Role of Microbial Proteases in Pathogenesis

Maeda, Hiroshi

doi:10.1111/j.1348-0421.1996.tb01129.x

Cited by 117 publications

(67 citation statements)

References 67 publications

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“…It has been postulated that the massive tissue disintegration caused by these metalloproteases, especially those possessing a collagenase function, may help bacteria cross the host barrier and gain access to deeper tissues (20,34,38). If InhA2 protease was to have a collagenase activity, like that of InhA, the passage of bacteria across the basement membrane underlining the midgut epithelium of the insect would be facilitated, thus explaining the synergy with the crystals following infection by the oral route.…”

Section: Discussionmentioning

confidence: 99%

“…Although the precise cause of the lethality was not elucidated, it is likely that the InhA metalloprotease interacts with a particularly important component of the host, leading to death. As illustrated by many examples (34,38), various zinc metalloproteases from pathogens actively contribute to pathology. Indeed, in addition to damaging the host tissues during infection, these proteases act as toxic factors that degrade many blood components and enhance vascular permeability, cytotoxicity, etc.…”

Section: Discussionmentioning

confidence: 99%

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The InhA2 Metalloprotease of Bacillus thuringiensis Strain 407 Is Required for Pathogenicity in Insects Infected via the Oral Route

2002

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The entomopathogenic bacterium Bacillus thuringiensis is known to secrete a zinc metalloprotease (InhA) that specifically cleaves antibacterial peptides produced by insect hosts. We identified a second copy of the inhA gene, named inhA2, in B. thuringiensis strain 407 Cry ؊ . The inhA2 gene encodes a putative polypeptide showing 66.2% overall identity with the InhA protein and harboring the zinc-binding domain (HEXXH), which is characteristic of the zinc-requiring metalloproteases. We used a transcriptional inhA2-lacZ fusion to show that inhA2 expression is induced at the onset of the stationary phase and is overexpressed in a Spo0A minus background. The presence of a reverse Spo0A box in the promoter region of inhA2 suggests that Spo0A directly regulates the transcription of inhA2. To determine the role of the InhA and InhA2 metalloproteases in pathogenesis, we used allelic exchange to isolate single and double mutant strains for the two genes. Spores and vegetative cells of the mutant strains were as virulent as those of the parental strain in immunized Bombyx mori larvae infected by the intrahemocoelic route. Exponential phase cells of all the strains displayed the same in vitro potential for colonizing the vaccinated hemocoel. We investigated the synergistic effect of the mutant strain spores on the toxicity of Cry1C proteins against Galleria mellonella larvae infected via the oral pathway. The spores of ⌬inhA2 mutant strain were ineffective in providing synergism whereas those of the ⌬inhA mutant strain were not. These results indicate that the B. thuringiensis InhA2 zinc metalloprotease has a vital role in virulence when the host is infected via the oral route.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The InhA2 Metalloprotease of Bacillus thuringiensis Strain 407 Is Required for Pathogenicity in Insects Infected via the Oral Route

2002

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“…Moreover, surface-associated proteolytic activity seems to be mainly due to a thiol-protease (32). Proteases are a well-known virulence factor for some important pathogenic bacteria (22), and especially the cysteine proteases produced by Porphyromonas gingivalis or Streptococcus pyogenes (21,35).…”

Section: Discussionmentioning

confidence: 99%

“…The cwp84 gene, located immediately downstream from cwp66, encodes a putative 84-kDa protein with a characteristic signal peptide, whose functional domain displays homologies with several cysteine proteases, and shows the conserved Pept_C1 domain of the papain family. Since extracellular proteases have been described to be virulence factors in many bacteria (22), we cloned and expressed the corresponding gene to permit functional studies. We studied the transcription of slpA, cwp66, and cwp84 genes by Northern blotting to determine whether these genes are expressed as part of an operon.…”

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confidence: 99%

Transcription and Analysis of Polymorphism in a Cluster of Genes Encoding Surface-Associated Proteins ofClostridium difficile

et al. 2003

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Recent investigations of the Clostridium difficile genome have revealed the presence of a cluster of 17 genes, 11 of which encode proteins with similar two-domain structures, likely to be surface-anchored proteins. Two of these genes have been proven to encode proteins involved in cell adherence: slpA encodes the precursor of the two proteins of the S-layer, P36 and P47, whereas cwp66 encodes the Cwp66 adhesin. To gain further insight into the function of this cluster, we further focused on slpA, cwp66, and cwp84, the latter of which encodes a putative surface-associated protein with homology to numerous cysteine proteases. It displayed nonspecific proteolytic activity when expressed as a recombinant protein in Escherichia coli. Polymorphism of cwp66 and cwp84 genes was analyzed in 28 strains, and transcriptional organization of the three genes was explored by Northern blots. The slpA gene is strongly transcribed during the entire growth phase as a bicistronic transcript; cwp66 is transcribed only in the early exponential growth phase as a polycistronic transcript encompassing the two contiguous genes upstream. The putative proteins encoded by the cotranscribed genes have no significant homology with known proteins but may have a role in adherence. No correlation could be established between sequence patterns of Cwp66 and Cwp84 and virulence of the strains. The cwp84 gene is strongly transcribed as a monocistronic message. This feature, together with the highly conserved sequence pattern of cwp84, suggests a significant role in the physiopathology of C. difficile for the Cwp84 protease, potentially in the maturation of surface-associated adhesins encoded by the gene cluster.

