Parallel Evolution of Group B Streptococcus Hypervirulent Clonal Complex 17 Unveils New Pathoadaptive Mutations.

Group B Streptococcus (GBS) is a commensal of the gastrointestinal and genitourinary tracts, while a prevailing cause of neonatal disease worldwide. Of the various clonal complexes (CCs), CC17 is overrepresented in GBS-infected newborns for reasons that are still largely unknown. Here, we report a comprehensive genomic analysis of 626 CC17 isolates collected worldwide, identifying the genetic traits behind their successful adaptation to humans and the underlying differences between carriage and clinical strains. Comparative analysis with 923 GBS genomes belonging to CC1, CC19, and CC23 revealed that the evolution of CC17 is distinct from that of other human-adapted lineages and recurrently targets functions related to nucleotide and amino acid metabolism, cell adhesion, regulation, and immune evasion. We show that the most distinctive features of disease-specific CC17 isolates were frequent mutations in the virulence-associated CovS and Stk1 kinases, underscoring the crucial role of the entire CovRS regulatory pathway in modulating the pathogenicity of GBS. Importantly, parallel and convergent evolution of major components of the bacterial cell envelope, such as the capsule biosynthesis operon, the pilus, and Rib, reflects adaptation to host immune pressures and should be taken into account in the ongoing development of a GBS vaccine. The presence of recurrent targets of evolution not previously implicated in virulence also opens the way for uncovering new functions involved in host colonization and GBS pathogenesis. IMPORTANCE The incidence of group B Streptococcus (GBS) neonatal disease continues to be a significant cause of concern worldwide. Strains belonging to clonal complex 17 (CC17) are the most frequently responsible for GBS infections in neonates, especially among late-onset disease cases. Therefore, we undertook the largest genomic study of GBS CC17 strains to date to decipher the genetic bases of their remarkable colonization and infection ability. We show that crucial functions involved in different steps of the colonization or infection process of GBS are distinctly mutated during the adaptation of CC17 to the human host. In particular, our results implicate the CovRS two-component regulator of virulence in the differentiation between carriage- and disease-associated isolates. Not only does this work raise important implications for the ongoing development of a vaccine against GBS but might also drive the discovery of key functions for GBS adaptation and pathogenesis that have been overlooked until now. Author Video: An author video summary of this article is available.

In Vitro Activity of Quaternary Ammonium Surfactants against Streptococcal, Chlamydial, and Gonococcal Infective Agents.

Quaternary ammonium compounds (QAC) are widely used, cheap, and chemically stable disinfectants and topical antiseptics with wide-spectrum antimicrobial activities. Within this group of compounds, we recently showed that there are significant differences between the pharmacodynamics of n-alkyl quaternary ammonium surfactants (QAS) with a short (C12) alkyl chain when in vitro toxicities toward bacterial and mammalian epithelial cells are compared. These differences result in an attractive therapeutic window that justifies studying short-chain QAS as prophylactics for sexually transmitted infections (STI) and perinatal vertically transmitted urogenital infections (UGI). We have evaluated the antimicrobial activities of short-chain (C12) n-alkyl QAS against several STI and UGI pathogens as well as against commensal Lactobacillus species. Inhibition of infection of HeLa cells by Neisseria gonorrhoeae and Chlamydia trachomatis was studied at concentrations that were not toxic to the HeLa cells. We show that the pathogenic bacteria are much more susceptible to QAS toxic effects than the commensal vaginal flora and that QAS significantly attenuate the infectivity of N. gonorrhoeae and C. trachomatis without affecting the viability of epithelial cells of the vaginal mucosa. N-Dodecylpyridinium bromide (C12PB) was found to be the most effective QAS. Our results strongly suggest that short-chain (C12) n-alkyl pyridinium bromides and structurally similar compounds are promising microbicide candidates for topical application in the prophylaxis of STI and perinatal vertical transmission of UGI.

Quaternary ammonium surfactant structure determines selective toxicity towards bacteria: mechanisms of action and clinical implications in antibacterial prophylaxis.

