The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform
Martin J Mcgavin - One of the best experts on this subject based on the ideXlab platform.
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Role of Lipase from Community-Associated Methicillin-Resistant Staphylococcus aureus Strain USA300 in Hydrolyzing Triglycerides into Growth-Inhibitory Free Fatty Acids
Journal of bacteriology, 2014Co-Authors: Brigitte Cadieux, Martin J Mcgavin, Vithooshan Vijayakumaran, Mark A. Bernards, David E. HeinrichsAbstract:Part of the human host innate immune response involves the secretion of bactericidal lipids on the skin and delivery of triglycerides into abscesses to control invading pathogens. Two Staphylococcus aureus lipases, named SAL1 and SAL2, were identified in the community-associated methicillin-resistant S. aureus strain USA300, which, presumably, are produced and function to degrade triglycerides to release free fatty acids. We show that the SAL2 lipase is one of the most abundant proteins secreted by USA300 and is proteolytically processed from the 72-kDa proSAL2 to the 44-kDa mature SAL2 by the metalloprotease Aureolysin. We show that spent culture supernatants had lipase activity on both short- and long-chain fatty acid substrates and that deletion of gehB, encoding SAL2, resulted in the complete loss of these activities. With the use of gas chromatography-mass spectrometry, we show that SAL2 hydrolyzed trilinolein to linoleic acid, a fatty acid with known antistaphylococcal properties. When added to cultures of USA300, trilinolein and, to a lesser extent, triolein inhibited growth in a SAL2-dependent manner. This effect was shown to be due to the enzymatic activity of SAL2 on these triglycerides, since the catalytically inactive SAL2 Ser412Ala mutant was incapable of hydrolyzing the triglycerides or yielding delayed growth in their presence. Overall, these results reveal that SAL2 hydrolyzes triglycerides of both short- and long-chain fatty acids and that the released free fatty acids have the potential to cause significant delays in growth, depending on the chemical nature of the free fatty acid.
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Production and distribution of selected MW2 (USA400) virulence factors during phagocytosis by human PMNs
2011Co-Authors: Christopher Burlak, Carl H Hammer, Mary-ann Robinson, Adeline R Whitney, Martin J Mcgavin, Barry N Kreiswirth, Frank R DeleoAbstract:Copyright information:Taken from "Global analysis of community-associated methicillin-resistant exoproteins reveals molecules produced and during infection"Cellular Microbiology 2007;9(5):1172-1190.Published online 01 May 2007PMCID:PMC2064037.Journal compilation © 2007 Blackwell Publishing Ltd; No claim to original US government works Following phagocytosis of MW2, Aureolysin (Aur), SspA and SspB were visualized by confocal laser-scanning microscopy. White arrowheads indicate bacteria. Yellow arrowheads indicate areas enriched with the protein of interest. The image labelled ‘Merge’ illustrates distribution of neutrophil actin-related protein (ARP, green) and nuclei (blue). DIC, differential interference contrast
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rapid autocatalytic activation of the m4 metalloprotease Aureolysin is controlled by a conserved n terminal fungalysin thermolysin propeptide domain
Molecular Microbiology, 2008Co-Authors: Nicholas N. Nickerson, Vineet Joag, Martin J McgavinAbstract:SUMMARY The Staphylococcus aureus proteolytic cascade consists of a metalloprotease Aureolysin (Aur), which activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease. As with other M4 metalloproteases, including elastase of Pseudomonas aeruginosa, the propeptide of proAur contains an N-terminal fungalysin-thermolysin-propeptide (FTP) domain. Autocatalytic activation of proAur was initiated by processing at T85 downward arrowL(86) in the FTP domain. This differed from the mechanism described for proElastase, where the FTP domain has an RY motif in place of TL(86), and processing occurred at the junction of the propeptide and metalloprotease domains, which remained as an inactive complex during passage across the outer membrane. When TL(86) in the FTP domain was replaced with RY, an intact N-terminal propeptide was secreted, but the M4 metalloprotease domain was degraded. Consequently, this segment of the FTP domain promotes intramolecular processing of proAur while bestowing a chaperone function, but discourages processing within the FTP domain of proElastase, where activation must be co-ordinated with passage across a second membrane. We conclude that the FTP domain of proAur is adapted to facilitate a rapid autocatalytic activation mechanism, consistent with the role or proAur as initiator of the staphylococcal proteolytic cascade.
