Interaction of <i>S</i>. Typhimurium ST19 and ST313 with macrophage cell lines.

<p><b>(A)</b> J774 murine macrophages were infected with four strains of <i>S</i>. Typhimurium ST19 (I77, I41, S52 and I89), five strains of ST313 (D65, Q55, S11, S12 and A13), <i>S</i>. Typhimurium SL1344 (ST19), <i>S</i>. Typhi Ty2 and <i>S</i>. Paratyphi A ATCC9150. The ability of these strains to be phagocytosed by J774 macrophage cells was determined 30 min p.i. <b>(B)</b> U937 human macrophages were infected with four strains of <i>S</i>. Typhimurium ST19 (I77, I41, S52 and I89), five strains of ST313 (D65, Q55, S11, S12 and A13), <i>S</i>. Typhimurium SL1344 (ST19), <i>S</i>. Typhi Ty2 and <i>S</i>. Paratyphi A ATCC9150 in a gentamicin protection assay. Percent survival was expressed as the number of intracellular bacteria at 24 h p.i. divided by the number of intracellular bacteria at 0 h p.i. times 100. <b>(C)</b><i>S</i>. Typhimurium I77 (ST19) and D65 (ST313) were used to infect human THP-1 cells. <i>S</i>. Typhimurium SL1344 (ST19) and <i>S</i>. Typhimurium D23580 (ST313) were used as controls for the assay. Percent survival was expressed as the number of intracellular bacteria at 24 h p.i. divided by the number of intracellular bacteria at 0 h p.i. times 100. Results are expressed as mean ± SD from at least 3 independent experiments. ** represents P <0.01.</p

Flagella expression in <i>S</i>. Typhimurium ST19 and ST313 strains.

<p><b>(A)</b> Motility assay using <i>S</i>. Typhimurium ST313 (D65, Q55, S11, S12) and <i>S</i>. Typhimurium ST19 (I77, S52, I41, I89). <b>(B)</b> Bacterial supernatant of <i>S</i>. Typhimurium (1) I77 (ST19), (2) D65 (ST313), (3) D65 Δ<i>guaBA</i> and (4) D65 Δ<i>guaBA</i> Δ<i>clpX</i> were analyzed by Western blot using a monoclonal antibody against <i>S</i>. Typhimurium flagellin. <b>(C)</b> Human HEK293 cells stably transfected with a firefly luciferase reporter gene under control of the NF-κB promoter were infected in triplicate with live or heat-killed wild-type <i>S.</i> Typhimurium I77 (ST19), D65 (ST313), D65 Δ<i>guaBA</i> and D65 Δ<i>guaBA</i> Δ<i>clpX</i> which overexpresses flagellin. Culture supernatants from all the strains were also used to activate NF-κB expression. HEK293 cells were harvested at 4 h after infection and luciferase activity (RLU) determined. Luciferase activity from infected cells is expressed relative to that obtained from uninfected cells. <b>(D)</b> Cytotoxicity assay on THP-1 cells infected with <i>S.</i> Typhimurium I77 (ST19), D65 (ST313), and D65 Δ<i>guaBA</i> and D65 Δ<i>guaBA</i> Δ<i>clpX</i> at 24 h p.i. * represents P<0.05.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Survival of <i>S</i>. Typhimurium ST313 and ST19 within primary mouse and human macrophages.

<p>Peritoneal macrophages from BALB/c mice <b>(A)</b> or CD-1 mice <b>(B)</b> or PBMCs from human donors <b>(C)</b> were infected with four strains of <i>S</i>. Typhimurium ST19 (I77, I41, S52 and I89), five strains of ST313 (D65, Q55, S11, S12 and A13), <i>S</i>. Typhimurium SL1344 (ST19), <i>S</i>. Typhi Ty2 and <i>S</i>. Paratyphi A ATCC9150 at a MOI of 10∶1. Percent survival was expressed as the number of intracellular bacteria at 24 h p.i. divided by the number of intracellular bacteria at 0 h p.i. times 100. Results are expressed as mean ± SD from at least 3 independent experiments. *** represents P<0.001.</p

NFκB expression in THP-1 cells infected with <i>S</i>. Typhimurium ST313 and ST19.

<p><b>(A)</b> Induction of phosphorylated p38 in THP-1 cells infected with <i>S</i>. Typhimurium D65 (ST313) or I77 (ST19) 30 min p.i. by Western blot analysis using polyclonal rabbit-anti-phosphorylated p38. <b>(B)</b> Densitometry quantification of P-p38 at 30 min p.i. of THP-1 cells by ImageJ software. <b>(C)</b> Induction of P-p65 in THP-1 cells infected with <i>S</i>. Typhimurium D65 (ST313) or I77 (ST19) at 60 min p.i. <b>(D)</b> Densitometry quantification of P-p65 in THP-1 cells by ImageJ software at 60 min p.i. with <i>S</i>. Typhimurium D65 (ST313) or I77 (ST19).</p

Toxicity assay to determine the effect of <i>S</i>. Typhimurium ST313 and ST19 on THP-1 macrophages.

<p>Human THP-1 cells were infected with <i>S</i>. Typhimurium I77 (ST19) or <i>S</i>. Typhimurium D65 (ST313) at a MOI of 10∶1 or left uninfected. Guava ViaCount was used to determine the number of viable, mid-apoptotic and apoptotic cells 24 h p.i. Results are expressed as mean ± SD from 3 independent experiments. ** represents P<0.01.</p

Cytokine expression of THP-1 cells infected with <i>S</i>. Typhimurium ST313 and ST19.

<p>Human THP-1 cells were infected with <i>S</i>. Typhimurium I77 (ST19), D65 (ST313) or left uninfected. <b>(A)</b> Total mRNA was isolated from the cells and expression profiles for different proinflammatory cytokines, IL-1β, IL-8 and TNF-α were analyzed by qRT-PCR at 3 h p.i. <b>(B)</b> IL-1β, IL-8 and TNF-α gene expression at 8 h p.i. (<b>C)</b> IL-8 and TNF-α protein expression analyzed at 3 h p.i. <b>(D)</b> IL-8 and TNF-α protein expression analyzed at 8 h p.i.</p

Passive immunization with monoclonal FliC antibodies and protection against lethal challenge.

<p>CD-1 mice (n = 10 /group) were intravenously administered PBS or 50 μg of either a monoclonal IgG specific for <i>S</i>. Enteritidis FliC (CA6IE2) or a monoclonal IgG specific for <i>S</i>. Typhimurium FliC (AH12IE6). Two to 3 hours later, they were infected intraperitoneally with 1 x 10⁵ CFU of <i>S</i>. Typhimurium D65, and monitored for 14 days recording mortality and overall health. P value by log-rank survival analysis versus control mice administered CA6IE2.</p

Comparison of SBA titers of <i>S</i>. Enteritidis anti-FliC polyclonal antibodies against heterologous <i>Salmonella</i> serovars.

<p>SBA titer produced by pooled sera (n = 10) from mice immunized with <i>S</i>. Enteritidis phase 1 flagellin against the target strains <i>S</i>. Enteritidis S15, <i>S</i>. Typhimurium D65, <i>S</i>. Paratyphi A ATCC9150, and <i>S</i>. Typhi Ty2. Dashed line = detection limit. Data shown is the average and standard deviation of triplicate wells, and representative of two independent experiments. P values were assessed by paired t-test.</p

Monoclonal IgG antibodies used in this study

<p>Monoclonal IgG antibodies used in this study</p