Phylogenetic Analysis of Chlamydia trachomatis Tarp and Correlation with Clinical Phenotype

Chlamydia trachomatis is the leading cause of infectious blindness worldwide and is the most commonly reported pathogen causing sexually transmitted infections. Tarp (translocated actin recruiting phosphoprotein), a type III secreted effector that mediates actin nucleation, is central to C. trachomatis infection. The phylogenetic analysis of tarP from reference strains as well as ocular, genital, and lymphogranuloma venereum (LGV) clinical isolates demonstrated an evolutionary relationship with disease phenotype, with LGV and ocular isolates branched into clades that were separate from the urogenital isolates. The sequence analysis of Tarp indicated a high degree of variability and identified trends within clinical groupings. Tarps from LGV strains contained the highest number of tyrosine-rich repeat regions (up to nine) and the fewest (two) predicted actin binding domains. The converse was noted for Tarp proteins from ocular isolates that contained up to four actin binding domains and as few as one tyrosine-rich repeat region. The results suggest that Tarp is among the few known genes to play a role in C. trachomatis adaptations to specific niches within the host.Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections (STIs) and preventable blindness worldwide (52). The species C. trachomatis consists of more than 15 serologically defined variants, or serovars, associated with different disease states and anatomical sites of infection (31, 52, 69, 70). Despite a high degree of synteny among the genomes of C. trachomatis strains, the different serovars exhibit a remarkable degree of tissue tropism (55). Distinct diseases caused by C. trachomatis include trachoma (serotypes A to C), sexually transmitted diseases (serotypes D to K), and lymphogranuloma venereum (LGV; L1 to L3). Infections by the ocular and urogenital strains, collectively referred to as the trachoma biovar, are restricted to mucosal epithelial cells of the conjunctivae and genital tracts, respectively. The LGV biovar is more invasive and disseminates via the infection of macrophages to regional lymph nodes, where they establish a chronic granulomatis disease (52).The serological typing of C. trachomatis strains is based on the major outer membrane protein (MOMP) (11, 71-73). Despite MOMP being the immunodominant surface antigen, the phylogenetic categorization of MOMP is not concordant with pathobiotypes or tissue tropism (1, 6, 8, 24, 44, 50, 58). Numerous typing techniques have been applied to C. trachomatis to better understand the epidemiology and pathogenesis of disease. In addition to serological typing, the sequencing of ompA (which encodes MOMP) can detect numerous trachoma and serovar genotypes. Restriction fragment length polymorphism (RFLP) (26), pulsed-field gel electrophoresis (PFGE) (48), and multilocus sequence typing (MLST) (39) analysis of ompA have been used to discriminate C. trachomatis strains within serotypes but correlate poorly with disease phenotype.Although the diseases associated with C. trachomatis serovars are unique, the completed genome sequence of three serovars representing ocular, urogenital, and LGV strains exhibit more than 99% identity (14, 56, 66) with a high degree of synteny in gene order and content. The observed differences in tissue tropism and pathobiology of C. trachomatis serovars therefore likely are due to a relatively small number of loci. One 50-kb region of the chlamydial genome, termed the plasticity zone, exhibits a much higher degree of divergence than the remainder of the genome (47). In C. trachomatis serovar D, the plasticity zone encompasses the region between ycfV (CT152) and dbsB (CT177) (47). Within this plasticity zone are at least two loci that have been correlated with tissue tropism and disease. One of these loci, first identified in Chlamydia muridarum (47), includes three copies of a putative cytotoxin with homology to the large clostridial toxins. In C. trachomatis serovar D, only a single, disrupted partial cytotoxin-like gene is expressed (3, 13). This locus is highly polymorphic, in that all urogenital serovars express both the UDP-glucose binding and glucosyltransferase domains of the toxin, while the ocular serovars, with the exception of serovar B, encode only the UDP-glucose binding domain, and the LGV strains have both domains entirely deleted (13). Also within the plasticity zone is the trpAB operon, which encodes tryptophan synthase and a repressor. The trpAB operon distinguishes genital and LGV strains from ocular strains, as only genital and LGV strains produce a functional synthase (12, 22, 53). The phylogenetic analysis of the C. trachomatis polymorphic membrane protein (pmp) genes, which are dispersed throughout the genome, has revealed that six of the nine pmp genes (pmpB, pmpC, pmpF, pmpG, pmpH, and pmpI) from 18 serovars correlate with disease groups (29, 30, 59).C. trachomatis, like many Gram-negative bacterial pathogens, utilizes a type III secretion system to deliver an arsenal of bacterial gene-encoded effector proteins directly into the cytosol of the host cell (4, 16, 17, 23). Tarp (translocated actin recruiting protein [CT456]), a type III secreted effector, directly induces actin polymerization at the site of chlamydial invasion (16, 37). Tarp has been found in all C. trachomatis serovars to date in addition to orthologs present in Chlamydophila pneumoniae, Chlamydophila caviae, and C. muridarum (15). The biochemical and sequence analyses of Tarp have revealed three functionally distinct domains consisting of an N-terminal tyrosine-rich repeat region, a proline-rich domain, and C-terminal Wasp homology 2 (WH2) actin binding domains (15, 37, 38).Overall, there are variations in the number of tyrosine-rich repeats and actin binding domains of Tarp between different C. trachomatis serovars examined to date (15, 16, 37). This suggests that Tarp is adapting to the selective pressures in the host and therefore is a good candidate protein to examine for tissue tropism. Here, we present a phylogenetic analysis of tarP from reference strains as well as ocular, urogenital, and LGV clinical isolates and demonstrate a correlation between tarP and clinical phenotype. We demonstrate that LGV isolates contain the greatest number of tyrosine-rich repeat regions and the fewest actin binding domains, which is in contrast to the ocular isolates that contain up to four predicted actin binding domains and the fewest tyrosine-rich repeats. Taken together, these findings identify Tarp as another C. trachomatis gene that varies in relation to disease and tissue tropism.     