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Rapid autocatalytic activation of the M4 metalloprotease Aureolysin is controlled by a conserved N‐terminal fungalysin‐thermolysin‐propeptide domain
Molecular microbiology, 2008Co-Authors: Nicholas N. Nickerson, Vineet Joag, Martin J McgavinAbstract:The Staphylococcus aureus proteolytic cascade consists of a metalloprotease Aureolysin (Aur), which activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease. As with other M4 metalloproteases, including elastase of Pseudomonas aeruginosa, the propeptide of proAur contains an N-terminal fungalysin-thermolysin-propeptide (FTP) domain. Autocatalytic activation of proAur was initiated by processing at T85 downward arrowL(86) in the FTP domain. This differed from the mechanism described for proElastase, where the FTP domain has an RY motif in place of TL(86), and processing occurred at the junction of the propeptide and metalloprotease domains, which remained as an inactive complex during passage across the outer membrane. When TL(86) in the FTP domain was replaced with RY, an intact N-terminal propeptide was secreted, but the M4 metalloprotease domain was degraded. Consequently, this segment of the FTP domain promotes intramolecular processing of proAur while bestowing a chaperone function, but discourages processing within the FTP domain of proElastase, where activation must be co-ordinated with passage across a second membrane. We conclude that the FTP domain of proAur is adapted to facilitate a rapid autocatalytic activation mechanism, consistent with the role or proAur as initiator of the staphylococcal proteolytic cascade.
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Activation of the SspA Serine Protease Zymogen of Staphylococcus aureus Proceeds through Unique Variations of a Trypsinogen-like Mechanism and Is Dependent on Both Autocatalytic and Metalloprotease-specific Processing
The Journal of biological chemistry, 2007Co-Authors: Nicholas N. Nickerson, Lata Prasad, Latha Jacob, Louis T. J. Delbaere, Martin J McgavinAbstract:The serine and cysteine proteases SspA and SspB of Staphylococcus aureus are secreted as inactive zymogens, zSspA and zSspB. Mature SspA is a trypsin-like glutamyl endopeptidase and is required to activate zSspB. Although a metalloprotease Aureolysin (Aur) is in turn thought to contribute to activation of zSspA, a specific role has not been demonstrated. We found that pre-zSspA is processed by signal peptidase at ANA(29) downward arrow, releasing a Leu(30) isoform that is first processed exclusively through autocatalytic intramolecular cleavage within a glutamine-rich propeptide segment, (40)QQTQSSKQQTPKIQ(53). The preferred site is Gln(43) with secondary processing at Gln(47) and Gln(53). This initial processing is necessary for optimal and subsequent Aur-dependent processing at Leu(58) and then Val(69) to release mature SspA. Although processing by Aur is rate-limiting in zSspA activation, the first active molecules of Val(69)SspA promote rapid intermolecular processing of remaining zSspA at Glu(65), producing an N-terminal (66)HANVILP isoform that is inactive until removal of the HAN tripeptide by Aur. Modeling indicated that His(66) of this penultimate isoform blocks the active site by hydrogen bonding to Ser(237) and occlusion of substrate. Binding of glutamate within the active site of zSspA is energetically unfavorable, but glutamine fits into the primary specificity pocket and is predicted to hydrogen bond to Thr(232) proximal to Ser(237), permitting autocatalytic cleavage of the glutamine-rich propeptide segment. These and other observations suggest that zSspA is activated through a trypsinogen-like mechanism where supplementary features of the propeptide must be sequentially processed in the correct order to allow efficient activation.
Mark S. Smeltzer - One of the best experts on this subject based on the ideXlab platform.
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SarA plays a predominant role in controlling the production of extracellular proteases in the diverse clinical isolates of Staphylococcus aureus LAC and UAMS-1.
Virulence, 2020Co-Authors: Aura M. Ramirez, Karen E. Beenken, Stephanie D. Byrum, Alan J. Tackett, Lindsey N. Shaw, Brittney D. Gimza, Mark S. SmeltzerAbstract:Using DNA affinity chromatography we demonstrate that the S. aureus regulatory proteins MgrA, Rot, SarA, and SarS bind DNA baits derived from the promoter regions associated with the genes encoding Aureolysin, ScpAB, SspABC, and SplA-F. Three of four baits also bound SarR and SarZ, the exception in both cases being the ScpAB-associated bait. Using the USA300, methicillin-resistant strain LAC and the USA200, methicillin-sensitive strain UAMS-1, we generated mutations in the genes encoding each of these proteins alone and in combination with sarA and examined the impact on protease production, the accumulation of high molecular weight proteins, and biofilm formation. These studies confirmed that multiple regulatory loci are involved in limiting protease production to a degree that impacts all of these phenotypes, but also demonstrate that sarA plays a predominant role in this regard. Using sarA mutants unable to produce individual proteases alone and in combination with each other, we also demonstrate that the increased production of Aureolysin and ScpA is particularly important in defining the biofilm-deficient phenotype of LAC and UAMS-1 sarA mutants, while Aureolysin alone plays a key role in defining the reduced accumulation of alpha toxin and overall cytotoxicity as assessed using both osteoblasts and osteoclasts.
