Search

This research examines self-efficacy and internships. The purpose is to uncover if internships impact a student's occupational self-efficacy and if there are gender differences in the effect of an internship on self-efficacy? Over a period of 1 year, pre- and post-surveys were sent to college juniors and seniors working in internship positions for academic credit at a Mid-Atlantic public university. The findings have salient strengths, extending the understanding of the relationships between internships, gender, and occupations self-efficacy. Findings reveal that internships had a positive impact, but that gender differences were present in the effect of the internship.

Several research studies found that managers lacked a basic understanding of a balanced scorecard (BSC) approach to evaluate an organization. Because of this, the authors saw an opportunity to reorganize a course in order to provide students with an opportunity to address this issue. Therefore, a recent pilot study was conducted to ascertain student understanding of the functional areas of business. Students were assigned a BSC project in a foundational course, and completed a survey to establish if the BSC enhanced their knowledge and understanding of business disciplines within an organization. Based on students' perceptions, it was concluded that a better understanding of how businesses operate holistically was attained.

Flagellar motility has long been regarded as an important virulence factor. In Vibrio cholerae, the single polar flagellum is essential for motility as well as for proper attachment and colonization. In this study, we demonstrate that the novel flagellar protein FlgT is involved in anchoring the flagellum to the V. cholerae cell. A screen for novel colonization factors by use of TnphoA mutagenesis identified flgT. An in-frame deletion of flgT established that FlgT is required for attachment, colonization, and motility. Transmission electron microscopy revealed that while the flgT mutant is capable of assembling a phenotypically normal flagellum, the flgT population is mostly aflagellate compared to the wild-type population. Further analyses indicated that the flagellum of the flgT mutant is released into the culture supernatant from the cell upon completion of assembly. Additionally, hook basal body complexes appear to be released along with the filament. These results indicate that FlgT functions to stabilize the flagellar apparatus at the pole of the cell. doi: 10.1128/JB.01562-09

Vibrio cholerae is the etiologic agent of cholera in humans. Intestinal colonization occurs in a stepwise fashion, initiating with attachment to the small intestinal epithelium. This attachment is followed by expression of the toxin-coregulated pilus, microcolony formation, and cholera toxin (CT) production. We have recently characterized a secreted attachment factor, GlcNAc binding protein A (GbpA), which functions in attachment to environmental chitin sources as well as to intestinal substrates. Studies have been initiated to define the regulatory network involved in GbpA induction. At low cell density, GbpA was detected in the culture supernatant of all wild-type (WT) strains examined. In contrast, at high cell density, GbpA was undetectable in strains that produce HapR, the central regulator of the cell density-dependent quorum-sensing system of V. cholerae. HapR represses the expression of genes encoding regulators involved in V. cholerae virulence and activates the expression of genes encoding the secreted proteases HapA and PrtV. We show here that GbpA is degraded by HapA and PrtV in a time-dependent fashion. Consistent with this, [DELTA]hapA [DELTA]ortV strains attach to chitin beads more efficiently than either the WT or a [DELTA]hapA [DELTA]ortV [DELTA]gbpA strain. These results suggest a model in which GbpA levels fluctuate in concert with the bacterial production of proteases in response to quorum-sensing signals. This could provide a mechanism for GbpA-mediated attachment to, and detachment from, surfaces in response to environmental cues. doi:10.1128/JB.00747-09

