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Allelic replacement of the streptococcal cysteine protease SpeB in a Δsrv mutant background restores biofilm formation.

Roberts AL, Holder RC, Reid SD - BMC Res Notes (2010)

Bottom Line: To address this question, we constructed a ΔsrvΔspeB double mutant through allelic replacement (MGAS5005ΔsrvΔspeB) and tested its ability to form biofilms in vitro.Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation.The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of speB/SpeB.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, 27157, USA. sreid@wfubmc.edu.

ABSTRACT

Background: Group A Streptococcus (GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease. Thus, the organism has evolved to colonize a number of physiologically distinct host sites. One such mechanism to aid colonization is the formation of a biofilm. We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), results in a mutant strain exhibiting a significant reduction in biofilm formation. Unlike the parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is constitutively produced by the srv mutant (MGAS5005Δsrv) suggesting Srv contributes to the control of SpeB production. Given that SpeB is a potent protease, we hypothesized that the biofilm deficient phenotype of the srv mutant was due to the constitutive production of SpeB. In support of this hypothesis, we have previously demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of MGAS5005Δsrv to form biofilms. Still, it was unclear if the loss of biofilm formation by MGAS5005Δsrv was due only to the constitutive production of SpeB or to other changes inherent in the srv mutant strain. To address this question, we constructed a ΔsrvΔspeB double mutant through allelic replacement (MGAS5005ΔsrvΔspeB) and tested its ability to form biofilms in vitro.

Findings: Allelic replacement of speB in the srv mutant background restored the ability of this strain to form biofilms under static and continuous flow conditions. Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation.

Conclusions: The constitutive production of SpeB by the srv mutant strain is responsible for the significant reduction of biofilm formation previously observed. The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of speB/SpeB.

No MeSH data available.


Related in: MedlinePlus

Addition of purified active SpeB inhibits biofilm formation. MGAS5005, MGAS5005ΔspeB and MGAS5005ΔsrvΔspeB were either untreated or treated with 1 μg/mL of purified SpeB (Toxin Technology, Inc., Sarasota, FL) 3 times at time 0, 6 h, and 12 h. Biofilm was measured at 18 h using CV staining as previously discussed. The level of reduction in biofilm formation was statistically significant ((***) P < 0.0001) compared to the untreated samples. MGAS5005Δsrv, with constitutive production of SpeB, is presented for comparison.
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Figure 4: Addition of purified active SpeB inhibits biofilm formation. MGAS5005, MGAS5005ΔspeB and MGAS5005ΔsrvΔspeB were either untreated or treated with 1 μg/mL of purified SpeB (Toxin Technology, Inc., Sarasota, FL) 3 times at time 0, 6 h, and 12 h. Biofilm was measured at 18 h using CV staining as previously discussed. The level of reduction in biofilm formation was statistically significant ((***) P < 0.0001) compared to the untreated samples. MGAS5005Δsrv, with constitutive production of SpeB, is presented for comparison.

Mentions: To prove that SpeB alone is capable of disrupting GAS biofilm formation, we added purified active SpeB (Toxin Technology, Inc., Sarasota, FL)(final concentration 1 μg/mL) 3 times over the course of static biofilm development (0, 6 h, and 12 h). CV staining was performed on treated and untreated samples at 18 h post-seeding (Figure 4). SpeB addition resulted in a significant decrease in measurable biofilm of all treated strains to levels comparable to MGAS5005Δsrv (Figure 4).


Allelic replacement of the streptococcal cysteine protease SpeB in a Δsrv mutant background restores biofilm formation.

Roberts AL, Holder RC, Reid SD - BMC Res Notes (2010)

Addition of purified active SpeB inhibits biofilm formation. MGAS5005, MGAS5005ΔspeB and MGAS5005ΔsrvΔspeB were either untreated or treated with 1 μg/mL of purified SpeB (Toxin Technology, Inc., Sarasota, FL) 3 times at time 0, 6 h, and 12 h. Biofilm was measured at 18 h using CV staining as previously discussed. The level of reduction in biofilm formation was statistically significant ((***) P < 0.0001) compared to the untreated samples. MGAS5005Δsrv, with constitutive production of SpeB, is presented for comparison.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992062&req=5

Figure 4: Addition of purified active SpeB inhibits biofilm formation. MGAS5005, MGAS5005ΔspeB and MGAS5005ΔsrvΔspeB were either untreated or treated with 1 μg/mL of purified SpeB (Toxin Technology, Inc., Sarasota, FL) 3 times at time 0, 6 h, and 12 h. Biofilm was measured at 18 h using CV staining as previously discussed. The level of reduction in biofilm formation was statistically significant ((***) P < 0.0001) compared to the untreated samples. MGAS5005Δsrv, with constitutive production of SpeB, is presented for comparison.
Mentions: To prove that SpeB alone is capable of disrupting GAS biofilm formation, we added purified active SpeB (Toxin Technology, Inc., Sarasota, FL)(final concentration 1 μg/mL) 3 times over the course of static biofilm development (0, 6 h, and 12 h). CV staining was performed on treated and untreated samples at 18 h post-seeding (Figure 4). SpeB addition resulted in a significant decrease in measurable biofilm of all treated strains to levels comparable to MGAS5005Δsrv (Figure 4).

Bottom Line: To address this question, we constructed a ΔsrvΔspeB double mutant through allelic replacement (MGAS5005ΔsrvΔspeB) and tested its ability to form biofilms in vitro.Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation.The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of speB/SpeB.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, 27157, USA. sreid@wfubmc.edu.

ABSTRACT

Background: Group A Streptococcus (GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease. Thus, the organism has evolved to colonize a number of physiologically distinct host sites. One such mechanism to aid colonization is the formation of a biofilm. We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), results in a mutant strain exhibiting a significant reduction in biofilm formation. Unlike the parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is constitutively produced by the srv mutant (MGAS5005Δsrv) suggesting Srv contributes to the control of SpeB production. Given that SpeB is a potent protease, we hypothesized that the biofilm deficient phenotype of the srv mutant was due to the constitutive production of SpeB. In support of this hypothesis, we have previously demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of MGAS5005Δsrv to form biofilms. Still, it was unclear if the loss of biofilm formation by MGAS5005Δsrv was due only to the constitutive production of SpeB or to other changes inherent in the srv mutant strain. To address this question, we constructed a ΔsrvΔspeB double mutant through allelic replacement (MGAS5005ΔsrvΔspeB) and tested its ability to form biofilms in vitro.

Findings: Allelic replacement of speB in the srv mutant background restored the ability of this strain to form biofilms under static and continuous flow conditions. Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation.

Conclusions: The constitutive production of SpeB by the srv mutant strain is responsible for the significant reduction of biofilm formation previously observed. The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of speB/SpeB.

No MeSH data available.


Related in: MedlinePlus