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Impact of individual extracellular proteases on Staphylococcus aureus biofilm formation in diverse clinical isolates and their isogenic sarA mutants.
MicrobiologyOpen, 2014Co-Authors: Allister J. Loughran, Danielle N. Atwood, Allison C. Anthony, Nada S Harik, Horace J. Spencer, Karen E. Beenken, Mark S. SmeltzerAbstract:We demonstrate that the purified Staphylococcus aureus extracellular proteases Aureolysin, ScpA, SspA, and SspB limit biofilm formation, with Aureolysin having the greatest impact. Using protease-deficient derivatives of LAC, we confirmed that this is due to the individual proteases themselves. Purified Aureolysin, and to a lesser extent ScpA and SspB, also promoted dispersal of an established biofilm. Mutation of the genes encoding these proteases also only partially restored biofilm formation in an FPR3757 sarA mutant and had little impact on restoring virulence in a murine bacteremia model. In contrast, eliminating the production of all of these proteases fully restored both biofilm formation and virulence in a sarA mutant generated in the closely related USA300 strain LAC. These results confirm an important role for multiple extracellular proteases in S. aureus pathogenesis and the importance of sarA in repressing their production. Moreover, purified Aureolysin limited biofilm formation in 14 of 15 methicillin-resistant isolates and 11 of 15 methicillin-susceptible isolates, while dispersin B had little impact in UAMS-1, LAC, or 29 of 30 contemporary isolates of S. aureus. This suggests that the role of sarA and its impact on protease production is important in diverse strains of S. aureus irrespective of their methicillin resistance status.
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A Secreted Bacterial Protease Tailors the Staphylococcus aureus Virulence Repertoire to Modulate Bone Remodeling during Osteomyelitis
Cell host & microbe, 2013Co-Authors: James E. Cassat, Mark S. Smeltzer, Neal D. Hammer, J. Preston Campbell, Meredith A. Benson, Daniel S. Perrien, Lara N. Mrak, Victor J. Torres, Eric P. SkaarAbstract:Osteomyelitis is a common manifestation of invasive Staphylococcus aureus infection. Pathogen-induced bone destruction limits antimicrobial penetration to the infectious focus and compromises treatment of osteomyelitis. To investigate mechanisms of S. aureus-induced bone destruction, we developed a murine model of osteomyelitis. Microcomputed tomography of infected femurs revealed that S. aureus triggers profound alterations in bone turnover. The bacterial regulatory locus sae was found to be critical for osteomyelitis pathogenesis, as Sae-regulated factors promote pathologic bone remodeling and intraosseous bacterial survival. Exoproteome analyses revealed the Sae-regulated protease Aureolysin as a major determinant of the S. aureus secretome and identified the phenol-soluble modulins as Aureolysin-degraded, osteolytic peptides that trigger osteoblast cell death and bone destruction. These studies establish a murine model for pathogen-induced bone remodeling, define Sae as critical for osteomyelitis pathogenesis, and identify protease-dependent exoproteome remodeling as a major determinant of the staphylococcal virulence repertoire.
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The impact of protease inhibitors on the activity of extracellular proteases.
2013Co-Authors: Karen E. Beenken, Alexander R Horswill, Lindsey N. Shaw, Lara N. Mrak, Linda M. Griffin, Agnieszka K. Zielinska, Kelly C. Rice, Kenneth W. Bayles, Mark S. SmeltzerAbstract:Supernatants from the wild-type strains (WT) and their isogenic sarA mutants with (SPI) and without (S) protease inhibitors were harvested from overnight cultures and standardized with respect to each other prior to zymographic analysis using both casein (top) and gelatin gels (bottom). Based on relative activity with casein vs. gelatin, molecular size, and known polymorphisms within the corresponding genes/proteins [49], the presumed identity of specific proteases are SspA (1), Aureolysin (2), ScpA (3) and SspB (4). The identity of other proteases remains unknown.
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Impact of sarA, saeRS, and extracellular proteases on accumulation of FnbA and biofilm formation.
2013Co-Authors: Lara N. Mrak, Danielle N. Atwood, Karen E. Beenken, Linda M. Griffin, Agnieszka K. Zielinska, Ian N. Mrak, Chia Y. Lee, Mark S. SmeltzerAbstract:Top: Relative amounts of surface-anchored FnbA were assessed in Newman (New), its saeS-repaired derivative (P18L), and its saeRS mutant (sae) after introduction of an intact copy of fnbA on a plasmid. Newman without this plasmid was included as a negative control. The impact of mutating sarA was assessed in each of these strains together with the impact of mutating the gene encoding Aureolysin (aur), sspABC (ssp) or sae on the phenotype of the sarA mutants. Bottom: Biofilm formation was assessed by microtiter plate assay in Newman and P18L as well as their sarA and sarA/ssp derivatives after the introduction of pFnbA.
