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Chlamydia muridarum actively grows in murine mucosae and is a representative model of human chlamydial genital tract disease. In contrast, C. trachomatis infections in mice are limited and rarely cause disease. The factors that contribute to these differences in host adaptation and specificity remain elusive. Overall genomic similarity leads to challenges in the understanding of these significant differences in tropism. A region of major genetic divergence termed the plasticity zone (PZ) has been hypothesized to contribute to the host specificity. To evaluate this hypothesis, lateral gene transfer was used to generate multiple hetero-genomic strains that are predominately C. trachomatis but have replaced regions of the PZ with those from C. muridarum. In vitro analysis of these chimeras revealed C. trachomatis-like growth as well as poor mouse infection capabilities. Growth-independent cytotoxicity phenotypes have been ascribed to three large putative cytotoxins (LCT) encoded in the C. muridarum PZ. However, analysis of PZ chimeras supported that gene products other than the LCTs are responsible for cytopathic and cytotoxic phenotypes. Growth analysis of associated chimeras also led to the discovery of an inclusion protein, CTL0402 (CT147), and homolog TC0424, which was critical for the integrity of the inclusion and preventing apoptosis.

The factors that control the growth and infectious processes for Chlamydia are still poorly understood. This study used recently developed genetic tools to determine the regulon for one of the key transcription factors encoded by Chlamydia, sigma 54. Surrogate and computational analyses provide additional support for the hypothesis that sigma 54 plays a key role in controlling the expression of many components critical to converting and enabling the infectious capability of Chlamydia. These components include those that remodel the membrane for the extracellular environment and incorporation of an arsenal of type III secretion effectors in preparation for infecting new cells., Chlamydia bacteria are obligate intracellular organisms with a phylum-defining biphasic developmental cycle that is intrinsically linked to its ability to cause disease. The progression of the chlamydial developmental cycle is regulated by the temporal expression of genes predominantly controlled by RNA polymerase sigma (σ) factors. Sigma 54 (σ54) is one of three sigma factors encoded by Chlamydia for which the role and regulon are unknown. CtcC is part of a two-component signal transduction system that is requisite for σ54 transcriptional activation. CtcC activation of σ54 requires phosphorylation, which relieves inhibition by the CtcC regulatory domain and enables ATP hydrolysis by the ATPase domain. Prior studies with CtcC homologs in other organisms have shown that expression of the ATPase domain alone can activate σ54 transcription. Biochemical analysis of CtcC ATPase domain supported the idea of ATP hydrolysis occurring in the absence of the regulatory domain, as well as the presence of an active-site residue essential for ATPase activity (E242). Using recently developed genetic approaches in Chlamydia to induce expression of the CtcC ATPase domain, a transcriptional profile was determined that is expected to reflect the σ54 regulon. Computational evaluation revealed that the majority of the differentially expressed genes were preceded by highly conserved σ54 promoter elements. Reporter gene analyses using these putative σ54 promoters reinforced the accuracy of the model of the proposed regulon. Investigation of the gene products included in this regulon supports the idea that σ54 controls expression of genes that are critical for conversion of Chlamydia from replicative reticulate bodies into infectious elementary bodies.

