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A putative cytotoxic serine protease from Salmonella typhimurium UcB5 recovered from undercooked burger.

ABSTRACT: A putative virulence exoprotease designated as UcB5 was successfully purified from the bacterium Salmonella typhimurium to the electrophoretic homogeneity with 13.2-fold and 17.1% recovery by hydrophobic, ion-exchange, and gel permeation chromatography using Phenyl-Sepharose 6FF, DEAE-Sepharose CL-6B, and Sephadex G-75, respectively. By applying SDS-PAGE, the molecular weight was confirmed at 35 kDa. The optimal temperature, pH, and isoelectric point were 35 °C, 8.0, 5.6 ± 0.2, respectively. UcB5 was found to have a broad substrate specificity against almost all the tested chromogenic substrates with maximal affinity against N-Succ-Ala-Ala-Pro-Phe-pNA achieving K_m of 0.16 mM, K_cat/K_m of 3.01 × 10⁵ S^-1 M^-1, and amidolytic activity of 28.9 µmol min^-1 L^-1. It was drastically inhibited by TLCK, PMSF, SBTI, and aprotinin while, DTT, β-mercaptoethanol, 2,2'-bipyridine, o-phenanthroline, EDTA, and EGTA had no effect, which suggested a serine protease-type. Also, it has shown a broad substrate specificity against a broad range of natural proteins including serum proteins. A cytotoxicity and electron microscopy study revealed that UcB5 could cause subcellular proteolysis that finally led to liver necrosis. For this, future research should focus on using a combination of external antiproteases and antimicrobial agents for the treatment of microbial diseases instead of using drugs alone.

SUBMITTER: Kotb E

PROVIDER: S-EPMC9998444 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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A putative cytotoxic serine protease from Salmonella typhimurium UcB5 recovered from undercooked burger.

Kotb Essam E El-Nogoumy Baher A BA Alqahtani Haifa A HA Ahmed Asmaa A AA Al-Shwyeh Hussah A HA Algarudi Sakina M SM Almahasheer Hanan H

Scientific reports 20230309 1

A putative virulence exoprotease designated as UcB5 was successfully purified from the bacterium Salmonella typhimurium to the electrophoretic homogeneity with 13.2-fold and 17.1% recovery by hydrophobic, ion-exchange, and gel permeation chromatography using Phenyl-Sepharose 6FF, DEAE-Sepharose CL-6B, and Sephadex G-75, respectively. By applying SDS-PAGE, the molecular weight was confirmed at 35 kDa. The optimal temperature, pH, and isoelectric point were 35 °C, 8.0, 5.6 ± 0.2, respectively. UcB ...[more]

PMID: 36894576

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Project description:Salmonella Typhimurium is the leading cause of human non-typhoidal gastroenteritis in the US. S. Kentucky is one the most commonly recovered serovars from commercially processed poultry carcasses. This study compared the genotypic and phenotypic properties of two Salmonella enterica strains Typhimurium (ST221_31B) and Kentucky (SK222_32B) recovered from commercially processed chicken carcasses using whole genome sequencing, phenotype characterizations and an intracellular killing assay. Illumina MiSeq platform was used for sequencing of two Salmonella genomes. Phylogenetic analysis employing homologous alignment of a 1,185 non-duplicated protein-coding gene in the Salmonella core genome demonstrated fully resolved bifurcating patterns with varying levels of diversity that separated ST221_31B and SK222_32B genomes into distinct monophyletic serovar clades. Single nucleotide polymorphism (SNP) analysis identified 2,432 (ST19) SNPs within 13 Typhimurium genomes including ST221_31B representing Sequence Type ST19 and 650 (ST152) SNPs were detected within 13 Kentucky genomes including SK222_32B representing Sequence Type ST152. In addition to serovar-specific conserved coding sequences, the genomes of ST221_31B and SK222_32B harbor several genomic regions with significant genetic differences. These included phage and phage-like elements, carbon utilization or transport operons, fimbriae operons, putative membrane associated protein-encoding genes, antibiotic resistance genes, siderophore operons, and numerous hypothetical protein-encoding genes. Phenotype microarray results demonstrated that ST221_31B is capable of utilizing certain carbon compounds more efficiently as compared to SK222_3B; namely, 1,2-propanediol, M-inositol, L-threonine, α-D-lactose, D-tagatose, adonitol, formic acid, acetoacetic acid, and L-tartaric acid. ST221_31B survived for 48 h in macrophages, while SK222_32B was mostly eliminated. Further, a 3-fold growth of ST221_31B was observed at 24 hours post-infection in chicken granulosa cells while SK222_32B was unable to replicate in these cells. These results suggest that Salmonella Typhimurium can survive host defenses better and could be more invasive than Salmonella Kentucky and provide some insights into the genomic determinants responsible for these differences.

