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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 mutating individual protease genes/operons. Proteolytic activity was assessed in FPR3757 (WT) with and without addition of the indicated protease, its sarA mutant, and derivatives of the sarA mutant with mutations inactivating the indicated protease genes. Top panel: To ensure the use of physiologically relevant amounts of purified proteases in the context of the amounts produced by the isogenic sarA mutant, each purified protease was examined individually at a concentration of 250 nmol/L, which was the highest concentration used in our protease add-back experiments. Bottom panels: The protease phenotype of the WT strain was compared to that of its sarA mutant carrying mutations in the indicated protease genes. As discussed in the text, the sspA mutation is polar, thus eliminating production of both SspA and SspB. Purified proteases were also included as additional controls.
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fig03: Impact of mutating individual protease genes/operons. Proteolytic activity was assessed in FPR3757 (WT) with and without addition of the indicated protease, its sarA mutant, and derivatives of the sarA mutant with mutations inactivating the indicated protease genes. Top panel: To ensure the use of physiologically relevant amounts of purified proteases in the context of the amounts produced by the isogenic sarA mutant, each purified protease was examined individually at a concentration of 250 nmol/L, which was the highest concentration used in our protease add-back experiments. Bottom panels: The protease phenotype of the WT strain was compared to that of its sarA mutant carrying mutations in the indicated protease genes. As discussed in the text, the sspA mutation is polar, thus eliminating production of both SspA and SspB. Purified proteases were also included as additional controls.

Mentions: Given our interest in orthopedic infection and the role of biofilms in these infections (Brady et al. 2008), we initially focused our efforts investigating biofilm formation of the USA200, CC30, MSSA strain UAMS-1, which was isolated from the bone of an osteomyelitis patient during surgical debridement (Smeltzer et al. 1997). Under in vitro conditions, consistent biofilm formation with this strain, as well as almost all others we examined, was dependent on supplementation of the medium with salt and glucose and coating the substrate with human plasma (Beenken et al. 2003). Here, we confirm that this is also true with the USA300, CC8, MRSA strain LAC and that, in both of these strains, biofilm formation under these optimized in vitro conditions is dramatically reduced by mutation of sarA (Fig. 1). To the extent that plasma coating had the most dramatic impact on promoting biofilm formation in both of these strains, which were chosen because they are phenotypically and genetically distinct by comparison to each other including their methicillin resistance status (Cassat et al. 2005, 2006), this suggests the existence of a common, protein-dependent mechanism of biofilm formation in diverse contemporary clinical isolates of S. aureus. Based on this, we examined the impact of adding purified aureolysin, ScpA, SspA, and SspB (BioCentrum) on preventing biofilm formation. Results confirmed that all four of these proteases limit biofilm formation in both LAC (MRSA) and UAMS-1 (MSSA) (Fig. 2). We also confirmed by zymogram that the amount of each protease required to achieve this effect is lower than the amounts observed in the isogenic sarA mutant (Fig. 3), thus suggesting that these experiments are likely to be physiologically relevant at least in the context of defining the biofilm-deficient phenotype of the respective sarA mutants.


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 mutating individual protease genes/operons. Proteolytic activity was assessed in FPR3757 (WT) with and without addition of the indicated protease, its sarA mutant, and derivatives of the sarA mutant with mutations inactivating the indicated protease genes. Top panel: To ensure the use of physiologically relevant amounts of purified proteases in the context of the amounts produced by the isogenic sarA mutant, each purified protease was examined individually at a concentration of 250 nmol/L, which was the highest concentration used in our protease add-back experiments. Bottom panels: The protease phenotype of the WT strain was compared to that of its sarA mutant carrying mutations in the indicated protease genes. As discussed in the text, the sspA mutation is polar, thus eliminating production of both SspA and SspB. Purified proteases were also included as additional controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Impact of mutating individual protease genes/operons. Proteolytic activity was assessed in FPR3757 (WT) with and without addition of the indicated protease, its sarA mutant, and derivatives of the sarA mutant with mutations inactivating the indicated protease genes. Top panel: To ensure the use of physiologically relevant amounts of purified proteases in the context of the amounts produced by the isogenic sarA mutant, each purified protease was examined individually at a concentration of 250 nmol/L, which was the highest concentration used in our protease add-back experiments. Bottom panels: The protease phenotype of the WT strain was compared to that of its sarA mutant carrying mutations in the indicated protease genes. As discussed in the text, the sspA mutation is polar, thus eliminating production of both SspA and SspB. Purified proteases were also included as additional controls.
Mentions: Given our interest in orthopedic infection and the role of biofilms in these infections (Brady et al. 2008), we initially focused our efforts investigating biofilm formation of the USA200, CC30, MSSA strain UAMS-1, which was isolated from the bone of an osteomyelitis patient during surgical debridement (Smeltzer et al. 1997). Under in vitro conditions, consistent biofilm formation with this strain, as well as almost all others we examined, was dependent on supplementation of the medium with salt and glucose and coating the substrate with human plasma (Beenken et al. 2003). Here, we confirm that this is also true with the USA300, CC8, MRSA strain LAC and that, in both of these strains, biofilm formation under these optimized in vitro conditions is dramatically reduced by mutation of sarA (Fig. 1). To the extent that plasma coating had the most dramatic impact on promoting biofilm formation in both of these strains, which were chosen because they are phenotypically and genetically distinct by comparison to each other including their methicillin resistance status (Cassat et al. 2005, 2006), this suggests the existence of a common, protein-dependent mechanism of biofilm formation in diverse contemporary clinical isolates of S. aureus. Based on this, we examined the impact of adding purified aureolysin, ScpA, SspA, and SspB (BioCentrum) on preventing biofilm formation. Results confirmed that all four of these proteases limit biofilm formation in both LAC (MRSA) and UAMS-1 (MSSA) (Fig. 2). We also confirmed by zymogram that the amount of each protease required to achieve this effect is lower than the amounts observed in the isogenic sarA mutant (Fig. 3), thus suggesting that these experiments are likely to be physiologically relevant at least in the context of defining the biofilm-deficient phenotype of the respective sarA mutants.

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