Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector

Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide “proof of principle” that it is possible to “knock out” selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.     

Chlamydia trachomatis GlgA Is Secreted into Host Cell Cytoplasm

Glycogen has been localized both inside and outside Chlamydia trachomatis organisms. We now report that C. trachomatis glycogen synthase (GlgA) was detected in both chlamydial organism-associated and -free forms. The organism-free GlgA molecules were localized both in the lumen of chlamydial inclusions and in the cytosol of host cells. The cytosolic GlgA displayed a distribution pattern similar to that of a known C. trachomatis-secreted protease, CPAF. The detection of GlgA was specific since the anti-GlgA antibody labeling was only removed by preabsorption with GlgA but not CPAF fusion proteins. GlgA was detectable at 12h and its localization into host cell cytosol only became apparent at 24h after infection. The cytosolic localization of GlgA was conserved among all C. trachomatis serovars. However, the significance of the GlgA secretion into host cell cytoplasm remains unclear since, while expression of chlamydial GlgA in HeLa cells increased glycogen stores, it did not affect a subsequent infection with C. trachomatis. Similar to several other C. trachomatis-secreted proteins, GlgA is immunogenic in women urogenitally infected with C. trachomatis, suggesting that GlgA is expressed and may be secreted into host cell cytosol during C. trachomatis infection in humans. These findings have provided important information for further understanding C. trachomatis pathogenic mechanisms.     

Association of Veterinary Third-Generation Cephalosporin Use with the Risk of Emergence of Extended-Spectrum-Cephalosporin Resistance in Escherichia coli from Dairy Cattle in Japan

The use of extended-spectrum cephalosporins in food animals has been suggested to increase the risk of spread of Enterobacteriaceae carrying extended-spectrum β-lactamases to humans. However, evidence that selection of extended-spectrum cephalosporin–resistant bacteria owing to the actual veterinary use of these drugs according to criteria established in cattle has not been demonstrated. In this study, we investigated the natural occurrence of cephalosporin-resistant Escherichia coli in dairy cattle following clinical application of ceftiofur. E. coli isolates were obtained from rectal samples of treated and untreated cattle (n = 20/group) cultured on deoxycholate-hydrogen sulfide-lactose agar in the presence or absence of ceftiofur. Eleven cefazoline-resistant isolates were obtained from two of the ceftiofur-treated cattle; no cefazoline-resistant isolates were found in untreated cattle. The cefazoline-resistant isolates had mutations in the chromosomal ampC promoter region and remained susceptible to ceftiofur. Eighteen extended-spectrum cephalosporin–resistant isolates from two ceftiofur-treated cows were obtained on ceftiofur-supplemented agar; no extended-spectrum cephalosporin–resistant isolates were obtained from untreated cattle. These extended-spectrum cephalosporin–resistant isolates possessed plasmid-mediated β-lactamase genes, including bla _CTX-M-2 (9 isolates), bla _CTX-M-14 (8 isolates), or bla _CMY-2 (1 isolate); isolates possessing bla _CTX-M-2 and bla _CTX-M-14 were clonally related. These genes were located on self-transmissible plasmids. Our results suggest that appropriate veterinary use of ceftiofur did not trigger growth extended-spectrum cephalosporin–resistant E. coli in the bovine rectal flora; however, ceftiofur selection in vitro suggested that additional ceftiofur exposure enhanced selection for specific extended-spectrum cephalosporin–resistant β-lactamase-expressing E. coli clones     

IFN-γ-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy

Chlamydial infection of the host cell induces Gamma interferon (IFNγ), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNγ-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen''s elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNγ-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNγ, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5−/− MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNγ-induced Atg5−/− cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6−/−) MEFs, in which chlamydial growth is enhanced, do not respond to IFNγ even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction.     

