pr-39 and Salmonella-Infections--Animal

PR-39 is a porcine antimicrobial peptide that kills bacteria with a mechanism that does not involve cell lysis. Here, we demonstrate that Salmonella enterica serovar Typhimurium can rapidly acquire mutations that reduce susceptibility to PR-39. Resistant mutants appeared at a rate of 0.4 x 10(-6) per cell per generation. These mutants were about four times more resistant than the wild type and showed a greatly reduced rate of killing. Genetic analysis revealed mutations in the putative transport protein SbmA as being responsible for the observed resistance. These sbmA mutants were as fit as the wild-type parental strain as measured by growth rates in culture medium and mice and by long-term survival in stationary phase. These results suggest that resistance to certain antimicrobial peptides can rapidly develop without an obvious fitness cost for the bacteria and that resistance development could become a threat to the efficacy of antimicrobial peptides if used in a clinical setting.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Proteins; Cathelicidins; Drug Resistance, Multiple, Bacterial; Female; Genetic Complementation Test; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Models, Genetic; Mutagenesis, Insertional; Mutation; Salmonella Infections, Animal; Salmonella typhimurium; Sequence Deletion

Cathelicidins are a family of antimicrobial peptides prominent in the host defense mechanisms of several mammalian species. In addition to their antimicrobial activities, these peptides have been implicated in wound healing, angiogenesis, and other innate immune mechanisms. To investigate the regulatory mechanisms of cathelicidin gene expression, we conducted in vitro experiments evaluating the bone marrow cell expression of two porcine cathelicidins, PR-39 and protegrin, and cloned and evaluated the promoter sequence of PR-39. In addition, we evaluated in vivo kinetics of cathelicidin gene expression in pigs during an infection with Salmonella enterica serovar Typhimurium. Lipopolysaccharide (LPS) increased PR-39 and protegrin mRNA expression, which was ameliorated by polymyxin B. Concentrations of PR-39 in supernatants from bone marrow cell cultures were increased 10-fold after LPS stimulation. Similarly, interleukin-6 (IL-6) and all-trans retinoic acid (RA) markedly induced cathelicidin gene expression. To verify the transcriptional activation of the PR-39 gene by these agents, we made a PR-39 promoter-luciferase construct containing the full-length PR-39 promoter driving luciferase gene expression and transiently transfected PK-15 epithelial cells. RA and IL-6 increased luciferase activity in PK-15 cells transfected with the PR-39 promoter-luciferase reporter. Similarly, Salmonella-challenged pigs showed increased expression of PR-39 and protegrin mRNA in bone marrow cells at 6 and 24 h postchallenge. Taken together, these findings show that bacterial products (LPS), IL-6, RA, and Salmonella infection enhance the expression of the cathelicidins, PR-39 and protegrin, in bone marrow progenitor cells, and we suggest that extrinsic modulation of this innate host defense mechanism may be possible.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Base Sequence; Cathelicidins; Cells, Cultured; Gene Expression Regulation; Interleukin-6; Lipopolysaccharides; Molecular Sequence Data; Promoter Regions, Genetic; Salmonella Infections, Animal; Salmonella typhimurium; Swine; Tretinoin

PR-39 is a multifunctional neutrophil peptide involved in host defense and inflammation. To investigate the involvement of PR-39 in a Salmonella choleraesuis infection, a PR-39 enzyme immunoassay was developed. The concentrations of PR-39 in serum were 13.6 +/- 1.9 ng/ml before challenge and increased (P < 0.01) threefold by 10 to 14 days postinfection. Peripheral blood neutrophil counts paralleled the changes in the concentrations of PR-39 in serum, both returned to basal values by 4 weeks postinfection. These findings suggest that the concentrations of serum PR-39 reflect the involvement of this antibacterial peptide in the host's response to an S. choleraesuis infection.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Blood Bactericidal Activity; Enzyme-Linked Immunosorbent Assay; Goats; Mice; Mice, Inbred BALB C; Neutrophil Activation; Neutrophils; Peptides; Salmonella Infections, Animal; Stimulation, Chemical; Swine; Tetradecanoylphorbol Acetate