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SaeRS-dependent inhibition of biofilm formation in Staphylococcus aureus Newman.

Cue D, Junecko JM, Lei MG, Blevins JS, Smeltzer MS, Lee CY - PLoS ONE (2015)

Bottom Line: The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms.Although a number of candidate proteins were identified, none were found to be the actual inhibitor.RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, United States of America.

ABSTRACT
The SaeRS two-component regulatory system of Staphylococcus aureus is known to affect the expression of many genes. The SaeS protein is the histidine kinase responsible for phosphorylation of the response regulator SaeR. In S. aureus Newman, the sae system is constitutively expressed due to a point mutation in saeS, relative to other S. aureus strains, which results in substitution of proline for leucine at amino acid 18. Strain Newman is unable to form a robust biofilm and we report here that the biofilm-deficient phenotype is due to the saeSP allele. Replacement of the Newman saeSP with saeSL, or deletion of saeRS, resulted in a biofilm-proficient phenotype. Newman culture supernatants were observed to inhibit biofilm formation by other S. aureus strains, but did not affect biofilm formation by S. epidermidis. Culture supernatants of Newman saeSL or Newman ΔsaeRS had no significant effect on biofilm formation. The inhibitory factor was inactivated by incubation with proteinase K, but survived heating, indicating that the inhibitory protein is heat-stable. The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms. Replacement of saeSL with saeSP in the biofilm-proficient S. aureus USA300 FPR3757 resulted in the loss of biofilm formation. Culture supernatants of USA300 FPR3757 saeSP, did not inhibit biofilm formation by other staphylococci, suggesting that the inhibitory factor is produced but not secreted in the mutant strain. A number of biochemical methods were utilized to isolate the inhibitory protein. Although a number of candidate proteins were identified, none were found to be the actual inhibitor. In an effort to reduce the number of potential inhibitory genes, RNA-Seq analyses were done with wild-type strain Newman and the saeSL and ΔsaeRS mutants. RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.

No MeSH data available.


Related in: MedlinePlus

Strain-dependent production of the biofilm inhibitory protein.(A) Biofilm formation by strain USA300 FPR 3757 and derivatives. Wells were inoculated with the indicated USA 300 FBR 3757 derivatives. +saePQRSL and +saePQRSP indicate the presence of plasmids pCWSAE50 or pCWSAE51, respectively. (B) Anti-biofilm activity of USA300 derivatives. Culture supernatants from the strains listed to the right of the figure were tested for inhibition of biofilm formation by strain UAMS-1.
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pone.0123027.g004: Strain-dependent production of the biofilm inhibitory protein.(A) Biofilm formation by strain USA300 FPR 3757 and derivatives. Wells were inoculated with the indicated USA 300 FBR 3757 derivatives. +saePQRSL and +saePQRSP indicate the presence of plasmids pCWSAE50 or pCWSAE51, respectively. (B) Anti-biofilm activity of USA300 derivatives. Culture supernatants from the strains listed to the right of the figure were tested for inhibition of biofilm formation by strain UAMS-1.

Mentions: As stated previously, strain Newman has a point mutation in saeS that results in constitutive activation of the SaeRS two-component system. Our data suggested that a consequence of this constitutive activation was the inability to form a biofilm. To determine if the saeSP allele would affect other S. aureus strains, a SaeRS constitutively active strain was constructed in the USA300 FPR 3757 (USA300) background. First, the ΔsaePQRS::kan mutation was introduced into wild-type strain USA300 FPR 3757 via phage transduction. Next, either the saePQRS operon from strain Newman (saePQRSP) or from strain 8325–4 (saePQRSL) was introduced into USA300 ΔsaePQRS::kan, using pCWSAE51 or pCWSAE50, respectively. The vector carrying the sae operons is a single copy integration vector that integrates within the hlb gene at the phage Ø13 attachment site [24]. The benefit of using an integration vector is stable maintenance of the construct in the chromosome allowing biological assays to be performed without the use of antibiotics to maintain the plasmid. Resulting strains were tested for biofilm production and culture supernatants from planktonic cultures were subsequently tested for anti-biofilm activity against strain UAMS-1. As shown in Fig 4A, wild-type strain USA300 and the ΔsaePQRS::kan mutant formed a biofilm, and complementation of ΔsaePQRS::kan with saePQRS from strain 8325–4 (denoted saePQRSL) resulted in no change in the biofilm phenotype. Interestingly, complementation of ΔsaePQRS::kan with saePQRS from strain Newman (denoted saePQRSP) resulted in an apparent reduction in biofilm formation, further confirming our hypothesis that constitutive activation of SaeRS limits biofilm formation. To assess production of the secreted biofilm inhibitory protein, USA300 and derivatives were grown to stationary phase and culture supernatants were harvested, filter sterilized and added to biofilm assays of strain UAMS-1. Culture supernatants from strain USA300 expressing saePQRSP did not contain any apparent anti-biofilm activity (Fig 4B), which suggests the inhibitory factor is not secreted by USA300. It is possible that the inhibitory protein is associated with the cell surface in USA300, but is released into the environment by Newman. Newman does encode some truncated versions of cell-wall anchored proteins that, due to the truncation, would no longer be anchored [25,28]. Identification of the inhibitory protein should aid in understanding this result. It is worth noting that another USA300 isolate (USA300 LAC) does secrete a DNA nuclease that can inhibit biofilm of other staphylococci [29]. However, as discussed below, the nuclease is distinct from the inhibitory factor produced by Newman.