EseG,an Effector of the Type III Secretion System of Edwardsiella tarda,Triggers Microtubule Destabilization

Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. A type III secretion system (T3SS) was recently shown to contribute to pathogenesis, since deletions of various T3SS genes increased the 50% lethal dose (LD₅₀) by about 1 log unit in the blue gourami infection model. In this study, we report EseG as the first identified effector protein of T3SS. EseG shares partial homology with two Salmonella T3SS effectors (SseG and SseF) over a conserved domain (amino acid residues 142 to 192). The secretion of EseG is dependent on a functional T3SS and, in particular, requires the chaperone EscB. Experiments using TEM-1 β-lactamase as a fluorescence-based reporter showed that EseG was translocated into HeLa cells at 35°C. Fractionation of infected HeLa cells demonstrated that EseG was localized to the host membrane fraction after translocation. EseG is able to disassemble microtubule structures when overexpressed in mammalian cells. This phenotype may require a conserved motif of EseG (EseG_142-192), since truncated versions of EseG devoid of this motif lose their ability to cause microtubule destabilization. By demonstrating the function of EseG, our study contributes to the understanding of E. tarda pathogenesis. Moreover, the approach established in this study to identify type III effectors can be used to identify and characterize more type III and possible type VI effectors in Edwardsiella.Edwardsiella tarda is a Gram-negative bacterium associated with septicemia and fatal infections in many animals, including fish and humans (18, 36, 48). In humans, it causes gastrointestinal infections as well as extraintestinal infections such as myonecrosis, bacteremia, septic arthritis, and wound infections (18). Using a comparative proteomics approach, our group reported the presence of two secretion systems, namely, a type III secretion system (T3SS) and a type VI secretion system (T6SS), which are vital for E. tarda pathogenesis (44, 45, 46, 54).T3SSs are contact-dependent translocation systems that have been found in many Gram-negative bacteria of animals, commensals, and plant-symbiotic rhizobia (3, 31). They have many components, such as secretion and translocon apparatuses, effectors, regulators, and chaperones. The protein secretion machinery directs the secretion and translocation of many bacterial effectors into host cells. The concerted action of these effectors stimulates or interferes with host cellular processes, thereby dictating the terms of bacterium-host cell interactions (8). In Salmonella spp., more than 30 effectors are secreted by two different T3SSs. These effectors are involved in several diverse functions, such as forced entry into epithelial cells, intracellular replication inside vacuoles, and suppression of cellular immune responses (16, 29, 50). In Yersinia species, six Yop effectors have been identified, and they have been found to disrupt vital signaling cascades that are required for innate immunity (20, 41). T3SS effectors are generally less conserved than other components of T3SSs, and they perform unique functions adapted to a pathogen''s virulence strategy.The T3SS facilitates the survival of E. tarda in phagocytes and HEp-2 cells (19, 33, 34, 45) and contributes to virulence in vivo (45). The deletion of various single T3SS genes, such as escC, eseB, eseD, or escA, increased the 50% lethal dose (LD₅₀) by approximately 1 log unit in blue gourami (49, 55). EscC has been shown to act as the chaperone for EseB and EseD, whereas EscA is the chaperone for EseC (49, 