Chlamydia trachomatis infections have an immense impact on public health; however, understanding the basic biology and pathogenesis of this organism has been stalled by the limited repertoire of genetic tools. This report describes the successful adaptation of an important tool that has been lacking in Chlamydia studies: transposon mutagenesis. This advance enabled the generation of 105 insertional mutants, demonstrating that numerous gene products are not essential for in vitro growth. Mammalian infections using these mutants revealed that several gene products are important for infections in vivo. Moreover, this tool enabled the investigation and discovery of a gene critical for lateral gene transfer; a process fundamental to the evolution of bacteria and likely for Chlamydia as well. The development of transposon mutagenesis for Chlamydia has broad impact for the field and for the discovery of genes associated with selected phenotypes, providing an additional avenue for the discovery of molecular mechanisms used for pathogenesis and for a more thorough understanding of this important pathogen., Transposon mutagenesis is a widely applied and powerful genetic tool for the discovery of genes associated with selected phenotypes. Chlamydia trachomatis is a clinically significant, obligate intracellular bacterium for which many conventional genetic tools and capabilities have been developed only recently. This report describes the successful development and application of a Himar transposon mutagenesis system for generating single-insertion mutant clones of C. trachomatis. This system was used to generate a pool of 105 transposon mutant clones that included insertions in genes encoding flavin adenine dinucleotide (FAD)-dependent monooxygenase (C. trachomatis 148 [ct148]), deubiquitinase (ct868), and competence-associated (ct339) proteins. A subset of Tn mutant clones was evaluated for growth differences under cell culture conditions, revealing that most phenocopied the parental strain; however, some strains displayed subtle and yet significant differences in infectious progeny production and inclusion sizes. Bacterial burden studies in mice also supported the idea that a FAD-dependent monooxygenase (ct148) and a deubiquitinase (ct868) were important for these infections. The ct339 gene encodes a hypothetical protein with limited sequence similarity to the DNA-uptake protein ComEC. A transposon insertion in ct339 rendered the mutant incapable of DNA acquisition during recombination experiments. This observation, along with in situ structural analysis, supports the idea that this protein is playing a role in the fundamental process of lateral gene transfer similar to that of ComEC. In all, the development of the Himar transposon system for Chlamydia provides an effective genetic tool for further discovery of genes that are important for basic biology and pathogenesis aspects.

Functional genetic analysis of Chlamydia has been a challenge due to the historical genetic intractability of Chlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for Chlamydia muridarum, a mouse-adapted Chlamydia species that is widely used in Chlamydia infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing C. muridarum transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions in glgB, pmpI, pmpA, and pmpD were investigated further for in vitro and in vivo phenotypes, including growth, inclusion morphology, and attachment to host cells. The glgB mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 pmp mutants, pmpI was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum. The generation of a complete library of C. muridarum mutants will ultimately enable comprehensive identification of the functional genetic requirements for Chlamydia infection in vivo. IMPORTANCE Historical issues with genetic manipulation of Chlamydia have prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adapted Chlamydia species that is widely used for in vivo investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis. We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized in vitro phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.

Numerous Chlamydia trachomatis proteins have been identified as potential subunit vaccines, of which the major outer-membrane protein (MOMP) has, so far, proven the most efficacious. Recently, subunit A of the V-type ATP synthase (ATPase; TC0582) complex was shown to elicit partial protection against infection. Computational modeling of a neighboring gene revealed a novel subunit of the V-type ATPase (TC0583). To determine if this newly identified subunit could induce protection and/or enhance the partial protection provided by subunit A alone, challenge studies were performed using a combination of these recombinant proteins. The TC0583 subunit alone and concurrently with TC0582, was used to vaccinate BALB/c mice utilizing CpG-1826 and Montanide ISA 720 VG as adjuvants. Vaccinated animals were challenged intranasally with Chlamydia muridarum and the course of the infection was followed. Mice immunized with individual antigens showed minimal alleviation of body weight reduction; however, mice immunized with TC0583 and TC0582 in combination, displayed weight loss levels close to those observed with MOMP. Importantly, immunization with a combination of recombinant subunit proteins reduced chlamydial inclusion forming units by approximately a log-fold. These protection levels support that, these highly conserved Chlamydia proteins, in combination with other antigens, may serve as potential vaccine candidates.

A major limitation in the identification of novel antichlamydial compounds is the paucity of effective methods for large-scale compound screening. The immunofluorescence assay is the preferred approach for accurate quantification of the intracellular growth of Chlamydia . In this study, an immunofluorescence image-based method (termed image-based automated chlamydial identification and enumeration [iBAChIE]) was customized for fully automated quantification of Chlamydia infection using the freely available open-source image analysis software program CellProfiler and the complementary data exploration software program CellProfiler Analyst. The method yielded enumeration of different species and strains of Chlamydia highly comparably to the conventional manual methods while drastically reducing the analysis time. The inhibitory capability of established antichlamydial activity was also evaluated. Overall, these data support that iBAChIE is a highly effective tool for automated quantification of Chlamydia infection and assessment of antichlamydial activities of molecules. Furthermore, iBAChIE is expected to be amenable to high-throughput screening studies for inhibitory compounds and fluorescently labeled molecules to study host-pathogen interactions.

