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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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Related in: MedlinePlus

Impact of purified extracellular proteases in LAC protease-deficient derivatives. The indicated proteases were added to the BFM used in biofilm assays using derivatives of LAC unable to produce any extracellular protease (LAC Δprotease) or unable to produce any extracellular protease other than those encoded within the spl operon (LAC Δprotease/spl+). Triangles indicate decreasing concentrations from 250 to 16 nmol/L, with “C” indicating the control assay without the addition of any exogenous protease. Asterisks indicate the lowest concentration of each protease at which a statistically significant difference was observed relative to this control.
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fig04: Impact of purified extracellular proteases in LAC protease-deficient derivatives. The indicated proteases were added to the BFM used in biofilm assays using derivatives of LAC unable to produce any extracellular protease (LAC Δprotease) or unable to produce any extracellular protease other than those encoded within the spl operon (LAC Δprotease/spl+). Triangles indicate decreasing concentrations from 250 to 16 nmol/L, with “C” indicating the control assay without the addition of any exogenous protease. Asterisks indicate the lowest concentration of each protease at which a statistically significant difference was observed relative to this control.

Mentions: Although all four of these proteases had an inhibitory effect, the impact of each individual protease was not equivalent, with equivalent molar amounts of aureolysin having the greatest inhibitory effect in both UAMS-1 and LAC (Fig. 2). However, the position of aureolysin at the top of the S. aureus protease activation cascade (Shaw et al. 2004; Mootz et al. 2013) leaves open the possibility that aureolysin is not more active but rather that its absence simply decreases the activity of the downstream proteases SspA and/or SspB. To address this, we repeated the experiments using a derivative of LAC unable to produce any of the recognized extracellular proteases. The results confirmed the inhibitory effect of aureolysin, ScpA, SspA, and SspB, but even under these circumstances aureolysin was found to have the greatest relative effect (i.e., the greatest inhibition at the lowest concentration) (Fig. 4). Because we did not have access to purified preparations of the spl-encoded proteases, we also repeated these experiments using a derivative of LAC capable of producing only the spl operon proteases. The presence or absence of spl proteases had no effect on biofilm formation in this assay (Fig. 4). More importantly, these results suggest that the inhibitory effect of each protease is mediated directly by the individual proteases themselves and that aureolysin does in fact have a greater impact on limiting biofilm formation than any of the other extracellular proteases. Additionally, the inhibitory impact of SspA was less apparent in the total protease-deficient derivative of LAC (Fig. 4) than in LAC itself (Fig. 2). Because the wild-type strain produces SspB while the total protease mutant does not, this suggests that the impact of SspA is at least partly due to its impact on activation of SspB.


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 purified extracellular proteases in LAC protease-deficient derivatives. The indicated proteases were added to the BFM used in biofilm assays using derivatives of LAC unable to produce any extracellular protease (LAC Δprotease) or unable to produce any extracellular protease other than those encoded within the spl operon (LAC Δprotease/spl+). Triangles indicate decreasing concentrations from 250 to 16 nmol/L, with “C” indicating the control assay without the addition of any exogenous protease. Asterisks indicate the lowest concentration of each protease at which a statistically significant difference was observed relative to this control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig04: Impact of purified extracellular proteases in LAC protease-deficient derivatives. The indicated proteases were added to the BFM used in biofilm assays using derivatives of LAC unable to produce any extracellular protease (LAC Δprotease) or unable to produce any extracellular protease other than those encoded within the spl operon (LAC Δprotease/spl+). Triangles indicate decreasing concentrations from 250 to 16 nmol/L, with “C” indicating the control assay without the addition of any exogenous protease. Asterisks indicate the lowest concentration of each protease at which a statistically significant difference was observed relative to this control.
Mentions: Although all four of these proteases had an inhibitory effect, the impact of each individual protease was not equivalent, with equivalent molar amounts of aureolysin having the greatest inhibitory effect in both UAMS-1 and LAC (Fig. 2). However, the position of aureolysin at the top of the S. aureus protease activation cascade (Shaw et al. 2004; Mootz et al. 2013) leaves open the possibility that aureolysin is not more active but rather that its absence simply decreases the activity of the downstream proteases SspA and/or SspB. To address this, we repeated the experiments using a derivative of LAC unable to produce any of the recognized extracellular proteases. The results confirmed the inhibitory effect of aureolysin, ScpA, SspA, and SspB, but even under these circumstances aureolysin was found to have the greatest relative effect (i.e., the greatest inhibition at the lowest concentration) (Fig. 4). Because we did not have access to purified preparations of the spl-encoded proteases, we also repeated these experiments using a derivative of LAC capable of producing only the spl operon proteases. The presence or absence of spl proteases had no effect on biofilm formation in this assay (Fig. 4). More importantly, these results suggest that the inhibitory effect of each protease is mediated directly by the individual proteases themselves and that aureolysin does in fact have a greater impact on limiting biofilm formation than any of the other extracellular proteases. Additionally, the inhibitory impact of SspA was less apparent in the total protease-deficient derivative of LAC (Fig. 4) than in LAC itself (Fig. 2). Because the wild-type strain produces SspB while the total protease mutant does not, this suggests that the impact of SspA is at least partly due to its impact on activation of SspB.

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