How Staphylococcus aureus biofilms develop their characteristic structure

Abstract

Biofilms cause significant problems in the environment and during the treatment of infections. However, the molecular mechanisms underlying biofilm formation are poorly understood. There is a particular lack of knowledge about biofilm maturation processes, such as biofilm structuring and detachment, which are deemed crucial for the maintenance of biofilm viability and the dissemination of cells from a biofilm. Here, we identify the phenol-soluble modulin (PSM) surfactant peptides as key biofilm structuring factors in the premier biofilm-forming pathogen Staphylococcus aureus. We provide evidence that all known PSM classes participate in structuring and detachment processes. Specifically, absence of PSMs in isogenic S. aureus psm deletion mutants led to strongly impaired formation of biofilm channels, abolishment of the characteristic waves of biofilm detachment and regrowth, and loss of control of biofilm expansion. In contrast, induced expression of psm loci in preformed biofilms promoted those processes. Furthermore, PSMs facilitated dissemination from an infected catheter in a mouse model of biofilm-associated infection. Moreover, formation of the biofilm structure was linked to strongly variable, quorum sensing-controlled PSM expression in biofilm microenvironments, whereas overall PSM production remained constant to ascertain biofilm homeostasis. Our study describes a mechanism of biofilm structuring in molecular detail, and the general principle (i.e., quorum-sensing controlled expression of surfactants) seems to be conserved in several bacteria, despite the divergence of the respective biofilm-structuring surfactants. These findings provide a deeper understanding of biofilm development processes, which represents an important basis for strategies to interfere with biofilm formation in the environment and human disease.

Fig. 1.

Impact of PSMs and Agr on the structuring of static S. aureus biofilms. Static biofilms were grown in eight-well chambered coverglass plates for 48 h. (A–D) Biofilm parameters were measured in at least 20 randomly chosen biofilm CLSM images of the same extension. Horizontal bars depict the mean. Statistical analysis is by t tests vs. the corresponding values of the WT samples, which were grown and measured separately for every mutant comparison. Only one WT analysis is shown for brevity; however, statistical analysis was performed vs. the corresponding WT samples grown in parallel, which were very similar in all cases. ****P < 0.0001. Values for 24-h biofilms were also measured, and differences were similar. (E) Example 48-h CLSM biofilm images. Extensions and scales are the same in every image (total x extension, 230 μm; total y extension, 230 μm).

Fig. 2.

PSMs are responsible for quorum-sensing controlled waves of detachment in dynamic S. aureus biofilms. Dynamic (flow cell) biofilm formation was measured over 5 d, and three randomly chosen biofilm CLSM images were analyzed for the total biovolume at regular intervals. Arrows mark the detachment waves occurring in the WT sample after the third day of biofilm formation.

Fig. 3.

Impact of PSMs and Agr on the structuring of dynamic (flow cell) S. aureus biofilms. Dynamic (flow cell) biofilms were grown for 72 h. (A–D) Biofilm parameters were measured at 72 h in at least 14 randomly chosen biofilm CLSM images of the same extension. Horizontal bars depict the mean. Statistical analysis is by t tests vs. the corresponding values of the WT samples, which were grown and measured separately for every mutant comparison. Only one WT analysis is shown for brevity; however, statistical analysis was performed vs. the corresponding WT samples grown in parallel, which were very similar in all cases. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Values for 24- and 48-h biofilms were also measured, and differences were similar. (E) Example 48-h CLSM biofilm images. Extensions and scales are the same in every image (total x extension, 230 μm; total y extension, 230 μm).

Fig. 4.

Induction of PSM expression leads to biofilm detachment in dynamic S. aureus biofilms. Dynamic biofilms of the LAC psm triple mutant (Δα/Δβ/Δhld) harboring the indicated plasmids were grown in flow cells for 24 h (t = 0) in tryptic soy broth without glucose. Then, expression of the respective psm genes, which were cloned in the pTX series of plasmids under a xylose-inducible promoter (see Table S1 for all oligonucleotides used), was induced by switching the media to tryptic soy broth without glucose/0.5% xylose. In control samples, the media were not switched. (A) Total and (B) average biovolumes were measured at 24 and 48 h after in five randomly chosen biofilm CLSM images of the same extension. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; t tests vs. corresponding control (pTX16) samples.

Fig. 5.

Expression of psm and agr promoters in static and dynamic S. aureus biofilms. (A) Agr quorum-sensing control circuit and regulation of target genes. Modified from ref. . The quorum-sensing circuit is shown at the top. AgrB modifies and exports the AgrD Agr autoinducing peptide precursor, which activates the histidine kinase AgrC. Activated (phosphorylated) AgrA binds to the P2, P3, psmα, and psmβ promoters. The P2 promoter controls expression of RNAII, comprising the agrB, agrD, agrC, and agrA transcripts, which form the basis of the Agr quorum-sensing autofeedback mechanism. Most Agr targets other than the psm genes are regulated by the P3-controlled RNAIII, which also encodes the PSM δ-toxin (hld gene). (B–D) CLSM visualization of promoter expression in (B) static and (C and D) dynamic biofilms using promoter-egfp transcriptional fusions in strain LAC; x and y axes dimensions are 160 × 160 μm in all images. In B, top and side views are shown of corresponding images. In C, example images are shown that depict typical average promoter expression observed for the respective promoters; at different time points or locations in the biofilm, promoter expression was strongly different, which is shown as an example for the psmβ promoter in D.

Fig. 6.

Mouse model of biofilm-associated infection. Two catheter pieces coated with the same strain of USA300 (LAC) WT or isogenic psm mutant bacteria (∼3 × 10⁵ cfu per catheter piece) were inserted in the left and right dorsum of Nu/Nu mice. At day 7, mice were euthanized, and disseminated bacteria were counted. Samples with considerable bacterial numbers are shown. Only very small numbers of bacteria were found in the organs. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs. LAC (one-way ANOVA with Bonferroni posttests).