Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system

Abstract

Mutations in rcsC that result in constitutive colanic acid capsule synthesis were obtained in Salmonella enterica serovar Typhimurium. Most rcsC alleles were dominant; however, recessive rcsC alleles were also found, in agreement with the postulated double role (positive and negative) of RcsC on the activation of the RcsB/C phosphorelay system. Salmonella rcsC mutants with constitutive activation of the Rcs system are severely attenuated for virulence in BALB/c mice and their degree of attenuation correlates with the level of Rcs activation. Partial relief of attenuation by a gmm mutation indicates that capsule overproduction is one of the factors leading to avirulence in constitutively activated rcsC mutants. INTRODUCTIONThe Rcs phosphorelay system was initially characterized in Escherichia coli as a two-component positive regulator of colanic acid capsule synthesis encoded by rcsC and rcsB (Brill et al., 1988;Gottesman et al., 1985;Stout & Gottesman, 1990). The sensor protein RcsC, a hybrid histidine kinase, and the transcriptional activator RcsB are the main components of the system, which also includes a second transcriptional activator, RcsA (Stout et al., 1991) and an intermediate phosphotransmitter, YojN (Takeda et al., 2001). The Rcs system is also a negative regulator of the flagellar master operon flhDC (Francez-Charlot et al., 2003), whose products are necessary for the expression of genes involved in flagellum synthesis, motility and chemotaxis.The first environmental signal shown to strongly and rapidly induce colanic acid synthesis was osmotic upshift (Sledjeski & Gottesman, 1996). This effect was mediated by the Rcs system. More recently it has been shown that the Rcs phosphorelay can be activated when wild-type cells are grown at 20 uC in the presence of glucose and a high concentration of Zn 2+ (Hagiwara et al., 2003). Furthermore, several genetic conditions that result in alterations in envelope composition or integrity are known to activate the Rcs system. For example, a mutation in the mdoH gene involved in the biosynthesis of membrane-derived oligosaccharides (Ebel et al., 1997) Here, we describe the isolation of Salmonella rcsC mutants with constitutive activation of the Rcs system, and the characterization of amino acid substitutions that lead to different degrees of activation of the system. Virulence trials with constitutively activated rcsC mutants provide a direct demonstration of the link between activation of the Rcs system and avirulence in mice. We also show that virulence attenuation associated with activation of the RcsB/C system is partially due to colanic acid overproduction. METHODSBacterial strains, bacteriophages and strain construction.S. enterica serovar Typhimurium strains used in this study are described in Table 1. Unless otherwise indicated, the strains derive from the mouse-virulent strain 14028. Transductional crosses using phage P22 HT 105/1 int201 (Schmieger, 1972) were used for strain construction operations. The transduction protocol was described by Maloy (199...

The Virulence of aDickeya dadantii3937 Mutant Devoid of Osmoregulated Periplasmic Glucans Is Restored by Inactivation of the RcsCD-RcsB Phosphorelay

“…This is in agreement with the low level of RcsCD-RcsB activation needed for full virulence in S. enterica (14,20,31) and with the restoration of virulence in D. dadantii for opgG strains harboring the null mutation rcsC or rcsB. Three dominant mutations were isolated in the barA gene encoding the sensor protein of the BarA-UvrY phosphorelay system of E. coli.…”

“…The RcsCDRcsB phosphorelay regulates motility by negatively regulating the flhDC master operon (19,20) 10 7 ) of the wild-type strain (EC3937) (spot 1), the opgG strain (NFB3500) (spot 2), the opgG rcsC2 strain (NFB3591) (spot 3), and the rcsC2 strain (NFB3611) (spot 4) were inoculated into holes on potato tubers. Disease symptoms were observed after 72 h of incubation at 30°C.…”

“…The RcsCD-RcsB phosphorelay was first identified in E. coli as a positive regulator of exopolysaccharide synthesis (27). Further studies demonstrated the role of this phosphorelay in regulation of motility, cell division, and virulence in various enterobacterial species, such as E. coli, Salmonella enterica, and P. carotovorum (2,8,19,20,28,31). In D. dadantii, this phosphorelay also controls these cellular processes.…”

Genome Expression Analyses Revealing the Modulation of the Salmonella Rcs Regulon by the Attenuator IgaA

“…These RcsB proteins were produced from plasmids under the arabinose-inducible P BAD promoter ( (Table 5). Since both the MD0842-J4 and MD0855-J10 strains upregulate colanic acid capsule genes and the yjbEFGH operon, we see as unlikely a negative effect of exopolysaccharide overproduction on virulence (28,45). On the other hand, MD0842-J4 and MD0855-J10 displayed different levels of expression of the virulence genes spvAB, a change that could explain the observed phenotypes.…”

“…Spontaneous mutations consisting of deletions leading to a diminished production of RcsC or RcsD are also known to suppress IgaA essentiality (42). Important traits of serovar Typhimurium, such as its virulence potential, depend on the fine-tuning of the activity of the RcsCDB system (17,28,45). IgaA and the RcsCDB system are present in the same genera of enteric bacteria (17,20,34), and this concurrence strongly supports the functional link existing between IgaA and the RcsCDB system.…”