Jan Potempa - One of the best experts on this subject based on the ideXlab platform.
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Staphylococcal proteases aid in evasion of the human complement system.
Journal of innate immunity, 2013Co-Authors: Monika Jusko, Jan Potempa, Grzegorz Dubin, Lindsey N. Shaw, Tomasz Kantyka, Ewa Bielecka, Halie K. Miller, Magdalena Kalinska, Peter Garred, Anna M. BlomAbstract:Staphylococcus aureus is an opportunistic pathogen that presents severe health care concerns due to the prevalence of multiple antibiotic-resistant strains. New treatment strategies are urgently needed, which requires an understanding of disease causation mechanisms. Complement is one of the first lines of defense against bacterial pathogens, and S. aureus expresses several specific complement inhibitors. The effect of extracellular proteases from this bacterium on complement, however, has been the subject of limited investigation, except for a recent report regarding cleavage of the C3 component by Aureolysin (Aur). We demonstrate here that four major extracellular proteases of S. aureus are potent complement inhibitors. Incubation of human serum with the cysteine proteases staphopain A and staphopain B, the serine protease V8 and the metalloproteinase Aur resulted in a drastic decrease in the hemolytic activity of serum, whereas two staphylococcal serine proteases D and E, had no effect. These four proteases were found to inhibit all pathways of complement due to the efficient degradation of several crucial components. Furthermore, S. aureus mutants lacking proteolytic enzymes were found to be more efficiently killed in human blood. Taken together, the major proteases of S. aureus appear to be important for pathogen-mediated evasion of the human complement system.
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The human fibrinolytic system is a target for the staphylococcal metalloprotease Aureolysin.
The Biochemical journal, 2008Co-Authors: Nathalie Beaufort, Jan Potempa, Piotr Wojciechowski, Christian P. Sommerhoff, Grzegorz Szmyd, Grzegorz Dubin, Sigrun Eick, Josef Kellermann, Manfred Schmitt, Viktor MagdolenAbstract:The major opportunistic pathogen Staphylococcus aureus utilizes the human fibrinolytic system for invasion and spread via plasmin(ogen) binding and non-proteolytic activation. Because S. aureus secretes several proteases recently proposed as virulence factors, we explored whether these enzymes could add to the activation of the host's fibrinolytic system. Exposure of human pro-urokinase [pro-uPA (where uPA is urokinase-type plasminogen activator)] to conditioned growth media from staphylococcal reference strains results in an EDTA-sensitive conversion of the single-chain zymogen into its two-chain active form, an activity not observed in an Aureolysin-deficient strain. Using purified Aureolysin, we verified the capacity of this thermolysin-like metalloprotease to activate pro-uPA, with a 2.6 x 10(3) M(-1) x s(-1) catalytic efficiency. Moreover, activation also occurs in the presence of human plasma, as well as in conditioned growth media from clinical isolates. Finally, we establish that Aureolysin (i) converts plasminogen into angiostatin and mini-plasminogen, the latter retaining its capacity to be activated by uPA and to hydrolyse fibrin, (ii) degrades the plasminogen activator inhibitor-1, and (iii) abrogates the inhibitory activity of alpha(2)-antiplasmin. Altogether, we propose that, in parallel with the staphylokinase-dependent activation of plasminogen, Aureolysin may contribute significantly to the activation of the fibrinolytic system by S. aureus, and thus may promote bacterial spread and invasion.
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The human fibrinolytic system is a target for the staphylococcal metalloprotease Aureolysin
Biochemical Journal, 2008Co-Authors: Nathalie Beaufort, Jan Potempa, Piotr Wojciechowski, Christian P. Sommerhoff, Grzegorz Szmyd, Grzegorz Dubin, Sigrun Eick, Josef Kellermann, Manfred Schmitt, Viktor MagdolenAbstract:The major opportunistic pathogen Staphylococcus aureus utilizes the human fibrinolytic system for invasion and spread via plasmin(ogen) binding and non-proteolytic activation. Because S. aureus secretes several proteases recently proposed as virulence factors, we explored whether these enzymes could add to the activation of the host's fibrinolytic system. Exposure of human pro-urokinase (pro-uPA) to conditioned growth media from staphylococcal reference strains results in an EDTA-sensitive conversion of the single-chain zymogen into its two-chain active form, an activity not observed in an Aureolysin-deficient strain. Using purified Aureolysin, we verified the capacity of this thermolysin-like metalloprotease to activate pro-uPA, with a 2.6 x 10 3}M -1}s -1} catalytic efficiency. Moreover, activation also occurs in the presence of human plasma, as well as in conditioned growth media from clinical isolates. Finally, we establish that Aureolysin (i) converts plasminogen into angiostatin and mini-plasminogen, the latter retaining its capacity to be activated by uPA and to hydrolyze fibrin, (ii) degrades the plasminogen activator inhibitor-1, and (iii) abrogates the inhibitory activity of α 2}-antiplasmin. Altogether, we propose that, in parallel to the staphylokinase-dependent activation of plasminogen, Aureolysin may contribute significantly to the activation of the fibrinolytic system by S. aureus, and thus may promote bacterial spread and invasion.
