Multidrug efflux pumps are integral membrane proteins known to actively excrete antibiotics. The macrolide-specific pump MacAB, the only ABC-type drug efflux pump in Salmonella, has previously been linked to virulence in mice. The molecular mechanism of this link between macAB and infection is unclear. We demonstrate that macAB plays a role in the detoxification of reactive oxygen species (ROS), compounds that salmonellae are exposed to at various stages of infection. macAB is induced upon exposure to H2O2 and is critical for survival of Salmonella enterica serovar Typhimurium in the presence of peroxide. Furthermore, we determined that macAB is required for intracellular replication inside J774.A1 murine macrophages but is not required for survival in ROS-deficient J774.D9 macrophages. macAB mutants also had reduced survival in the intestine in the mouse colitis model, a model characterized by a strong neutrophilic intestinal infiltrate where bacteria may experience the cytotoxic actions of ROS. Using an Amplex red-coupled assay, macAB mutants appear to be unable to induce protection against exogenous H2O2 in vitro, in contrast to the isogenic wild type. In mixed cultures, the presence of the wild-type organism, or media preconditioned by the growth of the wild-type organism, was sufficient to rescue the macAB mutant from peroxide-mediated killing. Our data indicate that the MacAB drug efflux pump has functions beyond resistance to antibiotics and plays a role in the protection of Salmonella against oxidative stress. Intriguingly, our data also suggest the presence of a soluble anti-H2O2 compound secreted by Salmonella cells through a MacAB-dependent mechanism.

Salmonella enterica serotype Typhimurium can move through liquid using swimming motility, and across a surface by swarming motility. We generated a library of targeted deletion mutants in Salmonella Typhimurium strain ATCC14028, primarily in genes specific to Salmonella, that we have previously described. In the work presented here, we screened each individual mutant from this library for the ability to move away from the site of inoculation on swimming and swarming motility agar. Mutants in genes previously described as important for motility, such as flgF, motA, cheY are do not move away from the site of inoculation on plates in our screens, validating our approach. Mutants in 130 genes, not previously known to be involved in motility, had altered movement of at least one type, 9 mutants were severely impaired for both types of motility, while 33 mutants appeared defective on swimming motility plates but not swarming motility plates, and 49 mutants had reduced ability to move on swarming agar but not swimming agar. Finally, 39 mutants were determined to be hypermotile in at least one of the types of motility tested. Both mutants that appeared non-motile and hypermotile on plates were assayed for expression levels of FliC and FljB on the bacterial surface and many of them had altered levels of these proteins. The phenotypes we report are the first phenotypes ever assigned to 74 of these open reading frames, as they are annotated as ‘hypothetical genes’ in the Typhimurium genome.

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the type VII collagen gene (COL7A1). Nearly all cases of dominant DEB are caused by glycine substitution mutations occurring within the triple helical region of type VII collagen, and most of the mutations are unique to individual families. In this study, we identified a patient of Hispanic-Mexican origin with a mild form of DEB, which resulted from a de novo dominant glycine substitution, G2043R, in exon 73 of COL7A1. We also investigated a Scottish family with a three-generation pedigree of dominant DEB, in whom the same glycine to arginine substitution mutation was demonstrated. This particular mutation has also been detected previously in three other families with dominant DEB: one Italian, one Hungarian and one Norwegian. Given the widespread geographical distribution of this mutation and the demonstration of its occurrence as a de novo event, G2043R therefore represents the first example of a mutational hotspot in dominant DEB. Interestingly, although both the Mexican and Scottish families we studied had some clinical features in keeping with the Pasini form of the disorder, there was considerable interfamilial variability as well as intrafamilial diversity in the affected individuals.

Cattle are naturally infected with Salmonella enterica serotype Typhimurium and exhibit pathological features of enteric salmonellosis that closely resemble those in humans. Cattle are the most relevant model of gastrointestinal disease resulting from nontyphoidal Salmonella infection in an animal with an intact microbiota. We utilized this model to screen a library of targeted single-gene deletion mutants to identify novel genes of Salmonella Typhimurium required for survival during enteric infection. Fifty-four candidate mutants were strongly selected, including numerous mutations in genes known to be important for gastrointestinal survival of salmonellae. Three genes with previously unproven phenotypes in gastrointestinal infection were tested in bovine ligated ileal loops. Two of these mutants, STM3602 and STM3846, recapitulated the phenotype observed in the mutant pool. Complementation experiments successfully reversed the observed phenotypes, directly linking these genes to the colonization defects of the corresponding mutant strains. STM3602 encodes a putative transcriptional regulator that may be involved in phosphonate utilization, and STM3846 encodes a retron reverse transcriptase that produces a unique RNA-DNA hybrid molecule called multicopy single-stranded DNA. The genes identified in this study represent an exciting new class of virulence determinants for further mechanistic study to elucidate the strategies employed by Salmonella to survive within the small intestines of cattle.