55). EseB, EseC, and EseD, which are secreted in large quantities by E. tarda, are homologous to Salmonella sp. SseB, SseC, and SseD, respectively (45). The SseB, SseC, and SseD proteins are secreted by the T3SS of Salmonella pathogenicity island 2 (SPI-2) and predominantly assemble into complexes (SseBCD) that function as a translocon for effector proteins (32). EseB, EseC, and EseD can also form a protein complex (EseBCD) after secretion (55), suggesting that the EseBCD complex functions as a translocon component, rather than as T3SS effectors. To our knowledge, no T3SS effectors have been identified in E. tarda yet.The E. tarda T3SS has almost a full set of genes that are homologous to those of SPI-2 (45). A bioinformatics analysis of E. tarda T3SS proteins revealed that EseG bears some similarity to two Salmonella effectors, SseG and SseF. SseG is an effector of SPI-2 that targets intracellular Salmonella to the Golgi network, where the close association of bacterial cells allows Salmonella to multiply efficiently (40). SseF, another SPI-2 effector, is involved in positioning and maintaining the Salmonella-containing vacuoles (SCV) in a juxtanuclear position in infected epithelial cells (1). The translocated Salmonella effector proteins SseF and SseG interact with each other and are required for Salmonella to establish an intracellular replication niche (12).In this study, we used a modified TEM-1 β-lactamase system (TEM) to show that EseG is an E. tarda T3SS effector that is localized in the host membrane fraction after translocation. A functional study showed that EseG interacts with α-tubulin and destabilizes the microtubule through a conserved domain. The ΔeseG mutant showed slightly reduced virulence in blue gourami.     

Characterization of CPAF Critical Residues and Secretion during Chlamydia trachomatis Infection

CPAF (chlamydial protease-like activity factor), a Chlamydia serine protease, is activated via proximity-induced intermolecular dimerization that triggers processing and removal of an inhibitory peptide occupying the CPAF substrate-binding groove. An active CPAF is a homodimer of two identical intramolecular heterodimers, each consisting of 29-kDa N-terminal and 35-kDa C-terminal fragments. However, critical residues for CPAF intermolecular dimerization, catalytic activity, and processing were defined in cell-free systems. Complementation of a CPAF-deficient chlamydial organism with a plasmid-encoded CPAF has enabled us to characterize CPAF during infection. The transformants expressing CPAF mutated at intermolecular dimerization, catalytic, or cleavage residues still produced active CPAF, although at a lower efficiency, indicating that CPAF can tolerate more mutations inside Chlamydia-infected cells than in cell-free systems. Only by simultaneously mutating both intermolecular dimerization and catalytic residues was CPAF activation completely blocked during infection, both indicating the importance of the critical residues identified in the cell-free systems and exploring the limit of CPAF''s tolerance for mutations in the intracellular environment. We further found that active CPAF was always detected in the host cell cytoplasm while nonactive CPAF was restricted to within the chlamydial inclusions, regardless of how the infected cell samples were treated. Thus, CPAF translocation into the host cell cytoplasm correlates with CPAF enzymatic activity and is not altered by sample treatment conditions. These observations have provided new evidence for CPAF activation and translocation, which should encourage continued investigation of CPAF in chlamydial pathogenesis.     