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Staphylococcus aureus infection triggers production of neutralizing, V8 protease-specific antibodies.
FEMS immunology and medical microbiology, 2008Co-Authors: Ann-marie Calander, Jan Potempa, Grzegorz Dubin, Andrej TarkowskiAbstract:Staphylococcus aureus infection triggers polyclonal B-cell activation. It was sought to further characterize the hypergammaglobulinemia seen in Staphylococcus aureus infection, focusing on the significance of protease-specific B-cell responses. Sera from mice infected with Staphylococcus aureus wild-type strain 8325-4 and two of its isogenic mutants devoid of protease expression were analyzed for the occurrence of polyclonal B-cell activation and the presence of specific antibodies against a set of exoproteases and superantigens. Furthermore, the functional properties of anti-V8-protease antibodies were analyzed in vitro. Polyclonal activation was manifested by increased levels of total serum IgG and IgM in all infected animals and by antibodies to staphylococcal toxins and Aureolysin whether these antigens were present in the inoculate or not. Importantly, Staphylococcus aureus mutant lacking the V8-protease did not trigger a response against this enzyme. In contrast, strains expressing the V8-protease elicited V8-protease antibodies, proving the antigen-specific nature of this response. In vitro tests revealed that these antibodies had the capacity to inhibit the V8 protease activity in a dose-dependent manner. It was concluded that exposure to Staphylococcus aureus, in addition to a massive polyclonal B-cell response, gives rise to production of exoprotease-specific antibodies displaying functional properties.
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DOI:10.1111/j.1574-695X.2007.00371.x
2007Co-Authors: Ann-marie Cal, Jan Potempa, Grzegorz Dubin, Andrej Tarkowski, Correspondence Ann-marie CalanderAbstract:proteases; extracellular proteases; V8; SSPA. Staphylococcus aureus infection triggers polyclonal B-cell activation. It was sought to further characterize the hypergammaglobulinemia seen in Staphylococcus aureus infection, focusing on the significance of protease-specific B-cell responses. Sera from mice infected with Staphylococcus aureus wild-type strain 8325-4 and two of its isogenic mutants devoid of protease expression were analyzed for the occurrence of polyclonal B-cell activation and the presence of specific antibodies against a set of exoproteases and superantigens. Furthermore, the functional properties of anti-V8-protease antibodies were analyzed in vitro. Polyclonal activation was manifested by increased levels of total serum IgG and IgM in all infected animals and by antibodies to staphylococcal toxins and Aureolysin whether these antigens were present in the inoculate or not. Importantly, Staphylococcus aureus mutant lacking the V8-protease did not trigger a respons
Nicholas N. Nickerson - One of the best experts on this subject based on the ideXlab platform.
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rapid autocatalytic activation of the m4 metalloprotease Aureolysin is controlled by a conserved n terminal fungalysin thermolysin propeptide domain
Molecular Microbiology, 2008Co-Authors: Nicholas N. Nickerson, Vineet Joag, Martin J McgavinAbstract:SUMMARY The Staphylococcus aureus proteolytic cascade consists of a metalloprotease Aureolysin (Aur), which activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease. As with other M4 metalloproteases, including elastase of Pseudomonas aeruginosa, the propeptide of proAur contains an N-terminal fungalysin-thermolysin-propeptide (FTP) domain. Autocatalytic activation of proAur was initiated by processing at T85 downward arrowL(86) in the FTP domain. This differed from the mechanism described for proElastase, where the FTP domain has an RY motif in place of TL(86), and processing occurred at the junction of the propeptide and metalloprotease domains, which remained as an inactive complex during passage across the outer membrane. When TL(86) in the FTP domain was replaced with RY, an intact N-terminal propeptide was secreted, but the M4 metalloprotease domain was degraded. Consequently, this segment of the FTP domain promotes intramolecular processing of proAur while bestowing a chaperone function, but discourages processing within the FTP domain of proElastase, where activation must be co-ordinated with passage across a second membrane. We conclude that the FTP domain of proAur is adapted to facilitate a rapid autocatalytic activation mechanism, consistent with the role or proAur as initiator of the staphylococcal proteolytic cascade.