Vibrio cholerae is highly motile by the action of a single polar flagellum. The loss of motility reduces the infectivity of V. cholerae, demonstrating that motility is an important virulence factor. FIrC is the sigma-54-dependent positive regulator of flagellar genes. Recently, the genes VC2206 (flgP) and VC2207 (flgO) were identified as being regulated by FlrC via a microarray analysis of anflrC mutant (D. C. Morris, F. Peng, J. R. Barker, and K. E. Klose, J. Bacteriol. 190:231-239, 2008). FlgP is reported to be an outer membrane lipoprotein required for motility that functions as a colonization factor. The study reported here focuses on the characterization of flgO, the first gene in the flgOP operon. We show that FlgO and FlgP are important for motility, as strains with mutations in the flgOP genes have reduced motility phenotypes. The flgO and flgP mutant populations display fewer motile cells as well as reduced numbers of flagellated cells. The flagella produced by the flgO and flgP mutant strains are shorter in length than the wild-type flagella, which can be restored by inhibiting rotation of the flagellum. FlgO is an outer membrane protein that localizes throughout the membrane and not at the flagellar pole. Although FlgO and FlgP do not specifically localize to the flagellum, they are required for flagellar stability. Due to the nature of these motility defects, we established that the flagellum is not sufficient for adherence; rather, motility is the essential factor required for attachment and thus colonization by V. cholerae O1 of the classical biotype. This study reveals a novel mechanism for which the outer membrane proteins FlgO and FIgP function in motility to mediate flagellar stability and influence attachment and colonization.

A novel protective monoclonal antibody (mAb) that recognizes a lipopolysaccharide (LPS) epitope common between serotypes Ogawa and Inaba of the O1 serogroup of Vibrio cholerae was characterized and the potential to develop peptide mimics of this protective LPS epitope was investigated, mAb 72.1 recognizes both Ogawa and Inaba LPS and it is vibriocidal and protective in passive immunization against infection by strains of both serotypes. The cDNA-derived amino acid sequence of mAb 72.1 is closely related to the previously characterized mAb ZAC-3, which is thought to recognize an epitope in the lipid A core region of O1 LPS. In an attempt to develop a peptide mimic-based vaccine against V. cholerae, phage display libraries were screened with mAb 72.1 and 11 peptide mimics were identified. Remarkably, all of the peptide sequences identified from linear phage display libraries contained two cysteine residues, suggesting that mAb 72.1 preferentially binds to peptides constrained with a disulphide bond. One of the peptide mimics was immunologically characterized. Although immunization of mice with this peptide mimic conjugated to KLH elicited antibodies against the peptide itself, these antibodies did not cross-react with Ogawa or Inaba LPS. Effectiveness of a peptide mimic as a vaccine may depend on how well the peptide can mimic the carbohydrate interactions when binding to the anti-carbohydrate antibody. Thus, investigating how peptides and LPS bind to mAb 72.1 may be useful in improving current peptide mimics or designing more effective peptide mimics. Identification and characterization of novel protective anti-LPS antibodies may be useful in studying protective epitopes of LPS, which may help develop LPS-based therapeutics against V. cholerae.

Colonization of the human small intestine by Vibrio cholerae requires the type IV toxin-coregulated pilus (TCP). TcpF, which is encoded within the tcp operon, is secreted from the bacterial cell by the TCP apparatus and is also essential for colonization. Bacteria lacking tcpF are deficient in colonization, and anti-TcpF antibodies are protective in the infant mouse cholera model. In order to elucidate the regions of the protein that are required for secretion through the TCP apparatus and for its function in colonization, random mutagenesis of tcpF was performed. Analysis of these mutants suggests that multiple regions throughout the protein influence extracellular secretion and that determinants near the C terminus are important for the function of TcpF in colonization. The TcpF proteins of certain environmental V. cholerae isolates with 31% to 66% identity to pathogenic V. cholerae TcpF showed higher similarity in regions identified as secretion determinants but diverged in regions found to be important for colonization. These environmental TcpF proteins are secreted from the pathogenic strain; however, they do not mediate colonization in the infant mouse model. Here we provide genetic evidence pointing toward regions of TcpF that influence secretion, as well as regions that play an important role in in vivo colonization.

