In this study we characterized aro mutants of Salmonella enterica serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the aroA, aroD, and aroC mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate aro gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the aroD mutant in serovar Typhimurium indicated that the reason for this sensitivity might be the upregulation of murA. To confirm this, we artificially overexpressed murA from a multicopy plasmid, and this overexpression caused sensitivity of the strain to albumen and EDTA but not to serum and ovotransferrin. We concluded that attenuation of aro mutants is caused not only by their inability to synthesize aromatic metabolites but also by their defect in cell wall and outer membrane functions associated with decreased resistance to components of innate immune response.

Veterinary Research Institute Hudcova 70 621 00 Brno Czech Republic

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Bar-Tana, J., B. J. Howlett, and R. Hertz. 1980. Ubiquinone synthetic pathway in flagellation of Salmonella typhimurium. J. Bacteriol. 143637-643. PubMed PMC

Bennett, G. M., A. Seaver, and P. H. Calcott. 1981. Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses. Appl. Environ. Microbiol. 42843-849. PubMed PMC

Brown, E. D., E. I. Vivas, C. T. Walsh, and R. Kolter. 1995. MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli. J. Bacteriol. 1774194-4197. PubMed PMC

Conlan, J. W., and R. J. North. 1992. Early pathogenesis of infection in the liver with the facultative intracellular bacteria Listeria monocytogenes, Francisella tularensis, and Salmonella typhimurium involves lysis of infected hepatocytes by leukocytes. Infect. Immun. 605164-5171. PubMed PMC

Cooper, G. L., L. M. Venables, M. J. Woodward, and C. E. Hormaeche. 1994. Vaccination of chickens with strain CVL30, a genetically defined Salmonella enteritidis aroA live oral vaccine candidate. Infect. Immun. 624747-4754. PubMed PMC

Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 976640-6645. PubMed PMC

Duncan, K., S. Chaudhuri, M. S. Campbell, and J. R. Coggins. 1986. The overexpression and complete amino acid sequence of Escherichia coli 3-dehydroquinase. Biochem. J. 238475-483. PubMed PMC

Duncan, K., and J. R. Coggins. 1986. The serC-aroA operon of Escherichia coli. A mixed function operon encoding enzymes from two different amino acid biosynthetic pathways. Biochem. J. 23449-57. PubMed PMC

Eschenburg, S., W. Kabsch, M. L. Healy, and E. Schonbrunn. 2003. A new view of the mechanisms of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate-3-phosphate synthase (AroA) derived from X-ray structures of their tetrahedral reaction intermediate states. J. Biol. Chem. 27849215-49222. PubMed

Everest, P., J. Ketley, S. Hardy, G. Douce, S. Khan, J. Shea, D. Holden, D. Maskell, and G. Dougan. 1999. Evaluation of Salmonella typhimurium mutants in a model of experimental gastroenteritis. Infect. Immun. 672815-2821. PubMed PMC

Galloway, S. M., and C. R. Raetz. 1990. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis. J. Biol. Chem. 2656394-6402. PubMed

Govoni, G., and P. Gros. 1998. Macrophage NRAMP1 and its role in resistance to microbial infections. Inflamm. Res. 47277-284. PubMed

Halsey, T. A., A. Vazquez-Torres, D. J. Gravdahl, F. C. Fang, and S. J. Libby. 2004. The ferritin-like Dps protein is required for Salmonella enterica serovar Typhimurium oxidative stress resistance and virulence. Infect. Immun. 721155-1158. PubMed PMC

Hoiseth, S. K., and B. A. D. Stocker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live oral vaccines. Nature 291238-239. PubMed

Hoiseth, S. K., and B. A. Stocker. 1985. Genes aroA and serC of Salmonella typhimurium constitute an operon. J. Bacteriol. 163355-361. PubMed PMC

Horii, T., T. Kimura, K. Sato, K. Shibayama, and M. Ohta. 1999. Emergence of fosfomycin-resistant isolates of Shiga-like toxin-producing Escherichia coli O26. Antimicrob. Agents Chemother. 43789-793. PubMed PMC

Hormaeche, C. E., H. S. Joysey, L. Desilva, M. Izhar, and B. A. Stocker. 1991. Immunity conferred by Aro− Salmonella live vaccines. Microb. Pathog. 10149-158. PubMed

Lowe, D. C., T. C. Savidge, D. Pickard, L. Eckmann, M. F. Kagnoff, G. Dougan, and S. N. Chatfield. 1999. Characterization of candidate live oral Salmonella typhi vaccine strains harboring defined mutations in aroA, aroC, and htrA. Infect. Immun. 67700-707. PubMed PMC

