Roles of the Outer Membrane Protein AsmA of Salmonella enterica in the Control of marRAB Expression and Invasion of Epithelial Cells

Abstract

A genetic screen for suppressors of bile sensitivity in DNA adenine methylase (dam) mutants of Salmonella enterica serovar Typhimurium yielded insertions in an uncharacterized locus homologous to the Escherichia coli asmA gene. Disruption of asmA suppressed bile sensitivity also in phoP and wec mutants of S. enterica and increased the MIC of sodium deoxycholate for the parental strain ATCC 14028. Increased levels of marA mRNA were found in asmA, asmA dam, asmA phoP, and asmA wec strains of S. enterica, suggesting that lack of AsmA activates expression of the marRAB operon. Hence, asmA mutations may enhance bile resistance by inducing gene expression changes in the marRAB-controlled Mar regulon. In silico analysis of AsmA structure predicted the existence of one transmembrane domain. Biochemical analysis of subcellular fractions revealed that the asmA gene of S. enterica encodes a protein of ϳ70 kDa located in the outer membrane. Because AsmA is unrelated to known transport and/or efflux systems, we propose that activation of marRAB in asmA mutants may be a consequence of envelope reorganization. Competitive infection of BALB/c mice with asmA ؉ and asmA isogenic strains indicated that lack of AsmA attenuates Salmonella virulence by the oral route but not by the intraperitoneal route. Furthermore, asmA mutants showed a reduced ability to invade epithelial cells in vitro.

Identification of the Salmonella enterica damX Gene Product, an Inner Membrane Protein Involved in Bile Resistance

“…Previous studies have shown that bile sensitivity of S. enterica dam mutants is suppressed by two classes of recessive mutations: (i) in the mismatch repair genes mutH, mutL, and mutS (18) and (ii) in the asmA gene (17). The MIC analyses represented in Table 1 indicated that lack of AsmA suppresses DOC sensitivity in a damX background, while inactivation of the MutHLS system does not.…”

“…This is a relevant difference between dam and damX mutants despite the fact that both mutant types are bile sensitive. Lack of AsmA has previously been shown to activate the Mar regulon, which may in turn activate efflux pumps and other protective devices (17). As a consequence, asmA mutations suppress bile sensitivity in genetic backgrounds as diverse as dam, phoP, and wec (17).…”

“…The importance of the cell envelope as a barrier for bile salts is illustrated by the variety of envelope alterations that cause bile sensitivity. The current list includes mutations that alter the outer membrane (17,20), the lipopolysaccharide (5,16), and the enterobacterial common antigen (22). It is thus interesting that DamX, an inner membrane protein, is likewise required for bile resistance.…”

“…The primers used for introduction of the 3ϫ FLAG epitope were 5Ј GTG GGC AAA ACC GTT GCA TCA GGT TCA GGC CGA TCT GAA AGA CTA CAA AGA CCA TGA CGG 3Ј and 5Ј GTC AGC GAC ACT CAC AGG CAA TTG CAG GAC ACA GCC TGG ACA TAT GAA TAT CCT CCT TAG 3Ј. Protein purification, cell fractionation (cytosol, inner membrane, and outer membrane), and Western analysis of the resolved protein extracts were carried out as described elsewhere (17). DamX migrated as a 70-kDa protein under denaturing conditions, as previously described for E. coli (11).…”

The DamX protein ofEscherichia coliandSalmonella enterica

“…10,11 (ii) Sensitivity to bile in S. enterica dam mutants is suppressed by inactivation of either the MutHLS system or the AsmA protein. 22,23 In S. enterica damX mutants, however, bile sensitivity is suppressed by inactivation of AsmA, but not by inactivation of the MutHLS system, suggesting that the causes of bile sensitivity in dam and damX mutants are different.…”