OBJECTIVES: Broad-spectrum antimicrobial activity of quaternary ammonium surfactants (QAS) makes them attractive and cheap topical prophylactic options for sexually transmitted infections and perinatal vertically transmitted urogenital infections. Although attributed to their high affinity for biological membranes, the mechanisms behind QAS microbicidal activity are not fully understood. We evaluated how QAS structure affects antimicrobial activity and whether this can be exploited for use in prophylaxis of bacterial infections. METHODS: Acute toxicity of QAS to in vitro models of human epithelial cells and bacteria were compared to identify selective and potent bactericidal agents. Bacterial cell viability, membrane integrity, cell cycle and metabolism were evaluated to establish the mechanisms involved in selective toxicity of QAS. RESULTS: QAS toxicity normalized relative to surfactant critical micelle concentration showed n-dodecylpyridinium bromide (C12PB) to be the most effective, with a therapeutic index of ∼10 for an MDR strain of Escherichia coli and >20 for Neisseria gonorrhoeae after 1 h of exposure. Three modes of QAS antibacterial action were identified: impairment of bacterial energetics and cell division at low concentrations; membrane permeabilization and electron transport inhibition at intermediate doses; and disruption of bacterial membranes and cell lysis at concentrations close to the critical micelle concentration. In contrast, toxicity to mammalian cells occurs at higher concentrations and, as we previously reported, results primarily from mitochondrial dysfunction and apoptotic cell death. CONCLUSIONS: Our data show that short chain (C12) n-alkyl pyridinium bromides have a sufficiently large therapeutic window to be good microbicide candidates.

Chlamydia trachomatis In Vivo to In Vitro Transition Reveals Mechanisms of Phase Variation and Down-Regulation of Virulence Factors.

Research on the obligate intracellular bacterium Chlamydia trachomatis demands culture in cell-lines, but the adaptive process behind the in vivo to in vitro transition is not understood. We assessed the genomic and transcriptomic dynamics underlying C. trachomatis in vitro adaptation of strains representing the three disease groups (ocular, epithelial-genital and lymphogranuloma venereum) propagated in epithelial cells over multiple passages. We found genetic features potentially underlying phase variation mechanisms mediating the regulation of a lipid A biosynthesis enzyme (CT533/LpxC), and the functionality of the cytotoxin (CT166) through an ON/OFF mechanism. We detected inactivating mutations in CT713/porB, a scenario suggesting metabolic adaptation to the available carbon source. CT135 was inactivated in a tropism-specific manner, with CT135-negative clones emerging for all epithelial-genital populations (but not for LGV and ocular populations) and rapidly increasing in frequency (~23% mutants per 10 passages). RNA-sequencing analyses revealed that a deletion event involving CT135 impacted the expression of multiple virulence factors, namely effectors known to play a role in the C. trachomatis host-cell invasion or subversion (e.g., CT456/Tarp, CT694, CT875/TepP and CT868/ChlaDub1). This reflects a scenario of attenuation of C. trachomatis virulence in vitro, which may take place independently or in a cumulative fashion with the also observed down-regulation of plasmid-related virulence factors. This issue may be relevant on behalf of the recent advances in Chlamydia mutagenesis and transformation where culture propagation for selecting mutants/transformants is mandatory. Finally, there was an increase in the growth rate for all strains, reflecting gradual fitness enhancement over time. In general, these data shed light on the adaptive process underlying the C. trachomatis in vivo to in vitro transition, and indicates that it would be prudent to restrict culture propagation to minimal passages and check the status of the CT135 genotype in order to avoid the selection of CT135-negative mutants, likely originating less virulent strains.

Complete Genome Sequence of Chlamydia trachomatis Ocular Serovar C Strain TW-3.

Chlamydia trachomatis is the etiological agent of trachoma, the leading infectious cause of blindness worldwide. We report here the first complete and annotated genome of a C. trachomatis trachoma-causing serovar C strain (strain TW-3). The chromosome and plasmid are 1,043,554 bp and 7,501 bp in length, respectively.

Accuracy of prenatal culture in predicting intrapartum group B streptococcus colonization status.

OBJECTIVE: To evaluate the positive predictive value (PPV) of group B Streptococcus (GBS) cultures at 35-37 weeks of gestation relative to GBS colonization status at delivery. METHODS: Rectovaginal swabs from 221 women at labor in four Lisbon hospitals were collected for GBS screening according to the CDC guidelines. RESULTS: The PPV was 24.4%. IAP was administered to 100% of prenatally GBS positive women. There was no case of early onset GBS disease (EOD). CONCLUSIONS: Poor accuracy of prenatal cultures in identifying true candidates for IAP highlights the need for Portuguese clinical and laboratory guidelines to prevent EOD and antibiotic overtreatment of pregnant women.