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Rapid autocatalytic activation of the M4 metalloprotease Aureolysin is controlled by a conserved N‐terminal fungalysin‐thermolysin‐propeptide domain
Molecular microbiology, 2008Co-Authors: Nicholas N. Nickerson, Vineet Joag, Martin J McgavinAbstract:The Staphylococcus aureus proteolytic cascade consists of a metalloprotease Aureolysin (Aur), which activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease. As with other M4 metalloproteases, including elastase of Pseudomonas aeruginosa, the propeptide of proAur contains an N-terminal fungalysin-thermolysin-propeptide (FTP) domain. Autocatalytic activation of proAur was initiated by processing at T85 downward arrowL(86) in the FTP domain. This differed from the mechanism described for proElastase, where the FTP domain has an RY motif in place of TL(86), and processing occurred at the junction of the propeptide and metalloprotease domains, which remained as an inactive complex during passage across the outer membrane. When TL(86) in the FTP domain was replaced with RY, an intact N-terminal propeptide was secreted, but the M4 metalloprotease domain was degraded. Consequently, this segment of the FTP domain promotes intramolecular processing of proAur while bestowing a chaperone function, but discourages processing within the FTP domain of proElastase, where activation must be co-ordinated with passage across a second membrane. We conclude that the FTP domain of proAur is adapted to facilitate a rapid autocatalytic activation mechanism, consistent with the role or proAur as initiator of the staphylococcal proteolytic cascade.
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Activation of the SspA Serine Protease Zymogen of Staphylococcus aureus Proceeds through Unique Variations of a Trypsinogen-like Mechanism and Is Dependent on Both Autocatalytic and Metalloprotease-specific Processing
The Journal of biological chemistry, 2007Co-Authors: Nicholas N. Nickerson, Lata Prasad, Latha Jacob, Louis T. J. Delbaere, Martin J McgavinAbstract:The serine and cysteine proteases SspA and SspB of Staphylococcus aureus are secreted as inactive zymogens, zSspA and zSspB. Mature SspA is a trypsin-like glutamyl endopeptidase and is required to activate zSspB. Although a metalloprotease Aureolysin (Aur) is in turn thought to contribute to activation of zSspA, a specific role has not been demonstrated. We found that pre-zSspA is processed by signal peptidase at ANA(29) downward arrow, releasing a Leu(30) isoform that is first processed exclusively through autocatalytic intramolecular cleavage within a glutamine-rich propeptide segment, (40)QQTQSSKQQTPKIQ(53). The preferred site is Gln(43) with secondary processing at Gln(47) and Gln(53). This initial processing is necessary for optimal and subsequent Aur-dependent processing at Leu(58) and then Val(69) to release mature SspA. Although processing by Aur is rate-limiting in zSspA activation, the first active molecules of Val(69)SspA promote rapid intermolecular processing of remaining zSspA at Glu(65), producing an N-terminal (66)HANVILP isoform that is inactive until removal of the HAN tripeptide by Aur. Modeling indicated that His(66) of this penultimate isoform blocks the active site by hydrogen bonding to Ser(237) and occlusion of substrate. Binding of glutamate within the active site of zSspA is energetically unfavorable, but glutamine fits into the primary specificity pocket and is predicted to hydrogen bond to Thr(232) proximal to Ser(237), permitting autocatalytic cleavage of the glutamine-rich propeptide segment. These and other observations suggest that zSspA is activated through a trypsinogen-like mechanism where supplementary features of the propeptide must be sequentially processed in the correct order to allow efficient activation.
Lindsey N. Shaw - One of the best experts on this subject based on the ideXlab platform.
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Unraveling the Impact of Secreted Proteases on Hypervirulence in Staphylococcus aureus.