Serum resistance, an important virulence determinant of Borrelia burgdorferi sensu lato strains belonging to the Borrelia afzelii and B. burgdorferi sensu stricto genotypes, is related to binding of the complement inhibitor factor H to the spirochete surface protein outer surface protein E (OspE) and its homologues. In this study, we show that the C-terminal short consensus repeats 18–20 of both human and mouse factor H bind to OspE. Analogously, factor H-related protein 1, a distinct plasma protein with three short consensus repeat domains homologous to those in factor H, bound to OspE. Deleting 15-aa residues (region V) from the C terminus of the OspE paralog P21 (a 20.7-kDa OspE-paralogous surface lipoprotein in the B. burgdorferi sensu stricto 297 strain) abolished factor H binding. However, C-terminal peptides from OspE, P21, or OspEF-related protein P alone and the C-terminal deletion mutants of P21 inhibited factor H binding to OspE only partially when compared with full-length P21 or its N-terminal mutant. Alanine substitution of amino acids in peptides from the key binding regions of the OspE family indicated that several lysine residues are required for factor H binding. Thus, the borrelial OspE family proteins bind the C inhibitor factor H via multiple sites in a lysine-dependent manner. The C-terminal site V (Ala151-Lys166) is necessary, but not sufficient, for factor H binding in both rodents and humans. Identification of the necessary binding sites forms a basis for the development of vaccines that block the factor H-OspE interaction and thereby promote the killing of Borreliae.

Borrelia burgdorferi spirochetes can circumvent the vertebrate host’s immune system for long periods of time. B. burgdorferi sensu stricto and B. afzelii, but not B. garinii, bind the complement inhibitor factor H to protect themselves against complement-mediated opsonophagocytosis and killing. We found that factor H binding and complement resistance are due to inducible expression of a wide repertoire of outer surface protein E (OspE) lipoproteins variably called OspE, p21, ErpA, and ErpP. Individual Borrelia strains carry multiple plasmid-encoded OspE paralogs. Together the OspE homologs were found to constitute an array of proteins that bind factor H via multiple C-terminal domains that are exposed outwards from the Borrelial surface. Charged residue substitutions in the key binding regions account for variations between OspE family members in the optimal binding pH, temperature, and ionic strength. This may help the spirochetes to adapt into various host environments. Our finding that multiple plasmid-encoded OspE proteins act as virulence factors of Borrelia can provide new tools for the prevention and treatment of borreliosis.

Vaginally delivered microbicides are being developed to offer women self-initiated protection against transmission of sexually transmitted infections such as Chlamydia trachomatis . A small molecule, DS-96, rationally designed for high affinity to Escherichia coli lipid A, was previously demonstrated to bind and neutralize lipopolysaccharide (LPS) from a wide variety of Gram-negative bacteria (D. Sil et al., Antimicrob. Agents Chemother. 51: 2811–2819, 2007, doi:10.1128/AAC.00200-07). Aside from the lack of the repeating O antigen, chlamydial lipooligosaccharide (LOS) shares general molecular architecture features with E. coli LPS. Importantly, the portion of lipid A where the interaction with DS-96 is expected to take place is well conserved between the two organisms, leading to the hypothesis that DS-96 inhibits Chlamydia infection by binding to LOS and compromising the function. In this study, antichlamydial activity of DS-96 was examined in cell culture. DS-96 inhibited the intercellular growth of Chlamydia in a dose-dependent manner and offered a high level of inhibition at a relatively low concentration (8 μM). The data also revealed that infectious elementary bodies (EBs) were predominantly blocked at the attachment step, as indicated by the reduced number of EBs associated with the host cell surface following pretreatment. Of those EBs that were capable of attachment, the vast majority was unable to gain entry into the host cell. Inhibition of EB attachment and entry by DS-96 suggests that Chlamydia LOS is critical to these processes during the developmental cycle. Importantly, given the low association of host toxicity previously reported by Sil et al., DS-96 is expected to perform well in animal studies as an active antichlamydial compound in a vaginal microbicide.