Transformation of Sexually Transmitted Infection-Causing Serovars of Chlamydia trachomatis Using Blasticidin for Selection

Plasmid-free Chlamydia trachomatis serovar L2 organisms have been transformed with chlamydial plasmid-based shuttle vectors pGFP::SW2 and pBRCT using β-lactamase as a selectable marker. However, the recommendation of amoxicillin, a β-lactam antibiotics, as one of the choices for treating pregnant women with cervicitis due to C. trachomatis infection has made the existing shuttle vectors unsuitable for transforming sexually transmitted infection (STI)-causing serovars of C. trachomatis. Thus, in the current study, we modified the pGFP::SW2 plasmid by fusing a blasticidin S deaminase gene to the GFP gene to establish blasticidin resistance as a selectable marker and replacing the β-lactamase gene with the Sh ble gene to eliminate the penicillin resistance. The new vector termed pGFPBSD/Z::SW2 was used for transforming plasmid-free C. trachomatis serovar D organisms. Using blasticidin for selection, stable transformants were obtained. The GFP-BSD fusion protein was detected in cultures infected with the pGFPBSD/Z::SW2-trasnformed serovar D organisms. The transformation restored the plasmid property to the plasmid-free serovar D organisms. Thus, we have successfully modified the pGFP::SW2 transformation system for studying the biology and pathogenesis of other STI-causing serovars of C. trachomatis.     

Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39

The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy. The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D. Differences between C.trachomatis strains were focused on an ~50 kb ‘plasticity zone’ near the termination origins. In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli 0157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region. The C.pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes. AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C.pneumoniae. Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene. Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region. The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites. In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens.     

Transition of bla OXA-58-like to bla OXA-23-like in Acinetobacter baumannii Clinical Isolates in Southern China: An 8-Year Study

Background

The prevalence of carbapenem-resistant Acinetobacter baumannii in hospitals has been increasing worldwide. This study aims to investigate the carbapenemase genes and the clonal relatedness among A. baumannii clinical isolates in a Chinese hospital.

Methods

Carbapenemase genes and the upstream locations of insertion sequences were detected by polymerase chain reaction (PCR), and the clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.

Results

A total of 231 nonduplicate carbapenemase gene-harboring A. baumannii clinical isolates recovered from Shenzhen People’s Hospital, were investigated between 2002 and 2009. bla _OXA-23-like, bla _OXA-58-like, bla _OXA-40-like, and ISAba1-bla _OXA-51-like were identified in 119, 107, 1, and 4 isolates, respectively. IS1008-ΔISAba3, ISAba3, and ISAba1 were detected upstream of the bla _OXA-58-like gene in 69, 35, and 3 isolates, respectively. All bla _OXA-23-like genes but one had an upstream insertion of ISAba1. bla _OXA-58-like was the most common carbapenemase gene in A.baumannii before 2008, thereafter bla _OXA-23-like became rapidly prevalent and replaced bla _OXA-58-like in 2009. The majority of bla _OXA-58-like-carrying isolates showed lower level of resistance to imipenem and meropenem (minimum inhibitory concentrations (MICs), 1 μg/ml to 16 μg/ml), compared with the majority of bla _OXA-23-like-carrying isolates (MICs, 16 μg/ml to 64 μg/ml for both imipenem and meropenem). All 231 bla _OXA carbapenemase gene-harboring isolates belonged to 14 PFGE types (A–N), and three dominant clones A, J, and H accounted for 43.3%, 42.0%, and 8.2% of the tested isolates, respectively. Clone A (sequence type ST92/ST208) with bla _OXA-58-like was the most prevalent before 2008. Clone H (ST229) with bla _OXA-23-like became striking between 2007 and 2008. Clone J (ST381) with bla _OXA-23-like rapidly spread and replaced clones A and H in 2009.

Conclusion

This study is the first to reveal that the distinct bla _OXA-23-like-carrying A. baumannii ST381 displaced the previously prevalent bla _OXA-58-like-carrying A. baumannii ST92/ST208, resulting in the rapidly increasing resistance to carbapenems in A. baumannii in Shenzhen People’s Hospital in 2009.     

Fluorescence lifetime imaging unravels C. trachomatis metabolism and its crosstalk with the host cell

Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ₂-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ₂-NAD(P)H increase inside the chlamydial inclusion. τ₂-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ₂-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity.     