mBio, 2021Co-Authors: Brittney D. Gimza, Jessica K Jackson, Andrew M Frey, Bridget G Budny, Dale Chaput, Devon N Rizzo, Lindsey N. ShawAbstract:Staphylococcus aureus controls the progression of infection through the coordinated production of extracellular proteases, which selectively modulate virulence determinant stability. This is evidenced by our previous finding that a protease-null strain has a hypervirulent phenotype in a murine model of sepsis, resulting from the unchecked accumulation of virulence factors. Here, we dissect the individual roles of these proteases by constructing and assessing the pathogenic potential of a combinatorial protease mutant library. When strains were constructed bearing increasing numbers of secreted proteases, we observed a variable impact on infectious capacity, where some exhibited hypervirulence, while others phenocopied the wild-type. The common thread for hypervirulent strains was that each lacked both Aureolysin and staphopain A. Upon assessment, we found that the combined loss of these two enzymes alone was necessary and sufficient to engender hypervirulence. Using proteomics, we identified a number of important secreted factors, including SPIN, LukA, Sbi, SEK, and PSMα4, as well as an uncharacterized chitinase-related protein (SAUSA300_0964), to be overrepresented in both the aur scpA and the protease-null mutants. When assessing the virulence of aur scpA SAUSA300_0964 and aur scpA lukA mutants, we found that hypervirulence was completely eliminated, whereas aur scpA spn and aur scpA sek strains elicited aggressive infections akin to the protease double mutant. Collectively, our findings shed light on the influence of extracellular proteases in controlling the infectious process and identifies SAUSA300_0964 as an important new component of the S. aureus virulence factor arsenal.IMPORTANCE A key feature of the pathogenic success of S. aureus is the myriad virulence factors encoded within its genome. These are subject to multifactorial control, ensuring their timely production only within an intended infectious niche. A key node in this network of control is the secreted proteases of S. aureus, who specifically and selectively modulate virulence factor stability. In our previous work we demonstrated that deletion of all 10 secreted proteases results in hypervirulence, since virulence factors exist unchecked, leading to overly aggressive infections. Here, using a combinatorial collection of protease mutants, we reveal that deletion of Aureolysin and staphopain A is necessary and sufficient to elicit hypervirulence. Using proteomic techniques, we identify the collection of virulence factors that accumulate in hypervirulent protease mutants, and demonstrate that a well-known toxin (LukA) and an entirely novel secreted element (SAUSA300_0964) are the leading contributors to deadly infections observed in protease-lacking strains.
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SarA plays a predominant role in controlling the production of extracellular proteases in the diverse clinical isolates of Staphylococcus aureus LAC and UAMS-1.
Virulence, 2020Co-Authors: Aura M. Ramirez, Karen E. Beenken, Stephanie D. Byrum, Alan J. Tackett, Lindsey N. Shaw, Brittney D. Gimza, Mark S. SmeltzerAbstract:Using DNA affinity chromatography we demonstrate that the S. aureus regulatory proteins MgrA, Rot, SarA, and SarS bind DNA baits derived from the promoter regions associated with the genes encoding Aureolysin, ScpAB, SspABC, and SplA-F. Three of four baits also bound SarR and SarZ, the exception in both cases being the ScpAB-associated bait. Using the USA300, methicillin-resistant strain LAC and the USA200, methicillin-sensitive strain UAMS-1, we generated mutations in the genes encoding each of these proteins alone and in combination with sarA and examined the impact on protease production, the accumulation of high molecular weight proteins, and biofilm formation. These studies confirmed that multiple regulatory loci are involved in limiting protease production to a degree that impacts all of these phenotypes, but also demonstrate that sarA plays a predominant role in this regard. Using sarA mutants unable to produce individual proteases alone and in combination with each other, we also demonstrate that the increased production of Aureolysin and ScpA is particularly important in defining the biofilm-deficient phenotype of LAC and UAMS-1 sarA mutants, while Aureolysin alone plays a key role in defining the reduced accumulation of alpha toxin and overall cytotoxicity as assessed using both osteoblasts and osteoclasts.
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Mapping the Global Network of Extracellular Protease Regulation in Staphylococcus aureus.
mSphere, 2019Co-Authors: Brittney D. Gimza, Bridget G Budny, Maria I. Larias, Lindsey N. ShawAbstract:ABSTRACT A primary function of the extracellular proteases of Staphylococcus aureus is to control the progression of infection by selectively modulating the stability of virulence factors. Consequently, a regulatory network exists to titrate protease abundance/activity to influence the accumulation, or lack thereof, of individual virulence factors. Herein, we comprehensively map this system, exploring the regulation of the four protease loci by known and novel factors. In so doing, we determined that seven major elements (SarS, SarR, Rot, MgrA, CodY, SaeR, and SarA) form the primary network of control, with the latter three being the most powerful. We note that expression of Aureolysin is largely repressed by these factors, while the spl operon is subject to the strongest upregulation of any protease loci, particularly by SarR and SaeR. Furthermore, when exploring scpA expression, we find it to be profoundly influenced in opposing fashions by SarA (repressor) and SarR (activator). We also present the screening of >100 regulator mutants of S. aureus, identifying 7 additional factors (ArgR2, AtlR, MntR, Rex, XdrA, Rbf, and SarU) that form a secondary circuit of protease control. Primarily, these elements serve as activators, although we reveal XdrA as a new repressor of protease expression. With the exception or ArgR2, each of the new effectors appears to work through the primary network of regulation to influence protease production. Collectively, we present a comprehensive regulatory circuit that emphasizes the complexity of protease regulation and suggest that its existence speaks to the importance of these enzymes to S. aureus physiology and pathogenic potential. IMPORTANCE The complex regulatory role of the proteases necessitates very tight coordination and control of their expression. While this process has been well studied, a major oversight has been the consideration of proteases as a single entity rather than as 10 enzymes produced from four different promoters. As such, in this study, we comprehensively characterized the regulation of each protease promoter, discovering vast differences in the way each protease operon is controlled. Additionally, we broaden the picture of protease regulation using a global screen to identify novel loci controlling protease activity, uncovering a cadre of new effectors of protease expression. The impact of these elements on the activity of proteases and known regulators was characterized by producing a comprehensive regulatory circuit that emphasizes the complexity of protease regulation in Staphylococcus aureus.