In previous studies we have characterized the cp32/18 loci in Borrelia burgdorferi 297 which encode OspE and OspF orthologs and a third group of lipoproteins which possess OspE/F-like leader peptides (Elps). To further these studies, we have comprehensively analyzed their patterns of expression throughout the borrelial enzootic cycle. Serial dilution reverse transcription-PCR analysis indicated that although a shift in temperature from 23 to 37°C induced transcription for all nine genes analyzed, this effect was often markedly enhanced in mammalian host-adapted organisms cultivated within dialysis membrane chambers (DMCs) implanted within the peritoneal cavities of rats. Indirect immunofluorescence assays performed on temperature-shifted, in vitro-cultivated spirochetes and organisms in the midguts of unfed and fed ticks revealed distinct expression profiles for many of the OspE-related, OspF-related, and Elp proteins. Other than BbK2.10 and ElpA1, all were expressed by temperature-shifted organisms, while only OspE, ElpB1, OspF, and BbK2.11 were expressed in the midguts of fed ticks. Additionally, although mRNA was detected for all nine lipoprotein-encoding genes, two of these proteins (BbK2.10 and ElpA1) were not expressed by spirochetes cultivated in vitro, within DMCs, or by spirochetes within tick midguts. However, the observation that B. burgdorferi -infected mice generated specific antibodies against BbK2.10 and ElpA1 indicated that these antigens are expressed only in the mammalian host and that a form of posttranscriptional regulation is involved. Analysis of the upstream regions of these genes revealed several differences between their promoter regions, the majority of which were found in the −10 and −35 hexamers and the spacer regions between them. Also, rather than undergoing simultaneous upregulation during tick feeding, these genes and the corresponding lipoproteins appear to be subject to progressive recruitment or enhancement of expression as B. burgdorferi is transmitted from its tick vector to the mammalian host. These findings underscore the potential relevance of these molecules to the pathogenic events of early Lyme disease.

The variable (V) region of an antibody or immunoglobulin (Ig) molecule contains within its structure the antigen-binding site. The contact site on the antigen that binds to the Ig V region is the epitope. Epitopes associated with an antigen make contact with the V region at a site referred to as the paratope. In addition, the V region of the antibody molecule contains immunogenic sites that are termed idiotopes. A collection of different idiotopes contributes to the idiotype (Id) of the antibody molecule. The Id of an antibody molecule is the antigenic determinant(s) associated with the V region. Other antigenic determinants expressed by Ig molecules include isotypes and allotypes. Isotypic determinants are common to all members of a given species. The isotype is responsible for the Ig class and subclass differences that are observed. Allotypes are intraspecies polymorphisms expressed on Ig molecules and are inherited as codominant alleles in classical Mendelian fashion. Allotypic determinants are comparable with the allelic polymorphisms found on red blood cells that constitute the blood group antigens. An allotype is to an Ig molecule as ABO blood group antigens are to red blood cells. The discovery of Id can be directly traced to investigators who were involved in the search for new Ig allotype markers. The term Id was originally proposed by Oudin 35 to designate the antigenic specificity recognized by a series of antiantibodies as being unique or peculiar and not representing a rare form of allotype. Its basis is from the Greek roots idios (individual) and typos (form). Originally, an Id was designated as an antigenic determinant uniquely expressed on an antibody molecule. The first reports on the antigenic individuality of Ig molecules were based on studies with human myeloma proteins. 28,43 This concept was later extended to a series of induced antibodies in humans 25 and in rabbits. 36 In these early studies, Ids were found to be unique or private to those particular antibody molecules expressing a given specificity in a single individual. These unique or private Ids have been referred to as IdI. Shared Ids, however, have also been observed in a variety of species with antibodies generated to numerous antigens. These Ids have been called shared, public, cross-reactive, or IdX determinants. A genetic distinction exists between shared and private Ids. A shared Id represents a V region phenotypic marker and tends to be inherited, whereas private Ids are not. Shared Ids that are expressed in one inbred strain of mouse and absent in a second strain (intraspecies) were useful in demonstrating V region linkage to Ig constant region genes. 48 Shared Ids can also be expressed on antibodies to a given antigen in different species (interspecies). The detection of an interspecies Id is one of the defining serologic characteristics used in the identification of an internal image Id-based strategy that may have vaccine potential (Fig. 1). This article focuses on the use of Ig V regions, including Id, anti-Id, anticlonotypes, and Id engineering as putative vaccines and vaccine strategies for infectious diseases. We restrict our discussion to Id systems involving antigenic determinants associated with potentially pathogenic organisms.