Complement C3 opsonization of Chlamydia trachomatis facilitates uptake in human monocytes

Chlamydia trachomatis is an obligate intracellular bacterium that causes severe infections, which can lead to infertility and ectopic pregnancy. Although both innate and adaptive immune responses are elicited during chlamydial infection the bacterium succeeds to evade host defense mechanisms establishing chronic infections. Thus, studying the host–pathogen interaction during chlamydial infection is of importance to understand how C. trachomatis can cause chronic infections. Both the complement system and monocytes play essential roles in anti-bacterial defense, and, therefore, we investigated the interaction between the complement system and the human pathogens C. trachomatis D and L2.Complement competent serum facilitated rapid uptake of both chlamydial serovars into monocytes. Using immunoelectron microscopy, we showed that products of complement C3 were loosely deposited on the bacterial surface in complement competent serum and further characterization demonstrated that the deposited C3 product was the opsonin iC3b. Using C3-depleted serum we confirmed that complement C3 facilitates rapid uptake of chlamydiae into monocytes in complement competent serum. Complement facilitated uptake did not influence intracellular survival of C. trachomatis or C. trachomatis-induced cytokine secretion. Hence, C. trachomatis D and L2 activate the complement system leading to chlamydial opsonization by iC3b and subsequent phagocytosis, activation and bacterial elimination by human monocytes.     

Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis inBacillus subtilis

A total of 20Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.     

The Conserved Tarp Actin Binding Domain Is Important for Chlamydial Invasion

The translocated actin recruiting phosphoprotein (Tarp) is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.     

Type II Fatty Acid Synthesis Is Essential for the Replication of Chlamydia trachomatis

The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC₅₀ of 0.9 μm at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis.     

Activation of neutrophils by Chlamydia trachomatis-infected epithelial cells is modulated by the chlamydial plasmid

The obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial agent of sexually transmitted disease world-wide. Chlamydia trachomatis primarily infects epithelial cells of the genital tract but the infection may be associated with ascending infection. Infection-associated inflammation can cause tissue damage resulting in female infertility and ectopic pregnancy. The precise mechanism of inflammatory tissue damage is unclear but earlier studies implicate the chlamydial cryptic plasmid as well as responding neutrophils. We here rebuilt the interaction of Chlamydia trachomatis-infected epithelial cells and neutrophils in-vitro. During infection of human (HeLa) or mouse (oviduct) epithelial cells with Chlamydia trachomatis, a soluble factor was produced that attracted neutrophils and prolonged neutrophil survival, independently of Toll-like receptor signaling but dependent on the chlamydial plasmid. A number of cytokines, but most strongly GM-CSF, were secreted at higher amounts from cells infected with plasmid-bearing, compared to plasmid-deficient, bacteria. Blocking GM-CSF removed the secreted pro-survival activity towards neutrophils. A second, neutrophil TNF-stimulatory activity was detected in supernatants, requiring MyD88 or TRIF independently of the plasmid. The results identify two pro-inflammatory activities generated during chlamydial infection of epithelial cells and suggest that the epithelial cell, partly through the chlamydial plasmid, can initiate a myeloid immune response and inflammation.     

Chlamydia trachomatis and Anti-MUC1 Serology and Subsequent Risk of High-Grade Serous Ovarian Cancer: A Population-Based Case–Control Study in Northern Sweden

BACKGROUND: Chlamydia trachomatis salpingitis causes inflammatory damage to the fallopian tube and could potentially cause initiation and progression of high-grade serous ovarian cancer (HGSC). Furthermore, C. trachomatis infection may stimulate mucin 1 (MUC1) protein production, possibly affecting anti-MUC1 antibody levels. The aim of this study was to examine if serology indicating past infection with C. trachomatis as well as anti-MUC1 production was associated with subsequent risk of HGSC. MATERIALS AND METHODS: In a prospective nested case–control study within the Northern Sweden Health and Disease Study and the Northern Sweden Maternity Cohort, the prevalence of chlamydial and anti-MUC1 antibodies was analyzed in blood samples drawn more than one year before diagnosis from 92 women with HGSC and 359 matched controls. Matching factors were age, date at blood draw, and sampling cohort. Plasma C. trachomatis IgG was analyzed using commercial micro-immunofluorescence test; chlamydial Heat Shock Protein 60 IgG (cHSP60) and anti-MUC1 IgG were analyzed with ELISA technique. RESULTS: The prevalence of C. trachomatis IgG and cHSP60 IgG antibodies, as well as the level of anti-MUC1 IgG was similar in women with HGSC and controls (16.3% vs. 17.0%, P = 0.87; 27.2% vs. 28.5%, P = 0.80; median 0.24 vs. 0.25, P = 0.70). Anti-MUC1 IgG and cHSP60 IgG levels were correlated (r = 0.169; P < 0.001). CONCLUSIONS: The findings of this prospective nested case–control study did not support an association between C. trachomatis infection, as measured by chlamydial serology, or anti-MUC1 IgG antibodies, and subsequent risk of HGSC.     

