The Salmonella enterica serotype typhimurium effector proteins SipA, SopA, SopB, SopD, and SopE2 act in concert to induce diarrhea in calves

Abstract

Salmonella enterica serotype Typhimurium requires a functional type III secretion system encoded by Salmonella pathogenicity island 1 (SPI1) to cause diarrhea. We investigated the role of genes encoding secreted target proteins of the SPI1-associated type III secretion system for enteropathogenicity in calves. Salmonella serotype Typhimurium strains having mutations in sptP, avrA, sspH1, or slrP induced fluid secretion in the bovine ligated ileal loop model at levels similar to that of the wild type. In contrast, mutations in sipA, sopA, sopB, sopD, or sopE2 significantly reduced fluid accumulation in bovine ligated ileal loops at 8 h postinfection. A strain carrying mutations in sipA, sopA, sopB, sopD, and sopE2 (sipA sopABDE2 mutant) caused the same level of fluid accumulation in bovine ligated ileal loops as a strain carrying a mutation in sipB, a SPI1 gene required for the translocation of effector proteins into host cells. A positive correlation was observed between the severity of histopathological lesions detected in the ileal mucosa and the levels of fluid accumulation induced by the different mutants. After oral infection of calves, the Salmonella serotype Typhimurium sipAsopABDE2 mutant caused only mild diarrhea and was more strongly attenuated than strains having only single mutations. These data demonstrate that SipA, SopA, SopB, SopD, and SopE2 are major virulence factors responsible for diarrhea during Salmonella serotype Typhimurium infection of calves.

FIG. 1.

Mutations in effector genes (solid arrows) which are required for fluid accumulation in calves. The open arrows indicate the positions of genes in the surrounding DNA region. Numbers indicate the positions of insertions or deletions relative to the first nucleotide (+1) in the open reading frame. The bars indicate internal fragments of sopD and sopE2 that were cloned into suicide vectors to inactivate these genes.

FIG. 2.

Ability of Salmonella serotype Typhimurium strains carrying mutations in SPI1 effector genes to induce secretory and inflammatory changes in bovine ligated ileal loops at 8 h postinfection. (A) Data for fluid accumulation in loops shown as percentages of the fluid secretion elicited by the wild type (ATCC 14028 for M119 and IR715 for ZA10, ZA11, ZA13, ZA14, and STN39). The 100% line indicates the amount of fluid accumulation elicited by the Salmonella serotype Typhimurium wild type. An asterisks indicates that the fluid accumulation was significantly lower than the wild-type response (P < 0.05). (B) Inflammatory responses scored on a scale from 1 to 5 according the criteria described in Materials and Methods. WT, wild type.

FIG. 3.

Ability of Salmonella serotype Typhimurium mutants carrying multiple mutations in SPI1 effector genes to induce secretory and inflammatory changes in bovine ligated ileal loops at 8 h postinfection. (A) Data for fluid accumulation in loops shown as percentages of the fluid secretion elicited by the wild type (IR715). The 100% line indicates the amount of fluid accumulation elicited by the Salmonella serotype Typhimurium wild type. All mutants shown elicited significantly less fluid accumulation than the wild type (IR715) (P < 0.05). The results are means from experiments performed with three animals, in which each strain was tested in two loops/animal. The bars indicate means ± standard deviations. The same letter above two bars indicates that the amounts of fluid accumulation elicited by the mutants are not significantly different. Different letters above two bars indicate that the amounts of fluid accumulation elicited by the mutants are significantly different (P < 0.05). (B) Inflammatory changes in infected loops assessed by microscopic examination of hematoxylin- and eosin-stained thin sections of infected tissue. wt, wild type.

FIG. 4.

In vivo complementation in bovine ligated ileal loops at 8 h postinfection of strains carrying a sipA deletion with the cloned sipA gene. (A) Data for fluid accumulation in loops shown as percentages of the fluid secretion elicited by the wild type (IR715). The results are means from three experiments in which each strain was tested in at least two loops/animal. The bars indicate means ± standard deviations. The same letter above two bars indicates that the amounts of fluid accumulation elicited by the mutants are not significantly different. Different letters above two bars indicate that the amounts of fluid accumulation elicited by the mutants are significantly different (P < 0.05). (B) Inflammatory changes in infected loops assessed by microscopic examination of hematoxylin- and eosin-stained thin sections of infected tissue. wt, wild type.

FIG. 5.

Ability of SPI1 mutants to cause diarrhea and lethal morbidity in calves infected orally with a dose of 10¹⁰ CFU per animal. Lethal morbidity caused by the wild type or by Salmonella serotype Typhimurium mutants over time is shown in the upper graphs. The lower graphs show the severity of diarrhea over time, which was scored on a scale from 1 to 5, as follows: 1, normal feces; 2, soft feces with loss of distinct conformation; 3, diarrhea, loose feces with reduced solid matter; 4, diarrhea, aqueous feces with markedly reduced or little solid matter, or fibrin; 5, diarrhea, aqueous feces with no solid matter, fibrin and blood.

FIG. 6.

Change in the plasma sodium concentration during oral infection of calves with Salmonella serotype Typhimurium strains. The graph shows the difference in the plasma sodium concentration between a sample collected on day 2 postinfection and a preinfection sample. Solid circles indicate that the plasma sodium concentration on day 2 postinfection was below the normal range. The open circle indicates that the plasma sodium concentration on day 2 postinfection was within the normal range. WT, wild type.

FIG. 7.

Recovery of Salmonella serotype Typhimurium strains from the feces of calves after oral infection with a dose of 10¹⁰ CFU per animal. The bars indicate the mean ± standard deviation for four calves. An asterisks indicates that the difference between the wild type (IR715) and an individual mutant was significant. wt, wild type.

FIG. 8.

Representative examples of the gross pathology and histopathology of Peyer's patches and the terminal ileum of calves inoculated orally with 10¹⁰ CFU of different Salmonella serotype Typhimurium strains. (A) Severe acute fibrinopurulent necrotizing enteritis with segmental or continuous pseudomembrane formation in a calf infected with wild-type strain IR715 (similar pathological changes were observed in calves infected with the sopA mutant, the sopD mutant, or the sopE2 mutant). Bar = 1 cm. (B) Marked subacute fibrinopurulent necrotizing enteritis often confined to the Peyer's patches of the terminal ileum of a calf infected with strain ZA10 (sipA). Bar = 1 cm. (C) Normal Peyer's patches and ileum of a calf infected with strain ZA21 (sipAsopABDE). Bar = 1 cm. (D to F) Hematoxylin- and eosin-stained sections of Peyer's patches of calves infected with IR715, ZA10 (sipA), and ZA21 (sipAsopABDE), respectively. The short arrows indicate areas of lymphoid depletion; the long arrows indicate various degrees of fibrinopurulent necrotizing ileitis at the mucosal surface. Bars = 200 μm.