Contribution of SPI-4 genes to the virulence of Salmonella enterica.

Salmonella pathogenicity island-4 (SPI-4) is a 27-kb region that carries six genes designated siiABCDEF. SiiC, SiiD, and SiiF form a type I secretion apparatus for the secretion of SiiE, a huge (approximately 600 kDa) protein contributing to the colonization of the bovine intestines. Here it is shown that loss of SPI-4 attenuates the oral virulence of Salmonella enterica serovars Typhimurium and Enteritidis in mice. Fifty percent lethal doses were elevated in both serovars upon the loss of SPI-4. Moreover, delta SPI-4 mutants were outcompeted in systemic organs by their wild-type strains in a cochallenge model. Contribution of SPI-4 to virulence appeared less pronounced in the S. Enteritidis strain, which was justified by lower levels of the secreted protein SiiE in this strain in comparison with S. Typhimurium. Competition assays with isogenic mutants lacking individual genes of the island showed that all six genes were required for full virulence of S. Typhimurium. Delta siiA and delta siiB mutants were, nevertheless, able to secrete SiiE to culture supernatants. The amount of secreted SiiE was, however, reduced in these two mutants compared with the wild-type strain. Furthermore, a down-regulation of SiiE levels is shown in structural and regulatory lipopolysaccharide mutants exhibiting the deep-rough phenotype.

Natural course of LGI1 encephalitis: 3-5 years of follow-up without immunotherapy.

Antibodies against LGI1 (leucin-rich glioma-inactivated 1 protein) are associated with limbic encephalitis (LE), which is characterized by a favorable outcome following immunotherapy. Here, we present two cases, where antibodies against LGI1 were detected in the sera 36 and 53 months after acute LE, respectively, and none of the patients received immunotherapy. LE showed characteristics of LGI1 encephalitis in both cases, including low sodium content in the sera; disorientation, hallucination, short-term memory loss; and epileptic seizures. One patient had faciobrachial tonic seizures. MRI indicated bilateral inflammation of the hippocampus in one case. We reviewed longitudinal clinical and MRI data covering 53 and 36 months after LE without immunotherapy, respectively. Both patients became seizure-free and spontaneously recovered with mild/moderate cognitive impairment. No relapses have been observed. Follow-up brain MRI indicated early hippocampal sclerosis and global brain atrophy in one case characterized by more pronounced cognitive deficit. Memory and verbal fluency were affected most during the natural course of LGI1 encephalitis. LGI1 encephalitis had a monophasic course and spontaneously improved, suggesting that a relatively benign natural course may contribute to the favorable outcome observed after immunotherapy. Our data also indicate that LGI1 antibodies can be present in the sera without clinical disease activity.

Investigation of the active site of the extracellular beta-D-xylosidase from Aspergillus carbonarius.

The catalytic amino acid residues of the extracellular beta-D-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) from Aspergillus carbonarius was investigated by the pH dependence of reaction kinetic parameters and chemical modifications of the enzyme. The pH dependence curves gave apparent pK values of 2.7 and 6.4 for the free enzyme, while pK value of 4.0 was obtained for the enzyme-substrate complex using p-nitrophenyl beta-D-xyloside as a substrate. These results suggested that a carboxylate group and a protonated group--presumably a histidine residue--took part in the binding of the substrate but only a carboxylate group was essential in the substrate cleavage. Carbodiimide- and Woodward's reagent K-mediated chemical modifications of the enzyme also supported that a carboxylate residue, located in the active center, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.4, proving that a carboxylate residue relevant for hydrolysis was modified. During modification V(max) decreased to 10% of that of the unmodified enzyme and K(m) remained unchanged, supporting that the modified carboxylate group participated in the cleavage and not in the binding of the substrate. We synthesized and tested a new, potential affinity label, N-bromoacetyl-beta-d-xylopyranosylamine for beta-D-xylosidase. The A. carbonarius beta-D-xylosidase was irreversible inactivated by N-bromoacetyl-beta-D-xylopyranosylamine. The competitive inhibitor beta-D-xylopyranosyl azide protected the enzyme from inactivation proving that the inactivation took place in the active center. Kinetic analysis indicated that one molecule of reagent was necessary for inactivation of one molecule of the enzyme.