Enteric pathogens have developed several resistance mechanisms to survive the antimicrobial action of bile. We investigated the transcriptional profile of Vibrio cholerae Ol El Tor strain C6706 under virulence geneinducing conditions in the presence and absence of bile. Microarray analysis revealed that the expression of 119 genes was affected by bile. The mRNA levels of genes encoding proteins involved in transport were increased in the presence of bile, whereas the mRNA levels of genes encoding proteins involved in pathogenesis and chemotaxis were decreased. This study identified genes encoding transcriptional regulators from the TetR family (vexR and breR) and multidrug efflux pumps from the resistance-nodulation-cell division superfamily (vexB and vexD [herein renamed breB]) that were induced in response to bile. Further analysis regarding vexAB and breAB expression in the presence of various antimicrobial compounds established that vexAB was induced in the presence of bile, sodium dodecyl sulfate, or novobiocin and that the induction of breAB was specific to bile. BreR is a direct repressor of the breAB promoter and is able to regulate its own expression, as demonstrated by transcriptional and electrophoretic mobility shift assays (EMSA). The expression of breR and breAB is induced in the presence of the bile salts cholate, deoxycholate, and chenodeoxycholate, and EMSA showed that deoxycholate is able to abolish the formation of BreR-[P.sub.breR] complexes. We propose that deoxycholate is able to interact with BreR and induce a conformational change that interferes with the DNA binding ability of BreR, resulting in breAB and breR expression. These results provide new insight into a transcriptional regulator and a transport system that likely play essential roles in the ability of V. cholerae to resist the action of bile in the host.

Virulence gene expression in Vibrio cholerae is dependent upon a complex transcriptional cascade that is influenced by both specific and global regulators in response to environmental stimuli. Here, we report that the global regulator integration host factor (IHF) positively affects virulence gene expression in V. cholerae. Inactivation of ihfA and ihfB, the genes encoding the IHF subunits, decreased the expression levels of the two main virulence factors tcpA and ctx and prevented toxin-coregulated pilus and cholera toxin production. IHF was found to directly bind to and bend the tcpA promoter region at an IHF consensus site centered at position -162 by using gel mobility shift assays and DNase I footprinting experiments. Deletion or mutation of the tcpA IHF consensus site resulted in the loss of IHF binding and additionally disrupted the binding of the repressor H-NS. DNase I footprinting revealed that H-NS protection overlaps with both the IHF and the ToxT binding sites at the tcpA promoter. In addition, disruption of ihfA in an hns or toxT mutant background had no effect on tcpA expression. These results suggest that IHF may function at the tcpA promoter to alleviate H-NS repression.

Vibrio cholerae, the causative agent of the human disease cholera, uses cell-to-cell communication to control pathogenicity and biofilm formation (1,2). This process, known as quorum sensing, relies on the secretion [...]

Quorum sensing in Vibrio cholerae involves signaling between two-component sensor protein kinases and the response regulator LuxO to control the expression of the master regulator HapR. HapR, in turn, plays a central role in regulating a number of important processes, such as virulence gene expression and biofilm formation. We have determined the crystal structure of HapR to 2.2-[Angstrom] resolution. Its structure reveals a dimeric, two-domain molecule with an all-helical structure that is strongly conserved with members of the TetR family of transcriptional regulators. The N-terminal DNA-binding domain contains a helix-turn-helix DNA-binding motif and alteration of certain residues in this domain completely abolishes the ability of HapR to bind to DNA, alleviating repression of both virulence gene expression and biofilm formation. The C-terminal dimerization domain contains a unique solvent accessible tunnel connected to an amphipathic cavity, which by analogy with other TetR regulators, may serve as a binding pocket for an as-yet-unidentified ligand.

The toxin-coregulated pilus (TCP) is one of the major virulence factors of Vibrio cholerae. Biogenesis of this type 4 pilus (Tfp) requires a number of structural components encoded by the tcp operon. TcpT, the cognate putative ATPase, is required for TCP biogenesis and all TCP-mediated functions. We studied the stability and localization of TcpT in cells containing in-frame deletions in each of the tcp genes. TcpT was detectable in each of the biogenesis mutants except the [DELTA]tcpT strain. TcpT was localized to the inner membrane (IM) in a TcpR-dependent manner. TcpR is a predicted bitopic inner membrane protein of the TCP biogenesis apparatus. Using metal affinity pull-down experiments, we demonstrated interaction between TcpT and TcpR. Using Escherichia coli as a heterologous system, we investigated direct interaction between TcpR and TcpT. We report that TcpR is sufficient for TcpT IM localization per se; however, stable IM localization of TcpT requires an additional V. cholerae-specific factor(s). A LexA-based two-hybrid system was utilized to define interaction domains of the two proteins. We demonstrate a strong interaction between the cytoplasmic domain of TcpR and the N-terminal 100 amino acid residues of TcpT. We also demonstrated the ability of the C-terminal domain of TcpT to multimerize.