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“…Proteolytic processing of BK alters the affinity of the ligand and its receptor. Bradykinin generated as a result of bacterial infections induces pain, edema, vasodilation, hypotension, and shock (16,20). Bradykinins have also been shown to enhance the invasiveness of V. vulnificus by facilitating the transvascular translocation of the bacterium (17,19,21).…”

Section: Resultsmentioning

confidence: 99%

Specificity of a Vibrio vulnificus Aminopeptidase toward Kinins and Other Peptidyl Substrates

Richards

Núñez

2006

J Bacteriol

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Recently, phosphoglucose isomerase with a lysyl aminopeptidase (PGI-LysAP) activity was identified in Vibrio vulnificus. In this paper, we demonstrate the proteolytic cleavage of human-derived peptides by PGILysAP of V. vulnificus using three approaches: (i) a quantitative fluorescent ninhydrin assay for free lysine, (ii) matrix-assisted laser desorption ionization-two-stage time of flight mass spectrometry (MALDI-TOF-TOF), and (iii) Tricine gel electrophoresis. PGI-LysAP hydrolyzed bradykinin, Lys-bradykinin, Lys-(des-Arg 9 )-bradykinin, neurokinin A, Met-Lys-bradykinin, histatin 8, and a myosin light chain fragment. We detected the proteolytic release of free L-lysine from peptide digests using a rapid, simple, sensitive, and quantitative fluorescent ninhydrin assay, and results were confirmed by MALDI-TOF-TOF. The use of the fluorescent ninhydrin assay to quantitatively detect free lysine hydrolyzed from peptides is the first application of its kind and serves as a paradigm for future studies. The visualization of peptide hydrolysis was accomplished by Tricine gel electrophoresis. Proteolytic processing of kinins alters their affinities toward specific cellular receptors and initiates signal transduction mechanisms responsible for inflammation, vasodilation, and enhanced vascular permeability. By applying novel approaches to determine the proteolytic potential of bacterial enzymes, we demonstrate that PGI-LysAP has broad exopeptidase activity which may enhance V. vulnificus invasiveness by altering peptides involved in signal transduction pathways.Vibrio vulnificus is a naturally occurring marine bacterium indigenous to temperate and tropical estuarine environments. It has two routes of transmission: wound infections and the ingestion of contaminated seafood, especially raw oysters. During summer months, nearly 100% of the oysters harvested from the Gulf of Mexico contain V. vulnificus (9). Liver cirrhosis, hemochromatosis, diabetes, kidney disease, and immune disorders predispose individuals to V. vulnificus infection via the food-borne route (4, 24); however, even seemingly healthy individuals are susceptible to V. vulnificus through wound infections (24). Symptoms include fever, chills, hypotension, and the development of secondary bullous skin lesions. Septicemia is common, and death can occur within 24 h after contact with the pathogen (8, 24). Mortality rates for V. vulnificus infections are approximately 60% in the United States (15) but are somewhat higher in Asian countries (14,25).Over the past two decades, numerous virulence factors have been identified in V. vulnificus based on cell culture and animal models; however, the validity of these models in assessing virulence has come into question (7). A case in point involves V. vulnificus protease, a well-characterized metalloprotease (23). In the mouse model, injection of this protease causes skin necrosis; however, knockout of the gene in V. vulnificus does not attenuate the symptoms in Vibrio-infected laboratory animals (34).Recently, we isolated a...

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Role of Microbial Proteases in Pathogenesis

Cited by 117 publications

References 67 publications

The InhA2 Metalloprotease of Bacillus thuringiensis Strain 407 Is Required for Pathogenicity in Insects Infected via the Oral Route

The InhA2 Metalloprotease of Bacillus thuringiensis Strain 407 Is Required for Pathogenicity in Insects Infected via the Oral Route

Transcription and Analysis of Polymorphism in a Cluster of Genes Encoding Surface-Associated Proteins ofClostridium difficile

Specificity of a Vibrio vulnificus Aminopeptidase toward Kinins and Other Peptidyl Substrates

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