Serological Investigation of Chlamydia trachomatis Heat Shock Protein 10

The humoral immune response to Chlamydia trachomatis 10-kDa heat shock protein (Chsp10) in populations of Russian and French origin was studied by using a recombinant Chsp10 enzyme-linked immunosorbent assay. A physiological but not a serological correlation of Chsp10 exposure with Chsp60 exposure was observed in the Russian population. In the French population studied, there was a significant association between detection of anti-r-Chsp10 immunoglobulin G (IgG) antibodies and chronic genital tract infections. Chsp10 residues 50 to 67 were found to contain an immunodominant although not universal B epitope. Cross-reactions with Chlamydia pneumoniae or Escherichia coli GroES protein are limited but may occur. Our study suggests that detection of anti-Chsp10 IgG antibodies is associated with chronicity of C. trachomatis genital tract infection and does not parallel that of anti-Chsp60 IgG antibodies.     

Expression of the Effector Protein IncD in Chlamydia trachomatis Mediates Recruitment of the Lipid Transfer Protein CERT and the Endoplasmic Reticulum-Resident Protein VAPB to the Inclusion Membrane

Chlamydia trachomatis is an obligate intracellular human pathogen responsible for ocular and genital infections. To establish its membrane-bound intracellular niche, the inclusion, C. trachomatis relies on a set of effector proteins that are injected into the host cells or inserted into the inclusion membrane. We previously proposed that insertion of the C. trachomatis effector protein IncD into the inclusion membrane contributes to the recruitment of the lipid transfer protein CERT to the inclusion. Due to the genetically intractable status of C. trachomatis at that time, this model of IncD-CERT interaction was inferred from ectopic expression of IncD and CERT in the host cell. In the present study, we investigated the impact of conditionally expressing a FLAG-tagged version of IncD in C. trachomatis. This genetic approach allowed us to establish that IncD-3×FLAG localized to the inclusion membrane and caused a massive recruitment of the lipid transfer protein CERT that relied on the PH domain of CERT. In addition, we showed that the massive IncD-dependent association of CERT with the inclusion led to an increased recruitment of the endoplasmic reticulum (ER)-resident protein VAPB, and we determined that, at the inclusion, CERT-VAPB interaction relied on the FFAT domain of CERT. Altogether, the data presented here show that expression of the C. trachomatis effector protein IncD mediates the recruitment of the lipid transfer protein CERT and the ER-resident protein VAPB to the inclusion.     

Induction of type III secretion by cell-free Chlamydia trachomatis elementary bodies

Chlamydiae secrete type III effector proteins at two distinct stages of their developmental cycle. Elementary bodies (EBs) secrete at least one pre-formed effector protein, Tarp, across the host plasma membrane from an extracellular location. Once internalized, a set of newly transcribed proteins are secreted to modify the inclusion membrane. In an effort to better understand the triggers for chlamydial type III secretion and develop means to identify new effectors, we investigated various inducers of T3SS in other Gram-negative bacterial systems to determine if they were able to activate chlamydial type III secretion from EBs using Tarp secretion as an indicator of activation. Chlamydial EBs are induced to secrete Tarp by exposure to FBS, BSA, or sphingolipid and cholesterol-rich liposomes (SCRLs). The induction by FBS and BSA, but not SCRL, is enhanced in the presence of the calcium-chelator, EGTA. This secretion was temperature dependent and inhibited by paraformaldehyde fixation of the EBs.     

Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors,Secretion of Translocators,and Pathogenesis in Fish

The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD₅₀) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens.     

