Increased antigen specific T cell numbers in the absence of altered migration or division rates as a result of mucosal cholera toxin administration

Cholera toxin (CT) is a mucosal adjuvant capable of inducing strong immune responses to co-administered antigens following oral or intranasal immunization of mice. To date, the direct effect of CT on antigen-specific CD4(+) T cell migration and proliferation profiles in vivo is not well characterized. In this study, the effect of CT on the migration pattern and proliferative responses of adoptively transferred, CD4(+) TCR transgenic T cells in orally or intranasally vaccinated mice, was analyzed by flow cytometry. GFP-expressing or CFSE-labeled OT-II lymphocytes were adoptively transferred to naïve C57BL/6 mice, and mice were subsequently vaccinated with OVA with or without CT via the oral or intranasal route. CT did not alter the migration pattern of antigen-specific T cells, regardless of the route of immunization, but increased the number of transgenic CD4(+) T cells in draining lymphoid tissue. This increase in the number of transgenic CD4(+) T cells was not due to cells undergoing more rounds of cellular division in vivo, suggesting that CT may exert an indirect adjuvant effect on CD4(+) T cells. The findings reported here suggest that CT functions as a mucosal adjuvant by increasing the number of antigen specific CD4(+) T cells independent of their migration pattern or kinetics of cellular division.Grant support was received from the National Health and Medical Research Council of Australia (NHMRC). OLW is a recipient of an R.D. Wright Career Development Award

The Major Surface-Associated Saccharides of Klebsiella pneumoniae Contribute to Host Cell Association

Analysing the pathogenic mechanisms of a bacterium requires an understanding of the composition of the bacterial cell surface. The bacterial surface provides the first barrier against innate immune mechanisms as well as mediating attachment to cells/surfaces to resist clearance. We utilised a series of Klebsiella pneumoniae mutants in which the two major polysaccharide layers, capsule and lipopolysaccharide (LPS), were absent or truncated, to investigate the ability of these layers to protect against innate immune mechanisms and to associate with eukaryotic cells. The capsule alone was found to be essential for resistance to complement mediated killing while both capsule and LPS were involved in cell-association, albeit through different mechanisms. The capsule impeded cell-association while the LPS saccharides increased cell-association in a non-specific manner. The electrohydrodynamic characteristics of the strains suggested the differing interaction of each bacterial strain with eukaryotic cells could be partly explained by the charge density displayed by the outermost polysaccharide layer. This highlights the importance of considering not only specific adhesin:ligand interactions commonly studied in adherence assays but also the initial non-specific interactions governed largely by the electrostatic interaction forces

MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices

Salmonella enterica Serovar Typhimurium Infection of Dendritic Cells Leads to Functionally Increased Expression of the Macrophage-Derived Chemokine

Gene expression in murine dendritic cells (DCs) infected with green fluorescent protein-expressing Salmonella enterica serovar Typhimurium BRD509 was studied by mRNA differential display. Infected DCs were sorted from uninfected cells by flow cytometry. The mRNA expression patterns of infected and uninfected cells revealed a number of differentially expressed transcripts, which included the macrophage-derived chemokine (MDC). Up-regulation of MDC transcription in infected DCs was confirmed by Northern blotting, and the kinetics of MDC expression was examined by real-time reverse transcription-PCR, with which 31- and 150-fold increases were detected at 2 and 6 h postinfection, respectively. The increased release by DCs of MDC into culture media was detected by an enzyme-linked immunosorbent assay. The biological activity of MDC was investigated in in vitro and in vivo assays. In vitro, supernatants from S. enterica serovar Typhimurium-infected DCs were chemoattractive to T cells, and neutralization of MDC in these supernatants inhibited T-cell migration. Passive transfer of anti-MDC antibody to mice infected with BRD509 revealed that neither growth of the bacterium nor resistance of the mice to reinfection was affected and that in vivo inhibition of MDC did not affect T-cell responses, as measured by the gamma interferon ELISPOT method 3 days after challenge infection

Congenic NOD mouse strains exhibit different diabetes incidences and localize <i>Idd5</i>.<i>4</i>.

<p>The cumulative incidence of diabetes was determined for age-matched cohorts for NOD and NOD.B6-Chr1 R7 females (A) and males (B); and age-matched cohorts for NOD, NOD.B6-Chr1 R0 and NOD.B6-Chr1 R2 females (C) and males (D). Congenic NOD mouse strains were homozygous for their respective B6-derived intervals. Pairwise comparisons of diabetes incidence curves were performed using the log-rank test: (A) ** P = 0.001; (B) *** P = 4.7x10^-6. For panel (C) and (D), the P values were corrected for multiple testing (i.e. three comparisons): (C) 1: Holm-adjusted P = 0.09, 2: Holm-adjusted P = 0.09, 3**: P = 0.0002; (D) 1: Holm-adjusted P = 0.15, 2: Holm-adjusted P > 0.2, 3*: Holm adjusted P = 0.04.</p

Schematic diagram of mouse chromosome 1 and congenic intervals.

<p>Congenic strains names are abbreviated: R0 = NOD.B6-Chr1^{D1Mit48-D1Mit348}Pigr^-/-, R2 = NOD.B6-Chr1^{Pigr-D1Mit348}Pigr^-/-, R7 = NOD.B6-Chr1^{<i>D1Mit48-D1Mit495</i>}. Diabetes incidence for congenic strains is described relative to NOD mice (>NOD or < NOD, based on Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121979#pone.0121979.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121979#pone.0121979.g003" target="_blank">3</a>). <i>Idd5</i>.<i>4^a</i> represents the B10-derived interval defined by Hunter <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121979#pone.0121979.ref025" target="_blank">25</a>]; <i>Idd5</i>.<i>4^b</i> represents the B6-derived interval, defined by the R7 congenic strain, that confers increased susceptibility to diabetes; <i>IddX</i> represents the B6-derived interval harboring the <i>Pigr</i> null allele, defined by the R2 congenic strain, that confers protection against diabetes. Marker and gene positions are based on NCBI Bld37, mm9.</p