Methner, U., P. A. Barrow, D. Gregorova, and I. Rychlik. 2004. Intestinal colonization-inhibition and virulence of Salmonella phoP, rpoS, and ompC deletion mutants in chickens. Vet. Microbiol. 9837-43. PubMed

Nnalue, N. A. 1990. Mice vaccinated with a non-virulent, aromatic-dependent mutant of Salmonella choleraesuis die from challenge with its virulent parent but survive challenge with Salmonella typhimurium. J. Med. Microbiol. 31225-233. PubMed

Perez-Gutierrez, C., C. M. Llompart, M. Skurnik, and J. A. Bengoechea. 2007. Expression of the Yersinia enterocolitica pYV-encoded type III secretion system is modulated by lipopolysaccharide O-antigen status. Infect. Immun. 751512-1516. PubMed PMC

Robertsson, J. A., A. A. Lindberg, S. Hoiseth, and B. A. Stocker. 1983. Salmonella typhimurium infection in calves: protection and survival of virulent challenge bacteria after immunization with live or inactivated vaccines. Infect. Immun. 41742-750. PubMed PMC

Rychlik, I., L. Cardova, M. Sevcik, and P. A. Barrow. 2000. Flow cytometry characterisation of Salmonella typhimurium mutants defective in proton translocating proteins and stationary-phase growth phenotype. J. Microbiol. Methods 42255-263. PubMed

Rychlik, I., G. Martin, U. Methner, M. Lovell, L. Cardova, A. Sebkova, M. Sevcik, J. Damborsky, and P. A. Barrow. 2002. Identification of Salmonella enterica serovar Typhimurium genes associated with growth suppression in stationary-phase nutrient broth cultures and in the chicken intestine. Arch. Microbiol. 178411-420. PubMed

Shaio, M. F., and H. Rowland. 1985. Bactericidal and opsonizing effects of normal serum on mutant strains of Salmonella typhimurium. Infect. Immun. 49647-653. PubMed PMC

Silva, E. N., G. H. Snoeyenbos, O. M. Weinack, and C. F. Smyser. 1981. Studies on the use of 9R strain of Salmonella gallinarum as a vaccine in chickens. Avian Dis. 2538-52. PubMed

Splichal, I., I. Trebichavsky, A. Splichalova, and P. A. Barrow. 2005. Protection of gnotobiotic pigs against Salmonella enterica serotype Typhimurium by rough mutant of the same serotype is accompanied by the change of local and systemic cytokine response. Vet. Immunol. Immunopathol. 103155-161. PubMed

Stevenson, G., B. Neal, D. Liu, M. Hobbs, N. H. Packer, M. Batley, J. W. Redmond, L. Lindquist, and P. Reeves. 1994. Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster. J. Bacteriol. 1764144-4156. PubMed PMC

Stinavage, P., L. E. Martin, and J. K. Spitznagel. 1989. O antigen and lipid A phosphoryl groups in resistance of Salmonella typhimurium LT-2 to nonoxidative killing in human polymorphonuclear neutrophils. Infect. Immun. 573894-3900. PubMed PMC

Stritzker, J., J. Janda, C. Schoen, M. Taupp, S. Pilgrim, I. Gentschev, P. Schreier, G. Geginat, and W. Goebel. 2004. Growth, virulence, and immunogenicity of Listeria monocytogenes aro mutants. Infect. Immun. 725622-5629. PubMed PMC

Tacket, C. O., M. B. Sztein, G. A. Losonsky, S. S. Wasserman, J. P. Nataro, R. Edelman, D. Pickard, G. Dougan, S. N. Chatfield, and M. M. Levine. 1997. Safety of live oral Salmonella typhi vaccine strains with deletions in htrA and aroC aroD and immune response in humans. Infect. Immun. 65452-456. PubMed PMC

Tacket, C. O., M. B. Sztein, S. S. Wasserman, G. Losonsky, K. L. Kotloff, T. L. Wyant, J. P. Nataro, R. Edelman, J. Perry, P. Bedford, D. Brown, S. Chatfield, G. Dougan, and M. M. Levine. 2000. Phase 2 clinical trial of attenuated Salmonella enterica serovar Typhi oral live vector vaccine CVD 908-htrA in U.S. volunteers. Infect. Immun. 681196-1201. PubMed PMC

Trebichavsky, I., A. Splichalova, I. Rychlik, H. Hojna, Y. Muneta, Y. Mori, and I. Splichal. 2006. Attenuated aroA Salmonella enterica serovar Typhimurium does not induce inflammatory response and early protection of gnotobiotic pigs against parental virulent LT2 strain. Vaccine 244285-4289. PubMed

Tusher, V. G., R. Tibshirani, and G. Chu. 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 985116-5121. PubMed PMC

phoP, SPI1, SPI2 and aroA mutants of Salmonella Enteritidis induce a different immune response in chickens