Human Mannose-Binding Protein Inhibits Infection of HeLa Cells by Chlamydia trachomatis

The role that collectin (mannose-binding protein) may play in the host’s defense against chlamydial infection was investigated. Recombinant human mannose-binding protein was used in the inhibition of cell culture infection by Chlamydia trachomatis (C/TW-3/OT, E/UW-5/Cx, and L₂/434/Bu), Chlamydia pneumoniae (AR-39), and Chlamydia psittaci (6BC). Mannose-binding protein (MBP) inhibited infection of all chlamydial strains by at least 50% at 0.098 μg/ml for TW-3 and UW-5, and at 6.25 μg/ml for 434, AR-39, and 6BC. The ability of MBP to inhibit infection with strain L₂ was not affected by supplementation with complement or addition of an L₂-specific neutralizing monoclonal antibody. Enzyme-linked immunosorbent assay and dot blot analyses showed MBP bound to the surface of the organism to exert inhibition, which appeared to block the attachment of radiolabeled organisms to HeLa cells. Immunoblotting and affinity chromatography indicated that MBP binds to the 40-kDa glycoprotein (the major outer membrane protein) on the outer surface of the chlamydial elementary body. Hapten inhibition assays with monosaccharides and defined oligosaccharides showed that the inhibitory effects of MBP were abrogated by mannose or high-mannose type oligomannose-oligosaccharide. The latter carbohydrate is the ligand of the 40-kDa glycoprotein of C. trachomatis L₂, which is known to mediate attachment, suggesting that the MBP binds to high mannose moieties on the surface of chlamydial organisms. These results suggest that MBP plays a role in first-line host defense against chlamydial infection in humans.     

Conserved type III secretion system exerts important roles in Chlamydia trachomatis

Upon infection, Chlamydiae alter host cellular functions in a variety of ways. Chlamydial infection prevents host cell apoptosis, induces re-organization of the actin cytoskeleton and alters host cellular signaling mechanisms. Chlamydia is among the many pathogenic Gram-negative bacteria that employ the type III secretion system (T3SS) to overcome host defenses and exploit available resources. T3SS are used by many Gram-negative bacterial pathogens to manipulate eukaryotic host cells through the delivery of effector proteins into their cytosol and membranes. T3SS is an evolutionarily refined, virulence determinant of Gram-negative bacteria where more than 20 proteins form an apparatus, generally termed injectisome, to achieve the vectorial secretion and translocation of anti-host effector proteins. This review describes challenges and recent advances that have revealed how Chlamydia trachomatis utilizes diversification to produce a conserved T3SS that exerts an important role in Chlamydia trachomatis.     

Chlamydia trachomatis Polymorphic Membrane Protein D Is an Oligomeric Autotransporter with a Higher-Order Structure

