Lipopolysaccharide-Binding Alkylpolyamine DS-96 Inhibits Chlamydia trachomatis Infection by Blocking Attachment and Entry

This is the published version. Copyright © 2014, American Society for Microbiology. All Rights Reserved.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

Simple Resazurin-Based Microplate Assay for Measuring Chlamydia Infections

This is the publisher's version. Copyright 2013 by the American Society for Microbiology.The conventional method for quantification of Chlamydia infection using fluorescence microscopy typically involves time- and labor-intensive manual enumeration, which is not applicable for a large-scale analysis required for an inhibitory compound screen. In this study, an alamarBlue (resazurin) assay was adopted to measure Chlamydia infection by measuring the redox capability of infected host cells in a 96-well format. The assay provided measurements comparable to those of the conventional microscopy method while drastically reducing the time required for analysis

Expression, purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of a Chlamydia trachomatis OmpR/PhoB-subfamily response regulator homolog, ChxR

This is the published version. Copyright 2009 by the International Union of Crystallography.Two-component signal transduction systems in bacteria are a primary mechan­ism for responding to environmental stimuli and adjusting gene expression accordingly. Generally in these systems a sensor kinase phosphorylates a response regulator that regulates transcription. Response regulators contain two domains: a receiver domain and an effector domain. The receiver domain is typically phosphorylated and as a result facilitates the DNA-binding and transcriptional activity of the effector domain. The OmpR/PhoB subfamily is the largest of the response-regulator subfamilies and is primarily defined by the winged helix-turn-helix DNA-binding motif within the effector domain. The overall structure of effector domains is highly conserved and contains three defined elements that are critical for transcriptional regulation: a DNA major-groove binding helix, a DNA minor-groove binding wing and a transcriptional activation loop. These functional elements are often diverse in sequence and conformation and reflect the functional differences observed between individual subfamily members. ChxR from Chlamydia trachomatis is an atypical OmpR/PhoB response regulator homolog that has transcriptional activity in the absence of phos­phorylation. To facilitate the precise identification of the functional elements of the ChxR effector domain, this protein was cloned, expressed, purified and crystallized. Crystals were obtained from two separate mother liquors, producing two morphologically distinct crystals. The space group of both crystals was P43212 (or its enantiomorph P41212) with isomorphous unit-cell parameters; the crystals diffracted to 2.2-2.5 Å resolution

Sigma 54-Regulated Transcription Is Associated with Membrane Reorganization and Type III Secretion Effectors during Conversion to Infectious Forms of Chlamydia trachomatis

This work is licensed under a Creative Commons Attribution 4.0 International License.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.NIH T32 GM008545AI126785NIH (AI126785)P20GM113117P20GM10363

Chlamydia trachomatis CT771 (nudH) is an asymmetric Ap4A hydrolase

Asymmetric diadenosine 5′,5′″-P1,P4-tetraphosphate (Ap4A) hydrolases are members of the Nudix superfamily that asymmetrically cleave the metabolite Ap4A into ATP and AMP while facilitating homeostasis. The obligate intracellular mammalian pathogen Chlamydia trachomatis possesses a single Nudix family protein, CT771. As pathogens that rely on a host for replication and dissemination typically have one or zero Nudix family proteins, this suggests that CT771 could be critical for chlamydial biology and pathogenesis. We identified orthologs to CT771 within environmental Chlamydiales that share active site residues suggesting a common function. Crystal structures of both apo- and ligand-bound CT771 were determined to 2.6 Å and 1.9 Å resolution, respectively. The structure of CT771 shows a αβα-sandwich motif with many conserved elements lining the putative Nudix active site. Numerous aspects of the ligand-bound CT771 structure mirror those observed in the ligand-bound structure of the Ap4A hydrolase from Caenorhabditis elegans. These structures represent only the second Ap4A hydrolase enzyme member determined from eubacteria and suggest that mammalian and bacterial Ap4A hydrolases might be more similar than previously thought. The aforementioned structural similarities, in tandem with molecular docking, guided the enzymatic characterization of CT771. Together, these studies provide the molecular details for substrate binding and specificity, supporting the analysis that CT771 is an Ap4A hydrolase (nudH)

Computational modeling of TC0583 as a putative component of the Chlamydia muridarum V-type ATP synthase complex and assessment of its protective capabilities as a vaccine antigen.

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

Structural and Biochemical Characterization of Chlamydia trachomatis Hypothetical Protein CT263 Supports That Menaquinone Synthesis Occurs through the Futalosine Pathway

BACKGROUND: Specific pathways and components for respiration in Chlamydia are poorly understood. RESULTS: The C. trachomatis hypothetical protein CT263 crystal structure displays strong structural similarity with 5′-methylthioadenosine nucleosidase enzymes. CONCLUSION: Bioinformatic analyses and enzymatic characterization of CT263 suggest menaquinone biosynthesis proceeds through the futalosine pathway in Chlamydiaceae. SIGNIFICANCE: Unique structural aspects of the CT263 active site can be leveraged to modify existing transition state inhibitors

Biophysical Characterization of Chlamydia trachomatis CT584 Supports Its Potential Role as a Type III Secretion Needle Tip Protein

This is the published version. Copyright American Chemical SocietyChlamydia are obligate intracellular bacterial pathogens that cause a variety of diseases. Likemany Gram-negative bacteria, they employ type III secretion systems (T3SS) for invasion, establishing and maintaining their unique intracellular niche, and possibly cellular exit. Computational structure prediction indicated that ORF CT584 is homologous to other T3SS needle tip proteins. Tip proteins have been shown to be localized to the extracellular end of the T3SS needle and play a key role in controlling secretion of effector proteins. We have previously demonstrated that T3SS needle tip proteins from different bacteria share many biophysical characteristics. To support the hypothesis that CT584 is a T3SS needle tip protein, biophysical properties of CT584 were explored as a function of pH and temperature, using spectroscopic techniques. Far-UV circular dichroism, Fourier transform infrared spectroscopy, UV absorbance spectroscopy, ANS extrinsic fluorescence, turbidity, right angle static light scattering, and analytical ultracentrifugation were all employed to monitor the secondary, tertiary, quaternary, and aggregation behavior of this protein. An empirical phase diagram approach is also employed to facilitate such comparisons. These analyses demonstrate that CT584 shares many biophysical characteristics with other T3SS needle tip proteins. These data support the hypothesis that CT584 is a member of the same functional family, although future biologic analyses are required

Transposon Mutagenesis in Chlamydia trachomatis Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer

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.S.D.L., Z.E.D., K.S.H., S.B., R.J.S., and P.S.H. were funded by NIH (AI126785)J.W. and P.S.H. were supported by NIH AI125929. P.S.H. was also supported by P20GM113117Support for genomic sequencing was supplemented by P20GM10363

Changes in Temporal and Spatial Patterns of Outer Surface Lipoprotein Expression Generate Population Heterogeneity and Antigenic Diversity in the Lyme Disease Spirochete, Borrelia burgdorferi

This is the published version. Copyright 2015 by the American Society for Microbiology.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