Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

Abstract

In Escherichia coli, the waaP (rfaP) gene product was recently shown to be responsible for phosphorylation of the first heptose residue of the lipopolysaccharide (LPS) inner core region. WaaP was also shown to be necessary for the formation of a stable outer membrane. These earlier studies were performed with an avirulent rough strain of E. coli (to facilitate the structural chemistry required to properly define waaP function); therefore, we undertook the creation of a waaP mutant of Salmonella enterica serovar Typhimurium to assess the contribution of WaaP and LPS core phosphorylation to the biology of an intracellular pathogen. The S. enterica waaP mutant described here is the first to be both genetically and structurally characterized, and its creation refutes an earlier claim that waaP mutations in S. enterica must be leaky to maintain viability. The mutant was shown to exhibit characteristics of the deep-rough phenotype, despite its ability to produce a full-length core capped with O antigen. Further, phosphoryl modifications in the LPS core region were shown to be required for resistance to polycationic antimicrobials. The waaP mutant was significantly more sensitive to polymyxin in both wild-type and polymyxin-resistant backgrounds, despite the decreased negative charge of the mutant LPSs. In addition, the waaP mutation was shown to cause a complete loss of virulence in mouse infection models. Taken together, these data indicate that WaaP is a potential target for the development of novel therapeutic agents.

The inner-core lipopolysaccharide biosynthetic waaE gene: function and genetic distribution among some Enterobacteriaceae b bThe GenBank accession number for the waaE gene sequences of P. mirabilis CECT170, Y. enterocolitica R102 and Ent. aerogenes CECT684 reported in this paper are AY075039, AY075041 and AY075040, respectively.

“…Interestingly, both the WaaP and WaaE proteins are involved in substitution of ,-HeppI at the O-4 position by a phosphate or pyrophosphoethanolamine (Yethon et al, 1998(Yethon et al, , 2000 and by a β--Glcp residue, respectively. The phenotype of the waaE mutants shares some similarities to E. coli F470 and Sal.…”

“…In addition, in Sal. enterica serovar Typhimurium a waaP mutant showed reduced virulence in vivo (Yethon et al, 2000).…”

Lipopolysaccharide Endotoxins

“…In fact, for some time it was thought that mutants lacking heptose-P residues were not viable unless the core was truncated. Discovery and biochemical characterization of the LPS kinase WaaP (see below) allowed construction of defined mutants lacking core phosphorylation but retaining a complete carbohydrate backbone and, where relevant, the O polysaccharide (191,192). In the laboratory, waaP mutants show the same growth rate as the parent.…”

Down-Regulation of Key Virulence Factors Makes the Salmonella enterica Serovar Typhimurium rfaH Mutant a Promising Live-Attenuated Vaccine Candidate

“…The LPS mutations shown to have the most pronounced effect on intracellular survival had formerly been described to cause the deep-rough phenotype (60,62). To test how intracellular yield of the LPS mutants correlates with susceptibilities to various environmental stressors, MICs of a detergent (SDS), an oxidative agent (H 2 O 2 ), and a small cationic antimicrobial peptide (polymyxin B) were determined.…”