Fig 1.
Vi increases serum resistance of Salmonella and reduces killing of Salmonella by peripheral blood phagocytes.
Killing of isogenic Vi-expressing and non-expressing S. Typhimurium (A) and S. Typhi (B) strains by ten individual adult sera characterized for anti-Salmonella antibody content in S1 Fig. For blood cell killing, isogenic Vi-expressing and non-expressing S. Typhimurium (C) and S. Typhi (D) were opsonized with antibodies from ten adult sera and C6-deficient serum then added to washed peripheral blood cells. Killing of Salmonella strains expressing (dashed lines) or not expressing (dotted lines) Vi capsule was recorded at 45, 90 and 180 minutes. C6-deficient serum was used as the source of complement, because it permits opsonization with C3b, but does not result in membrane attack complex formation. Solid lines in A and B are Vi+ and Vi- bacteria incubated with heat-inactivated sera (HiS). Solid lines in C and D represent Salmonella incubated with PBS and added to blood cells, or opsonized with serum and added to RPMI. Lines represent mean values using sera from ten adults in triplicate experiments. Error bars represent SEM.
Fig 2.
Vi capsule protects against cell damage following serum exposure.
Cellular integrity of and damage to Salmonella was assessed by transmission electron microscopy (TEM) after exposure to PBS or human sera. TEM images of isogenic (A and E) Vi+ S. Typhimurium, (B and F) Vi- S. Typhimurium, (C and G) Vi+ S. Typhi and (D and H) Vi- S. Typhi after incubation with either PBS (A–D respectively) or with undiluted human serum from Donor 1 (E–H respectively) for ten minutes. Representative images shown.
Fig 3.
Vi expression decreases antibody and complement binding to Salmonella in the absence of specific antibodies.
Levels of (A and B) IgG, (C and D) C3 and (E and F) C5b-9 MAC on Vi-expressing and non-expressing Salmonella Typhimurium (STM) and S. Typhi (STY) after incubation with sera containing anti-Vi IgG (A, C and E) or lacking anti-Vi IgG (B, D and F) were measured by flow cytometry. Levels of IgM binding to Vi+/- Salmonella for all ten sera were also assessed (G). All sera contained IgM anti-Vi antibodies. Bars represent mean +/- SEM
Fig 4.
Sera depleted of Salmonella-specific antibodies effect variable killing of different Salmonella strains.
Killing of Vi+ S. Typhimurium (STM) (A) and S. Typhi (STY) (B) and Vi- S. Typhimurium (C) and S. Typhi (D) by donor 1 serum adsorbed of antibodies with Vi+ and Vi- S. Typhimurium and S. Typhi or unadsorbed serum. Dashed lines represent killing of Salmonella by sera adsorbed using the homologous Salmonella strain. Dotted lines represent killing by unadsorbed serum. Experiments were performed in triplicate and error bars represent SEM. Initial concentration of bacteria in each assay was 106 CFU/ml.
Fig 5.
Media osmolarity affects the binding of human anti-Vi and anti-O:9 antibodies to wild-type S. Typhi.
Five wild-type S. Typhi isolates, one from each geographical site to ensure the greatest geographical variation among strains studied, were cultured in LB broth containing 9, 85 or 500mM NaCl and incubated with either purified human anti-Vi (A) or anti-O:9 (B) antibodies or PBS. Levels of antibody binding to S. Typhi were assessed by flow cytometry. Values are mean of three separate experiments. Bars represent mean +/- SEM.
Fig 6.
Bactericidal and opsonic activity of anti-Vi and anti-O:9 antibodies against S. Typhi depends on medium osmolarity.
The bactericidal (A–C) and opsonic (D–F) activity of purified human anti-Vi (red lines) and anti-O:9 (blue lines) antibodies supplemented with human complement was tested against wild-type S. Typhi. S. Typhi isolates were cultured in LB Broth containing 9 (high Vi expression, A and D), 85 (B and E) or 500mM (low Vi expression, C and F) NaCl. Isolates tested at 9mM or 500mM NaCl concentrations were the same and included in the 14 isolates tested at 85mM NaCl. They were selected from each geographical site to ensure the greatest geographical variation among strains studied. Each line represents an individual S. Typhi isolate. Data represent mean of three separate experiments +/- SEM.
Fig 7.
MAC deposition in the presence of anti-Vi, but not anti-O:9 antibodies, is associated with bactericidal activity.
Levels of C5b-9 MAC deposition on the surface of five wild-type S. Typhi isolates cultured at 9, 85 and 500mM NaCl in the presence of purified human anti-Vi antibodies (A) or anti-O:9 antibodies (B) were measured by flow cytometry. For each of the five isolates at each concentration of sodium chloride, killing in the serum bactericidal assay was plotted against the level of MAC deposition for anti-Vi antibodies (C) or anti-O:9 antibodies (D). Data are mean of three separate experiments. Bars represent mean +/- SEM.