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. 2017 Dec 1;83(24):e01709-17.
doi: 10.1128/AEM.01709-17. Print 2017 Dec 15.

Murein Hydrolase LytF of Streptococcus sanguinis and the Ecological Consequences of Competence Development

Nyssa Cullin  1   2 , Sylvio Redanz  2 , Kirsten J Lampi  3 , Justin Merritt  2 , Jens Kreth  4
Affiliations

Murein Hydrolase LytF of Streptococcus sanguinis and the Ecological Consequences of Competence Development

Nyssa Cullin et al. Appl Environ Microbiol. .

Abstract

The overall health of the oral cavity is dependent on proper homeostasis between health-associated bacterial colonizers and bacteria known to promote dental caries. Streptococcus sanguinis is a health-associated commensal organism, a known early colonizer of the acquired tooth pellicle, and is naturally competent. We have shown that LytF, a competence-controlled murein hydrolase, is capable of inducing the release of extracellular DNA (eDNA) from oral bacteria. Precipitated LytF and purified LytF were used as treatments against planktonic cultures and biofilms. Larger amounts of eDNA were released from cultures treated with protein samples containing LytF. Additionally, LytF could affect biofilm formation and cellular morphology. Biofilm formation was significantly decreased in the lytF-complemented strain, in which increased amounts of LytF are present. The same strain also exhibited cell morphology defects in both planktonic cultures and biofilms. Furthermore, the LytF cell morphology phenotype was reproducible in wild-type cells using purified LytF protein. In sum, our findings demonstrate that LytF can induce the release of eDNA from oral bacteria, and they suggest that, without proper regulation of LytF, cells display morphological abnormalities that contribute to biofilm malformation. In the context of the oral biofilm, LytF may play important roles as part of the competence and biofilm development programs, as well as increasing the availability of eDNA.IMPORTANCEStreptococcus sanguinis, a commensal organism in the oral cavity and one of the pioneer colonizers of the tooth surface, is associated with the overall health of the oral environment. Our laboratory showed previously that, under aerobic conditions, S. sanguinis can produce H2O2 to inhibit the growth of bacterial species that promote dental caries. This production of H2O2 by S. sanguinis also induces the release of eDNA, which is essential for proper biofilm formation. Under anaerobic conditions, S. sanguinis does not produce H2O2 but DNA is still released. Determining how S. sanguinis releases DNA is thus essential to understand biofilm formation in the oral cavity.

Keywords: Streptococcus.

