A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence.

Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA- and lcpB- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA- and lcpB- strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence.

Dietary NaCl regulates renal aminopeptidase N: relevance to hypertension in the Dahl rat.

Aminopeptidase N (APN) is an abundant metallohydrolase in the brush border of kidney proximal tubule cells that degrades angiotensin III (Ang III) to angiotensin IV (Ang IV) and, along with dipeptidylaminopeptidase, degrades Ang IV. We examined the impact of a high-salt diet on renal APN activity and transcript abundance in the Sprague-Dawley and Dahl salt-sensitive (SS/Jr) rat strains. APN transcript abundance and protein abundance were approximately 2-fold greater (P<0.05; n=6) in the kidneys of Sprague-Dawley and Lewis rats ingesting 8% versus 0.3% salt diets, suggesting that increased aminopeptidase activity may contribute to decreased renal sodium uptake during adaptation to a high-salt diet. In contrast, renal APN transcript abundance and activity were the same in Dahl SS/Jr rats ingesting 8.0% versus 0.3% salt diets. The APN gene was mapped, using a radiation-hybrid panel, to known quantitative loci on chromosome 1 for blood pressure in the Dahl SS/Jr rat. The results suggest that the APN gene is a good candidate for salt-sensitivity in the Dahl SS/Jr rat.

Dietary salt regulates renal SGK1 abundance: relevance to salt sensitivity in the Dahl rat.

Serum and glucocorticoid-induced kinase 1 (SGK1) activates the epithelial sodium channel (eNaC) in tubules. We examined renal SGK1 abundance in salt-adaptation and in salt-sensitive hypertension. Sprague-Dawley and Dahl salt-sensitive rats were placed on either 8% or 0.3% NaCl diets for 10 days. Plasma aldosterone levels were approximately 2.5-fold greater on 0.3% versus 8% NaCl diets in both rat strains. Both serum and glucocorticoid-induced kinase 1 transcript and protein abundance were less (P<0.01) in Sprague-Dawley rats and greater (P<0.01) in Dahl salt-sensitive rats on 8% versus 0.3% NaCl diets. The cDNA sequences of serum and glucocorticoid-induced kinase 1 in both strains of rat were the same. The present results provide evidence that the abundance of serum and glucocorticoid-induced kinase 1 in rat kidney may play a role in salt adaptation and the pathogenesis of hypertension and suggests that aldosterone is not the primary inducer of SGK1 in the Sprague-Dawley rat.