Chlamydia trachomatis is a globally important obligate intracellular bacterial pathogen that is a leading cause of sexually transmitted disease and blinding trachoma. Effective control of these diseases will likely require a preventative vaccine. C. trachomatis polymorphic membrane protein D (PmpD) is an attractive vaccine candidate as it is conserved among C. trachomatis strains and is a target of broadly cross-reactive neutralizing antibodies. We show here that immunoaffinity-purified native PmpD exists as an oligomer with a distinct 23-nm flower-like structure. Two-dimensional blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the oligomers were composed of full-length PmpD (p155) and two proteolytically processed fragments, the p73 passenger domain (PD) and the p82 translocator domain. We also show that PmpD undergoes an infection-dependent proteolytic processing step late in the growth cycle that yields a soluble extended PD (p111) that was processed into a p73 PD and a novel p30 fragment. Interestingly, soluble PmpD peptides possess putative eukaryote-interacting functional motifs, implying potential secondary functions within or distal to infected cells. Collectively, our findings show that PmpD exists as two distinct forms, a surface-associated oligomer exhibiting a higher-order flower-like structure and a soluble form restricted to infected cells. We hypothesize that PmpD is a multifunctional virulence factor important in chlamydial pathogenesis and could represent novel vaccine or drug targets for the control of human chlamydial infections.Chlamydia trachomatis is a mucosotropic obligate intracellular gram-negative pathogen that is a leading cause of sexually transmitted and ocular infections. Infection can result in serious sequelae such as infertility and blindness (54, 56) and an increased risk of human immunodeficiency virus infection and transmission (38). The pathophysiology of chlamydial infection is associated with the pathogen''s propensities to cause persistent infection and to suppress host immunity (3). A vaccine is needed to control chlamydial diseases; however, progress toward this goal will not be forthcoming until more is known about the virulence factors that mediate persistence and immune evasion.Chlamydiae are characterized by a unique biphasic developmental cycle that modulates between an extracellular, metabolically inactive, infectious elementary body (EB) and an intracellular, metabolically active, noninfectious reticulate body (RB) (34). Their obligate intracellular niche and the lack of a tractable genetic system present unique challenges in the study of chlamydial biology and pathogenesis. To overcome these hurdles, chlamydial genomes from a diverse spectrum of host-specific strains have been sequenced. Comparative genomics have shown considerable homology among various chlamydial species and have provided important insights into shared and species-specific virulence factors (7, 24, 41, 42, 46, 49).The type V or autotransporter (AT) secretion pathway is the most widespread secretion mechanism employed by gram-negative bacteria to deliver virulence factors involved in initiating infection, disease progression, and immune evasion (reviewed in references 11 and 21). AT proteins are characterized by three domains, (i) a signal sequence (SS), (ii) a diverse N-terminal passenger domain (PD) that confers effector function, and (iii) a conserved C-terminal translocator domain (TD). The TD inserts into the outer membrane (OM) by assembling into a β-barrel pore that facilitates PD translocation to the bacterial surface. The PD remains tethered to the TD or is cleaved and either is released or remains noncovalently associated with the OM. Well-characterized examples of ATs found on the bacterial cell surface as monomers or oligomers are Neisseria meningitidis NalP (37) and Helicobacter pylori VacA (31), respectively.C. trachomatis has a nine-member AT family (20), termed polymorphic membrane proteins (Pmps), whose role(s) in chlamydial pathogenesis has yet to be defined. The pmp paralogs (pmpA to pmpI) constitute 3.2% of the ∼1-Mb genome and are found at three chromosomal loci composed of two gene clusters (pmpA to pmpC and pmpE to pmpI) and the genetically isolated gene pmpD (46). Notably, PmpD is the second most highly conserved Pmp, exhibiting 99.2% amino acid identity among C. trachomatis serovars (16). Despite relatively low abundance in the chlamydial OM, Pmps are major immunogens and may be important virulence factors (29). C. trachomatis PmpD is a target of broadly cross-reactive neutralizing antibodies (Abs), which makes it an attractive vaccine candidate for the prevention of human infections (10).Previous reports have described proteolytic processing of C. pneumoniae and C. trachomatis PmpD (25, 52). Furthermore, recombinant C. pneumoniae PmpD has been suggested to function as an adhesin capable of inducing proinflammatory cytokine production (35, 52). Nothing is known about the native structure of C. trachomatis PmpD or the potential significance of its structure to chlamydial pathogenesis. Here we show that C. trachomatis PmpD is present on the organism''s surface as an oligomer with a higher-order flower-like structure. Moreover, we describe novel infection-dependent proteolytic processing of PmpD that produces soluble fragments with predicted eukaryotic motifs, implying a multifunctional protein important to chlamydial pathogenesis.     

TestPack Chlamydia, a new rapid assay for the direct detection of Chlamydia trachomatis.

TestPack Chlamydia (Abbott Laboratories) is a rapid enzyme immunoassay for the direct antigen detection of Chlamydia trachomatis in endocervical specimens. The assay is self-contained, requires no specialized equipment, and yields results in less than 30 min. The clinical performance of TestPack Chlamydia versus chlamydial cell culture was evaluated with a total of 1,694 paired endocervical specimens. Discordant samples were further investigated by immunofluorescent staining and by Chlamydiazyme immunoassay, with confirmatory procedures. The sensitivity of TestPack Chlamydia with less-than-48-h-old specimens was 76.5%, while culture sensitivity was 86.7%. TestPack Chlamydia specificity was determined to be 99.5%. These results indicate that TestPack Chlamydia is an accurate test for chlamydial infection, with a positive predictive value of 96.2%. This assay is suitable for low-volume chlamydial testing in physician offices, clinics, and smaller laboratories.     