Effect of long-term laboratory propagation on Chlamydia trachomatis genome dynamics.

It is assumed that bacterial strains maintained in the laboratory for long time shape their genome in a different fashion from the nature-circulating strains. Here, we analyzed the impact of long-term in vitro propagation on the genome of the obligate intracellular pathogen Chlamydia trachomatis. We fully-sequenced the genome of a historical prototype strain (L2/434/Bu) and a clinical isolate (E/CS88), before and after one-year of serial in vitro passaging (up to 3500 bacterial generations). We observed a slow adaptation of C. trachomatis to the in vitro environment, which was essentially governed by four mutations for L2/434/Bu and solely one mutation for E/CS88, corresponding to estimated mutation rates from 3.84 × 10(-10) to 1.10 × 10(-9) mutations per base pair per generation. In a speculative basis, the mutations likely conferred selective advantage as: (i) mathematical modeling showed that selective advantage is mandatory for frequency increase of a mutated clone; (ii) transversions and non-synonymous mutations were overrepresented; (iii) two non-synonymous mutations affected the genes CTL0084 and CTL0610, encoding a putative transferase and a protein likely implicated in transcription regulation respectively, which are families known to be highly prone to undergone laboratory-derived advantageous mutations in other bacteria; and (iv) the mutation for E/CS88 is located likely in the regulatory region of a virulence gene (CT115/incD) believed to play a role in subverting the host cell machinery. Nevertheless, we found no significant differences in the growth rate, plasmid load, and attachment/entry rate, between strains before and after their long-term laboratory propagation. Of note, from the mixture of clones in E/CS88 initial population, an inactivating mutation in the virulence gene CT135 evolved to 100% prevalence, unequivocally indicating that this gene is superfluous for C. trachomatis survival in vitro. Globally, C. trachomatis revealed a slow in vitro adaptation that only modestly modifies the in vivo-derived genomic evolutionary landscape.

Genomic features beyond Chlamydia trachomatis phenotypes: what do we think we know?

The obligate intracellular pathogen Chlamydia trachomatis is the causative agent of the blinding trachoma and the world's leading cause of bacterial sexually transmitted infections. Despite aggressive antibacterial control measures, C. trachomatis infections have been increasing, constituting a serious public health concern due to its morbidity and socioeconomic burden. Still, very little is known about the molecular basis underlying the phenotypic disparities observed among C. trachomatis serovars in terms of tissue tropism (ocular conjunctiva, epithelial-genitalia and lymph nodes), virulence (disease outcomes) and ecological success. This is in part due to the inexistence of straightforward tools to genetically manipulate Chlamydiae and host cell-free growth systems, hampering the elucidation of the biological role of loci. The recent release of tenths of full-genome C. trachomatis sequences depict a strains clustering scenario reflecting the organ/cell-type that they preferentially infect. However, the high degree of genomic conservation implies that few genetic features are involved in phenotypic dissimilarities. The purpose of this review is to gather the most relevant data dispersed throughout the literature concerning the genotypic evidences that support niche-specific phenotypes. This review focus on chromosomal dynamics phenomena like recombination and point-mutations, essentially involving outer and inclusion membrane proteins, type III secretion effectors, and hypothetical proteins with unknown function. The scrutiny of C. trachomatis loci involved in tissue tropism, pathogenesis and ecological success is crucial for the development of disease-specific prophylaxis.

Directional evolution of Chlamydia trachomatis towards niche-specific adaptation.

On behalf of the host-pathogen "arms race," a cutting-edge approach for elucidating genotype-phenotype relationships relies on the identification of positively selected loci involved in pathoadaptation. We studied the obligate intracellular bacterium Chlamydia trachomatis, for which same-species strains display a nearly identical core and pan genome, while presenting a wide range of tissue tropism and ecological success. We sought to evaluate the evolutionary patterns underlying species separation (divergence) and C. trachomatis serovar radiation (polymorphism) and to establish genotype-phenotype associations. By analyzing 60 Chlamydia strains, we detected traces of Muller's ratchet as a result of speciation and identified positively selected genes and codons hypothetically involved in the infection of different human cell types (e.g., columnar epithelial cells of ocular or genital mucosae and mononuclear phagocytes) and also events likely driving pathogenic and ecological success dissimilarities. In general, these genes code for proteins involved in immune response elicitation, proteolysis, and the subversion of host-cell functions, and also for proteins with unknown function(s). Several genes are potentially involved in more than one adaptive process, suggesting multiple functions or a distinct modus operandi for a specific function, and thus should be considered as crucial research targets. In addition, six of the nine genes encoding the putative antigen/adhesin polymorphic membrane proteins seem to be under positive selection along specific serovars, which sustains an essential biological role of this extra-large paralogue family in chlamydial pathobiology. This study provides insight into how evolutionary inferences illuminate ecological processes such as adaptation to different niches, pathogenicity, or ecological success driven by arms races.

