Journal of Microbiology

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VvpM, an Extracellular Metalloprotease of Vibrio vulnificus, Induces Apoptotic Death of Human Cells: Mi-Ae Lee , Jeong-A Kim , Yu Jin Yang , Mee-Young Shin , Soon-Jung Park , Kyu-Ho Lee; J. Microbiol. 2014;52(12):1036-1043. Published online November 3, 2014; DOI: https://doi.org/10.1007/s12275-014-4531-0

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A pathogenic bacterium, Vibrio vulnificus produces various extracellular proteases including the elastolytic metalloprotease, VvpE. In silico analysis of its genome revealed a VvpEhomologous protease, VvpM whose proteolytic activity was abolished by specific inhibitors against metalloproteases. To investigate whether this newly identified protease has pathogenic role in host interaction in addition to proteolytic role, human cell lines were incubated with recombinant VvpM (rVvpM). rVvpM-challenged cells showed typical morphological changes found in cells under apoptosis. Apoptotic cell death was further evidenced by estimating the Annexin V-stained cells, whose proportions were dependent upon the concentrations of rVvpM treated to human cells. To elucidate the signaling pathway for VvpM-induced apoptosis, three MAPKs were tested if their activation were mediated by rVvpM. ERK1/2 was phosphorylated by treatment of rVvpM and rVvpM-induced cell death was blocked by a specific inhibitor against ERK1/2. In rVvpM-treated cells, the cytosolic levels of cytochrome c were increased in a VvpM concentration- dependent manner, while the levels of cytochrome c in mitochondria were decreased. Cell deaths were accompanied by apparent cleavages of procaspases-9 and -3 to the active caspases-9 and -3, respectively. Therefore, this study demonstrates that an extracellular metalloprotease of V. vulnificus, VvpM induces apoptosis of human cells via a pathway consisting of ERK activation, cytochrome c release, and then activation of caspases-9 and -3.

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Two Forms of Vibrio vulnificus Metalloprotease VvpE are Secreted via the Type II General Secretion System: Jong Park , So-Yeon Ryu , Choon-Mee Kim , Sung-Heui Shin; J. Microbiol. 2008;46(3):338-343. Published online July 5, 2008; DOI: https://doi.org/10.1007/s12275-008-0058-6

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Abstract: Vibrio vulnificus has been known to secrete one form of metalloprotease VvpE (45 kDa) that is cleaved to 34 kDa-VvpE and 11 kDa-C-terminal propeptide via extracellular autoproteolysis. However, we found that extracellular secretion of both the 34 and 45 kDa forms of VvpE began in the early growth phase; moreover, 34 kDa-VvpE existed as the major form in V. vulnificus cell lysates and culture supernatants. In addition, extracellular secretion of both 34 and 45 kDa-VvpE was blocked by mutation of the pilD gene, which encodes for the type IV leader peptidase/N-methyltransferase of the type II general secretion system, and the blocked VvpE secretion was recovered by in trans-complementation of the wild-type pilD gene. These results indicate that 34 kDa-VvpE is the major form secreted along with 45 kDa-VvpE from the early growth phase via the PilD-mediated type II general secretion system.

Vibrio vulnificus Metalloprotease VvpE has no Direct Effect on Iron-uptake from Human Hemoglobin: Hui-Yu Sun , Song-Iy Han , Mi-Hwa Choi , Seong-Jung Kim , Choon-Mee Kim , Sung-Heui Shin; J. Microbiol. 2006;44(5):537-547.; DOI: https://doi.org/2443 [pii]

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Abstract: This study was designed to determine whether or not Vibrio vulnificus metalloprotease VvpE can promote iron uptake via the proteolytic cleavage of human hemoglobin. We found that V. vulnificus utilized hemoglobin as an iron source more efficiently via the vulnibactin-mediated iron-uptake system than via the HupA-mediated iron-uptake system and, of the proteases produced by V. vulnificus, VvpE was found to be the only protease capable of destroying hemoglobin. However, VvpE expression, on both the transcriptional and protein levels, was suppressed in iron-limited media. However, vvpE transcription, but not extracellular VvpE production, was reactivated by the addition of hemoglobin or inorganic iron into iron-limited media. Moreover, vvpE transcription began only in the late growth phase when V. vulnificus had already consumed most of the iron for growth. In addition, neither vvpE mutation nor in trans vvpE complementation affected the ability of V. vulnificus to acquire iron or to grow in iron-limited media or in cirrhotic ascites containing hemoglobin. Hemoglobin added into iron-limited media was not destroyed, but gradually formed an insoluble aggregate during culture; this aggregation of hemoglobin occurred regardless of vvpE mutation or complementation. These results indicate that VvpE is not required for efficient iron uptake from hemoglobin. On the contrary, hemoglobin or iron is required for efficient vvpE transcription. In addition, a discrepancy exists between vvpE transcription and extracellular VvpE production in iron-limited media containing inorganic iron or hemoglobin, which suggests that additional unknown posttranscriptional events may be involved in the extracellular production of VvpE.

Characteristics of trypsin-like protease and metalloprotease associated with mycelium differentiation of streptomyces albidoflavus SMF301: Kang, Sung Gyun , Kim, In Seop , Jeong, Byung Cheol , Ryu, Jae Gon , Rho, Yong Taik , Lee, Kye Joon; J. Microbiol. 1995;33(4):307-314.

