The Global Regulator ArcA Controls Resistance to Reactive Nitrogen and Oxygen Intermediates in Salmonella enterica Serovar Enteritidis

Abstract

Salmonella enterica serovar Enteritidis is a major cause of food-borne diseases associated with consumption of shell eggs. Clinical isolates of S. enterica serovar Enteritidis exhibit a wide spectrum of virulence in mice. A highly virulent isolate (SE2472) was previously shown to be more resistant in vitro than other clinical isolates to acidified sodium nitrite (ASN), a generator of reactive nitrogen and oxygen intermediates (RNI/ROI). SE2472 is also more resistant to S-nitrosoglutathione (GSNO) and hydrogen peroxide (H 2 O 2 ) than an ASN-susceptible isolate of S. enterica serovar Enteritidis (SE8743). To investigate the molecular basis for the RNI/ROI resistance of S. enterica serovar Enteritidis, we transformed a genomic DNA library of SE2472 into SE8743. A plasmid clone conferred upon SE8743 enhanced resistance to ASN, GSNO, and H 2 O 2 . The DNA insert in the clone encoded ArcA, a global regulator. An arcA mutant of SE2472 was constructed and was found to be more susceptible to GSNO and hydrogen peroxide but not more susceptible to ASN than wild-type SE2472. The susceptibility of the arcA mutant to GSNO and H 2 O 2 was complemented by a plasmid harboring arcA. The coding sequence of the arcA gene in SE2472 and the coding sequence of the arcA gene in SE8743 were identical, suggesting that the difference in resistance to RNI/ROI maybe due to the activity of genes regulated by ArcA. No significant difference in virulence between the wild type and the arcA mutant of SE2472 was observed in mice. These observations show that arcA is essential for resistance of S. enterica serovar Enteritidis to nitrosative and oxidative stress. However, additional genetic loci may contribute to the resistance to RNI/ROI and unusually high virulence for mice of SE2472.

Structural Analysis and Solution Studies of the Activated Regulatory Domain of the Response Regulator ArcA: A Symmetric Dimer Mediated by the α4-β5-α5 Face

“…16 ArcA, like the majority of RRs, is a transcription factor that, in addition to its N-terminal regulatory domain, contains a C-terminal helix-turn-helix DNA-binding domain. In recent years the ArcA-ArcB TCS has been implicated in the direct or indirect regulation of virulence and resistance in a number of clinically important human pathogens including Salmonella enterica serovar Enteritidis, 17 Haemophilus influenzae 18 and Vibrio cholerae. 19 ArcA is a member of the OmpR/PhoB subfamily of RR transcription factors, by far the largest subfamily of RRs, accounting for 15 of the 34 RRs presently identified in E. coli.…”

Oral delivery of RNase P ribozymes by Salmonella inhibits viral infection in mice

“…Four-to 6-wk-old CB17 SCID mice (Jackson Laboratory) were infected intraperitoneally with 1 × 10 4 pfu of MCMV and, at 36 h after infection, were inoculated with Salmonella intragastrically in oral delivery experiments. For intragastric inoculation of mice, animals were first anesthetized with isoflurane and then intragastrically inoculated with 0.1-0.2 mL PBS containing 1 × 10 8 cfu Salmonella, using a gavage needle (29). The oral inoculation procedure was repeated every 5 d. The gene delivery efficiency was evaluated by examining the GFP signal of the transfected cells in the tissues using fluorescence microscopy and by detecting the expression of M1GS RNAs in mouse tissues (e.g., livers) using Northern blot analysis.…”

Differential expression of Salmonella type III secretion system factors InvJ, PrgJ, SipC, SipD, SopA and SopB in cultures and in mice

“…The resulting PCR products were transformed into Salmonella ST14028s carrying plasmid pKD46. The tagged mutants were constructed using the l Red recombinase method (Datsenko & Wanner, 2000), following the procedures as described previously Lu et al, 2002Lu et al, , 2003. The non-polar strains were selected for their sensitivity to kanamycin and further confirmed using PCR.…”

“…were determined by plating of serially diluted organ homogenates. Protein extracts for Western blot analyses were prepared by a modification of a procedure described previously Lu et al, 2002Lu et al, , 1999. Briefly, the homogenates of the liver and ileum samples were centrifuged at 9000 g at 4 uC for 10 min.…”

“…and 37 uC for an additional 6 h, and then collected. Protein extract samples for Western blot analysis were prepared from the pellets and supernatants of bacterial cultures, following the procedure described previously Lu et al, 2002Lu et al, , 1999. The analyses were repeated at least three times.…”