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Mapping the Global Network of Extracellular Protease Regulation in Staphylococcus aureus
2019Co-Authors: Brittney D. Gimza, Bridget G Budny, Maria I. Larias, Lindsey N. ShawAbstract:AbstractA primary function of the extracellular proteases ofStaphylococcus aureusis to control the progression of infection by selectively modulating the stability of virulence factors. Consequently, a regulatory network exists to titrate protease abundance/activity, to influence accumulation, or lack thereof, of individual virulence factors. Herein, we comprehensively map this system, exploring regulation of the four protease loci by known and novel factors. In so doing, we determine that seven major elements (SarS, SarR, Rot, MgrA, CodY, SaeR, and SarA) form the primary network of control, with the latter three being the most powerful. We note that expression of Aureolysin is largely repressed by these factors, whilst thesploperon is subject to the strongest upregulation of any protease loci, particularly by SarR and SaeR. Furthermore, when exploringscpAexpression, we find it to be profoundly influenced in opposing fashions by SarA (repressor) and SarR (activator). We also present the screening of >100 regulator mutants ofS. aureus, identifying 7 additional factors (ArgR2, AtlR, MntR, Rex, XdrA, Rbf, and SarU) that form a secondary circuit of protease control. Primarily these elements serve as activators, although we reveal XdrA as a new repressor of protease expression. With the exception or ArgR2, each of the new effectors appear to work through the primary network of regulation to influence protease production. Collectively, we present a comprehensive regulatory circuit that emphasizes the complexity of protease regulation and suggest that its existence speaks to the importance of these enzymes toS. aureusphysiology and pathogenic potential.ImportanceThe complex regulatory role of the proteases necessitates very tight coordination and control of their expression. Whilst this process has been well studied, a major oversight has been the consideration of proteases as a single entity, rather than 10 enzymes produced from four different promoters. As such, in this study we comprehensively characterized the regulation of each protease promoter, discovering vast differences in the way each protease operon is controlled. Additionally, we broaden the picture of protease regulation using a global screen to identify novel loci controlling protease activity, uncovering a cadre of new effectors of protease expression. The impact of these elements on the activity of proteases and known regulators was characterized producing a comprehensive regulatory circuit that emphasizes the complexity of protease regulation inStaphylococcus aureus.
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Galectin-3 Is a Target for Proteases Involved in the Virulence of Staphylococcus aureus.
Infection and immunity, 2017Co-Authors: Jonas Elmwall, Lindsey N. Shaw, Jakub Kwiecinski, Abukar Ali, Veronica Osla, Wanzhong Wang, Karin Sävman, Elisabet Josefsson, Johan BylundAbstract:Staphylococcus aureus is a major cause of skin and soft tissue infection. The bacterium expresses four major proteases that are emerging as virulence factors: Aureolysin (Aur), V8 protease (SspA), staphopain A (ScpA), and staphopain B (SspB). We hypothesized that human galectin-3, a β-galactoside-binding lectin involved in immune regulation and antimicrobial defense, is a target for these proteases and that proteolysis of galectin-3 is a novel immune evasion mechanism. Indeed, supernatants from laboratory strains and clinical isolates of S. aureus caused galectin-3 degradation. Similar proteolytic capacities were found in Staphylococcus epidermidis isolates but not in Staphylococcus saprophyticus Galectin-3-induced activation of the neutrophil NADPH oxidase was abrogated by bacterium-derived proteolysis of galectin-3, and SspB was identified as the major protease responsible. The impact of galectin-3 and protease expression on S. aureus virulence was studied in a murine skin infection model. In galectin-3+/+ mice, SspB-expressing S. aureus caused larger lesions and resulted in higher bacterial loads than protease-lacking bacteria. No such difference in bacterial load or lesion size was detected in galectin-3-/- mice, which overall showed smaller lesion sizes than the galectin-3+/+ animals. In conclusion, the staphylococcal protease SspB inactivates galectin-3, abrogating its stimulation of oxygen radical production in human neutrophils and increasing tissue damage during skin infection.
