Study of the role of the htrB gene in Salmonella typhimurium virulence

Abstract

We have undertaken a study to investigate the contribution of the htrB gene to the virulence of pathogenic Salmonella typhimurium. An htrB::mini-Tn10 mutation from Escherichia coli was transferred by transduction to the mouse-virulent strain S. typhimurium SL1344 to create an htrB mutant. The S. typhimurium htrB mutant was inoculated into mice and found to be severely limited in its ability to colonize organs of the lymphatic system and to cause systemic disease in mice. A variety of experiments were performed to determine the possible reasons for this loss of virulence. Serum killing assays revealed that the S. typhimurium htrB mutant was as resistant to killing by complement as the wild-type strain. However, macrophage survival assays revealed that the S. typhimurium htrB mutant was more sensitive to the intracellular environment of murine macrophages than the wild-type strain. In addition, the bioactivity of the lipopolysaccharide (LPS) of the htrB mutant was reduced compared to that of the LPS from the parent strain as measured by both a Limulus amoebocyte lysate endotoxin quantitation assay and a tumor necrosis factor alpha bioassay. These results indicate that the htrB gene plays a role in the virulence of S. typhimurium.

Expression of a Porphyromonas gingivalis lipid A palmitylacyltransferase in Escherichia coli yields a chimeric lipid A with altered ability to stimulate interleukin-8 secretion

“…The levels of IL‐8 induced by LPS from the htrB mutant strain, however, were at least 10‐fold less than LPS from the wild‐type strain. This reduction in potency is similar to that described for htrB mutants in other bacteria (Jones et al ., 1997; Hone et al ., 1998; Ellis et al ., 2001; van der Ley et al ., 2001; Tong et al ., 2001; Post et al ., 2002; Swords et al ., 2002). LPS from the strain complemented with the P. gingivalis gene displayed intermediate behaviour, having a maximal value approximately half that of the wild‐type LPS.…”

“…For example, LPS from an E. coli strain containing a null mutation for msbB and lacking the expected secondary myristate on the 3′‐hydroxy‐myristate was found to have 10 000‐fold less ability to induce tumour necrosis factor‐α (TNFα) secretion from blood monocytes and at least 1000‐fold less ability to induce E‐selection upregulation in cultured endothelial cells (Darveau et al ., 1995). Strains of Haemophilus infuenzae , Neisseria meningitidis , Neisseria gonorrhoeae and Salmonella typhimurium containing mutations in htrB and lacking secondary fatty acids on the 2′‐hydroxy‐myristate have also been shown to have reduced inflammatory potential or virulence (Jones et al ., 1997; Vaara and Nurminen, 1999; Ellis et al ., 2001; van der Ley et al ., 2001; Tong et al ., 2001; Post et al ., 2002). Similarly, removal of both secondary fatty acids from purified E. coli LPS by a neutrophil acyloxyacyl hydrolase was shown to have a reduced ability to stimulate neutrophil adherence to endothelial cell layers as well as a reduced ability to promote a dermal Shwartzman reaction (Munford and Hall, 1986; Pohlman et al ., 1987).…”

“…It is important not only because of its role in the integrity of the bacterial cell envelope, but also because it is a potent toxin (endotoxin) and modulator of the host innate immune system. Both positive and negative modulations of the host response by LPS are believed to play a role in the development of various infectious conditions (Guo et al ., 1997; Jones et al ., 1997; Opal, 2002; Post et al ., 2002; Schroeder et al ., 2002; Swords et al ., 2002; Dixon et al ., 2004; Konopka et al ., 2004; Luo et al ., 2004). Most or all of this activity is contained in the lipid region of the molecule termed lipid A (Takada and Kotani, 1992; Rietschel et al ., 1994).…”

The Vi-capsule prevents Toll-like receptor 4 recognition of Salmonella

“…Furthermore, the S. Typhimurium msbB mutant was recovered in significantly lower numbers than all other S. Typhimurium strains tested. Intact lipid A has previously been shown to be important for macrophage survival of S. Typhimurium (Jones et al, 1997), which may explain the reduced recovery of the msbB mutant.…”

Expression of foreign LpxA acyltransferases in Neisseria meningitidis results in modified lipid A with reduced toxicity and retained adjuvant activity

“…With a functional assay at hand, it should now be feasible to study the structure-function relationship of RmpM in more detail using site-directed mutagenesis. Previously, reduction in the endotoxic activity of the LPS of N. meningitidis, E. coli, Salmonella typhimurium and Haemophilus influenzae has been accomplished by insertional inactivation of the genes encoding acyloxyacyltransferases, resulting in penta-or tetra-acylated lipid A species (Jones et al, 1997;Nichols et al, 1997;Hone et al, 1998;van der Ley et al, 2001). Here, we show that both extension and reduction of the length of the O-linked fatty acyl chains in N. meningitidis lipid A also led to a significant reduction in endotoxic activity.…”