which they might play a direct role in the pathological scarring seen as a consequence of Chlamydia infections. Data from these in vitro studies predict that infected oviduct epithelium contributes significantly to host innate and adaptive defenses but may also participate in the immunopathology seen with Chlamydia infections.
Subject(s)
Chlamydia Infections/metabolism , Chlamydia muridarum/metabolism , Fallopian Tubes/metabolism , Interleukin-12/metabolism , Protein Subunits/metabolism , Animals , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Epithelium/metabolism , Epithelium/microbiology , Fallopian Tubes/microbiology , Female , Humans , Mice , RNA, Messenger/metabolismABSTRACT
The BCL-2 family member BAX plays a critical role in regulating apoptosis. Surprisingly, bax-deficient mice display limited phenotypic abnormalities. Here we investigate the effect of BAX on infection by the sexually transmitted pathogen, Chlamydia muridarum (the mouse pneumonitis strain of Chlamydia trachomatis). Bax(-/-) cells are relatively resistant to Chlamydia-induced apoptosis, and fewer bacteria are recovered after two infection cycles from Bax(-/-) cells than from wild-type cells. These results suggest that BAX-dependent apoptosis may be used to initiate a new round of infection, most likely by releasing Chlamydia-containing apoptotic bodies from infected cells that could be internalized by neighboring uninfected cells. Nonetheless, infected Bax(-/-) cells die through necrosis, which is normally associated with inflammation, more often than infected wild-type cells. These studies were confirmed in mice infected intravaginally with C. muridarum; since the infection disappears more quickly from Bax(-/-) mice than from wild-type mice, secretion of proinflammatory cytokines is increased in Bax(-/-) mice, and large granulomas are present in the genital tract of Bax(-/-) mice. Taken together, these data suggest that chlamydia-induced apoptosis via BAX contributes to bacterial propagation and decreases inflammation. Bax deficiency results in lower infection and an increased inflammatory cytokine response associated with more severe pathology.
Subject(s)
Apoptosis , Chlamydia muridarum/metabolism , Inflammation , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , Animals , Chlamydia Infections/pathology , Female , Granuloma/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Necrosis , Proto-Oncogene Proteins/metabolism , Time Factors , bcl-2-Associated X ProteinABSTRACT
BACKGROUND: Complete genomic sequences of closely related organisms, such as the chlamydiae, afford the opportunity to assess significant strain differences against a background of many shared characteristics. The chlamydiae are ubiquitous intracellular parasites that are important pathogens of humans and other organisms. Tryptophan limitation caused by production of interferon-gamma by the host and subsequent induction of indoleamine dioxygenase is a key aspect of the host-parasite interaction. It appears that the chlamydiae have learned to recognize tryptophan depletion as a signal for developmental remodeling. The consequent non-cultivable state of persistence can be increasingly equated to chronic disease conditions. RESULTS: The genes encoding enzymes of tryptophan biosynthesis were the focal point of this study. Chlamydophila psittaci was found to possess a compact operon containing PRPP synthase, kynureninase, and genes encoding all but the first step of tryptophan biosynthesis. All but one of the genes exhibited translational coupling. Other chlamydiae (Chlamydia trachomatis, C. muridarum and Chlamydophila pneumoniae) lack genes encoding PRPP synthase, kynureninase, and either lack tryptophan-pathway genes altogether or exhibit various stages of reductive loss. The origin of the genes comprising the trp operon does not seem to have been from lateral gene transfer. CONCLUSIONS: The factors that accommodate the transition of different chlamydial species to the persistent (chronic) state of pathogenesis include marked differences in strategies deployed to obtain tryptophan from host resources. C. psittaci appears to have a novel mechanism for intercepting an early intermediate of tryptophan catabolism and recycling it back to tryptophan. In effect, a host-parasite metabolic mosaic has evolved for tryptophan recycling.