Salmonella Typhimurium is a Gram-negative bacterium that causes enterocolitis in humans and pigs. Lipopolysaccharide (LPS) is a component of the outer leaflet of Gram-negative bacteria that provokes endotoxin shock. LPS can be synthesized completely or incompletely and creates S (smooth) or R (rough) chemotypes. Toll-like receptors (TLR) 2, 4, and 9 initiate an inflammatory reaction to combat bacterial infections. We associated/challenged one-week-old gnotobiotic piglets with wild-type S. Typhimurium with S chemotype or its isogenic ∆rfa mutants with R chemotype LPS. The wild-type S. Typhimurium induced TLR2 and TLR4 mRNA expression but not TLR9 mRNA expression in the ileum and colon of one-week-old gnotobiotic piglets 24 h after challenge. The TLR2 and TLR4 stimulatory effects of the S. Typhimurium ∆rfa mutants were related to the completeness of their LPS chain. The transcription of IL-12/23 p40, IFN-γ, and IL-6 in the intestine and the intestinal and plasmatic levels of IL-12/23 p40 and IL-6 but not IFN-γ were related to the activation of TLR2 and TLR4 signaling pathways. The avirulent S. Typhimurium ∆rfa mutants are potentially useful for modulation of the TLR2 and TLR4 signaling pathways to protect the immunocompromised gnotobiotic piglets against subsequent infection with the virulent S. Typhimurium.

• MeSH
• Germ-Free Life physiology   MeSH
• Ileum metabolism   microbiology   MeSH
• Colon metabolism   microbiology   MeSH
• Swine, Miniature MeSH
• Mutation physiology   MeSH
• Swine MeSH
• Salmonella typhimurium genetics   isolation & purification   MeSH
• Salmonella Infections genetics   metabolism   pathology   MeSH
• Toll-Like Receptor 4 metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In this study, we were interested in the association of attenuated mutants of Salmonella enterica serovar Enteritidis with subpopulations of porcine white blood cells (WBC). The mutants included those with inactivated aroA, phoP, rfaL, rfaG, rfaC and fliC genes and a mutant with five major pathogenicity islands removed (ΔSPI1-5 mutant). Using flow cytometry, we did not observe any difference in the interactions of the wild-type S. Enteritidis, aroA and phoP mutants with WBC. ΔSPI1-5 and fliC mutants had a minor defect in their association with granulocytes and monocytes, but not with T- or B-lymphocytes. All three rfa mutants associated with granulocytes, monocytes and B-lymphocytes more than the wild-type S. Enteritidis did. Electron microscopy confirmed that the association correlated with the intracellular presence of S. Enteritidis and that the Salmonella-containing vacuole in the WBC infected with the rfa mutants, unlike all other strains, did not develop into a spacious phagosome. Intact lipopolysaccharide, but not the type III secretion system encoded by SPI-1, SPI-2 or the flagellar operon, is important for the initial interaction of S. Enteritidis with porcine leukocytes. This information can be used for the design of live Salmonella vaccines preferentially targeting particular cell types including cancer or tumor cells.

• MeSH
• Genes, Bacterial genetics   MeSH
• Glycosyltransferases genetics   MeSH
• Leukocytes microbiology   ultrastructure   MeSH
• Mutation genetics   MeSH
• Swine Diseases immunology   microbiology   pathology   MeSH
• Swine MeSH
• Salmonella enteritidis enzymology   genetics   immunology   MeSH
• Salmonella Infections, Animal immunology   microbiology   pathology   MeSH
• Salmonella Vaccines immunology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The key process for immune response development is the recognition of bacteria by the immune system of the host based on the sensing of pathogen-associated molecular patterns (PAMP). One of the most important PAMP is the lipopolysaccharide (LPS) molecule, a complex molecule present in the outer membrane of Gram negative bacteria. In this study we were interested in how different parts of the LPS of Salmonella enterica serovar Enteritidis are recognized by porcine neutrophils. To this aim, we constructed S. Enteritidis mutants with rfaL and rfaC genes disabled in the attachment of the O-antigen and in the synthesis of the inner oligosaccharide core of LPS, respectively. We found that in the absence of serum, both the rfa mutants associated with neutrophils and stimulated them for reactive oxygen species (ROS) production significantly more than the wild-type strain. Addition of polymyxin B, which neutralized lipid A, the endotoxic moiety of LPS, effectively decreased the association of the wild-type strain and the rfaC mutant with neutrophils, but not the rfaL mutant. This indicates that the oligosaccharide core newly exposed on the surface in the rfaL mutant, protected from interaction in the wild-type strain by the O-antigen but completely absent in the rfaC mutant, may represent a new ligand for porcine neutrophils that cannot be neutralized by polymyxin B.

• MeSH
• Lipopolysaccharides chemistry   MeSH
• Mutation MeSH
• Neutrophils immunology   microbiology   MeSH
• O Antigens chemistry   genetics   MeSH
• Polymyxin B pharmacology   MeSH
• Swine MeSH
• Reactive Oxygen Species metabolism   MeSH
• Salmonella enteritidis chemistry   genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH