Clinical isolates of Shiga toxin 1a-producing Shigella flexneri with an epidemiological link to recent travel to Hispañiola.

Shiga toxins (Stx) are cytotoxins involved in severe human intestinal disease. These toxins are commonly found in Shigella dysenteriae serotype 1 and Shiga-toxin-producing Escherichia coli; however, the toxin genes have been found in other Shigella species. We identified 26 Shigella flexneri serotype 2 strains isolated by public health laboratories in the United States during 2001-2013, which encode the Shiga toxin 1a gene (stx1a). These strains produced and released Stx1a as measured by cytotoxicity and neutralization assays using anti-Stx/Stx1a antiserum. The release of Stx1a into culture supernatants increased ≈100-fold after treatment with mitomycin C, suggesting that stx1a is carried by a bacteriophage. Infectious phage were found in culture supernatants and increased ≈1,000-fold with mitomycin C. Whole-genome sequencing of several isolates and PCR analyses of all strains confirmed that stx1a was carried by a lambdoid bacteriophage. Furthermore, all patients who reported foreign travel had recently been to Hispañiola, suggesting that emergence of these novel strains is associated with that region.

In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae.

The type II secretion system is a multi-protein complex that spans the cell envelope of Gram-negative bacteria and promotes the secretion of proteins, including several virulence factors. This system is homologous to the type IV pilus biogenesis machinery and contains five proteins, EpsG-K, termed the pseudopilins that are structurally homologous to the type IV pilins. The major pseudopilin EpsG has been proposed to form a pilus-like structure in an energy-dependent process that requires the ATPase, EpsE. A key remaining question is how the membrane-bound EpsG interacts with the cytoplasmic ATPase, and if this is a direct or indirect interaction. Previous studies have established an interaction between the bitopic inner membrane protein EpsL and EpsE; therefore, in this study we used in vivo cross-linking to test the hypothesis that EpsG interacts with EpsL. Our findings suggest that EpsL may function as a scaffold to link EpsG and EpsE and thereby transduce the energy generated by ATP hydrolysis to support secretion. The recent discovery of structural homology between EpsL and a protein in the type IV pilus system implies that this interaction may be conserved and represent an important functional interaction for both the type II secretion and type IV pilus systems.

Calcium is essential for the major pseudopilin in the type 2 secretion system.

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.

Identification of the TcpP-binding site in the toxT promoter of Vibrio cholerae and the role of ToxR in TcpP-mediated activation.

ToxR-dependent recruitment of TcpP to the toxT promoter facilitates toxT transcription in Vibrio cholerae, initiating a regulatory cascade that culminates in cholera toxin expression and secretion. Although TcpP usually requires ToxR to activate the toxT promoter, TcpP overexpression can circumvent the requirement for ToxR in this process. To define nucleotides critical for TcpP-dependent promoter recognition and activation, a series of toxT promoter derivatives with single-base-pair transversions spanning the TcpP-binding site were generated and used as plasmid-borne toxT-lacZ fusions, as DNA mobility shift targets, and as allelic replacements of the chromosomal toxT promoter. When present in ΔtoxR V. cholerae overexpressing TcpP, several transversions affecting nucleotides within two direct repeats present in the TcpP-binding region (TGTAA-N(6)-TGTAA) caused defects in TcpP-dependent toxT-lacZ fusion activation and toxin production. Electrophoretic mobility shift assays demonstrated that these same transversions reduced the affinity of the toxT promoter for TcpP. The presence of ToxR suppressed transcription activation defects associated with most, but not all, transversions. Particularly, the central thymine nucleotide of both pentameric repeats was essential for efficient toxT activation, even in the presence of ToxR. These results suggest that the toxT promoter recognition function provided by ToxR can facilitate the interaction of TcpP with the toxT promoter but is insufficient for promoter activation when the TcpP-binding site has been severely compromised by mutation. Thus, the interaction of TcpP with nucleotides of the direct repeat sequences appears to be a prerequisite for toxT promoter activation.

Docking and assembly of the type II secretion complex of Vibrio cholerae.

Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.

Chlamydia trachomatis dapF Encodes a Bifunctional Enzyme Capable of Both d-Glutamate Racemase and Diaminopimelate Epimerase Activities.

