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

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.

Figure 1

Construction of MGAS5005ΔsrvΔspeB. (A) speB flanking sequences were cloned upstream and downstream of the chloramphenicol resistance cassette cat (Cm^r) in pFW14. The resulting plasmid was transformed into MGAS5005Δsrv, and allelic replacement yielded MGAS5005ΔsrvΔspeB. (B) PCR of (I) MGAS5005, (II) MGAS5005Δsrv, (III) MGAS5005ΔspeB and (IV) MGAS5005ΔsrvΔspeB using primers srv internal FWD/REV (347 bp fragment) and internal speB FWD/REV (80 bp fragment) to verify deletion of the genes srv and speB within the MGAS5005 mutants. Ladder (L) is a 1 kB ladder.

Figure 2

Static crystal violet assays for the measurement of in vitro biofilm formation. MGAS5005, MGAS5005Δsrv, MGAS5005ΔspeB and MGAS5005ΔsrvΔspeB were grown in 6-well tissue culture treated polystyrene plates for 24 h (A), stained with crystal violet, and solubilized with ethanol. Each reported value for the CV assay is an average of at least 6 replicates and is adjusted by the dilution factor required to obtain a spectrophometric reading (A_{600 nm}) (P ≤ 0.0001, unpaired t-test). (B) Biofilm formation for each strain over time is shown out to 48 h.

Figure 3

MGAS5005ΔsrvΔspeB biofilm formation under continuous flow conditions. (A-C) Representative flow cell chambers containing 24 h grown cultures under a flow rate of ~ 0.7 mL/min of MGAS5005ΔsrvΔspeB, MGAS5005, and MGAS5005Δsrv, respectively. (A and B) Chambers inoculated with (A) MGAS5005ΔsrvΔspeB or (B) MGAS5005 were filled with dense viscous material indicative of GAS biofilms. (C) MGAS5005Δsrv was unable to form biofilms under flow conditions. Scanning electron microscopy of a 24 h (D) MGAS5005 and (E-G) a MGAS5005ΔsrvΔspeB continuous flow biofilm clearly depicts chains of cocci organized into a 3-dimensional structure encased in a matrix-like material.

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.

Figure 5

Hypothetical model of Srv/SpeB mediated GAS biofilm formation and dispersal. Following GAS exposure, Srv-mediated negative regulation of SpeB production would allow biofilm formation and colonization. As of yet unidentified environmental signals may reverse this control, promoting SpeB production and subsequent biofilm dispersal in order to facilitate dissemination/transmission of the organism. We hypothesize that this cycle is likely held in equilibrium such that controlled amounts of SpeB may be produced to allow dissemination without complete disruption of the GAS biofilm.