PMID:25257373

Citation	Loughran, AJ, Atwood, DN, Anthony, AC, Harik, NS, Spencer, HJ, Beenken, KE and Smeltzer, MS (2014) Impact of individual extracellular proteases on Staphylococcus aureus biofilm formation in diverse clinical isolates and their isogenic sarA mutants. Microbiologyopen 3:897-909
Abstract	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.
Links	PubMed PMC4263513 Online version:10.1002/mbo3.214
Keywords	Animals; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Biofilms; Cysteine Endopeptidases/genetics; Cysteine Endopeptidases/metabolism; Female; Humans; Mice; Mutation; Serine Endopeptidases/genetics; Serine Endopeptidases/metabolism; Staphylococcal Infections/microbiology; Staphylococcus aureus/enzymology; Staphylococcus aureus/genetics; Staphylococcus aureus/pathogenicity; Staphylococcus aureus/physiology; Trans-Activators/genetics; Trans-Activators/metabolism; Virulence
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
STAAU:AURE		GO:0090609: single-species submerged biofilm formation	ECO:0000315:		P		Figure 6 shows the impact of mutations in individual protease genes/operons on biofilm formation in vitro. The relative capacity to form a biofilm was assessed using a microtiter plate assay as previously described (Beenken et al. 2003) using FPR3757, its sarA mutant, and its sarA mutant carrying mutations in the indicated protease genes. Single asterisks indicate significance by comparison to the parent strain. Double asterisks indicate significance by comparison to the sarA mutant. As a control, biofilm formation was also assessed in LAC, its sarA mutant, and derivatives of thesarA mutant unable to produce aureolysin (Saur), unable to produce any extracellular protease (SP), or unable to produce any extracellular protease other than those encoded by the spl operon (SPspl+). Single asterisk indicates statistical significance by comparison to the sarA mutant. Double asterisks indicate significance by comparison to the Saur mutant.	complete CACAO 11739
STAAU:SARA		GO:0044010: single-species biofilm formation	ECO:0000314:		P		Figure 2 shows the impact of purified extracellular proteases aureolysin, SspA, ScpA, and SspB on biofilm formation.	complete CACAO 11382
STAAU:SARA		GO:0090605: submerged biofilm formation	ECO:0000315:		P		Figure 1 defines the optimal conditions for biofilm formation in vitro for both WT and isogenic sarA mutants.	complete CACAO 11381
STAAU:SARA		GO:0090605: submerged biofilm formation	ECO:0000314:		P		Figure 4 shows how purified extracellular proteases impact biofilm formation in LAC protease-deficient derivatives.	complete CACAO 11384
STAAU:SARA		GO:0098786: biofilm matrix disassembly	ECO:0000314:		P		Figure 5 shows the impact of purified extracellular proteases on dispersal of established biofilms.	complete CACAO 11385
STAAU:SARA		GO:1900232: negative regulation of single-species biofilm formation on inanimate substrate	ECO:0000315:		P		Figure 7 shows the impact that mutating individual protease genes/operons in vivo has on the ability of WT and its isogenic sarA mutant to produce proteases. The repression of protease production in turn promotes biofilm formation.	complete CACAO 11388
STAAU:SARA		GO:0044010: single-species biofilm formation	ECO:0000315:		P		Figure 8 shows the difference between biofilm formation in WT and sarA mutants.	complete CACAO 11389
STAAU:SARA		GO:0008658: penicillin binding	ECO:0000314:		F		Figure 9 shows the impact of purified aureolysin in contemporary clinical isolates as a function of methicillin resistance status. Methicillin is a member of the penicillin class.	complete CACAO 11390
STAAU:SARA		GO:0006508: proteolysis	ECO:0000316:	UniProtKB:P81177 UniProtKB:P0C1S6 UniProtKB:P0C1U6 UniProtKB:P0C1U8 UniProtKB:P81297	P		Figure 3 shows the impact of mutating individual protease genes or operons in the WT and sarA mutant as well as the derivatives of the sarA mutant in terms of proteolytic activity.	complete CACAO 11412
STAAU:SARA		GO:0090609: single-species submerged biofilm formation	ECO:0000314:		P		Figure 10 shows the relative impact of aureolysin and dispersin B on biofilm formation.	complete CACAO 11391
STAAU:SARA		GO:0090609: single-species submerged biofilm formation	ECO:0000316:	UniProtKB:P81177 UniProtKB:P0C1S6 UniProtKB:P0C1U6 UniProtKB:P0C1U8 UniProtKB:P81297	P		Figure 6 shows the impact of mutating individual protease genes or operons in both the WT and sarA mutant as well as the derivatives of the sarA mutant in terms of biofilm formation.	complete CACAO 11413
STAA3:SARA		GO:0045862: proteolysis	ECO:0000315:		P		Figure 3 shows the impact of mutating individual protease genes or operons in both the WT and sarA mutant in terms of proteolytic activity.	complete CACAO 11383
STAA3:SARA		GO:0090609: single-species submerged biofilm formation	ECO:0000315:		P		Figure 6 shows the impact of mutating individual protease genes or operons in both the WT and sarA mutant in terms of biofilm formation.	complete CACAO 11387
STAAU:SSPA		GO:0090609: single-species submerged biofilm formation	ECO:0000315:		P		Figure 6 shows the impact of mutations in individual protease genes/operons on biofilm formation in vitro. The relative capacity to form a biofilm was assessed using a microtiter plate assay as previously described (Beenken et al. 2003) using FPR3757, its sarA mutant, and its sarA mutant carrying mutations in the indicated protease genes. Single asterisks indicate significance by comparison to the parent strain. Double asterisks indicate significance by comparison to the sarA mutant. As a control, biofilm formation was also assessed in LAC, its sarA mutant, and derivatives of thesarA mutant unable to produce aureolysin (Saur), unable to produce any extracellular protease (SP), or unable to produce any extracellular protease other than those encoded by the spl operon (SPspl+). Single asterisk indicates statistical significance by comparison to the sarA mutant. Double asterisks indicate significance by comparison to the Saur mutant.	complete CACAO 11740
STAA3:A0A0G2Q3K2		GO:0090609: single-species submerged biofilm formation	ECO:0000315:		P		Figure 6 shows the impact of mutations in individual protease genes/operons on biofilm formation in vitro. The relative capacity to form a biofilm was assessed using a microtiter plate assay as previously described (Beenken et al. 2003) using FPR3757, its sarA mutant, and its sarA mutant carrying mutations in the indicated protease genes. Single asterisks indicate significance by comparison to the parent strain. Double asterisks indicate significance by comparison to the sarA mutant. As a control, biofilm formation was also assessed in LAC, its sarA mutant, and derivatives of thesarA mutant unable to produce aureolysin (Saur), unable to produce any extracellular protease (SP), or unable to produce any extracellular protease other than those encoded by the spl operon (SPspl+). Single asterisk indicates statistical significance by comparison to the sarA mutant. Double asterisks indicate significance by comparison to the Saur mutant.	complete CACAO 11741

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