Diversity, Structures, and Collagen-Degrading Mechanisms of Bacterial Collagenolytic Proteases

Zhang, Yuzhong; Ran, Ling; Li, Chunyang; Chen, Xiu-Lan

doi:10.1128/aem.00883-15

Cited by 108 publications

(103 citation statements)

References 97 publications

(102 reference statements)

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“…S2B). An exception among the class II proteases, this enzyme does not have a C-terminal PKD-like domain and thus cannot hydrolyze casein (28), explaining the lack of casein degradation in our P. damselae subsp. damselae collection.…”

Section: Figmentioning

confidence: 87%

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish

Vences

Rivas

Lemos

et al. 2017

Appl Environ Microbiol

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Photobacterium damselae subsp. damselae is a pathogen of marine animals, including fish of importance in aquaculture. The virulence plasmid pPHDD1, characteristic of highly hemolytic isolates, encodes the hemolysins damselysin (Dly) and phobalysin (PhlyP). Strains lacking pPHDD1 constitute the vast majority of the isolates from fish outbreaks, but genetic studies to identify virulence factors in plasmidless strains are scarce. Here, we show that the chromosome I-encoded hemolysin PhlyC plays roles in virulence and cell toxicity in pPHDD1-negative isolates of this pathogen. By combining the analyses of whole genomes and of gene deletion mutants, we identified two hitherto uncharacterized chromosomal loci encoding a phospholipase (PlpV) and a collagenase (ColP). PlpV was ubiquitous in the subspecies and exerted hemolytic activity against fish erythrocytes, which was enhanced in the presence of lecithin. ColP was restricted to a fraction of the isolates and was responsible for the collagen-degrading activity in this subspecies. Consistent with the presence of signal peptides in PlpV and ColP sequences, mutants for the type II secretion system (T2SS) genes epsL and pilD exhibited impairments in phospholipase and collagenase activities. Sea bass virulence experiments and cell culture assays demonstrated major contributions of PhlyC and PlpV to virulence and toxicity. IMPORTANCE This study constitutes genetic and genomic analyses of plasmidless strains of an emerging pathogen in marine aquaculture, Photobacterium damselae subsp. damselae. To date, studies on the genetic basis of virulence were restricted to the pPHDD1 plasmid-encoded toxins Dly and PhlyP. However, the vast majority of the recent isolates of this pathogen from fish farm outbreaks lack this plasmid. Here we demonstrate that the plasmidless strains produce two hitherto uncharacterized ubiquitous toxins encoded in chromosome I, namely, the hemolysin PhlyC and the phospholipase PlpV. We report the main roles of these two toxins in fish virulence and in cell toxicity. Our results constitute the basis for a better understanding of the virulence of a widespread marine pathogen.

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Section: Figmentioning

confidence: 87%

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish

Vences

Rivas

Lemos

et al. 2017

Appl Environ Microbiol

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“…It was suggested that these enzymes could contribute to the deterioration of structural integrity in root dentine (Zhang et al . , Duarte et al . ).…”

Section: Introductionmentioning

confidence: 99%

“…Collagenases, including metalloproteinases and serine proteases, are important virulence factors in a variety of pathogenic bacteria. It was suggested that these enzymes could contribute to the deterioration of structural integrity in root dentine (Zhang et al 2015, Duarte et al 2016.…”

Section: Introductionmentioning

confidence: 99%

Endodontic pathogens possess collagenolytic properties that degrade human dentine collagen matrix

et al. 2018

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Aim To measure collagenolytic protease activity from Enterococcus faecalis and Micrococcus luteus and their ability to degrade human dentinal collagen. Methodology Proteases activity of E. faecalis ATCC 29212, ATCC 47077 and M. luteus towards generic and specific human matrix metalloproteinase (MMP) substrates was measured using a fluorimetric assay. The ability of the bacteria to degrade dentinal collagen was tested by quantifying the amount of hydroxyproline released into the media following incubation of the bacteria or heat‐inactivated bacteria (HIN) with demineralized human dentine samples for 24 h and by scanning electron microscopy (SEM). Multifactorial anova and Tukey's post hoc test were used to analyse the data (P < 0.05). Results All strains had MMP‐like activities, but with different substrate affinity; E. faecalis ATCC 29212, ATCC 47077 and M. luteus had the greatest affinity towards MMP‐8 (7.75 ± 0.88 μmol L−1/3 × 106 CFU), MMP‐9 (33.86 ± 5.16 μmol L−1/3 × 106 CFU) and generic MMP (26.08 ± 4.48 μmol L−1/3 × 106 CFU), respectively. The amount of hydroxyproline released from demineralized dentine was similar (P > 0.05) for the three strains (range 1.8 ± 0.17 to 2.38 ± 0.39 μg 50 μL−1) and was significantly higher (P < 0.001) compared to their HIN counterparts (0.61 ± 0.22 μg 50 μL−1). SEM revealed increased collagen network degradation after incubation with bacteria versus HIN. Conclusions Endodontic pathogens possess collagenolytic protease properties that enable them to degrade dentinal collagen, potentially compromising the restoration‐tooth and sealer‐tooth interfaces. These collagenolytic protease properties could facilitate the migration of pathogenic bacteria into the root canal system and explain in part their role in root canal infections.

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“…MMP-12 CAT is a single domain protease, while collagenase I is multidomain [30], and thus MMP-12 CAT is less susceptible to autoproteolysis. MMP-12 exhibited excellent stability over 16 d storage (Figure 5E).…”

Section: Discussionmentioning

confidence: 99%

Matrix metalloproteinases as reagents for cell isolation

Knapinska

Amar

et al. 2016

Enzyme and Microbial Technology

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Cell isolation methods for therapeutic purposes have seen little advancement over the years. The original methods of stem cell and islet isolation using bacterial collagenases were developed in the early 1980s and are still used today. Bacterial collagenases are subject to autodegradation, and isolates obtained with these enzymes may be contaminated with endotoxins, reducing cell viability and contributing to toxicity in downstream applications. Here we describe a novel method for isolation of mesenchymal stem cells from adipose tissue (ADSC) utilizing recombinantly produced matrix metalloproteases (MMPs). The ADSCs isolated by MMPs displayed essentially identical morphological and phenotypical characteristics to cells isolated by bacterially-derived collagenase I and Liberase™. Samples isolated with MMPs and Liberase™ had comparable levels of CD73, CD90, and CD105. The adipogenic and osteogenic potential of the ADSCs isolated by MMPs was retained as compared to cells isolated with Liberase™. However, ADSCs isolated by Liberase™ displayed 6% contamination with other cells as per negative markers revealed by PE staining, as opposed to <1% for all MMP-treated samples. MMP-based cell isolation may contribute to optimization of transplantation technology.

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Diversity, Structures, and Collagen-Degrading Mechanisms of Bacterial Collagenolytic Proteases

Cited by 108 publications

References 97 publications

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish

Endodontic pathogens possess collagenolytic properties that degrade human dentine collagen matrix

Matrix metalloproteinases as reagents for cell isolation

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