VirF is an AraC family transcriptional activator that is required for the expression of virulence genes associated with invasion and cell-to-cell spread by Shigella flexneri , including multiple components of the type three secretion system (T3SS) machinery and effectors. We tested a small-molecule compound, SE-1 (formerly designated OSSL_051168), which we had identified as an effective inhibitor of the AraC family proteins RhaS and RhaR, for its ability to inhibit VirF. Cell-based reporter gene assays with Escherichia coli and Shigella , as well as in vitro DNA binding assays with purified VirF, demonstrated that SE-1 inhibited DNA binding and transcription activation (likely by blocking DNA binding) by VirF. Analysis of mRNA levels using real-time quantitative reverse transcription-PCR (qRT-PCR) further demonstrated that SE-1 reduced the expression of the VirF-dependent virulence genes icsA , virB , icsB , and ipaB in Shigella . We also performed eukaryotic cell invasion assays and found that SE-1 reduced invasion by Shigella . The effect of SE-1 on invasion required preincubation of Shigella with SE-1, in agreement with the hypothesis that SE-1 inhibited the expression of VirF-activated genes required for the formation of the T3SS apparatus and invasion. We found that the same concentrations of SE-1 had no detectable effects on the growth or metabolism of the bacterial cells or the eukaryotic host cells, respectively, indicating that the inhibition of invasion was not due to general toxicity. Overall, SE-1 appears to inhibit transcription activation by VirF, exhibits selectivity toward AraC family proteins, and has the potential to be developed into a novel antibacterial agent.

Chlamydia trachomatis is maintained through a complex bi-phasic developmental cycle that incorporates numerous processes that are poorly understood. This is reflective of the previous paucity of genetic tools available. The recent advent of a method for transforming Chlamydia has enabled the development of essential molecular tools to better study these medically important bacteria. Critical for the study of Chlamydia biology and pathogenesis, is a system for tightly controlled inducible gene expression. To accomplish this, a new shuttle vector was generated with gene expression controlled by the Tetracycline repressor and anhydryotetracycline. Evaluation of GFP expression by this system demonstrated tightly controlled gene regulation with rapid protein expression upon induction and restoration of transcription repression following inducer removal. Additionally, induction of expression could be detected relatively early during the developmental cycle and concomitant with conversion into the metabolically active form of Chlamydia. Uniform and strong GFP induction was observed during middle stages of the developmental cycle. Interestingly, variable induced GFP expression by individual organisms within shared inclusions during later stages of development suggesting metabolic diversity is affecting induction and/or expression. These observations support the strong potential of this molecular tool to enable numerous experimental analyses for a better understanding of the biology and pathogenesis of Chlamydia.