SaeRS-dependent inhibition of biofilm formation in Staphylococcus aureus Newman.

Cue D, Junecko JM, Lei MG, Blevins JS, Smeltzer MS, Lee CY - PLoS ONE (2015)

Strain-dependent production of the biofilm inhibitory protein.(A) Biofilm formation by strain USA300 FPR 3757 and derivatives. Wells were inoculated with the indicated USA 300 FBR 3757 derivatives. +saePQRSL and +saePQRSP indicate the presence of plasmids pCWSAE50 or pCWSAE51, respectively. (B) Anti-biofilm activity of USA300 derivatives. Culture supernatants from the strains listed to the right of the figure were tested for inhibition of biofilm formation by strain UAMS-1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390220&req=5

pone.0123027.g004: Strain-dependent production of the biofilm inhibitory protein.(A) Biofilm formation by strain USA300 FPR 3757 and derivatives. Wells were inoculated with the indicated USA 300 FBR 3757 derivatives. +saePQRSL and +saePQRSP indicate the presence of plasmids pCWSAE50 or pCWSAE51, respectively. (B) Anti-biofilm activity of USA300 derivatives. Culture supernatants from the strains listed to the right of the figure were tested for inhibition of biofilm formation by strain UAMS-1.
Mentions: As stated previously, strain Newman has a point mutation in saeS that results in constitutive activation of the SaeRS two-component system. Our data suggested that a consequence of this constitutive activation was the inability to form a biofilm. To determine if the saeSP allele would affect other S. aureus strains, a SaeRS constitutively active strain was constructed in the USA300 FPR 3757 (USA300) background. First, the ΔsaePQRS::kan mutation was introduced into wild-type strain USA300 FPR 3757 via phage transduction. Next, either the saePQRS operon from strain Newman (saePQRSP) or from strain 8325–4 (saePQRSL) was introduced into USA300 ΔsaePQRS::kan, using pCWSAE51 or pCWSAE50, respectively. The vector carrying the sae operons is a single copy integration vector that integrates within the hlb gene at the phage Ø13 attachment site [24]. The benefit of using an integration vector is stable maintenance of the construct in the chromosome allowing biological assays to be performed without the use of antibiotics to maintain the plasmid. Resulting strains were tested for biofilm production and culture supernatants from planktonic cultures were subsequently tested for anti-biofilm activity against strain UAMS-1. As shown in Fig 4A, wild-type strain USA300 and the ΔsaePQRS::kan mutant formed a biofilm, and complementation of ΔsaePQRS::kan with saePQRS from strain 8325–4 (denoted saePQRSL) resulted in no change in the biofilm phenotype. Interestingly, complementation of ΔsaePQRS::kan with saePQRS from strain Newman (denoted saePQRSP) resulted in an apparent reduction in biofilm formation, further confirming our hypothesis that constitutive activation of SaeRS limits biofilm formation. To assess production of the secreted biofilm inhibitory protein, USA300 and derivatives were grown to stationary phase and culture supernatants were harvested, filter sterilized and added to biofilm assays of strain UAMS-1. Culture supernatants from strain USA300 expressing saePQRSP did not contain any apparent anti-biofilm activity (Fig 4B), which suggests the inhibitory factor is not secreted by USA300. It is possible that the inhibitory protein is associated with the cell surface in USA300, but is released into the environment by Newman. Newman does encode some truncated versions of cell-wall anchored proteins that, due to the truncation, would no longer be anchored [25,28]. Identification of the inhibitory protein should aid in understanding this result. It is worth noting that another USA300 isolate (USA300 LAC) does secrete a DNA nuclease that can inhibit biofilm of other staphylococci [29]. However, as discussed below, the nuclease is distinct from the inhibitory factor produced by Newman.

Bottom Line: The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms.Although a number of candidate proteins were identified, none were found to be the actual inhibitor.RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, United States of America.

ABSTRACT
The SaeRS two-component regulatory system of Staphylococcus aureus is known to affect the expression of many genes. The SaeS protein is the histidine kinase responsible for phosphorylation of the response regulator SaeR. In S. aureus Newman, the sae system is constitutively expressed due to a point mutation in saeS, relative to other S. aureus strains, which results in substitution of proline for leucine at amino acid 18. Strain Newman is unable to form a robust biofilm and we report here that the biofilm-deficient phenotype is due to the saeSP allele. Replacement of the Newman saeSP with saeSL, or deletion of saeRS, resulted in a biofilm-proficient phenotype. Newman culture supernatants were observed to inhibit biofilm formation by other S. aureus strains, but did not affect biofilm formation by S. epidermidis. Culture supernatants of Newman saeSL or Newman ΔsaeRS had no significant effect on biofilm formation. The inhibitory factor was inactivated by incubation with proteinase K, but survived heating, indicating that the inhibitory protein is heat-stable. The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms. Replacement of saeSL with saeSP in the biofilm-proficient S. aureus USA300 FPR3757 resulted in the loss of biofilm formation. Culture supernatants of USA300 FPR3757 saeSP, did not inhibit biofilm formation by other staphylococci, suggesting that the inhibitory factor is produced but not secreted in the mutant strain. A number of biochemical methods were utilized to isolate the inhibitory protein. Although a number of candidate proteins were identified, none were found to be the actual inhibitor. In an effort to reduce the number of potential inhibitory genes, RNA-Seq analyses were done with wild-type strain Newman and the saeSL and ΔsaeRS mutants. RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.

No MeSH data available.


Related in: MedlinePlus