Molecular epidemiology of group B streptococcal meningitis in children beyond the neonatal period from Angola.

Streptococcus agalactiae is a major pathogen of neonates and immunocompromised adults. Prior studies have demonstrated that, beyond the neonatal period, S. agalactiae rarely causes invasive infections in children. However, during 2004-2005, S. agalactiae was the causative agent of 60 meningitis episodes in children aged 3 months to 12 years from Angola. To identify and study the specific causative genetic lineages of S. agalactiae childhood meningitis, which lack characterization to date, we conducted an extensive molecular analysis of the recovered isolates (n = 21). This constitutes what we believe to be the first molecular study of the population structure of invasive S. agalactiae isolates from Africa. A low genetic diversity was observed among the isolates, where the majority belonged to clonal complex (CC) 17 presenting the capsular subtype III-2 (86 % of cases) and marked by the intron group II GBSi1, which has previously been observed to be associated with neonatal hosts. The predominance of single-locus variants of sequence type (ST) 17 suggested the local diversification of this hypervirulent clone, which displayed novel alleles of the fbsB and sip virulence genes. The absence of the scpB-lmb region in two S. agalactiae isolates with the Ia/ST23 genotype is more typical of cattle than human isolates. Globally, these data provide novel information about the enhanced invasiveness of the CC17 genetic lineage in older children and suggest the local diversification of this clone, which may be related to the future emergence of a novel epidemic clone in Angola.

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<10⁻²°), 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.

Genotypes and antimicrobial-resistant phenotypes of Neisseria gonorrhoeae in Portugal (2004-2009).

OBJECTIVES: To determine the antibiotic phenotype and MAST-genotype distribution of Neisseria gonorrhoeae isolates in Portugal between 2004 and 2009, and to evaluate specific associations between MAST-genotypes and sexual orientation, age and antibiotic resistance. METHODS: A total of 236 N gonorrhoeae isolates were typed through N gonorrhoeae multiantigen sequence typing (NG-MAST). The degree of polymorphism and the phylogenetic relatedness among NG-MAST sequence types (STs) were evaluated with MEGA4 software on concatenated sequences of por and tbpb alleles. Etest was used to determine the susceptibility to ceftriaxone, ciprofloxacin, penicillin and spectinomycin. RESULTS: No isolates displayed resistance to spectinomycin and ceftriaxone, whereas 79.1% and 37.4% were resistant to penicillin and ciprofloxacin, respectively. A total of 104 different STs (one per 2.3 isolates) were found; the most common were ST210 (8.1%) and ST225 (7.6%). STs formed two major groups separated by 159.8 (SE 8.9) nucleotide differences, yielding several subgroups, one of them including the worldwide-prevalent ST225. The probability of ciprofloxacin resistance among isolates within this subgroup was 73.5-fold higher than for the remaining isolates. Indeed, for the genetically closest subgroup, which includes the most prevalent ST (ST210), only 8.0% of isolates were resistant to ciprofloxacin. There was a non-homogeneous distribution per year for ST225 (p<0.001), ST210 (p=0.011) and ST2 (p=0.007). CONCLUSIONS: The heterogeneous ST scenario may represent the 'tip of the iceberg', reflecting a high number of undiagnosed and unreported gonorrhoea cases. A laboratory-based national surveillance of N gonorrhoeae infections is necessary to provide a broader spectrum of isolates that will allow the sexual network situation in Portugal to be established.

Evolutionary dynamics of ompA, the gene encoding the Chlamydia trachomatis key antigen.

