scholarly journals Distinct Roles of the Repeat-Containing Regions and Effector Domains of the Vibrio vulnificus Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) Toxin

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Byoung Sik Kim ◽  
Hannah E. Gavin ◽  
Karla J. F. Satchell
Keyword(s):  
Cysteine Protease ◽  
Vibrio Vulnificus ◽  
Terminal Repeat ◽  
Protease Domain ◽  
Content Type ◽  
Effector Domains ◽  
Amino Terminal ◽  
Toxin Secretion ◽  
Cysteine Protease Domain ◽  
Carboxyl Terminal

ABSTRACTVibrio vulnificusis a seafood-borne pathogen that destroys the intestinal epithelium, leading to rapid bacterial dissemination and death. The most important virulence factor is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin comprised of effector domains in the center region flanked by long repeat-containing regions which are well conserved among MARTX toxins and predicted to translocate effector domains. Here, we examined the role of the repeat-containing regions using a modifiedV. vulnificusMARTX (MARTXVv) toxin generated by replacing all the internal effector domains with β-lactamase (Bla). Bla activity was detected in secretions from the bacterium and also in the cytosol of intoxicated epithelial cells. The modified MARTXVvtoxin without effector domains retained its necrotic activity but lost its cell-rounding activity. Further, deletion of the carboxyl-terminal repeat-containing region blocked toxin secretion from the bacterium. Deletion of the amino-terminal repeat-containing region had no effect on secretion but completely abolished translocation and necrosis. Neither secretion nor translocation was affected by enzymatically inactivating the cysteine protease domain of the toxin. These data demonstrate that the amino-terminal and carboxyl-terminal repeat-containing regions of the MARTXVvtoxin are necessary and sufficient for the delivery of effector domains and epithelial cell lysisin vitrobut that effector domains are required for other cytopathic functions. Furthermore, Ca2+-dependent secretion of the modified MARTXVvtoxin suggests that nonclassical RTX-like repeats found in the carboxyl-terminal repeat-containing region are functionally similar to classical RTX repeats found in other RTX proteins.IMPORTANCEUp to 95% of deaths from seafood-borne infections in the United States are due solely to one pathogen,V. vulnificus. Among its various virulence factors, the MARTXVvtoxin has been characterized as a critical exotoxin for successful pathogenesis ofV. vulnificusin mouse infection models. Similarly to MARTX toxins of other pathogens, MARTXVvtoxin is comprised of repeat-containing regions, central effector domains, and an autoprocessing cysteine protease domain. Yet how each of these regions contributes to essential activities of the toxins has not been fully identified for any of MARTX toxins. Using modified MARTXVvtoxin fused with β-lactamase as a reporter enzyme, the portion(s) responsible for toxin secretion from bacteria, effector domain translocation into host cells, rapid host cell rounding, and necrotic host cell death was identified. The results are relevant for understanding how MARTXVvtoxin serves as both a necrotic pore-forming toxin and an effector delivery platform.

scholarly journals The Porcine Reproductive and Respiratory Syndrome Virus nsp2 Cysteine Protease Domain Possesses both trans- and cis-Cleavage Activities

2009 ◽  
Vol 83 (18) ◽  
pp. 9449-9463 ◽  
Author(s):  
Jun Han ◽  
Mark S. Rutherford ◽  
Kay S. Faaberg
Keyword(s):  
Cysteine Protease ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Point Mutations ◽  
Respiratory Syndrome Virus ◽  
Protease Domain ◽  
Nonstructural Protein 2 ◽  
Cleavage Activity ◽  
Cysteine Protease Domain

ABSTRACT The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr47 to Cys240) and aa 47 to 323 (Tyr47 to Leu323), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys55- His124 catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys111, Cys142, and Cys147). The conserved aspartic acids (e.g., Asp89) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G1196|G1197 dipeptide.

scholarly journals Variable Virulence of Biotype 3Vibrio vulnificusdue to MARTX Toxin Effector Domain Composition

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Byoung Sik Kim ◽  
Hannah E. Gavin ◽  
Karla J. F. Satchell
Keyword(s):  
Food Chain ◽  
Vibrio Vulnificus ◽  
Infectious Agent ◽  
High Morbidity ◽  
Effector Domain ◽  
Content Type ◽  
Human Food ◽  
Effector Domains ◽  
Human Food Chain ◽  
Natural Hosts

