Comparative RNA-Seq based dissection of the regulatory networks and environmental stimuli underlying Vibrio parahaemolyticus gene expression during infection

Vibrio parahaemolyticus is the leading worldwide cause of seafood-associated gastroenteritis, yet little is known regarding its intraintestinal gene expression or physiology. To date, in vivo analyses have focused on identification and characterization of virulence factors—e.g. a crucial Type III secretion system (T3SS2)—rather than genome-wide analyses of in vivo biology. Here, we used RNA-Seq to profile V. parahaemolyticus gene expression in infected infant rabbits, which mimic human infection. Comparative transcriptomic analysis of V. parahaemolyticus isolated from rabbit intestines and from several laboratory conditions enabled identification of mRNAs and sRNAs induced during infection and of regulatory factors that likely control them. More than 12% of annotated V. parahaemolyticus genes are differentially expressed in the intestine, including the genes of T3SS2, which are likely induced by bile-mediated activation of the transcription factor VtrB. Our analyses also suggest that V. parahaemolyticus has access to glucose or other preferred carbon sources in vivo, but that iron is inconsistently available. The V. parahaemolyticus transcriptional response to in vivo growth is far more widespread than and largely distinct from that of V. cholerae, likely due to the distinct ways in which these diarrheal pathogens interact with and modulate the environment in the small intestine

Functional and biochemical characterization of pili in Corynebacterium diphtheriae

A variety of multi-subunit protein polymers on the bacterial cell surface known as pili or fimbriae play a pivotal role in the colonization of specific host tissues by many pathogens. Unlike gram-negative bacteria, the gram-positive pili are assembled by a distinct mechanism involving transpeptidases called sortase and this mechanism is conserved among gram-positive bacteria. Sortases cross-link individual pilin monomers and ultimately join the resulting covalent polymer to the cell wall peptidoglycan. A typical pilus consists of a major shaft protein and either one or two accessory minor pilins. Although much is known about the functional role of pili in pathogenesis in gram-negative bacteria, not much is known about the role of these pili in gram-positive bacteria, in particular in Corynebacterium diphtheriae which harbors three immunologically distinct pili. Functional and biochemical studies revealed the role of these pili in adhesion of the bacterium to host epithelial cells and demonstrated that these pili exhibit tissue tropism. Further analysis determined that the accessory pilins mediate this adhesion and are displayed on the cell surface even in the absence of the major shaft pilin. The current model of pilus assembly suggests that it is a biphasic process where polymerization catalyzed by the pilus specific sortase precedes the cell wall anchoring step which is catalyzed by the housekeeping sortase. Molecular genetic and biochemical studies demonstrated that SpaB, a minor pilin serves as a molecular switch between the polymerization and cell wall anchoring phase. Further analysis of the pilus assembly site revealed that the pilus specific sortase interacts with the housekeeping sortase to bring about pilus assembly. Using biochemical and functional studies, this thesis reveals the role of pili in corynebacterial attachment to host tissue and also dissects the pilus assembly site to gain insights into the assembly process.

A Cholera Conjugate Vaccine Containing O-specific Polysaccharide (OSP) of V. cholerae O1 Inaba and Recombinant Fragment of Tetanus Toxin Heavy Chain (OSP:rTTHc) Induces Serum, Memory and Lamina Proprial Responses against OSP and Is Protective in Mice

Background: Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). Methodology Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc). We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 μg), vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1), effect of an adjuvant, and route of immunization. Principle Findings Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In general, we found comparable immune responses in mice immunized with these variations, although memory B cell and vibriocidal responses were blunted in mice receiving the highest dose of vaccine (50 μg). We found no appreciable change in immune responses when the conjugate vaccine was administered in the presence or absence of immunoadjuvant alum. Administration of OSP:rTTHc resulted in 55% protective efficacy in a mouse survival cholera challenge model. Conclusion: We report development of an Inaba OSP:rTTHc conjugate vaccine that induces memory responses and protection against cholera in mice. Development of an effective cholera conjugate vaccine that induces high level and long-term immune responses against OSP would be beneficial, especially in young children who respond poorly to polysaccharide antigens

Plasma and memory B cell responses targeting O-specific polysaccharide (OSP) are associated with protection against Vibrio cholerae O1 infection among household contacts of cholera patients in Bangladesh