Chlamydia trachomatis is the trachoma agent and causes most bacterial sexually transmitted infections worldwide. Its major outer membrane protein (MOMP) is a well-known porin and adhesin and is the dominant antigen. So far, investigation of MOMP variability has been focused mainly on molecular epidemiological surveys. In contrast, we aimed to evaluate the impact of the host pressure on this key antigen by analyzing its evolutionary dynamics in 795 isolates from urogenital infections, taking into account the MOMP secondary structure and the sizes/positions of antigenic regions. One-third of the specimens showed a mutational drift from the corresponding genotype, where approximately 42% of the mutations had never been described. Amino acid alterations were sixfold more frequent within B-cell epitopes than in the remaining protein (P = 0.027), and some mutations were also found within or close to T-cell antigenic clusters. Interestingly, the two most ecologically successful genotypes, E and F, showed a mutation rate 60.3-fold lower than that of the other genotypes (P < 10(-8)), suggesting that their efficacy may be the result of a better fitness in dealing with the host immune system rather than of specific virulence factors. Furthermore, the variability exhibited by some genetic variants involved residues that are known to play a critical role during the membrane mechanical movements, contributing to a more stable and flexible porin conformation, which suggests some plasticity to deal with environmental pressure. Globally, these MOMP mutational trends yielded no mosaic structures or important phylogenetic changes, but instead yielded point mutations on specific protein domains, which may enhance pathogen's infectivity, persistence, and transmission.

Chlamydia trachomatis diversity viewed as a tissue-specific coevolutionary arms race.

BACKGROUND: The genomes of pathogens are thought to have evolved under selective pressure provided by the host in a coevolutionary arms race (the 'Red Queen's Hypothesis'). Traditionally, adaptation by pathogens is thought to rely not on whole chromosome dynamics but on gain/loss of specific genes, yielding differential abilities to infect distinct tissues. Thus, it is not known whether distinct host organs differently shape the genome of the same pathogen. We tested this hypothesis using Chlamydia trachomatis as model species, looking at 15 serovars that infect different organs: eyes, genitalia and lymph nodes. RESULTS: We analyzed over 51,000 base pairs from all serovars using various phylogenetic approaches and a non-phylogenetic indel-based algorithm to study the evolution of individual and concatenated loci. This survey comprised about 33% of all single nucleotide polymorphisms in C. trachomatis chromosomes. We present a model in which genome evolution indeed correlates with the cell type (epithelial versus lymph cells) and organ (eyes versus genitalia) that a serovar infects, illustrating an adaptation to physiologically distinct niches, and discarding genetic drift as the dominant evolutionary driving force. We show that radiation of serovars occurred primarily by accumulation of single nucleotide polymorphisms in intergenomic regions, housekeeping genes, and genes encoding hypothetical and cell envelope proteins. Furthermore, serovar evolution also correlates with ecological success, as the two most successful serovars showed a parallel evolution. CONCLUSION: We identified a single nucleotide polymorphism-based tissue-specific arms race for strains in the same species, reflecting global chromosomal dynamics. Studying such tissue-specific arms race scenarios is crucial for understanding pathogen-host interactions during the course of infectious diseases, in order to dissect pathogen biology and develop preventive and therapeutic strategies.

Comparative expression profiling of the Chlamydia trachomatis pmp gene family for clinical and reference strains.

BACKGROUND: Chlamydia trachomatis, an obligate intracellular pathogen, is a leading worldwide cause of ocular and urogenital diseases. Advances have been made in our understanding of the nine-member polymorphic membrane protein (Pmp) gene (pmp) family of C. trachomatis. However, there is only limited information on their biologic role, especially for biological variants (biovar) and clinical strains. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated expression for pmps throughout development for reference strains E/Bour and L2/434, representing different biovars, and for clinical E and L2 strains. Immunoreactivity of patient sera to recombinant (r)Pmps was also determined. All pmps were expressed at two hours. pmpA had the lowest expression but was up-regulated at 12 h for all strains, indicating involvement in reticulate body development. For pmpD, expression peaked at 36 h. Additionally, 57.7% of sera from infected and 0% from uninfected adolescents were reactive to rPmpD (p = 0.001), suggesting a role in immunogenicity. pmpF had the highest expression levels for all clinical strains and L2/434 with differential expression of the pmpFE operon for the same strains. Sera were nonreactive to rPmpF despite immunoreactivity to rMOMP and rPmpD, suggesting that PmpF is not associated with humoral immune responses. pmpFE sequences for clinical strains were identical to those of the respective reference strains. We identified the putative pmpFE promoter, which was, surprisingly, 100% conserved for all strains. Analyses of ribosomal binding sites, RNase E, and hairpin structures suggested complex regulatory mechanism(s) for this >6 Kb operon. CONCLUSIONS/SIGNIFICANCE: The dissimilar expression of the same pmp for different C. trachomatis strains may explain different strain-specific needs and phenotypic distinctions. This is further supported by the differential immunoreactivity to rPmpD and rPmpF of sera from patients infected with different strains. Furthermore, clinical E strains did not correlate with the E reference strain at the gene expression level, reinforcing the need for expansive studies of clinical strains.

