Bacillus anthracis-derived edema toxin (ET) counter-regulates movement of neutrophils and macromolecules through the endothelial paracellular pathway

<p>Abstract</p> <p>Background</p> <p>A common finding amongst patients with inhalational anthrax is a paucity of polymorphonuclear leukocytes (PMNs) in infected tissues in the face of abundant circulating PMNs. A major virulence determinant of anthrax is edema toxin (ET), which is formed by the combination of two proteins produced by the organism, edema factor (EF), which is an adenyl cyclase, and protective antigen (PA). Since cAMP, a product of adenyl cyclase, is known to enhance endothelial barrier integrity, we asked whether ET might decrease extravasation of PMNs into tissues through closure of the paracellular pathway through which PMNs traverse.</p> <p>Results</p> <p>Pretreatment of human microvascular endothelial cell(EC)s of the lung (HMVEC-L) with ET decreased interleukin (IL)-8-driven transendothelial migration (TEM) of PMNs with a maximal reduction of nearly 60%. This effect required the presence of both EF and PA. Conversely, ET did not diminish PMN chemotaxis in an EC-free system. Pretreatment of subconfluent HMVEC-Ls decreased transendothelial ¹⁴C-albumin flux by ~ 50% compared to medium controls. Coadministration of ET with either tumor necrosis factor-α or bacterial lipopolysaccharide, each at 100 ng/mL, attenuated the increase of transendothelial ¹⁴C-albumin flux caused by either agent alone. The inhibitory effect of ET on TEM paralleled increases in protein kinase A (PKA) activity, but could not be blocked by inhibition of PKA with either H-89 or KT-5720. Finally, we were unable to replicate the ET effect with either forskolin or 3-isobutyl-1-methylxanthine, two agents known to increase cAMP.</p> <p>Conclusions</p> <p>We conclude that ET decreases IL-8-driven TEM of PMNs across HMVEC-L monolayers independent of cAMP/PKA activity.</p

Towards a human oral vaccine for anthrax: the utility of a Salmonella Typhi Ty21a-based prime-boost immunization strategy

We previously demonstrated the ability of an orally administered attenuated Salmonella enterica serovar Typhimurium strain expressing the protective antigen (PA) of Bacillus anthracis to confer protection against lethal anthrax aerosol spore challenge [Stokes MG, Titball RW, Neeson BN, et al. Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis. Infect Immun 2007;75(April (4)):1827–34]. To extend the utility of this approach to humans we constructed variants of S. enterica serovar Typhi Ty21a, an attenuated typhoid vaccine strain licensed for human use, which expressed and exported PA via two distinct plasmid-based transport systems: the Escherichia coli HlyA haemolysin and the S. Typhi ClyA export apparatus. Murine immunogenicity studies confirmed the ability of these constructs, especially Ty21a expressing the ClyA-PA fusion protein, to stimulate strong PA-specific immune responses following intranasal immunization. These responses were further enhanced by a subsequent boost with either parenterally delivered recombinant PA or the licensed US human alum-adsorbed anthrax vaccine (AVA). Anthrax toxin neutralizing antibody responses using this prime-boost regimen were rapid, vigorous and broad in nature. The results of this study demonstrate the feasibility of employing a mucosal prime with a licensed Salmonella Typhi vaccine strain followed by a parenteral protein boost to stimulate rapid protective immunity against anthrax

A mouse model of human TLR4 D299G/T399I SNPs reveals mechanisms of altered LPS and pathogen responses

Two cosegregating single-nucleotide polymorphisms (SNPs) in human TLR4, an A896G transition at SNP rs4986790 (D299G) and a C1196T transition at SNP rs4986791 (T399I), have been associated with LPS hyporesponsiveness and differential susceptibility to many infectious or inflammatory diseases. However, many studies failed to confirm these associations, and transfection experiments resulted in conflicting conclusions about the impact of these SNPs on TLR4 signaling. Using advanced protein modeling from crystallographic data of human and murine TLR4, we identified homologous substitutions of these SNPs in murine Tlr4, engineered a knock-in strain expressing the D298G and N397I TLR4 SNPs homozygously, and characterized in vivo and in vitro responses to TLR4 ligands and infections in which TLR4 is implicated. Our data provide new insights into cellular and molecular mechanisms by which these SNPs decrease the TLR4 signaling efficiency and offer an experimental approach to confirm or refute human data possibly confounded by variables unrelated to the direct effects of the SNPs on TLR4 functionality

Inhibition of endogenous sialidase activity suppressed LPS-induced NFκB activation in HEK cells.