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Figures

FIG 1
FIG 1
Genetic context of lytF in S. sanguinis and S. gordonii, with lytF colored dark gray for each species. Note that open reading frames are approximate and are not drawn to scale. (A) Top, S. sanguinis genetic context, with lytF (ssa_0036) present on the negative strand and surrounded by purine biosynthesis genes; ssa_0036 is 1,950 bp and produces a 659-amino-acid protein, LytF. Bottom, S. gordonii genetic context, with lytF (sgo_2094) present on the positive strand. Two genes overlapping sgo_2094 are present, along with purine biosynthesis genes upstream and the competence-associated comAB operon downstream; sgo_2094 is 1,650 bp and produces a 549-amino-acid LytF. (B) Comparison of protein domains within LytF of S. sanguinis and LytF of S. gordonii. Both proteins contain the hydrolytic CHAP domain (light gray rectangles) at the C terminus. S. sanguinis LytF contains five Bsp regions (dark gray diamonds), while S. gordonii LytF contains three, highlighting the main difference in the proteins between the two species. Small black rectangles upstream of LytF indicate the cin box.
FIG 2
FIG 2
Induction of competence-associated genes with CSP addition. Cultures were grown to mid-log phase (OD600 of ∼0.4), 0.5 μg/ml CSP was added to the appropriate samples, and the cultures were further incubated for 2 h. RT-PCR was performed with 16S rRNA as the housekeeping gene. Expression levels are presented relative to levels in noninduced, no-CSP-added controls, which were set to 1. Data presented are the means and standard deviations of three independent experiments performed in duplicate on different days. (A) Cultures grown in TH medium at pH 6.8. (B) Cultures grown in TH medium at pH 7.4.
FIG 3
FIG 3
RACE results, showing the sequence of the identified lytF promoter. The agarose gel image shows the final RACE PCR product from the lytF promoter region that was ultimately sequenced, and the arrow indicates the band that correlates with the size of the lytF promoter with the 5′-RACE adapter.
FIG 4
FIG 4
Induction of competence-associated genes in lytF mutant strains. The relative expression of competence-associated genes in the lytF mutant and complemented strains was calculated with the levels in S. sanguinis SK36 control samples set to 1. Data presented are the means and standard deviations of three independent experiments performed in duplicate on different days.
FIG 5
FIG 5
LytF effects on transformation and cell division. (A) Cells were grown to an OD600 of ∼0.08 in TH medium with 2.5% heat-inactivated horse serum. Chromosomal DNA (kanamycin resistance) (37.6 ng/μl) and 0.5 μg/ml CSP were added, and cells were further incubated for 2 h. Cells were plated on selective and nonselective TH plates. Transformation efficiency was calculated as the number of kanamycin-resistant CFU relative to the CFU on nonselective agar. The decrease in ΔlytF transformation efficiency was statistically significant, compared to the wild-type value (**, P = 0.0099). Data represent the means and standard deviations of three independent experiments. (B) Cells were grown to mid-log phase, and images were obtained with oil immersion using an Olympus BX51 microscope, an Olympus DP72 digital camera, and cellSens 1.3 software. Images were adjusted for brightness and contrast and are representative of three independent experiments with similar outcomes. Chains from three images per strain were measured using ImageJ. Chain length differences between the strains were not statistically significant. Scale bars, 10 μm.
FIG 6
FIG 6
Altered cell morphologies of LytF mutant strains. (A) Cells were grown to mid-log phase and stained with 1 μg/ml vancomycin-BODIPY-FL conjugate. Images were obtained with oil immersion using an Olympus IX73 microscope, an Olympus DP72 digital camera, and cellSens standard software. Images were adjusted for brightness and contrast and are representative of multiple experiments with similar outcomes. (B) SEM was performed on 18-hour biofilms grown on Thermanox discs in CDM. Images are representative of at least three fields of view from two experiments performed on different days. Scale bars, 5 μm.
FIG 7
FIG 7
Inhibition of proper biofilm formation by excess LytF. Biofilms of S. sanguinis SK36, ΔlytF, and lytF-c strains were grown in CDM for 18 h, washed, and stained with 0.01% safranin. Biofilm formation was determined by measuring the absorbance of air-dried biofilms at 490 nm. The lytF-c biofilms showed a significant decrease in absorbance after safranin staining, indicating that biofilm formation was inhibited in this strain (****, P < 0.0001). Data represent averages and standard deviations from quadruplicate samples in three separate plate experiments.
FIG 8
FIG 8
S. sanguinis LytF effects as a murein hydrolase. Zymograms with either S. sanguinis or S. gordonii substrate cells show clearance bands where hydrolytic proteins contained in whole-cell suspensions of the indicated strains are active. Zymograms were photographed on a black background and are representative of at least three separate experiments performed on different days. Entire pictures were processed to increase the visibility of the bands.
FIG 9
FIG 9
Precipitation and purification of LytF from S. sanguinis. (A) SDS-PAGE analysis of proteins precipitated from culture supernatants with 60% ammonium sulfate saturation. The image is representative of at least three experiments performed on different days. (B) Zymogram assays with S. sanguinis substrate cells (left) or S. gordonii substrate cells (right). Both lytF-c protein samples (left lanes) and purified LytF (right lanes) showed hydrolysis of both species. Zymograms are representative of three separate experiments performed on different days. Images were processed to increase the visibility of the bands.
FIG 10
FIG 10
LytF induction of eDNA release. (A) Top, agarose gel showing precipitated eDNA samples. Bottom, relative amounts of eDNA released from S. sanguinis SK36 cultures after treatment for 15 min with precipitated proteins from the indicated samples. Compared to no treatment, cells treated with SK36 and lytF-c proteins released significantly more eDNA (*, P = 0.0211; **, P = 0.0060, respectively). Data represent the means and standard deviations of three independent experiments. (B) Relative amounts of eDNA released at 15 min from S. sanguinis (SS) or S. gordonii (SG) cultures, with or without pure LytF treatment (12.1 μg ml−1). LytF treatment significantly increased the amount of eDNA released from S. sanguinis cultures (**, P = 0.0019). n.s., not significant. Data represent the means and standard deviations of four independent experiments.
FIG 11
FIG 11
Excess LytF induction of morphological abnormalities. (A) S. sanguinis SK36 biofilms treated with precipitated proteins from the SK36 wild-type, lytF mutant, or lytF-complemented strains. Biofilms grown in the presence of LytF-containing protein samples had enlarged and irregular cells present within chains in the biofilms. Images were adjusted for brightness and contrast. Images are representative of at least three fields of view from two experiments performed on different days. Scale bars, 4 μm. (B) S. sanguinis SK36 biofilm grown in the presence of 12.1 μg ml−1 LytF. Cells within the biofilm again appeared enlarged and also displayed membrane blebs (white arrowheads). The dashed white box represents the area of the enlarged image to the right. Scale bar, 4 μm. The image is representative of multiple fields of view from one experiment. (C) Survival assay showing CFU of S. sanguinis SK36 grown in the presence of precipitated proteins from the indicated strains. Cultures were sonicated, serially diluted, and plated on BHI agar. Data represent the means and standard deviations of two individual experiments performed on different days.
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