Evolution of Chlamydia trachomatis diversity occurs by widespread interstrain recombination involving hotspots.

Chlamydia trachomatis is an obligate intracellular bacterium of major public health significance, infecting over one-tenth of the world's population and causing blindness and infertility in millions. Mounting evidence supports recombination as a key source of genetic diversity among free-living bacteria. Previous research shows that intracellular bacteria such as Chlamydiaceae may also undergo recombination but whether this plays a significant evolutionary role has not been determined. Here, we examine multiple loci dispersed throughout the chromosome to determine the extent and significance of recombination among 19 laboratory reference strains and 10 present-day ocular and urogenital clinical isolates using phylogenetic reconstructions, compatibility matrices, and statistically based recombination programs. Recombination is widespread; all clinical isolates are recombinant at multiple loci with no two belonging to the same clonal lineage. Several reference strains show nonconcordant phylogenies across loci; one strain is unambiguously identified as recombinantly derived from other reference strain lineages. Frequent recombination contrasts with a low level of point substitution; novel substitutions relative to reference strains occur less than one per kilobase. Hotspots for recombination are identified downstream from ompA, which encodes the major outer membrane protein. This widespread recombination, unexpected for an intracellular bacterium, explains why strain-typing using one or two genes, such as ompA, does not correlate with clinical phenotypes. Our results do not point to specific events that are responsible for different pathogenicities but, instead, suggest a new approach to dissect the genetic basis for clinical strain pathology with implications for evolution, host cell adaptation, and emergence of new chlamydial diseases.

Polymorphisms in the nine polymorphic membrane proteins of Chlamydia trachomatis across all serovars: evidence for serovar Da recombination and correlation with tissue tropism.

Chlamydia trachomatis is an intracellular bacterium responsible for ocular, respiratory, and sexually transmitted diseases. The genome contains a nine-member polymorphic membrane protein (Pmp) family unique to members of the order Chlamydiales. Genomic and molecular analyses were performed for the entire pmp gene family for the 18 reference serological variants (serovars) and genovariant Ja to identify specific gene and protein regions that differentiate chlamydial disease groups. The mean genetic distance among all serovars varied from 0.1% for pmpA to 7.0% for pmpF. Lymphogranuloma venereum (LGV) serovars were the most closely related for the pmp genes and were also the most divergent, compared to ocular and non-LGV urogenital disease groups. Phylogenetic reconstructions showed that for six of nine pmp genes (not pmpA, pmpD, or pmpE), the serovars clustered based on tissue tropism. The most globally successful serovars, E and F, clustered distantly from the urogenital group for five pmp genes. These pmp genes may confer a biologic advantage that may facilitate infection and transmission for E and F. Surprisingly, serovar Da clustered with the ocular group from pmpE to pmpI, which are located together in the chromosome, providing statistically significant evidence for intergenomic recombination and acquisition of a genetic composition that could hypothetically expand the host cell range of serovar Da. We also identified distinct domains for pmpE, pmpF, and pmpH where substitutions were concentrated and associated with a specific disease group. Thus, our data suggest a possible structural or functional role that may vary among pmp genes in promoting antigenic polymorphisms and/or diverse adhesions-receptors that may be involved in immune evasion and differential tissue tropism.

Correlating Chlamydia trachomatis infectious load with urogenital ecological success and disease pathogenesis.

