Figure 1.
Low agr activity is important for S. aureus biofilm formation.
(A–D) Biofilms were grown for 2 days in either 2% TSB or 2% TSB supplemented with 0.2% glucose (referred to as “TSBg”). Biofilm integrity and RFP fluorescence were monitored with CLSM. Three dimensional image reconstructions of a z series were created with Velocity software. CLSM images are representative of three separate experiments and each side of a grid square represents 20 µM. (A) AH596 (agr+) grown in TSB. (B) AH596 grown in TSBg. (C) AH871 (agr-) grown in TSB. (D) AH871 grown in TSBg. (E) Measurement of the agr P3-GFP reporter (pDB59) activity in strains AH596 and AH871 grown in broth culture in either TSB or TSBg. Error bars show standard error of the mean (SEM).
Figure 2.
Detachment of S. aureus biofilms with AIP.
Biofilms (strain AH500) were grown in flow cells for 2 days. Either (A) 1 mL of buffer (100 mM phosphate [pH 7], 50 mM NaCl, 1 mM TCEP) or (B) 1 mL of 20 µM AIP-I in buffer was diluted 1000-fold into the biofilm growth media. The biofilm integrity was monitored with CLSM for 2 more days. Each side of a grid square in the image reconstructions represents 20 µM. (C) Effect of AIP-I addition on number of detached bacteria in the effluent medium from flow cell biofilms. The plot depicts CFU/ml in effluents from biofilms, and the black squares (▪) represent AIP-I addition and the black circles (•) represent buffer addition to the biofilm. Graph shows the mean of 3 effluent collections from 1 experiment, error bars show SEM.
Figure 3.
Effect of AIP addition to biofilms from S. aureus strains representing different agr classes.
Biofilms were grown in flow cells for 2 days and indicated AIP was added (50 nM final concentration) to the growth media. Biofilm integrity was monitored with CLSM. Each side of a grid square in the image reconstructions represents 20 µM, and red color is from propidium iodide stain present in growth medium. (A) Biofilm of strain FRI1169 (agr Type I) treated with AIP-I. (B) Biofilm of strain SA502A (agr Type II) treated with AIP-II. (C) Biofilm of strain ATCC25923 (agr Type III) treated with AIP-III.
Figure 4.
Expression of agr P3 promoter in biofilms after AIP addition.
Dual-labeled biofilms (PsarA-RFP, PagrP3-GFP) were grown for 2 days, and AIP-I (50 nM final) was added to the growth media. Biofilm integrity and RFP/GFP fluorescence were monitored with CLSM at day 3 and 4. Greenish yellow color indicates expression of the agr P3-GFP reporter (pDB59). (A) Addition of AIP-I to an agr type I wild type strain (AH596) or (B) agr deficient strain (AH861). (C) Addition of interfering AIP-II to an agr type-I strain biofilm (AH596). CLSM image reconstructions are representative of three separate experiments and each side of a grid square represents 20 µM.
Figure 5.
Effect of changing growth conditions on agr-mediated biofilm detachment.
Dual-labeled biofilms (PsarA-RFP, PagrP3-GFP) of (A) agr positive strain AH596 and (B) agr mutant strain AH871 were grown for 2 days in TSBg. Glucose was removed from the growth media and the biofilm was grown an additional 2 days. Biofilm integrity and RFP/GFP fluorescence were monitored with CLSM. CLSM image reconstructions are representative of three separate experiments and each side of a grid square represents 20 µM.
Figure 6.
Susceptibility of biofilm and detached bacteria to rifampicin killing.
S. aureus SH1000 biofilm bacteria (black diamonds) were grown in flow cells containing removable coupons, allowing multiple replicate biofilms to be exposed to rifampicin and surviving CFU's to be determined. Detached bacteria (black circles) were collected from flow cell effluents of biofilms exposed to AIP-I. As a control, planktonic bacteria (black squares) were treated with the same level of rifampicin. Graph show the mean of three experiments; error bars show SEM.
Figure 7.
Role of ica locus in biofilm development.
(A) Microtiter biofilms of ica positive strain SH1000 and ica deletion mutant AH595. (B) Quantitation of microtiter biofilms. (C) Representative CLSM image of flow cells biofilms of strain AH595 grow in TSBg for 2 days. Each side of a grid square represents 20 µM, and red color is from propidium iodide stain present in growth medium.
Figure 8.
Effect of Proteinase K on biofilms and measurement of extracellular protease activity in AIP-detached biofilms.
(A) Proteinase K (proK, 2 µg/ml) was added to a 2 day old biofilm (strain AH500) and the biofilm integrity was monitored with CLSM at 6 and 12 hr. (B) Levels of protease activity detected in biofilm effluent collected from wild type (SH1000) or Δagr (SH1001) biofilms supplemented with indicated AIP's. Protease activity was referenced to wild-type without AIP addition. (C) The effect of inhibitors or activators on the proteolytic activity in an AIP-I detached biofilm effluent. Activity assay was supplemented with either the metalloprotease inhibitor EGTA (1 mM), serine protease inhibitor PMSF (10 µM), or the reducing agent DTT (1 mM). Error bars show SEM.
Figure 9.
Effect of a serine protease inhibitor and protease deficient mutants on AIP-I mediated biofilm detachment.
Columns show CLSM reconstructions of biofilms at day 2, day 3 and day 4. Biofilms were grown for 2 days and the growth media was supplemented with AIP-I or AIP-I+PMSF as indicated. Greenish yellow color indicates expression of the agr P3-GFP reporter, and the red color is from propidium iodide present in the growth medium. (A) Wild type biofilm (AH462) supplemented with 50 nM AIP-I. (B) Wild type biofilm (AH462) supplemented with 50 nM AIP-I and 10 µM PMSF. (C) Aureolysin (Δaur) mutant biofilm (AH789) supplemented with 50 nM AIP-I. (D) Aureolysin Spl (Δaur Δspl) double mutant biofilm (AH788) supplemented with 50 nM AIP-I. CSLM reconstructions are representative of three separate experiments and each side of a grid square represents 20 µM. Percent biomass detached was calculated by COMSTAT analysis comparing biomass at day 2 to biomass at day 4.
Figure 10.
Extracellular protease activity and biofilm formation of protease mutants.
(A) Relative protease levels detected in wild type and protease mutants grown in broth culture. Images show bacterial colonies and zones of clearing caused by protease activity on milk agar plates. (B–C) Biofilm formation of wild type and protease mutants in wells of microtiter plates. Graphs show quantitation of biofilm biomass attached to microtiter plate grown in either (B) TSBg or (C) TSB. Images below each graph are of crystal violet stained biofilms in wells of microtiter plates.
Table 1.
Strain and plasmid list