Identification of genes involved in polysaccharide-independent Staphylococcus aureus biofilm formation

Abstract

Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor alpha(2)-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.

Figure 1. Extracellular protease activity of biofilm mutants.

Protease activity in cell-free supernatants from cultures grown in TSB for 12 hours was measured with Azocoll reagent as described in Materials and Methods. Supernatant protease activity of the wild-type strain (SH1000) was set to 1 as a reference. The graph shows the mean of three samples; error bars show standard deviation. Blue bars indicate mutants that show consistently higher levels of protease activity.

Figure 2. Assessment of biofilm formation in transposon mutants.

Microtiter biofilm assays were performed in the absence (A) or presence (B) of the protease inhibitor α₂-macroglobulin in triplicate, and the percentages of biofilm formation for each mutant relative to that of the wild type are shown. Error bars show standard deviation. Color coding is as follows: black bars indicate mutants with normal extracellular protease activity, blue bars indicate high protease activity and biofilm recovery with α₂-macroglobulin, and red bars indicate the graS (37 C9) and imp (60 G7, 64 G10) mutations that did not recover with α₂-macroglobulin.

Figure 3. Extracellular nuclease activity of selected biofilm defective mutants.

Nuclease activity was measured as described in Materials and Methods and plotted relative to SH1000 levels. For the imp mutant, only the results of transposon mutant 60 G7 are shown. Results with imp mutant 64 G10 were indistinguishable (data not shown). Underneath the plotted nuclease activity results are representative images of colonies grown on methyl green DNase agar plates. Clearing zones around the colonies is indicative of increased nuclease activity. Error bars show standard deviation.

Figure 4. Time course of autolysis in selected biofilm mutants.

Planktonic cultures of seven transposon mutants harboring plasmid pAJ22, which expresses cytoplasmic β-galactosidase, were grown for 72 hrs. Every 12 hr during the time course samples were removed from cultures and β-galactosidase activity in cell free supernatants was measured (reported in Miller units). SH1000 with pAJ22 was included as a control (shown as black squares). Results shown were the average of two independent experiments done in triplicate and error bars show standard deviation.

Figure 5. Flow cell biofilm formation of graS and imp mutants in different strain backgrounds.

S. aureus strains SH1000 (A) and LAC* (B), and graS and imp mutations in each strain, were grown in a flow cell apparatus for two days. A z series of images was obtained with CLSM, reconstructed with Volocity software, and each side of a grid square is 20 µm in the image reconstruction. The addition of a complementing plasmid containing either the graRS or imp genes is shown in the last column.