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Impact of individual extracellular proteases on Staphylococcus aureus biofilm formation in diverse clinical isolates and their isogenic sarA mutants.

Loughran AJ, Atwood DN, Anthony AC, Harik NS, Spencer HJ, Beenken KE, Smeltzer MS - Microbiologyopen (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas.

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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 the sarA 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.
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fig06: 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 the sarA 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.

Mentions: Taken together, these results suggest that aureolysin, ScpA, and SspB play the most important roles in both biofilm formation and maintenance. To confirm these results, we also took the alternative approach of examining a S. aureus strain with mutations inactivating the genes encoding different proteases. Because the mutants were already available and genetically validated, these experiments were done using the USA300 strain FPR3757 and its isogenic sarA mutant rather than LAC, but these two USA300 strains have been shown to be essentially identical to each other (Kennedy et al. 2008). Zymogram analysis confirmed the protease phenotypes of these mutants, including the fact that the sspA mutant has a polar effect that abolishes the production of both SspA and SspB while the sspB mutant abolishes the production of SspB, but has no impact on the production of SspA (Fig. 3). The results also confirmed that mutation of the gene encoding aureolysin (aur), the sspABC operon, sspB, or the scpAB operon all enhanced biofilm formation in an FPR3757 sarA mutant, with mutation of aur having the greatest effect (Fig. 6). In contrast, mutation of the spl operon had little impact on biofilm formation in the FPR3757 sarA mutant. These results along with those seen in Figure 4 confirmed the same relative effects, suggesting that the spl-encoded proteases are unlikely to play a significant role in limiting biofilm formation in sarA mutants, at least under in vitro conditions. The observation that mutating the sspB gene was not statistically different to that of mutating the sspABC operon provides further support for the hypothesis that SspB plays the more important role in limiting biofilm formation by comparison to SspA. However, the impact of mutating each of these genes/operons, while statistically significant, was limited, an observation that provides further support for the hypothesis that multiple extracellular proteases contribute to the biofilm-deficient phenotype of sarA mutants (Fig. 6). Further support for this hypothesis comes from the observation that mutation of all of these protease genes was sufficient to fully restore biofilm formation in a LAC sarA mutant irrespective of the functional status of the spl operon (Fig. 6).


Impact of individual extracellular proteases on Staphylococcus aureus biofilm formation in diverse clinical isolates and their isogenic sarA mutants.

Loughran AJ, Atwood DN, Anthony AC, Harik NS, Spencer HJ, Beenken KE, Smeltzer MS - Microbiologyopen (2014)

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 the sarA 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263513&req=5

fig06: 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 the sarA 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.
Mentions: Taken together, these results suggest that aureolysin, ScpA, and SspB play the most important roles in both biofilm formation and maintenance. To confirm these results, we also took the alternative approach of examining a S. aureus strain with mutations inactivating the genes encoding different proteases. Because the mutants were already available and genetically validated, these experiments were done using the USA300 strain FPR3757 and its isogenic sarA mutant rather than LAC, but these two USA300 strains have been shown to be essentially identical to each other (Kennedy et al. 2008). Zymogram analysis confirmed the protease phenotypes of these mutants, including the fact that the sspA mutant has a polar effect that abolishes the production of both SspA and SspB while the sspB mutant abolishes the production of SspB, but has no impact on the production of SspA (Fig. 3). The results also confirmed that mutation of the gene encoding aureolysin (aur), the sspABC operon, sspB, or the scpAB operon all enhanced biofilm formation in an FPR3757 sarA mutant, with mutation of aur having the greatest effect (Fig. 6). In contrast, mutation of the spl operon had little impact on biofilm formation in the FPR3757 sarA mutant. These results along with those seen in Figure 4 confirmed the same relative effects, suggesting that the spl-encoded proteases are unlikely to play a significant role in limiting biofilm formation in sarA mutants, at least under in vitro conditions. The observation that mutating the sspB gene was not statistically different to that of mutating the sspABC operon provides further support for the hypothesis that SspB plays the more important role in limiting biofilm formation by comparison to SspA. However, the impact of mutating each of these genes/operons, while statistically significant, was limited, an observation that provides further support for the hypothesis that multiple extracellular proteases contribute to the biofilm-deficient phenotype of sarA mutants (Fig. 6). Further support for this hypothesis comes from the observation that mutation of all of these protease genes was sufficient to fully restore biofilm formation in a LAC sarA mutant irrespective of the functional status of the spl operon (Fig. 6).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas.

Show MeSH
Related in: MedlinePlus