P. Scott Hefty

Chlamydial Type III Secretion System Is Encoded on Ten Operons Preceded by Sigma 70-Like Promoter Elements

Many gram-negative bacterial pathogens employ type III secretion systems for infectious processes. Chlamydiae are obligate intracellular bacteria that encode a conserved type III secretion system that is likely requisite for growth. Typically, genes encoding type III secretion systems are located in a single locus; however, for chlamydiae these genes are scattered throughout the genome. Little is known regarding the gene regulatory mechanisms for this essential virulence determinant. To facilitate identification of cis-acting transcriptional regulatory elements, the operon structure was determined. This analysis revealed 10 operons that contained 37 genes associated with the type III secretion system. Linkage within these operons suggests a role in type III secretion for each of these genes, including 13 genes encoding proteins with unknown function. The transcriptional start site for each operon was determined. In conjunction with promoter activity assays, this analysis revealed that the type III secretion system operons encode sigma(70)-like promoter elements. Transcriptional initiation by a sigma factor responsible for constitutive gene expression indicates that undefined activators or repressors regulate developmental stage-specific expression of chlamydial type III secretion system genes.

Conditional Gene Expression in Chlamydia trachomatis Using the Tet System

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.

Small-Molecule Inhibitor of the Shigella flexneri Master Virulence Regulator VirF

ABSTRACT 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.

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

Chlamydia are obligate intracellular bacterial pathogens that cause a variety of diseases. Like many 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.

Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense

Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense. DOI: http://dx.doi.org/10.7554/eLife.21465.001

Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB

Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non‐canonical mechanisms may be employed. The rod‐shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein encoded by ORF CT009 interacts with MreB despite low sequence similarity to RodZ. The studies herein expand on those observations through protein structure, mutagenesis and cellular localization analyses. Structural analysis indicated that CT009 shares high level of structural similarity to RodZ, revealing the conserved orientation of two residues critical for MreB interaction. Substitutions eliminated MreB protein interaction and partial complementation provided by CT009 in RodZ deficient Escherichia coli. Cellular localization analysis of CT009 showed uniform membrane staining in Chlamydia. This was in contrast to the localization of MreB, which was restricted to predicted septal planes. MreB localization to septal planes provides direct experimental observation for the role of MreB in cell division and supports the hypothesis that it serves as a functional replacement for FtsZ in Chlamydia.

An Automated Image-Based Method for Rapid Analysis of Chlamydia Infection as a Tool for Screening Antichlamydial Agents

ABSTRACT 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.

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination.

UNLABELLED Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperature-shift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle. IMPORTANCE Study of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms.

Hypothetical protein CT398 (CdsZ) interacts with σ54 (RpoN)‐holoenzyme and the type III secretion export apparatus in Chlamydia trachomatis

A significant challenge to bacteriology is the relatively large proportion of proteins that lack sufficient sequence similarity to support functional annotation (i.e. hypothetical proteins). The aim of this study was to apply protein structural homology to gain insights into a candidate protein of unknown function (CT398) within the medically important, obligate intracellular bacterium Chlamydia trachomatis. C. trachomatis is a major human pathogen responsible for numerous infections throughout the world that can lead to blindness and infertility. A 2.12 Å crystal structure of hypothetical protein CT398 was determined that was comprised of N‐terminal coiled‐coil and C‐terminal Zn‐ribbon domains. The structure of CT398 displayed a high degree of structural similarity to FlgZ (Flagellar‐associated zinc‐ribbon domain protein) from Helicobacter pylori. This observation directed analyses of candidate protein partners of CT398, revealing interactions with two paralogous type III secretion system (T3SS) ATPase‐regulators (CdsL and FliH) and the alternative sigma factor RpoN (σ54). Furthermore, genetic introduction of a conditional expression, affinity‐tagged construct into C. trachomatis enabled the purification of a CT398‐RpoN‐holoenzyme complex, suggesting a potential role for CT398 in modulating transcriptional activity during infection. The interactions reported here, in tandem with previous FlgZ studies in H. pylori, indicate that CT398 functions as a regulator of several key areas of chlamydial biology throughout the developmental cycle. Accordingly, we propose that CT398 be named CdsZ (Contact‐dependent secretion‐associated zinc‐ribbon domain protein).

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. The obligate intracellular human pathogen Chlamydia trachomatis is the etiological agent of blinding trachoma and sexually transmitted disease. Genomic sequencing of Chlamydia indicated this medically important bacterium was not exclusively dependent on the host cell for energy. In order for the electron transport chain to function, electron shuttling between membrane-embedded complexes requires lipid-soluble quinones (e.g. menaquionone or ubiquinone). The sources or biosynthetic pathways required to obtain these electron carriers within C. trachomatis are poorly understood. The 1.58Å crystal structure of C. trachomatis hypothetical protein CT263 presented here supports a role in quinone biosynthesis. Although CT263 lacks sequence-based functional annotation, the crystal structure of CT263 displays striking structural similarity to 5′-methylthioadenosine nucleosidase (MTAN) enzymes. Although CT263 lacks the active site-associated dimer interface found in prototypical MTANs, co-crystal structures with product (adenine) or substrate (5′-methylthioadenosine) indicate that the canonical active site residues are conserved. Enzymatic characterization of CT263 indicates that the futalosine pathway intermediate 6-amino-6-deoxyfutalosine (kcat/Km = 1.8 × 103 m−1 s−1), but not the prototypical MTAN substrates (e.g. S-adenosylhomocysteine and 5′-methylthioadenosine), is hydrolyzed. Bioinformatic analyses of the chlamydial proteome also support the futalosine pathway toward the synthesis of menaquinone in Chlamydiaceae. This report provides the first experimental support for quinone synthesis in Chlamydia. Menaquinone synthesis provides another target for agents to combat C. trachomatis infection.