Peptidoglycan is a sugar/amino acid polymer unique to bacteria and essential for division and cell shape maintenance. The d-amino acids that make up its cross-linked stem peptides are not abundant in nature and must be synthesized by bacteria de novo d-Glutamate is present at the second position of the pentapeptide stem and is strictly conserved in all bacterial species. In Gram-negative bacteria, d-glutamate is generated via the racemization of l-glutamate by glutamate racemase (MurI). Chlamydia trachomatis is the leading cause of infectious blindness and sexually transmitted bacterial infections worldwide. While its genome encodes a majority of the enzymes involved in peptidoglycan synthesis, no murI homologue has ever been annotated. Recent studies have revealed the presence of peptidoglycan in C. trachomatis and confirmed that its pentapeptide includes d-glutamate. In this study, we show that C. trachomatis synthesizes d-glutamate by utilizing a novel, bifunctional homologue of diaminopimelate epimerase (DapF). DapF catalyzes the final step in the synthesis of meso-diaminopimelate, another amino acid unique to peptidoglycan. Genetic complementation of an Escherichia coli murI mutant demonstrated that Chlamydia DapF can generate d-glutamate. Biochemical analysis showed robust activity, but unlike canonical glutamate racemases, activity was dependent on the cofactor pyridoxal phosphate. Genetic complementation, enzymatic characterization, and bioinformatic analyses indicate that chlamydial DapF shares characteristics with other promiscuous/primordial enzymes, presenting a potential mechanism for d-glutamate synthesis not only in Chlamydia but also numerous other genera within the Planctomycetes-Verrucomicrobiae-Chlamydiae superphylum that lack recognized glutamate racemases.IMPORTANCE Here we describe one of the last remaining "missing" steps in peptidoglycan synthesis in pathogenic Chlamydia species, the synthesis of d-glutamate. We have determined that the diaminopimelate epimerase (DapF) encoded by Chlamydia trachomatis is capable of carrying out both the epimerization of DAP and the pyridoxal phosphate-dependent racemization of glutamate. Enzyme promiscuity is thought to be the hallmark of early microbial life on this planet, and there is currently an active debate as to whether "moonlighting enzymes" represent primordial evolutionary relics or are a product of more recent reductionist evolutionary pressures. Given the large number of Chlamydia species (as well as members of the Planctomycetes-Verrucomicrobiae-Chlamydiae superphylum) that possess DapF but lack homologues of MurI, it is likely that DapF is a primordial isomerase that functions as both racemase and epimerase in these organisms, suggesting that specialized d-glutamate racemase enzymes never evolved in these microbes.

Stx-Producing Shigella Species From Patients in Haiti: An Emerging Pathogen With the Potential for Global Spread.

Shiga toxins (Stx) are commonly produced by Shigella dysenteriae serotype 1 and Stx-producing Escherichia coli. However, the toxin genes have been detected in additional Shigella species. We recently reported the emergence of Stx-producing Shigella in travelers in the United States and France who had recently visited Hispaniola (Haiti and the Dominican Republic). In this study, we confirm this epidemiological link by identifying Stx-producing Shigella from Haitian patients attending clinics near Port-au-Prince. We also demonstrate that the bacteriophage encoding Stx is capable of dissemination to stx-negative Shigella species found in Haiti, suggesting that Stx-producing Shigella may become more widespread within that region.

Type II Secretion in Escherichia coli.

The type II secretion system (T2SS) is used by Escherichia coli and other gram-negative bacteria to translocate many proteins, including toxins and proteases, across the outer membrane of the cell and into the extracellular space. Depending on the bacterial species, between 12 and 15 genes have been identified that make up a T2SS operon. T2SSs are widespread among gram-negative bacteria, and most E. coli appear to possess one or two complete T2SS operons. Once expressed, the multiple protein components that form the T2S system are localized in both the inner and outer membranes, where they assemble into an apparatus that spans the cell envelope. This apparatus supports the secretion of numerous virulence factors; and therefore secretion via this pathway is regarded in many organisms as a major virulence mechanism. Here, we review several of the known E. coli T2S substrates that have proven to be critical for the survival and pathogenicity of these bacteria. Recent structural and biochemical information is also reviewed that has improved our current understanding of how the T2S apparatus functions; also reviewed is the role that individual proteins play in this complex system.

Activation of the unfolded protein response is necessary and sufficient for reducing topoisomerase IIalpha protein levels and decreasing sensitivity to topoisomerase-targeted drugs.

A wide range of chemotherapeutic agents has been identified that are active against solid tumors. However, resistance remains an important obstacle to the development of curative regimens. Whereas much attention has been paid to acquired drug resistance, a variety of physiological pathways also have been described that reduce the sensitivity of previously untreated tumors to cytotoxic antitumor agents. Treatment of cells with pharmacological agents that alter the environment of the endoplasmic reticulum (ER) and activate the unfolded protein response (UPR) can render cells resistant to topoisomerase II poisons. We describe experiments showing that activation of the mammalian ER stress response is both necessary and sufficient to decrease topoisomerase IIalpha protein levels and to render cells resistant to etoposide, a topoisomerase II-targeting drug. This is not caused by the elevated levels of BiP that are a hallmark of this response, because a cell line that has been engineered to overexpress BiP does not show increased resistance to etoposide. The UPR was shown to be required for altered drug sensitivity, because the BiP-overexpressing cell line, which is unable to activate the UPR, did not show decreased topoisomerase II levels or increased resistance to etoposide in response to stress conditions. The transient overexpression of an unfolded protein activated the UPR and led to the concomitant loss of topoisomerase IIalpha protein from the cells, demonstrating that UPR activation is sufficient for the changes in topoisomerase II levels that had been observed previously with pharmacological induction of the UPR.