Background: The mediators of protection against cholera, a severe dehydrating illness of humans caused by Vibrio cholerae, are unknown. We have previously shown that plasma IgA as well as memory B IgG cells targeting lipopolysaccharide (LPS) of Vibrio cholerae O1 correlate with protection against V. cholerae O1 infection among household contacts of cholera patients. Protection against cholera is serogroup specific, and serogroup specificity is defined by the O-specific polysaccharide (OSP) component of LPS. Therefore, we prospectively followed household contacts of cholera patients to determine whether OSP-specific immune responses present at the time of enrollment are associated with protection against V. cholerae infection. Methodology In this study, we enrolled two hundred forty two household contacts of one hundred fifty index patients who were infected with Vibrio cholerae. We determined OSP-specific memory B cells and plasma IgA, IgG and IgM antibody responses on study entry (day 2). Principle findings The presence of OSP-specific plasma IgA, IgM, and IgG antibody responses on study entry were associated with a decrease in the risk of infection in household contacts (IgA, p = 0.015; IgM, p = 0.01, and IgG, p = 0.024). In addition, the presence of OSP-specific IgG memory B cell responses in peripheral blood on study entry was also associated with a decreased risk of infection (44% reduction; 95% CI: 31.1 to 99.8) in contacts. No protection was associated with cholera toxin B subunit (CtxB)-specific memory B cell responses. Conclusion: These results suggest that immune responses that target OSP, both in plasma and memory responses, may be important in mediating protection against infection with V. cholerae O1

Plasma and memory B cell responses targeting O-specific polysaccharide (OSP) are associated with protection against <i>Vibrio cholerae</i> O1 infection among household contacts of cholera patients in Bangladesh

<div><p>Background</p><p>The mediators of protection against cholera, a severe dehydrating illness of humans caused by <i>Vibrio cholerae</i>, are unknown. We have previously shown that plasma IgA as well as memory B IgG cells targeting lipopolysaccharide (LPS) of <i>Vibrio cholerae</i> O1 correlate with protection against <i>V</i>. <i>cholerae</i> O1 infection among household contacts of cholera patients. Protection against cholera is serogroup specific, and serogroup specificity is defined by the O-specific polysaccharide (OSP) component of LPS. Therefore, we prospectively followed household contacts of cholera patients to determine whether OSP-specific immune responses present at the time of enrollment are associated with protection against <i>V</i>. <i>cholerae</i> infection.</p><p>Methodology</p><p>In this study, we enrolled two hundred forty two household contacts of one hundred fifty index patients who were infected with <i>Vibrio cholerae</i>. We determined OSP-specific memory B cells and plasma IgA, IgG and IgM antibody responses on study entry (day 2).</p><p>Principle findings</p><p>The presence of OSP-specific plasma IgA, IgM, and IgG antibody responses on study entry were associated with a decrease in the risk of infection in household contacts (IgA, p = 0.015; IgM, p = 0.01, and IgG, p = 0.024). In addition, the presence of OSP-specific IgG memory B cell responses in peripheral blood on study entry was also associated with a decreased risk of infection (44% reduction; 95% CI: 31.1 to 99.8) in contacts. No protection was associated with cholera toxin B subunit (CtxB)-specific memory B cell responses.</p><p>Conclusion</p><p>These results suggest that immune responses that target OSP, both in plasma and memory responses, may be important in mediating protection against infection with <i>V</i>. <i>cholerae</i> O1.</p></div

Immunoreactivity in human plasma of OSP:rTTHc, OSP:BSA, and TT.

<p>Immunoreactivity of OSP:rTTHc, OSP:BSA and TT was measured in day 2 versus day 7 plasma of patients with cholera versus typhoid fever in Dhaka, Bangladesh.</p

Memory B cell IgG responses in spleen at day 56 targeting O-specific polysaccharide (OSP) in various vaccine cohorts following intramuscular (IM) or intradermal (ID) immunization with various doses (based on OSP component) and/or loading ratios of OSP to TTHc, with or without adjuvantative alum.

<p>Mice were immunized on days 0, 21, and 42. Responder frequency reflects an increase of >5 times total IgG secreting cells with stimulation versus no stimulation, and >3.5 anti-OSP spots per 10⁵ splenocytes. An asterisk denotes a statistically significant difference (<i>P</i><0.05) in the mean response from the group immunized with only alum.</p

Survival likelihood of neonatal CD -1 mice following oral challenge with wild type <i>V</i>. <i>cholerae</i> O1 El Tor Inaba strain N16961.

<p>Three to five day old pups were orally gavaged with 50 μl of a preparation containing 5.8x10⁹ CFU of wild type <i>V</i>. <i>cholerae</i> N16961 mixed with a 1:250 dilution of pooled day 42 serum from mice intramuscularly immunized with conjugate vaccine (OSP:rTTHc) and immunoadjuvantative alum, or alum alone. Survival curves were compared by log rank testing.</p