Abstract
The extracellular biofilm matrix includes primarily DNA and exopolysaccharides (EPS), which function to maintain aggregate structures and to protect biofilms from antibiotics and the immune response. Both polymers are anionic and have cation binding activity, however the impact of this activity on biofilms is not fully understood. Host cell contact is considered the primary signal for activation of most type III secretion systems (T3SS), although calcium limitation is frequently used as a trigger of contact-independent T3SS expression. We hypothesized that alginate, which is a known calcium binding exopolysaccharide produced in mucoid Pseudomonas aeruginosa isolates, can activate the T3SS in biofilms. The addition of exogenous purified alginate to planktonic, non-mucoid PAO1 cultures induced expression of exoS, exoT and exoY-lux reporters of the T3SS in a concentration-dependent manner. Induction by alginate was comparable to induction by the calcium chelator NTA. We extended our analysis of the T3SS in flow chamber-cultivated biofilms, and showed that hyperproduction of alginate in mucA22 mucoid isolates resulted in induction of the exoS-gfp transcriptional reporter compared to non-mucoid paired isolates. We confirmed the transcriptional effects of alginate on the T3SS expression using a FlAsH fluorescence method and showed high levels of the ExoT-Cys4 protein in mucoid biofilms. Induction of the T3SS could be prevented in planktonic cultures and mucoid biofilms treated with excess calcium, indicating that Ca2+ chelation by the EPS matrix caused contact-independent induction. However, mucoid isolates generally had reduced exoS-lux expression in comparison to paired, non-mucoid isolates when grown as planktonic cultures and agar colonies. In summary, we have shown a mucoid biofilm-specific induction of the type III secretion system and highlight a difference between planktonic and biofilm cultures in the production of virulence factors.
Highlights
Biofilms are multicellular, surface-associated microbial communities encased in an extracellular matrix largely composed of extracellular DNA and exopolysaccharides (EPS) [1,2]
T3SS We first examined expression of a transcriptional exoS-lux fusion in wild type PAO1 under conditions with varying cation concentrations and observed that the T3SS was maximally expressed with low levels of Ca2+ (20 mM) and relatively high concentration of Mg2+ (0.1–2 mM) (Fig. 1A,C)
The T3SS was repressed with the addition of 2 mM Ca2+, confirming the role of calcium limitation in inducing expression of the T3SS
Summary
Surface-associated microbial communities encased in an extracellular matrix largely composed of extracellular DNA and exopolysaccharides (EPS) [1,2] Both DNA and EPS are structural components of the biofilm matrix that are required for attachment, aggregation and the later stage of biofilm maturation [1,3]. Exopolysaccharides perform capsule functions that include reducing phagocytosis by macrophages [5,6], limiting neutrophil migration, preventing the binding of complement factors and absorbing reactive oxygen species [6] Another property of the EPS matrix is to provide short-term protection as a diffusion barrier to antibiotics, not all antibiotics have reduced penetration through biofilms [7]
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