Author(s): Thomas J. Kirn [1, 2, 3]; Brooke A. Jude [1, 3]; Ronald K. Taylor (corresponding author) [1] Many bacteria that cause diseases must be able to survive inside and [...]

The toxin-coregulated pilus (TCP) of Vibrio cholerae and the soluble TcpF protein that is secreted via the TCP biogenesis apparatus are essential for intestinal colonization. The TCP biogenesis apparatus is composed of at least nine proteins but is largely uncharacterized. TcpC is an outer membrane lipoprotein required for TCP biogenesis that is a member of the secretin protein superfamily. In the present study, analysis of TcpC in a series of strains deficient in each of the TCP biogenesis proteins revealed that TcpC was absent specifically in a tcpQ mutant. TcpQ is a predicted periplasmic protein required for TCP biogenesis. Fractionation studies revealed that the protein is not localized to the periplasm but is associated predominantly with the outer membrane fraction. An analysis of the amount of TcpQ present in the series of tcp mutants demonstrated the inverse of the TcpC result (absence of TcpQ in a tcpC deletion strain). Complementation of the tcpQ deletion restored TcpC levels and TCP formation, and similarly, complementation of tcpC restored TcpQ. Metal affinity pull-down experiments performed using His-tagged TcpC or TcpQ demonstrated a direct interaction between TcpC and TcpQ. In the presence of TcpQ, TcpC was found to form a high-molecular-weight complex that is stable in 2% sodium dodecyl sulfate and at temperatures below 65[degrees]C, a characteristic of secretin complexes. Fractionation studies in which TcpC was overexpressed in the absence of TcpQ showed that TcpQ is also required for proper localization of TcpC to the outer membrane.

Results reveal that ToxT-dependent activation at tcpA promoter is mediated by the C-terminal domain of the RNA polymerase alpha subunit. ToxT interacts with Two adenine-rich regions of tcpA promoter assisted by the C-terminal domain of RNA polymerase alpha subunit.

Research has been conducted on quorum sensing in Vibrio cholerae. Results indicate that different redundant sensory systems operating in V. cholerae control virulence.

Results reveal that the TonB-facilitated proton-motive-force does not regulate the type 4 pilus biogenesis and type II secretion in Vibrio cholerae as shown by the mutants defective for TonB.

The mannose-sensitive hemagglutinin on the surface of Vibrio cholerae strains appears to be involved in promoting adherence to plankton. Attachment to plankton increases the growth and survival of the bacterium, thus contributing to its persistence in water.

Results suggest that the nucleoid-associated protein H-NS has negative influence on gene expression of the virulence cascade of Vibrio cholerae. Data further indicae that the repressive effects of H-NS gene is counteracted by the transcriptional activator proteins of the ToxR regulon.

Research was conducted to examine the Vibrio cholerae mannose-sensitive hemagglutinin (MSHA) type 4 pilus gene locus using genetic and transcriptional analyses. Type 4 pili have demonstrated their importance as bacteriophage receptors, host colonization factors and mediators of DNA transfer. Results that the MSHA locus is located between two Escherichia coli homologs and flanked by a 7-bp direct repeat indicate that the MSHA locus may have been acquired as a mobile genetic element.

A study was conducted to characterize how the integral membrane proteins, ToxR and ToxS, activate virulence genes using a two-layered genetic framework. Plasmid pTSK was obtained by transforming plasmid DNA into Salmonella typhimurium. Results indicated that the assembly and stability of the periplasmic domain of ToxR is influenced by ToxS. Findings also showed that the natural role of ToxS is to complex with ToxR.