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The Structure of Treponema pallidum Tp0624 Reveals a Modular Assembly of Divergently Functionalized and Previously Uncharacterized Domains.

ABSTRACT: Treponema pallidum subspecies pallidum is the causative agent of syphilis, a chronic, multistage, systemic infection that remains a major global health concern. The molecular mechanisms underlying T. pallidum pathogenesis are incompletely understood, partially due to the phylogenetic divergence of T. pallidum. One aspect of T. pallidum that differentiates it from conventional Gram-negative bacteria, and is believed to play an important role in pathogenesis, is its unusual cell envelope ultrastructure; in particular, the T. pallidum peptidoglycan layer is chemically distinct, thinner and more distal to the outer membrane. Established functional roles for peptidoglycan include contributing to the structural integrity of the cell envelope and stabilization of the flagellar motor complex, which are typically mediated by the OmpA domain-containing family of proteins. To gain insight into the molecular mechanisms that govern peptidoglycan binding and cell envelope biogenesis in T. pallidum we report here the structural characterization of the putative OmpA-like domain-containing protein, Tp0624. Analysis of the 1.70 Å resolution Tp0624 crystal structure reveals a multi-modular architecture comprised of three distinct domains including a C-terminal divergent OmpA-like domain, which we show is unable to bind the conventional peptidoglycan component diaminopimelic acid, and a previously uncharacterized tandem domain unit. Intriguingly, bioinformatic analysis indicates that the three domains together are found in all orthologs from pathogenic treponemes, but are not observed together in genera outside Treponema. These findings provide the first structural insight into a multi-modular treponemal protein containing an OmpA-like domain and its potential role in peptidoglycan coordination and stabilization of the T. pallidum cell envelope.

SUBMITTER: Parker ML

PROVIDER: S-EPMC5104382 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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The Structure of Treponema pallidum Tp0624 Reveals a Modular Assembly of Divergently Functionalized and Previously Uncharacterized Domains.

Parker Michelle L ML Houston Simon S Wetherell Charmaine C Cameron Caroline E CE Boulanger Martin J MJ

PloS one 20161110 11

Treponema pallidum subspecies pallidum is the causative agent of syphilis, a chronic, multistage, systemic infection that remains a major global health concern. The molecular mechanisms underlying T. pallidum pathogenesis are incompletely understood, partially due to the phylogenetic divergence of T. pallidum. One aspect of T. pallidum that differentiates it from conventional Gram-negative bacteria, and is believed to play an important role in pathogenesis, is its unusual cell envelope ultrastru ...[more]

PMID: 27832149

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Project description:Past work has shown that Treponema pallidum, the causative agent of syphilis, binds host fibronectin (FN). FN and other host proteins are believed to bind to rare outer membrane proteins (OMPs) of T. pallidum, and it is postulated that this interaction may facilitate cell attachment and mask antigenic targets on the surface. This research seeks to prepare a surface capable of mimicking the FN binding ability of T. pallidum in order to investigate the impact of FN binding with adsorbed Tp0483 on the host response to the surface. By understanding this interaction, it may be possible to develop more effective treatments for infection and possibly mimic the stealth properties of the bacteria. Functionalized self-assembled monolayers (SAMs) on gold were used to investigate rTp0483 and FN adsorption. Using a quartz crystal microbalance (QCM), rTp0483 adsorption and subsequent FN adsorption onto rTp0483 were determined to be higher on negatively charged carboxylate-terminated self-assembled monolayers (-COO(-) SAMs) compared to the other surfaces analyzed. Kinetic analysis of rTp0483 adsorption using surface plasmon resonance (SPR) supported this finding. Kinetic analysis of FN adsorption using SPR revealed a multistep event, where the concentration of immobilized rTp0483 plays a role in FN binding. An examination of relative QCM dissipation energy compared to the shift in frequency showed a correlation between the physical properties of adsorbed rTp0483 and SAM surface chemistry. In addition, AFM images of rTp0483 on selected SAMs illustrated a preference of rTp0483 to bind as aggregates. Adsorption on -COO(-) SAMs was more uniform across the surface, which may help further explain why FN bound more strongly. rTp0483 antibody studies suggested the involvement of amino acids 274-289 and 316-333 in binding between rTp0483 to FN, while a peptide blocking study only showed inhibition of binding with amino acids 316-333. Finally, surface adsorbed rTp0483 with FN bound significantly less anti-RGD and gelatin compared to FN adsorbed directly to -COO(-) SAMs, indicating that one or both binding regions may play a role in binding between rTp0483 and FN.

Project description:UNLABELLED:Members of the genus Vibrio include many pathogens of humans and marine animals that share genetic information via horizontal gene transfer. Hence, the Vibrio pan-genome carries the potential to establish new pathogenic strains by sharing virulence determinants, many of which have yet to be characterized. Here, we investigated the virulence properties of Vibrio proteolyticus, a Gram-negative marine bacterium previously identified as part of the Vibrio consortium isolated from diseased corals. We found that V. proteolyticus causes actin cytoskeleton rearrangements followed by cell lysis in HeLa cells in a contact-independent manner. In search of the responsible virulence factor involved, we determined the V. proteolyticus secretome. This proteomics approach revealed various putative virulence factors, including active type VI secretion systems and effectors with virulence toxin domains; however, these type VI secretion systems were not responsible for the observed cytotoxic effects. Further examination of the V. proteolyticus secretome led us to hypothesize and subsequently demonstrate that a secreted hemolysin, belonging to a previously uncharacterized clan of the leukocidin superfamily, was the toxin responsible for the V. proteolyticus-mediated cytotoxicity in both HeLa cells and macrophages. Clearly, there remains an armory of yet-to-be-discovered virulence factors in the Vibrio pan-genome that will undoubtedly provide a wealth of knowledge on how a pathogen can manipulate host cells. IMPORTANCE:The pan-genome of the genus Vibrio is a potential reservoir of unidentified toxins that can provide insight into how members of this genus have successfully risen as emerging pathogens worldwide. We focused on Vibrio proteolyticus, a marine bacterium that was previously implicated in virulence toward marine animals, and characterized its interaction with eukaryotic cells. We found that this bacterium causes actin cytoskeleton rearrangements and leads to cell death. Using a proteomics approach, we identified a previously unstudied member of the leukocidin family of pore-forming toxins as the virulence factor responsible for the observed cytotoxicity in eukaryotic cells, as well as a plethora of additional putative virulence factors secreted by this bacterium. Our findings reveal a functional new clan of the leukocidin toxin superfamily and establish this pathogen as a reservoir of potential toxins that can be used for biomedical applications.

Project description:Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

Dataset Information

The Structure of Treponema pallidum Tp0624 Reveals a Modular Assembly of Divergently Functionalized and Previously Uncharacterized Domains.

Publications

The Structure of Treponema pallidum Tp0624 Reveals a Modular Assembly of Divergently Functionalized and Previously Uncharacterized Domains.

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