<p>HEK293T cells were infected with recombinant adenoviruses expressing human Neu1 (Ad-Neu1) or Neu3 (Ad-Neu3), or with virus containing empty vector (Ad-GFP) and sialidase activity from cell lysates was assayed using 4-MUNANA as substrate in the absence or presence of the sialidase inhibitor 2-DN (250 µg/ml) (A). TLR4/CD14/MD2-transfected HEK293T cells were further infected with Ad-Neu1, Ad-Neu3 or Ad-GFP, for 2 days, stimulated with LPS (1 ng/ml) for 16 h, and evaluated for luciferase activity (B). TLR4/CD14/MD2-transfected HEK293T cells were incubated with 2-DN (250 µg/ml) or KDO (250 µg/ml) for 2 days, and stimulated with different concentrations of LPS for 16 h prior to analysis of cell lysates for luciferase activity (C). Results shown are representative of data from at least 3 independent experiments, each with similar results. ND: not done.</p

Effect of P714H, N35A/N173A, and N35A/N173A/N205A mutations on the ability of TLR4 to mediate LPS-induced activation.

<p>HEK293T cells were co-transfected with expression vectors encoding wild type TLR4 (WT) or P714H, N35A/N173A, and N35A/N173A/N205A mutants along with pEFBOS-MD2, pCDNA3-CD14, pELAM-luc, and pTK-<i>Renilla</i>-luc. Transfected cells were stimulated with LPS for 6 h, and firefly vs. renilla luciferase activities were measured in cell lysates. Data were processed using Student t-test. *p<0.005; **p<0.05 (vs. WT).</p

Sialylation of TLR4 and MD2.

<p>Recombinant human TLR4-His/MD2-His proteins expressed in the mammalian NS0 cell line were separated by SDS-PAGE and analyzed on immunoblot for binding to lectin SNA, MAAII and to anti-His antibody (A). Recombinant human TLR4-His/MD2-His expressed in NS0 cells (lane 1) or recombinant human MD2-His expressed in <i>E. coli</i> (lane 2) were separated by SDS-PAGE and probed by lectin blot with SNA (left) and on immunoblot with anti-His antibody (right) (B). Recombinant human CD14 (lane 1) were separated by SDS-PAGE and probed by lectin blot with SNA (left) and MAAII (right). The highly sialylated glycoprotein, fetuin (lane 2) and asialofetuin (lane 3) were included as positive and negative controls respectively (C). HEK293T cells were transfected with control pcDNA, or plasmids encoding TLR4-YFP alone or with MD2 and CD14 expression plasmids, and proteins from cell lysates were immunoprecipitated with ant-GFP antibody and probed on immunoblot with either anti-GFP antibody (top) or SNA (bottom) (D). Proteins from the medium of MD2-transfected cells were immunoprecipitated with anti-FLAG antibody and probed on immunoblot with anti-FLAG antibody (left) or SNA (right) (E). The expected molecular weights of MD2 and TLR4 are indicated by arrows. Results shown are representative of data from at least 2 independent experiments, each with similar results.</p

Optimal LPS-induced signaling when both TLR4/CD14 and MD2 treated with neuraminidase.

<p>HEK293T cells that were transfected as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032359#pone-0032359-g002" target="_blank">Figure 2</a> and TLR4/CD14-transfected cells were supplemented with NA-treated SNT from MD2 transfected cells (MD2*), heated-inactivated NA-treated SNT (MD2**), or SNT from empty vector transfected cells (pcDNA) before LPS stimulation (5 ng/ml). Cell lysates were harvested after 16 h for reporter assay (A). TLR4/CD14-transfected HEK293T cells were treated with NA, heat-inactivated NA (ΔNA), or were untreated (-) for one hour at 37°C, stimulated with LPS (5 ng/ml) for 16 h in the presence of SNT from MD2 transfected cells (sMD2), which had been pre-treated with NA agarose (NA), heated-inactivated NA agarose (ΔNA), or were untreated, and luciferase activities were determined in cell lysates (B). TLR4/MD2-transfected HEK293T cells were supplemented with NA treated recombinant human CD14 (rhCD14[NA]), untreated CD14 (rhCD14), or medium (none) prior to LPS stimulation (1–5 ng/ml). Cell lysates were harvested after 16 h for reporter assay (C). Results shown are representative of data from at least 3 independent experiments, each with similar results.</p