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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

saeRS regulates attachment but not dispersal.(A) Stationary phase cultures of Newman and the ΔsaePQRS::kan strain were diluted into biofilm medium, inoculated into a 24-well plate and allowed to attach for 1 h at 37°C before washing and staining with crystal violet. (B) Supernatants from Newman and Newman ΔsaePQRS::kan were diluted into biofilm media and the attachment phenotype of strain UAMS-1 was tested with and without supernatant supplementation. (C) Newman (row 2) and Newman ΔsaePQRS::kan (row 3) culture supernatants were added at the indicated times following inoculation with UAMS-1. Wells in row 1 were supplemented with sterile medium rather than culture supernatant.
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pone.0123027.g003: saeRS regulates attachment but not dispersal.(A) Stationary phase cultures of Newman and the ΔsaePQRS::kan strain were diluted into biofilm medium, inoculated into a 24-well plate and allowed to attach for 1 h at 37°C before washing and staining with crystal violet. (B) Supernatants from Newman and Newman ΔsaePQRS::kan were diluted into biofilm media and the attachment phenotype of strain UAMS-1 was tested with and without supernatant supplementation. (C) Newman (row 2) and Newman ΔsaePQRS::kan (row 3) culture supernatants were added at the indicated times following inoculation with UAMS-1. Wells in row 1 were supplemented with sterile medium rather than culture supernatant.

Mentions: To test whether the inhibitory protein affects the attachment step of biofilm formation, attachment assays were performed by diluting mid-log phase bacteria into biofilm media and inoculating plasma-coated 12-well plates. Bacteria were incubated for 1 h at 37°C, then were washed, fixed, and stained with crystal violet. Newman wild-type attached as distinct clumps, while the ΔsaePQRS::kan strain attached as a uniform layer of cells (Fig 3A). To confirm this effect, a strain UAMS-1 culture was supplemented with supernatants from either strain Newman or Newman ΔsaePQRS::kan and inoculated as stated above. Strain UAMS-1 cultures supplemented with supernatant from strain Newman exhibited the same clumping phenotype as strain Newman, whereas UAMS-1 incubated with the ΔsaePQRS::kan mutant supernatant attached as a uniform layer of cells (Fig 3B). These results suggested the SaeRS-regulated protein affects the attachment step of biofilm formation. To test a possible role of SaeRS in biofilm dispersal, strain UAMS-1 was inoculated into a plasma-coated microtiter plate at time 0. Every two hours for 10 hours post inoculation of strain UAMS-1, the media was changed to one of the following: 1) fresh biofilm media, 2) fresh biofilm media supplemented with Newman supernatant, or 3) fresh biofilm media supplemented with the ΔsaePQRS::kan mutant supernatant. At 24 h post inoculation, biofilms were washed, fixed, and stained with crystal violet. Media supplemented with strain Newman supernatant did not affect mature biofilms (Fig 3C), suggesting that the putative inhibitory protein does not have the capacity to disrupt a mature biofilm. Collectively, these data suggest that a SaeRSP-regulated protein inhibits the attachment step in biofilm formation.


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)

saeRS regulates attachment but not dispersal.(A) Stationary phase cultures of Newman and the ΔsaePQRS::kan strain were diluted into biofilm medium, inoculated into a 24-well plate and allowed to attach for 1 h at 37°C before washing and staining with crystal violet. (B) Supernatants from Newman and Newman ΔsaePQRS::kan were diluted into biofilm media and the attachment phenotype of strain UAMS-1 was tested with and without supernatant supplementation. (C) Newman (row 2) and Newman ΔsaePQRS::kan (row 3) culture supernatants were added at the indicated times following inoculation with UAMS-1. Wells in row 1 were supplemented with sterile medium rather than culture supernatant.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123027.g003: saeRS regulates attachment but not dispersal.(A) Stationary phase cultures of Newman and the ΔsaePQRS::kan strain were diluted into biofilm medium, inoculated into a 24-well plate and allowed to attach for 1 h at 37°C before washing and staining with crystal violet. (B) Supernatants from Newman and Newman ΔsaePQRS::kan were diluted into biofilm media and the attachment phenotype of strain UAMS-1 was tested with and without supernatant supplementation. (C) Newman (row 2) and Newman ΔsaePQRS::kan (row 3) culture supernatants were added at the indicated times following inoculation with UAMS-1. Wells in row 1 were supplemented with sterile medium rather than culture supernatant.
Mentions: To test whether the inhibitory protein affects the attachment step of biofilm formation, attachment assays were performed by diluting mid-log phase bacteria into biofilm media and inoculating plasma-coated 12-well plates. Bacteria were incubated for 1 h at 37°C, then were washed, fixed, and stained with crystal violet. Newman wild-type attached as distinct clumps, while the ΔsaePQRS::kan strain attached as a uniform layer of cells (Fig 3A). To confirm this effect, a strain UAMS-1 culture was supplemented with supernatants from either strain Newman or Newman ΔsaePQRS::kan and inoculated as stated above. Strain UAMS-1 cultures supplemented with supernatant from strain Newman exhibited the same clumping phenotype as strain Newman, whereas UAMS-1 incubated with the ΔsaePQRS::kan mutant supernatant attached as a uniform layer of cells (Fig 3B). These results suggested the SaeRS-regulated protein affects the attachment step of biofilm formation. To test a possible role of SaeRS in biofilm dispersal, strain UAMS-1 was inoculated into a plasma-coated microtiter plate at time 0. Every two hours for 10 hours post inoculation of strain UAMS-1, the media was changed to one of the following: 1) fresh biofilm media, 2) fresh biofilm media supplemented with Newman supernatant, or 3) fresh biofilm media supplemented with the ΔsaePQRS::kan mutant supernatant. At 24 h post inoculation, biofilms were washed, fixed, and stained with crystal violet. Media supplemented with strain Newman supernatant did not affect mature biofilms (Fig 3C), suggesting that the putative inhibitory protein does not have the capacity to disrupt a mature biofilm. Collectively, these data suggest that a SaeRSP-regulated protein inhibits the attachment step in biofilm formation.

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