The gene encoding a Lon protease homologue has been cloned from Brucella abortus. The putative Brucella abortus Lon shares > 60% amino acid identity with its Escherichia coli counterpart and the recombinant form of this protein restores the capacity of an Escherichia coli lon mutant to resist killing by ultraviolet irradiation and regulate the expression of a cpsB:lacZ fusion to wild-type levels. A sigma32 type promoter was identified upstream of the predicted lon coding region and Northern analysis revealed that transcription of the native Brucella abortus lon increases in response to heat shock and other environmental stresses. ATP-dependent proteolytic activity was also demonstrated for purified recombinant Lon. To evaluate the capacity of the Brucella abortus Lon homologue to function as a stress response protease, the majority of the lon coding region was removed from virulent strain Brucella abortus 2308 via allelic exchange. In contrast to the parent strain, the Brucella abortus lon mutant, designated GR106, was impaired in its capacity to form isolated colonies on solid medium at 41 degrees C and displayed an increased sensitivity to killing by puromycin and H2O2. GR106 also displayed reduced survival in cultured murine macrophages and significant attenuation in BALB/c mice at 1 week post infection compared with the virulent parental strain. Beginning at 2 weeks and continuing for 6 weeks post infection, however, GR106 and 2308 displayed equivalent spleen and liver colonization levels in mice. These findings suggest that the Brucella abortus Lon homologue functions as a stress response protease that is required for wild-type virulence during the initial stages of infection in the mouse model, but is not essential for the establishment and maintenance of chronic infection in this host.

The pathogenic role of the spv (Salmonella plasmid virulence) genes of Salmonella dublin was determined in the natural, bovine host. Since the lack of overt signs of enteritis or enterocolitis due to Salmonella infections in mice has limited the development of a convenient experimental system to study enteric disease, we used calves to study the contribution of the spv genes to S. dublin-induced salmonellosis. Since the SpvR transcriptional regulator is required for expression of the spvABCD operon, we constructed an spvR knockout mutation in a calf-virulent strain of S. dublin. Calves were infected with the wild-type strain, an spvR mutant, and an spvR mutant containing a complementing plasmid. Calves that were infected with the wild type or the complemented spvR mutant rapidly developed severe diarrhea and became moribund. Calves that were infected with the spvR mutant showed little or no clinical signs of systemic salmonellosis and developed only mild diarrhea. The survival and growth of the wild-type strain and the spvR mutant were determined by using blood-derived bovine monocytes. Wild-type S. dublin survived and grew inside cells, while the spvR mutant did not proliferate. These results suggest that the spv genes of S. dublin promote enhanced intracellular proliferation in intestinal tissues and at extraintestinal sites in the natural host.

Cholera is a devastating diarrhoeal disease caused by certain strains of serogroup O1/O139 Vibrio cholerae. Mobile genetic elements such as genomic islands (GIs) have been pivotal in the evolution of O1/O139 V. cholerae. Perhaps the most important GI involved in cholera disease is the V. cholerae pathogenicity island 1 (VPI-1). This GI contains the toxin-coregulated pilus (TCP) gene cluster that is necessary for colonization of the human intestine as well as being the receptor for infection by the cholera-toxin bearing CTX phage. In this study, we report a GI (designated GIVchS12) from a non-O1/O139 strain of V. cholerae that is present in the same chromosomal location as VPI-1, contains an integrase gene with 94% nucleotide and 100% protein identity to the VPI-1 integrase, and attachment (att) sites 100% identical to those found in VPI-1. However, instead of TCP and the other accessory genes present in VPI-1, GIVchS12 contains a CRISPR-Cas element and a type VI secretion system (T6SS). GIs similar to GIVchS12 were identified in other V. cholerae genomes, also containing CRISPR-Cas elements and/or T6SS’s. This study highlights the diversity of GIs circulating in natural V. cholerae populations and identifies GIs with VPI-1 recombination characteristics as a propagator of CRISPR-Cas and T6SS modules.

The O antigen of Brucella abortus has been described as a major virulence determinant based on the attenuated survival of fortuitously isolated rough variants. However, the lack of genetic definition of these mutants and the virulence of naturally occurring rough species, Brucella ovis and Brucella canis, has confused interpretation. To better characterize the role of O antigen in virulence and survival, transposon mutagenesis was used to generate B. abortus rough mutants defective in O-antigen presentation. Sequence analysis of DNA flanking the site of Tn5 insertion was used to verify insertion in genes encoding lipopolysaccharide (LPS) biosynthetic functions. Not surprisingly, each of the rough mutants was attenuated for survival in mice, but unexpected differences among the mutants were observed. In an effort to define the basis for the observed differences, the structure of the rough LPS and the sensitivity of these mutants to individual killing mechanisms were examined in vitro. All of the B. abortus rough mutants exhibited a 4- to 5-log-unit increase, compared to the smooth parental strain, in sensitivity to complement-mediated lysis. Little change was evident in the sensitivity of these organisms to hydrogen peroxide, consistent with an inability of O antigen to exclude relatively small molecules. Sensitivity to polymyxin B, which was employed as a model cationic, amphipathic peptide similar to defensins found in phagocytic cells, revealed survival differences among the rough mutants similar to those observed in the mouse. One mutant in particular exhibited hypersensitivity to polymyxin B and reduced survival in mice. This mutant was characterized by a truncated rough LPS. DNA sequence analysis of this mutant revealed a transposon interruption in the gene encoding phosphomannomutase (pmm), suggesting that this activity may be required for the synthesis of a full-length core polysaccharide in addition to O antigen. B. abortus O antigen appears to be essential for extra- and intracellular survival in mice.

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104 has become a widespread cause of human and other animal infection worldwide. The severity of clinical illness in S. enterica serovar Typhimurium DT104 outbreaks has led to the suggestion that this strain possesses enhanced virulence. In the present study, in vitro and in vivo virulence-associated phenotypes of several clinical isolates of S. enterica serovar Typhimurium DT104 were examined and compared to S. enterica serovar Typhimurium ATCC 14028s. The ability of these DT104 isolates to survive within murine peritoneal macrophages, invade cultured epithelial cells, resist antimicrobial actions of reactive oxygen and nitrogen compounds, and cause lethal infection in mice were assessed. Our results failed to demonstrate that S. enterica serovar Typhimurium DT104 isolates are more virulent than S. enterica serovar Typhimurium ATCC 14028s.