Chlamydia trachomatis infections: a clinical update

Chlamydia trachomatis has emerged as the most common sexually transmitted pathogen in the United States and in many other countries. Because of its increasing importance, the authors present an update on chlamydial infections.     

The immunology of Chlamydia trachomatis

Chlamydia trachomatis (C. trachomatis) is one of the most common sexually transmitted bacterial agents. What distinguishes it from other organisms is its intracellular reproductive cycle. Up to now, four antigens have been identified in the Chlamydia genus: genus-specific antigen as well as species-specific, type-specific and subspecies-specific. C. trachomatis is a powerful immunogen which stimulates the host's immunological processes. The intracellular parasitism of the bacteria is the basis for both symptomatic or asymptomatic infection as well as for chronic ones. The primary infection leads to a local inflammatory reaction due to penetration and reproduction of the bacteria in the epithelial cells and to IgA secretory antibody production. In most cases the host's reaction to the primary infection is transient and does not cause tissue damage. In the course of chronic infection or reinfection, the most important processes are those of delayed hypersensitivity, which lead to a fast and intense immunological reaction of specifically sensitized Th1 lymphocytes. This reaction leads to progressive damage of the epithelial cells and to cicatrization and fibrosis, which means irreversible complications. Interferon gamma is of special importance in the process of C. trachomatis infection. High concentrations of it inhibit the bacteria's reproductive cycle, while lower concentrations promote the development of atypical, non-contagious forms of Chlamydia of diminished metabolic activity and altered antigenicity. The chlamydial heat shock proteins are considered to be of great importance lately. Their molecular weights of 60 and 10 kDa are a powerful stimulant of immunological reactions and show significant homology (40-90%) to human and other bacterial heat shock proteins.     

Evaluation of a novel solid-phase immunoassay, Clearview Chlamydia, for the rapid detection of Chlamydia trachomatis.

Clearview Chlamydia (Unipath Limited, Bedford, United Kingdom) is a rapid immunoassay for the direct detection of Chlamydia trachomatis antigen. This assay was evaluated against the tissue culture method by using 376 paired endocervical specimens. The Clearview assay had a sensitivity of 93.5% and a specificity of 99% when it was compared with the tissue culture method. This assay does not require specialized equipment or trained personnel and yields results within 30 min from the time that a specimen is collected.     

Global Multilocus Sequence Type Analysis of Chlamydia trachomatis Strains from 16 Countries

The Uppsala University Chlamydia trachomatis multilocus sequence type (MLST) database (http://mlstdb.bmc.uu.se) is based on five target regions (non-housekeeping genes) and the ompA gene. Each target has various numbers of alleles—hctB, 89; CT058, 51; CT144, 30; CT172, 38; and pbpB, 35—derived from 13 studies. Our aims were to perform an overall analysis of all C. trachomatis MLST sequence types (STs) in the database, examine STs with global spread, and evaluate the phylogenetic capability by using the five targets. A total of 415 STs were recognized from 2,089 specimens. The addition of 49 ompA gene variants created 459 profiles. ST variation and their geographical distribution were characterized using eBURST and minimum spanning tree analyses. There were 609 samples from men having sex with men (MSM), with 4 predominating STs detected in this group, comprising 63% of MSM cases. Four other STs predominated among 1,383 heterosexual cases comprising, 31% of this group. The diversity index in ocular trachoma cases was significantly lower than in sexually transmitted chlamydia infections. Predominating STs were identified in 12 available C. trachomatis whole genomes which were compared to 22 C. trachomatis full genomes without predominating STs. No specific gene in the 12 genomes with predominating STs could be linked to successful spread of certain STs. Phylogenetic analysis showed that MLST targets provide a tree similar to trees based on whole-genome analysis. The presented MLST scheme identified C. trachomatis strains with global spread. It provides a tool for epidemiological investigations and is useful for phylogenetic analyses.