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Abstract: Trypsin like protease (TLP) and metalloprotease (MTP) were induced in associated with the mycelium differentiation in Streptomyces albidoflavus SMF301. TLP and MTP were purified and characterized from the culture. The molecular mass of TLP and MTP were estimated to be 32 kDa and 18 kDa, respectively. The molecular mass of TLP and MTP were estimated to be 32 kDa and 18 kDa, respectively. The optimum pH and temperature of TLP were 10 and 40℃. Those of MTP were 8 and 55 ℃. TLP was stable at alkaline pH (6-9) and unstable above 45℃ and MTP was stable at alkaline pH and unstable above 80℃. Km and Vmax values with benzoyl-arginyl p-nitroanilide of TLP were 139 uM, and 10 nmole of nitroanilide released per min per ㎍ protein, respectively. Km, and Vmax values with a synthetic substrate, leucine p-nitroanilide, or MTP were 58.9 uM, 3.47 nmol of nitroanilide released per min per/㎍ protein, respectively. TLP was inhibited competitively by leupeptin; the inhibition constant was 0.0031 uM. MTP was inhibited by EDTA, phenonthroline and bestatin.

Purification and Partial Characterization of a metalloprotease in Flammulina velutipes: Shin, Hyun Hee , Choi, Hye Seon; J. Microbiol. 1998;36(1):20-25.

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Abstract: Metalloprotease was detected in the fruit body of the edible mushroom Flammulina velutipe. Inactivation of the metalloprotease reduced mycelial growth signicantly, implying that metalliprotease is important for growth. A neutral metalloprotease was purified by hydrophobic, gel filtration, and anion exchange chromatography. The M_r of the protein was determined to be 30,000 by SDS-PAGE and 33,000 by gel filtration on a Sepjadex G-150 column, indicating that it is a monomer. Its first 11 N-terminal amino acids (P-Q-V-K-T-W-D-L-A) did not show any homology with any known protein in Database(GENEBANK, Swissprot). The enzyme was inhibited by EDTA, 1, 10-phenanthroline, and phosphoamidon but not by inhibitors specific for serine, aspartate and custeine protease. Addition of Zn^2+ and Co^2+ reversed the inhibition caused by 1,10-ohenanthroline. This protease hydrolyzed human fivrinogen, fibrin, azoalbumin, and azocasein as substrates. It showed cleavage preference for hydrophobic residues among tested synthetic substrates.

Characterization of Bacillus cereus SH-7 Extracellular Protease: Hak Kyu Yi , Young Jin Chum , Han Bok Kim; J. Microbiol. 1999;37(4):213-217.

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Abstract: An extracellular endopeptidase from Bacillus cereus SH-7 was purified to homogeneity. The protease was most active at pH 8 and 40 C, respectively. The molecular mass of the protease was 40 kDa on SDS-PAGE, and 120 kDa by gel filtration, suggesting that the native enzyme is composed of three homogeneous subunits. The K_m and V_max values of the protease for N-succinyl-(Ala)_2-Pro-Phe-p-nitroanilide were 11.11 mM and 170 nmol/mg of protein/min, respectively. The protease was also identified as a metalloprotease. The bioactivity of the SH-7 protease will need further study in the future.

Streptomyces griseus HH1, An A-factor Deficient Mutant, Produces Diminished Level of Trypsin and Increased Level of Metalloproteases: Jung-Mee Kim , Soon-Kwang Hong; J. Microbiol. 2000;38(3):160-168.

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Abstract: A-factor is a microbial hormone that can positively control cell differentiation leading to spore formation and secondary metabolite formation in Streptomyces griseus. To identify a protease that is deeply involved in the morphological and physiological differentiation of Streptomyces, the proteases produced by S. griseus IFO 13350 and its A-factor deficient mutant strain, S. griseus HH1, as well as S. griseus HH1 transformed with the afsA gene were studied. In general, S. griseus showed a higher degree of cell growth and protease activity in proportion to its ability to produce a higher amount of A-factor. In particular, the specific activity of the trypsin of S. griseus IFO 13350 was greatly enhanced more than twice compared with that of S. griseus HH1 in the later stage of growth. The specific activity of the metalloprotease of S. griseus HH1 was greatly enhanced more than twice compared with that of S. griseus IFO 13350, and this observation was reversed in the presence of thiostreptone. However, S. griseus HH1 transformed with the afsA gene showed a significantly decreased level of trypsin and metalloprotease activity compared with that of the HH1 strain. There was no significant difference between S. griseus IFO 13350 and HH1 strain in their chymotrypsin and thiol protease activity, yet the level of leu-aminopeptidase activity was 2 times higher in S. griseus HH1 than in strain IFO 13350. S. griseus HH1 harboring afsA showed a similar level of enzyme activity, however, all the three protease activities sharply increased and the thiol protease activity was critically increased at the end of the fermentation. When a serine protease inhibitor, pefabloc SC, and metalloprotease inhibitor, EDTA, were applied to strain IFO 13350 to examine the in vivo effects of the protease inhibitors on the morphological differentiation, the formation of aerial mycelium and spores was delayed by two or three days.

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