Project description:BackgroundIntestinal fibrosis is a common and serious complication of Crohn's disease characterized by the accumulation of fibroblasts, deposition of extracellular matrix, and formation of scar tissue. Although many factors including cytokines and proteases contribute to the development of intestinal fibrosis, the initiating mechanisms and the complex interplay between these factors remain unclear.MethodsChronic infection of mice with Salmonella enterica serovar Typhimurium was used to induce intestinal fibrosis. A murine protease-specific CLIP-CHIP microarray analysis was employed to assess regulation of proteases and protease inhibitors. To confirm up- or downregulation during fibrosis, we performed quantitative real-time polymerase chain reaction (PCR) and immunohistochemical stainings in mouse tissue and tissue from patients with inflammatory bowel disease. In vitro infections were used to demonstrate a direct effect of bacterial infection in the regulation of proteases.ResultsMice develop severe and persistent intestinal fibrosis upon chronic infection with Salmonella enterica serovar Typhimurium, mimicking the pathology of human disease. Microarray analyses revealed 56 up- and 40 downregulated proteases and protease inhibitors in fibrotic cecal tissue. Various matrix metalloproteases, serine proteases, cysteine proteases, and protease inhibitors were regulated in the fibrotic tissue, 22 of which were confirmed by quantitative real-time PCR. Proteases demonstrated site-specific staining patterns in intestinal fibrotic tissue from mice and in tissue from human inflammatory bowel disease patients. Finally, we show in vitro that Salmonella infection directly induces protease expression in macrophages and epithelial cells but not in fibroblasts.ConclusionsIn summary, we show that chronic Salmonella infection regulates proteases and protease inhibitors during tissue fibrosis in vivo and in vitro, and therefore this model is well suited to investigating the role of proteases in intestinal fibrosis.

Project description:The flagellar gene fliO of Salmonella typhimurium can be translated from an AUG codon that overlaps the termination codon of fliN (K. Ohnishi et al., J. Bacteriol. 179:6092-6099, 1997). However, it had been concluded on the basis of complementation analysis that in Escherichia coli a second start codon 60 bp downstream was the authentic one (J. Malakooti et al., J. Bacteriol. 176:189-197, 1994). This raised the possibility of tandem translational starts, such as occur for the chemotaxis gene cheA; this possibility was increased by the existence of a stem-loop sequence covering the second start, a feature also found with cheA. Protein translated from the first start codon was detected regardless of whether the second start codon was present; it was also detected when the stem-loop structure was disrupted or deleted. Translation from the second start codon, either as the natural one (GUG) or as AUG, was not detected when the first start and intervening sequence were intact. Nor was it detected when the first codon was attenuated (by conversion of AUGAUG to AUAAUA; in S. typhimurium there is a second, adjacent, AUG) or eliminated (by conversion to CGCCGC); disruption of the stem-loop structure still did not yield detectable translation from the second start. When the entire sequence up to the second start was deleted, translation from the second start was detected provided the natural codon GUG had been converted to AUG. A fliO null mutant could be fully complemented in swarm assays whenever the first start and intervening sequence were present, regardless of the state of the second start. Reasonably good complementation occurred when the first start and intervening sequence were absent provided the second start was intact, either as AUG or as GUG; thus translation from the GUG codon must have been occurring even though protein levels were too low to be detected. The translated intervening sequence is rather divergent between S. typhimurium and E. coli and corresponds to a substantial cytoplasmic domain prior to the sole transmembrane segment, which is highly conserved; the sequence following the second start begins immediately prior to that transmembrane segment. The significance of the data for FliO is discussed and compared to the equivalent data for CheA. Attention is also drawn to the fact that given an optimal ribosome binding site, AUA can serve as a fairly efficient start codon even though it seldom if ever appears to be used in nature.

Dataset Information

A putative cytotoxic serine protease from Salmonella typhimurium UcB5 recovered from undercooked burger.

Publications

A putative cytotoxic serine protease from Salmonella typhimurium UcB5 recovered from undercooked burger.

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