The association of infectious burden of Chlamydia trachomatis with patient characteristics and clinical disease may have implications for understanding disease pathogenesis. We examined chlamydial load from 171 urine samples where load was based on copy number of organisms per copy number of eukaryotic cells derived by real-time quantitative PCR. High- (E, F, G) and low-prevalence (Ia, H, J, Ja) genotypes in the population had similar loads, suggesting a similar propensity for replicating in vivo, despite their differential ecological success. Symptomatic and asymptomatic patients also had similar chlamydial loads, indicating that virulence differences are likely not associated with variations in replication. There was a significant difference in genotypes by age for F (<31 years; P = 0.031) and for H where the mean age was lower than for the most prevalent genotype, E (P=0.013). Also, men had a significantly lower load than women when the genotype was F (P=0.042), although there was no significant difference in load between partners. Patients with recurrent chlamydial infections had a significant reduction in load with each subsequent episode regardless of genotype (P=0.007), suggesting that immune defenses do not block chlamydial entry but may impact replication. Additionally, the probability of being infected with J was 7.7-fold higher in patients with prior chlamydial infections (P=0.016), and although the loads were lower when compared with patients without prior infection, the results did not reach statistical significance. These findings suggest that chlamydial burden could be an important marker for recurrence and host immune response, which would facilitate pathogenesis research.

Immunoreactivity and differential developmental expression of known and putative Chlamydia trachomatis membrane proteins for biologically variant serovars representing distinct disease groups.

Chlamydia trachomatis is an intracellular bacterium that causes ocular and urogenital diseases worldwide. Membrane proteins have only been partially characterized, and the discovery of a nine-member polymorphic membrane protein gene family has enhanced interest in defining their function. We previously reported two putative insertion sequence-like elements in pmpC for biovariant Ba and one each for G and L2, suggesting horizontal gene transfer. Because of this and the tissue tropism differences for these biovariants, we analyzed by quantitative real-time RT-PCR pmpC expression relative to immunogenic protein genes ompA, groEL and gseA throughout development. Sera from infected adolescents were reacted by immunoblot against recombinant (r)PmpC and rMOMP. ompA and groEL revealed different developmental transcriptome profiles among the biovariants. pmpC expression occurred at 2 h, peaked at 18 for L2 (at 24 for Ba and G), with the highest mRNA levels throughout development for L2. pmpC expression as a function of time paralleled ompA expression with higher mRNA levels compared with groEL later in development. Only sera from D-, E- and G-infected patients reacted to rPmpC; all infected patients reacted to rMOMP. pmpC expression during logarithmic growth suggests a role in membrane building and/or integrity, which is supported by the presence of a signal peptidase and C-terminal phenylalanine in PmpC. Because phylogenetic analyses of pmpC segregate serovars according to tissue tropism, we speculate that biovariant transcriptome differences may contribute to this tropism. The heterogeneous biovariant pmpC expression throughout development and differential PmpC immunoreactivity also suggest a role for pmpC in antigenic variation.

Recombination in the genome of Chlamydia trachomatis involving the polymorphic membrane protein C gene relative to ompA and evidence for horizontal gene transfer.

Genome sequencing of Chlamydia trachomatis serovar D has identified polymorphic membrane proteins (Pmp) that are a newly recognized protein family unique to the Chlamydiaceae family. Cumulative data suggest that these diverse proteins are expressed on the cell surface and might be immunologically important. We performed phylogenetic analyses and statistical modeling with 18 reference serovars and 1 genovariant of C. trachomatis to examine the evolutionary characteristics and comparative genetics of PmpC and pmpC, the gene that encodes this protein. We also examined 12 recently isolated ocular and urogenital clinical samples, since reference serovars are laboratory adapted and may not represent strains that are presently responsible for human disease. Phylogenetic reconstructions revealed a clear distinction for disease groups, corresponding to levels of tissue specificity and virulence of the organism. Further, the most prevalent serovars, E, F, and Da, formed a distinct clade. According to the results of comparative genetic analyses, these three genital serovars contained two putative insertion sequence (IS)-like elements with 10- and 15-bp direct repeats, respectively, while all other genital serovars contained one IS-like element. Ocular trachoma serovars also contained both insertions. Previously, no IS-like elements have been identified for Chlamydiaceae. Surprisingly, 7 (58%) of 12 clinical isolates revealed pmpC sequences that were identical to the sequences of other serovars, providing clear evidence for a high rate of whole-gene recombination. Recombination and the differential presence of IS-like elements among distinct disease and prevalence groups may contribute to genome plasticity, which may lead to adaptive changes in tissue tropism and pathogenesis over the course of the organism's evolution.