Mutation of the htrB gene in a virulent Salmonella typhimurium strain by intergeneric transduction: strain construction and phenotypic characterization

Abstract

The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A ␤-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV A , which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a welldefined murine typhoid model of animal pathogenesis. To this end, an r ؊ m ؉ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter-and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.Lipooligosaccharide (LOS), a major component of the outer membrane of nontypeable Haemophilus influenzae, is a complex molecule that requires the functions of many genes for proper assembly. One gene, htrB, has recently been shown to play a role in the acylation of the lipid A portion of the LOS of H. influenzae (19). The lipid A of H. influenzae is typically hexaacyl, containing ester-and amide-linked 4-hydroxymyristic acids. The two ␤-hydroxymyristic acids on the second glucosamine are replaced at their hydroxy group with myristic acid. In contrast, the lipid A of the H. influenzae htrB mutant is approximately 90% tetraacyl, with only four hydroxymyristic acid ester-and amide-linked fatty acids. This mutant lipid A is similar in structure to lipid IV A . The remaining 10% of the htrB lipid A is pentaacyl, with a single myristic acid substitution.Recent studies of nontypeable H. influenzae have indicated that changes in the LOS structure affect bacterial virulence. The similarity of the LOS from the nontypeable H. influenzae htrB mutant to lipid IV A suggests that it binds to the CD14 receptor but does not initiate signalling which results in a macrophage cytokine response (18). Experimental evidence has shown that LOS from the nontypeable H. influenzae htrB mutant has reduced toxicity in an infant-rat model and elicits less tumor necrosis factor alpha from human macrophages than does wild-type LOS (unpublished data). In addition, Somerville et al. (3...

Extragenic Suppressors of Growth Defects inmsbB Salmonella

“…2A, YS1 grows well on LB-0 agar at 30°C (and also at 23, 28, 37, and 42°C; data not shown). The phenotype of msbB Salmonella strains is in sharp contrast to the reported phenotype of mutants of htrB, which encodes another LPS biosynthetic enzyme, since htrB Salmonella has a temperature-sensitive growth defect on rich agar at or above 37°C (30).…”

“…The reported apparent lack of growth defects, in msbB E. coli K-12 or Salmonella, was quite surprising because the other reported lipid A mutations were either lethal or exhibited conditional (temperature-sensitive) phenotypes, such as that seen with htrB (14,30). The published data seem to suggest that the myristic acid moiety, added to lipid A by the MsbB enzyme, does not play a significant role in outer membrane barrier function, since msbB E. coli K-12 mutants were reported to have no growth phenotype other than increased deoxycholate resistance.…”

“…Under non-cold-shock conditions, the tightly regulated addition of fatty acids to the lipid A precursor is catalyzed by the enzymes HtrB (lauric acid [3]), MsbB (myristic acid [4]), and PagP (palmitic acid [9]). htrB Escherichia coli and Salmonella are nonpermissive for growth on rich agar at or above 37°C (14,30). However, this growth defect can be suppressed in E. coli with the msbB gene on a high-copy-number plasmid.…”

Two msbB Genes Encoding Maximal Acylation of Lipid A Are Required for Invasive Shigella flexneri to Mediate Inflammatory Rupture and Destruction of the Intestinal Epithelium

“…A mutation in the waaM (htrB) gene of Salmonella typhimurium severely altered the capacity to cause systemic dissemination and to colonize organs in mice. However, the conditional, temperature-dependent lethality of this mutation may have accounted for impairment (52). More recently, following discovery of the msbB gene function (23), a S. typhimurium waaN (msbB) mutant administered systemically to mice showed higher LD 50 and reached higher numbers in organs, confirming that death in the murine typhoid fever model depends greatly on the endotoxin activity of lipid A (53).…”

Function of Escherichia coli MsbA, an Essential ABC Family Transporter, in Lipid A and Phospholipid Biosynthesis

“…4 and 7). Mass spectrometry of lipid A isolated from Salmonella typhimurium strains lacking htrB likewise suggests the activation of compensatory lipid A acylation reactions when such mutants are grown at 30°C (44). Mutants lacking both htrB and these other acyltransferases may therefore be unable to grow at any temperature in the absence of a covering plasmid carrying msbA.…”