Chlamydia pneumoniae is a community-acquired respiratory pathogen that has been associated with the development of atherosclerosis. Analysis of the C. pneumoniae genome identified a gene (Cpn1046) homologous to eukaryotic aromatic amino acid hydroxylases. Aromatic amino acid hydroxylases (AroAA-H) hydroxylate phenylalanine, tyrosine, and tryptophan into tyrosine, dihydroxyphenylalanine (L-DOPA), and 5-hydroxytryptophan, respectively. Sequence analysis of Cpn1046 demonstrated that residues essential for AroAA-H enzymatic function are conserved and that a subset of Chlamydia species contain an AroAA-H homolog. The chlamydial AroAA-H are transcriptionally linked to a putative bacterial membrane transport protein. We determined that recombinant Cpn1046 is able to hydroxylate phenylalanine, tyrosine, and tryptophan with roughly equivalent activity for all three substrates. Cpn1046 is expressed within 24 h of infection, allowing C. pneumoniae to hydroxylae host stores of aromatic amino acids during the period of logarithmic bacterial growth. From these results we can conclude that C. pneumoniae, as well as a subset of other Chlamydia species, encode an AroAA-H that is able to use all three aromatic amino acids as substrates. The maintenance of this gene within a number of Chlamydia suggests that the enzyme may have an important role in shaping the metabolism or overall pathogenesis of these bacteria.

Presently, the rhesus macaque is the only nonhuman primate animal model utilized for the study of Lyme disease. While this animal model closely mimics human disease, rhesus macaques can harbor the herpes B virus, which is often lethal to humans; macaques also do not express the full complement of immunoglobulin G (IgG) subclasses found in humans. Conversely, baboons contain the full complement of IgG subclasses and do not harbor the herpes B virus. For these reasons, baboons have been increasingly utilized as the basis for models of infectious diseases and studies assessing the safety and immunogenicity of new vaccines. Here we analyzed the capability of baboons to become infected withBorrelia burgdorferi, the agent of Lyme disease. Combined culture and PCR analyses of tick- and syringe-infected animals indicated that baboons are a sufficient host forB. burgdorferi. Analysis of the antibody responses in infected baboons over a 48-week period revealed that antibodies are generated early during infection against many borrelial antigens, including the various OspE, OspF, and Elp paralogs that are encoded on the ubiquitous 32-kb circular plasmids (cp32s). By using the baboon sera generated by experimental infection it was determined that a combination of two cp32-encoded lipoproteins, OspE and ElpB1, resulted in highly specific and sensitive detection ofB. burgdorferiinfection. An expanded analysis, which included 39 different human Lyme disease patients, revealed that a combination of the OspE and ElpB1 lipoproteins could be the basis for a new serodiagnostic assay for Lyme disease. Importantly, this novel serodiagnostic test would be useful independent of prior OspA vaccination status.

Borrelia burgdorferi differentially expresses many of the OspE/F/Elp paralogs during tick feeding. These findings, combined with the recent report that stable B. burgdorferi infection of mammals occurs only after 53 h of tick attachment, prompted us to further analyze the expression of the OspE/F/Elp paralogs during this critical period of transmission. Indirect immunofluorescence analysis revealed that OspE, p21, ElpB1, ElpB2, and OspF/BbK2.11 are expressed in the salivary glands of ticks allowed to feed on mice for 53 to 58 h. Interestingly, many of the spirochetes in the salivary glands that expressed abundant amounts of these antigens were negative for OspA and OspC. Although prior reports have indicated that OspE/F/Elp orthologs are surface exposed, none of the individual lipoproteins or combinations of the lipoproteins protected mice from challenge infections. To examine why these apparently surface-exposed lipoproteins were not protective, we analyzed their genetic stability during infection and their cellular locations after cultivation in vitro and within dialysis membrane chambers, mimicking a mammalian host-adapted state. Combined restriction fragment length polymorphism and nucleotide sequence analyses revealed that the genes encoding these lipoproteins are stable for at least 8 months postinfection. Interestingly, cellular localization experiments revealed that while all of these proteins can be surface localized, there were significant populations of spirochetes that expressed these lipoproteins only in the periplasm. Furthermore, host-specific signals were found to alter the expression patterns and final cellular location of these lipoproteins. The combined data revealed a remarkable heterogeneity in populations of B. burgdorferi during tick transmission and mammalian infection. The diversity is generated not only by temporal changes in antigen expression but also by modulation of the surface lipoproteins during infection. The ability to regulate the temporal and spatial expression patterns of lipoproteins throughout infection likely contributes to persistent infection of mammals by B. burgdorferi .