Leucine-Responsive Regulatory Protein (Lrp) Acts as a Virulence Repressor in Salmonella enterica Serovar Typhimurium▿

Pathogenic bacteria have evolved gene regulation systems to facilitate successful colonization and pathogenesis in animal and plant hosts. Bacteria coordinate expression of virulence determinants by efficient switching of the activation/repression status of genes in response to specific environmental and nutritional cues. Global regulators sensing environmental signals such as iron, temperature, calcium, magnesium, osmolarity, anaerobiosis, pH, nutritional status, host products, and pheromones are involved in pathogenesis (20, 40, 50, 61, 65, 68, 77). Lrp is a global transcription regulator that affects expression of a number of genes in Escherichia coli, acting as both an activator and a repressor. The Lrp regulon in E. coli includes genes responsible for amino acid metabolism, carbon and energy metabolism, pilus synthesis, macromolecular biosynthesis, stress response, outer membrane proteins (OMPs), and gene regulators (12, 48, 89). Lrp in E. coli K-12 represses several genes, including CpxP, PhoP, and RpoS (48), that are known to regulate virulence traits in some pathogenic bacteria (30, 40, 47). Salmonella enterica serovar Typhimurium has an Lrp homologue with 99% amino acid sequence identity to the E. coli sequence (34). Thus, it is likely that Lrp will also act as a global regulator in Salmonella. Lrp-regulated genes in Salmonella include fimZ, for type 1 fimbria expression (67); ilvIH, for branched amino acid biosynthesis (93); hisJ, for d-histidine utilization (44); traJ, for conjugal transfer of virulence plasmid pSLT (13); and pef, for pSLT plasmid-encoded fimbriae (74). In addition, Lrp represses expression of the spvABCD operon (64), which is required for the establishment of a systemic infection by Salmonella in mice (41, 42). Lrp homologues appear to be widely distributed among bacteria and archaea as a modulator of genes involved in amino acid metabolism and related processes (11). In general, Lrp activates genes for biosynthetic enzymes and represses genes for catabolic enzymes (12, 73). In Vibrio cholerae and Xenorhabdus nematophil, Lrp is required for virulence gene expression (15, 60). Based on these observations, we hypothesize that pathogenic bacteria use Lrp to coordinate the expression of virulence traits in response to nutritional state (feast or famine) and host environments. A group of genes including cadA in Shigella spp. (25), csrRS in group A streptococcus (43), ptr1 in Leishmania major (17), and grvA (46), pcgL (70), and polynucleotide phosphorylase (95) in Salmonella have been identified as antivirulence genes. These genes encode antivirulence factors that repress virulence, and inactivation of these genes results in hypervirulence. It has been suggested that retention of these loci throughout evolution is beneficial for some pathogens in vivo since suppression of virulence at certain pathogenic stages is particularly important for host survival (33). Alternatively, some antivirulence genes might be retained for facilitating survival in nonhost environments (70). In the present study, we present the first evidence that the global regulator Lrp acts as an antivirulence coordinator in pathogenic Salmonella. Typically, to investigate gene functions, the phenotypes of the wild type and a gene-null mutation are compared. However, this approach would not reveal downregulated genes that are not critical for growth under laboratory conditions. Thus, we have also included a strain that overproduces Lrp. To address Lrp effects on Salmonella virulence, we present here the in vitro and in vivo virulence-related phenotypes of an lrp-null mutant, a mutant that constitutively expresses Lrp [lrp(Con)], and a mutant with arabinose-regulated Lrp expression.