ABSTRACTVibrio vulnificusis an environmental organism that causes septic human infections characterized by high morbidity and mortality. The annual incidence and global distribution of this pathogen are increasing as ocean waters warm. Clinical strains exhibit variations in the primary virulence toxin, suggesting a potential for the emergence of new strains with altered virulence properties. A clonal outbreak of tilapia-associated wound infections in Israel serves as a natural experiment for the sudden emergence of a newV. vulnificusstrain. The effector domain content of the multifunctional autoprocessing RTX (MARTX) toxin of the outbreak-associated biotype 3 (BT3) strains was previously shown to harbor a modification generated by recombination. The modification introduced an actin-induced adenylate cyclase effector domain (ExoY) and an effector domain that disrupts the Golgi organelle (DmX). Here, we report that the exchange of these effector domains for a putative progenitor biotype 1 toxin arrangement produces a toxin that slows the lysis kinetics of targeted epithelial cells but increases cellular rounding phenotypes in response to bacteria. In addition, replacing the biotype 3 toxin variant with the putative progenitor biotype 1 variant renders the resulting strain significantly more virulent in mice. This suggests that the exchange of MARTX effector domains during the emergence of BT3 generated a toxin with reduced toxin potency, resulting in decreased virulence of this outbreak-associated strain. We posit that selection for reduced virulence may serve as a route for this lethal infectious agent to enter the human food chain by allowing it to persist in natural hosts.IMPORTANCEVibrio vulnificusis a serious infection linked to climate change. The virulence capacity of these bacteria can vary by gene exchange, resulting in new variants of the primary virulence toxin. In this study, we tested whether the emergence of an epidemic strain ofV. vulnificuswith a novel toxin variant correlated with a change in virulence. We found that restoring the biotype 3 toxin variant to the putative progenitor-type toxin resulted in dramatically increased virulence, revealing that the emergence of the biotype 3 strain could be linked to virulence reduction. This reduced virulence, previously found also in the biotype 1 strain, suggests that reduced virulence may stimulate outbreaks, as strains have greater capacity to enter the human food chain through reduced impact to environmental hosts.

scholarly journals Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain

2007 ◽  
Vol 26 (10) ◽  
pp. 2552-2561 ◽  
Author(s):  
Kerri-Lynn Sheahan ◽  
Christina L Cordero ◽  
Karla J Fullner Satchell
Keyword(s):  
Vibrio Cholerae ◽  
Cysteine Protease ◽  
Protease Domain ◽  
Rtx Toxin ◽  
Cysteine Protease Domain

scholarly journals Small Molecule-Induced Allosteric Activation of the Vibrio cholerae RTX Cysteine Protease Domain

Science ◽  
2008 ◽  
Vol 322 (5899) ◽  
pp. 265-268 ◽  
Author(s):  
P. J. Lupardus ◽  
A. Shen ◽  
M. Bogyo ◽  
K. C. Garcia
Keyword(s):  
Vibrio Cholerae ◽  
Small Molecule ◽  
Cysteine Protease ◽  
Protease Domain ◽  
Allosteric Activation ◽  
Cysteine Protease Domain

scholarly journals The Cysteine Protease Domain of Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 2 Possesses Deubiquitinating and Interferon Antagonism Functions

2010 ◽  
Vol 84 (15) ◽  
pp. 7832-7846 ◽  
Author(s):  
Zhi Sun ◽  
Zhenhai Chen ◽  
Steven R. Lawson ◽  
Ying Fang
Keyword(s):  
Cysteine Protease ◽  
Nonstructural Protein ◽  
Cell Epitope ◽  
Wild Type Virus ◽  
Type I ◽  
Protease Domain ◽  
Live Virus ◽  
Recombinant Viruses ◽  
Nonstructural Protein 2 ◽  
Cysteine Protease Domain

ABSTRACT Porcine reproductive and respiratory syndrome (PRRS) virus nonstructural protein 2 (nsp2) contains a cysteine protease domain at its N terminus, which belongs to the ovarian tumor (OTU) protease family. In this study, we demonstrated that the PRRSV nsp2 OTU domain antagonizes the type I interferon induction by interfering with the NF-κB signaling pathway. Further analysis revealed that the nsp2 OTU domain possesses ubiquitin-deconjugating activity. This domain has the ability to inhibit NF-κB activation by interfering with the polyubiquitination process of IκBα, which subsequently prevents IκBα degradation. To determine whether the nsp2 protein antagonist function can be ablated from the virus, we introduced point mutations into the OTU domain region by use of reverse genetics. The D458A, S462A, and D465A mutations targeting on a B-cell epitope in the OTU domain region generated the viable recombinant viruses, and the S462A and D465A mutants were attenuated for growth in cell culture. The OTU domain mutants were examined to determine whether mutations in the nsp2 OTU domain region altered virus ability to inhibit NF-κB activation. The result showed that certain mutations lethal to virus replication impaired the ability of nsp2 to inhibit NF-κB activation but that the viable recombinant viruses, vSD-S462A and vSD-D465A, were unable to inhibit NF-κB activation as effectively as the wild-type virus. This study represents a fundamental step in elucidating the role of nsp2 in PRRS pathogenesis and provides an important insight in future modified live-virus vaccine development.

scholarly journals CPDadh: A new peptidase family homologous to the cysteine protease domain in bacterial MARTX toxins

2009 ◽  
pp. NA-NA
Author(s):  
Jimin Pei ◽  
Patrick J. Lupardus ◽  
K. Christopher Garcia ◽  
Nick V. Grishin
Keyword(s):  
Cysteine Protease ◽  
Protease Domain ◽  
Cysteine Protease Domain
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