Metagenomic quorum quenching enzymes affect biofilm formation of Candida albicans and Staphylococcus epidermidis

Abstract

Biofilm formation in the clinical environment is of increasing concern since a significant part of human infections is associated, and caused by biofilm establishment of (opportunistic) pathogens, for instance Candida albicans and Staphylococcus epidermidis. The rapidly increasing number of antibiotic-resistant biofilms urgently requires the development of novel and effective strategies to prevent biofilm formation ideally targeting a wide range of infectious microorganisms. Both, synthesis of extracellular polymeric substances and quorum sensing are crucial for biofilm formation, and thus potential attractive targets to combat undesirable biofilms.We evaluated the ability of numerous recently identified metagenome-derived bacterial quorum quenching (QQ) proteins to inhibit biofilm formation of C. albicans and S. epidermidis. Here, proteins QQ-5 and QQ-7 interfered with the morphogenesis of C. albicans by inhibiting the yeast-to-hyphae transition, ultimately leading to impaired biofilm formation. Moreover, QQ5 and QQ-7 inhibited biofilm formation of S. epidermidis; in case of QQ7 most likely due to induced expression of the icaR gene encoding the repressor for polysaccharide intercellular adhesin (PIA) synthesis, the main determinant for staphylococcal biofilm formation. Our results indicate that QQ-5 and QQ-7 are attractive potential anti-biofilm agents in the prevention and treatment of C. albicans and S. epidermidis mono-species biofilms, and potentially promising anti-biofilm drugs in also combating multi-species infections.

Fig 1. Effects of immobilized QQ proteins on the biofilm formation of C. albicans.

Phase-contrast images were taken with a Zeiss Axioscope microscope over a time period of 2 to 24 h. QQ proteins MBP-QQ-5 and MBP-QQ-7 as well as MBP were immobilized in 12 well culture dishes in comparision to the control. C. albicans cells (10⁹ yeast cells/mL) were incubated in YPD at 30°C to analyze yeast to hyphae formation. Scale bars represent 10 μm.

Fig 2. Relative transcript levels of dpp3 in C. albicans after incubation with QQ proteins.

Relative transcript levels of dpp3 were determined by quantitative RT-PCR analysis in C. albicans with at least three biological replicates each with three technical replicates. QQ proteins MBP-QQ-5 and MBP-QQ-7 as well as MBP were immobilized on the surface of culture dishes. C. albicans cells (10⁹ yeast cells/mL) were incubated for 2 h to 6 h at 30°C in YPD. Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts (calculated 2^-ΔΔCt values) of the control gene actin.

Fig 3. S. epidermidis biofilm formation under static conditions in presence of MBP-QQ-5, MBP-QQ-7 and MBP.

S. epidermidis was grown in 96 well plates in 200 μL culture medium in presence of purified MBP-QQ proteins MBP-QQ-5 (light grey bars) and MBP-QQ-7 (dark grey bars) at final concentrations of 10 μg, 50 μg and 100 μg. Purified MBP (black bar) was added as control to a final concentration of 100 μg and the determined absorbance set to 100%. Biofilm formation was quantified via crystal violet assay. Means of standard deviations of at least 4 independent biological replicates are indicated. Statistics (unpaired t test) were performed with GraphPad Prism 6 software with differences *p < 0.05, **p < 0.01 and ***p < 0.001 considered significant; n.s., not significant.

Fig 4. Decreased S. epidermidis biofilm formation on immobilized proteins MBP-QQ-5 and MBP-QQ-7.

Coverslips coated with proteins MBP-QQ-5, MBP-QQ-7and MBP as control were used in flow chambers (total volume of 1.3 mL) for S. epidermidis biofilm formation (3 x 10⁸ cells/ mL). After 1 h adhesion, flow chambers were incubated for 20 h (long-term experiment) with a flow rate of 20 mL/h using 3% TSB medium. Biofilms were stained with Syto9 and propidium iodide. Effects on biofilm formation were visualized using (A) CLSM and (B) SEM. (C) Biofilm characteristics thickness and volume were quantified using IMARIS software. Means of 2 independent biological replicates, each with 3 technical replicates are indicated. Unpaired t test was performed with GraphPad Prism 6 software with differences *p < 0.05, **p < 0.01 and ***p < 0.001 considered significant.

Fig 5. Excluding potential effects of QQ proteins on growth of S. epidermidis RP6A.

S. epidermidis RP6A was grown in Tryptic Soy Broth medium at 37°C and 120 rpm in 20 mL. Cultures were grown complemented with 100 μg MBP, QQ-5 or QQ-7 as well as without any supplement. The results represent a mean of three independent experiments. Error bars indicate standard deviations.

Fig 6. Impact of immobilized protein MBP-QQ-7 on S. epidermidis initial attachment.

Proteins MBP-QQ-7 and MBP as control were immobilized on coverslips, which were inserted into flow chambers with a total volume of 1.3 mL. After 1 h adhesion, flow chambers were incubated for 3 h (short-term experiment) at a flow rate of 20 mL/h with 3% TSB medium. Adhered cells were stained with Syto9 and propidium iodide and visualized using (A) CLSM and (B) SEM.

Fig 7. Transcript levels of icaR, lrgA and rnaIII in S. epidermidis in the presence of MBP-QQ-7.

S. epidermidis (3 x 10⁸ cells/mL in 3 mL TSB) were incubated in the presence of 500 μg/mL MBP-QQ-7 without shaking. After 3 h incubation, RNA was isolated and the relative transcript levels of icaR, lrgA and rnaIII were determined by quantitative RT-PCR analysis. Three biological replicates each with three technical replicates were performed to determine the standard deviation of the calculated 2^-ΔΔCt values. Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts of the control gene gyrB.