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.     

Host Adaptation of Chlamydia pecorum towards Low Virulence Evident in Co-Evolution of the ompA,incA, and ORF663 Loci

Chlamydia (C.) pecorum, an obligate intracellular bacterium, may cause severe diseases in ruminants, swine and koalas, although asymptomatic infections are the norm. Recently, we identified genetic polymorphisms in the ompA, incA and ORF663 genes that potentially differentiate between high-virulence C. pecorum isolates from diseased animals and low-virulence isolates from asymptomatic animals. Here, we expand these findings by including additional ruminant, swine, and koala strains. Coding tandem repeats (CTRs) at the incA locus encoded a variable number of repeats of APA or AGA amino acid motifs. Addition of any non-APA/AGA repeat motif, such as APEVPA, APAVPA, APE, or APAPE, associated with low virulence (P<10⁻⁴), as did a high number of amino acids in all incA CTRs (P = 0.0028). In ORF663, high numbers of 15-mer CTRs correlated with low virulence (P = 0.0001). Correction for ompA phylogram position in ORF663 and incA abolished the correlation between genetic changes and virulence, demonstrating co-evolution of ompA, incA, and ORF663 towards low virulence. Pairwise divergence of ompA, incA, and ORF663 among isolates from healthy animals was significantly higher than among strains isolated from diseased animals (P≤10⁻⁵), confirming the longer evolutionary path traversed by low-virulence strains. All three markers combined identified 43 unique strains and 4 pairs of identical strains among all 57 isolates tested, demonstrating the suitability of these markers for epidemiological investigations.     

Adaptive Evolution of the Chlamydia trachomatis Dominant Antigen Reveals Distinct Evolutionary Scenarios for B- and T-cell Epitopes: Worldwide Survey

Background

Chlamydia trachomatis is one of the most disseminated human pathogens, for which no vaccine is available yet. Understanding the impact of the host pressure on pathogen antigens is crucial, but so far it was only assessed for highly-restricted geographic areas. We aimed to evaluate the evolutionary picture of the chlamydial key antigen (MOMP), which is one of the leading multi-subunit vaccine candidates, in a worldwide basis.

Methodology/Principal Findings

Using genetics, molecular evolution methods and mathematical modelling, we analyzed all MOMP sequences reported worldwide, composed by 5026 strains from 33 geographic regions of five continents. Overall, 35.9% of variants were detected. The evolutionary pattern of MOMP amino acid gains/losses was found to differ from the remaining chromosome, reflecting the demanding constraints of this porin, adhesin and dominant antigen. Amino acid changes were 4.3-fold more frequent in host-interacting domains (P<10⁻¹²), specifically within B-cell epitopes (P<10⁻⁵), where 25% of them are at fixation (P<10⁻⁵). According to the typical pathogen-host arms race, this rampant B-cell antigenic variation likely represents neutralization escape mutants, as some mutations were previously shown to abrogate neutralization of chlamydial infectivity in vitro. In contrast, T-cell clusters of diverse HLA specificities are under purifying selection, suggesting a strategy that may lead to immune subversion. Moreover, several silent mutations are at fixation, generating preferential codons that may influence expression, and may also reflect recombination-derived ‘hitchhiking-effect’ from favourable nonsilent changes. Interestingly, the most prevalent C. trachomatis genotypes, E and F, showed a mutation rate 22.3-fold lower than that of the remainder (P<10⁻²⁰), suggesting more fitted antigenic profiles.

Conclusions/Significance

Globally, the adaptive evolution of the C. trachomatis dominant antigen is likely driven by its complex pathogenesis-related function and reflects distinct evolutionary antigenic scenarios that may benefit the pathogen, and thus should be taking into account in the development of a MOMP-based vaccine.