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. 2015 Oct 9;10(10):e0139803.
doi: 10.1371/journal.pone.0139803. eCollection 2015.

Antibodies Directed against Shiga-Toxin Producing Escherichia coli Serotype O103 Type III Secreted Proteins Block Adherence of Heterologous STEC Serotypes to HEp-2 Cells

Taseen S Desin  1 , Hugh G Townsend  1 , Andrew A Potter  1
Affiliations

Antibodies Directed against Shiga-Toxin Producing Escherichia coli Serotype O103 Type III Secreted Proteins Block Adherence of Heterologous STEC Serotypes to HEp-2 Cells

Taseen S Desin et al. PLoS One. .

Abstract

Shiga toxin-producing Escherichia coli (STEC) serotype O103 is a zoonotic pathogen that is capable of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The main animal reservoir for STEC is ruminants and hence reducing the levels of this pathogen in cattle could ultimately lower the risk of STEC infection in humans. During the process of infection, STECO103 uses a Type III Secretion System (T3SS) to secrete effector proteins (T3SPs) that result in the formation of attaching and effacing (A/E) lesions. Vaccination of cattle with STEC serotype O157 T3SPs has previously been shown to be effective in reducing shedding of STECO157 in a serotype-specific manner. In this study, we tested the ability of rabbit polyclonal sera against individual STECO103 T3SPs to block adherence of the organism to HEp-2 cells. Our results demonstrate that pooled sera against EspA, EspB, EspF, NleA and Tir significantly lowered the adherence of STECO103 relative to pre-immune sera. Likewise, pooled anti-STECO103 sera were also able to block adherence by STECO157. Vaccination of mice with STECO103 recombinant proteins induced strong IgG antibody responses against EspA, EspB, NleA and Tir but not against EspF. However, the vaccine did not affect fecal shedding of STECO103 compared to the PBS vaccinated group over the duration of the experiment. Cross reactivity studies using sera against STECO103 recombinant proteins revealed a high degree of cross reactivity with STECO26 and STECO111 proteins implying that sera against STECO103 proteins could potentially provide neutralization of attachment to epithelial cells by heterologous STEC serotypes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Inhibition of STECO103 adherence to HEp-2 cells by either (A) pooled sera against recombinant STECO103 T3SPs or (B and C) individual serum samples specific for STECO103 recombinant proteins EspB, EspF, EspG or NleA.
Anti-O103 refers to antibodies against a secreted fraction of T3SPs from STECO103. Values are expressed as median bacteria per cell from 8 replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Fig 2
Fig 2. Inhibition of STECO157 adherence to HEp-2 cells by pooled sera against recombinant STECO103 T3SPs.
(A) STECO157 adherence to HEp-2 cells was significantly lower in the presence of antibodies against STECO103 EspA, EspB, EspF, NleA and Tir. (B) STECO157 adherence was partially lower in the presence of STECO103 anti-EspA and anti-Tir or anti-EspB, anti-EspF and anti-NleA. Values are expressed as median bacteria per cell from 8 replicates. **, P < 0.01; ****, P < 0.0001.
Fig 3
Fig 3. STECO103 shedding in feces following oral challenge in mice.
Mice were immunized subcutaneously with either PBS (control) or a pool of STECO103 EspA, EspB, EspF, NleA and Tir followed by a booster immunization three weeks later. Two weeks after the second immunization, mice were orally challenged with 109 cfu of STECO103. N = 12 for both groups. Values are expressed as median cfu per gram of feces.
Fig 4
Fig 4. Cross-reactivity of STECO103 EspA, EspB, EspF, NleA and Tir specific rabbit polyclonal sera with the corresponding STECO26 and STECO111 recombinant proteins.
(A) Western blot using anti-EspA. Lane 1, marker; Lane 2, EspAO103 (20.5 kDa); Lane 3, EspAO26 (20.5 kDa); Lane 4, EspAO111(20.5 kDa). (B) Western blot using anti-EspB. Lane 1, marker; Lane 2, EspBO103 (33.1 kDa); Lane 3, EspBO26 (33.2 kDa); Lane 4, EspBO111 (32.8 kDa). (C) Western blot using anti-EspF. Lane 1, marker; Lane 2, EspFO103 (57 kDa); Lane 3, EspFO26 (40 kDa); Lane 4, EspFO111 (27 kDa). (D) Western blot using anti-NleA. Lane 1, marker; Lane 2, NleA O103 (44 kDa); Lane 3, NleA O26 (11.7 kDa); Lane 4, NleA O111 (47.5 kDa). (E) Western blot using anti- Tir. Lane 1, marker; Lane 2, Tir O103 (56 kDa); Lane 3, Tir O111 (56.9 kDa).
Fig 5
Fig 5. Cross-reactivity of STECO103 EspA, EspB, EspF, NleA and Tir specific mouse polyclonal sera with the corresponding STECO26 and STECO111 recombinant proteins.
Western blot using sera from mice immunized with a combination of STECO103 EspA, EspB, EspF, NleA and Tir to test the cross-reactivity with: (A) STECO26 proteins. Lane 1, marker; Lane 2, EspAO26 (20.5 kDa); Lane 3, EspBO26 (33.2 kDa); Lane 4, EspFO26 (40 kDa); and Lane 5, NleAO26 (11.7 kDa). (B) STECO111 proteins. Lane 1, marker; Lane 2, EspAO111 (20.5 kDa); Lane 3, EspBO111 (32.8 kDa); Lane 4, EspF O111 (27 kDa); Lane 5, NleA O111 (47.5 kDa); and Lane 6, TirO111 (56.9 kDa).
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