Biofilms and type III secretion are not mutually exclusive in Pseudomonas aeruginosa

Mikkelsen, Helga; Bond, Nicholas J.; Skindersoe, Mette E.; Givskov, Michael; Lilley, Kathryn S.; Welch, Martin

doi:10.1099/mic.0.025551-0

Cited by 41 publications

(51 citation statements)

References 69 publications

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“…Type III secretion is considered an archetypical acute virulence factor in P. aeruginosa. A number of previous studies, both in in vitro systems and in longitudinal studies of sputum from cystic fibrosis patients, have reported that the type III secretion system is downregulated in P. aeruginosa biofilms (3,47), although a study indicating that type III secretion is active in biofilms has also been reported (48). Recently it was reported that the translocon apparatus of the type III secretion system, but not the effectors themselves, is required for cell-associated aggregation of P. aeruginosa on the surface of polarized epithelial cells (49).…”

Section: Discussionmentioning

confidence: 98%

The Cyclic AMP-Vfr Signaling Pathway in Pseudomonas aeruginosa Is Inhibited by Cyclic Di-GMP

Almblad¹,

Harrison²,

Rybtke³

et al. 2015

J. Bacteriol.

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The opportunistic human pathogen Pseudomonas aeruginosa expresses numerous acute virulence factors in the initial phase of infection, and during long-term colonization it undergoes adaptations that optimize survival in the human host. Adaptive changes that often occur during chronic infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilmforming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (cAMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused by inhibition of adenylate cyclase production or activity and that it is not caused by activation of cAMP phosphodiesterase activity. In addition to the studies of the RSCVs, we present evidence that the deeper layers of wild-type P. aeruginosa biofilms have high c-di-GMP levels and low cAMP levels. IMPORTANCEOur work suggests that cross talk between c-di-GMP and cAMP signaling pathways results in downregulation of acute virulence factors in P. aeruginosa biofilm infections. Knowledge about this cross-regulation adds to our understanding of virulence traits and immune evasion by P. aeruginosa in chronic infections and may provide new approaches to eradicate biofilm infections.

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Section: Discussionmentioning

confidence: 98%

The Cyclic AMP-Vfr Signaling Pathway in Pseudomonas aeruginosa Is Inhibited by Cyclic Di-GMP

Almblad¹,

Harrison²,

Rybtke³

et al. 2015

J. Bacteriol.

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“…These results imply that the coordinate regulation of T3SS and biofilm formation may be more complex during formation of cell-associated aggregation. Indeed, some studies have shown that T3SS can be activated in a subset of biofilm-grown bacteria [7]–[9]. Our data suggest a model in which a few type III secretion-competent bacteria adhere to the apical surface of the epithelial barrier and elicit the release of one or more host cell factors (Figure S9).…”

Section: Discussionmentioning

confidence: 52%

“…The T3SS is a key virulence determinant in acute infections in vitro and in vivo [23], but most published studies suggest that it is downregulated in abiotic biofilms [6], [9], [24], [25]. To investigate the role of T3SS components in cell-associated aggregates, we infected MDCK cells stably expressing the PH domain of Akt fused to GFP (PH-Akt-GFP), a probe for the basolateral phospholipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) [26], with a panel of isogenic T3SS mutants.…”

Section: Resultsmentioning

confidence: 99%

“…For example, critical virulence factors associated with acute infections, such as the type III secretion system (T3SS)—a contact-dependent, needle-like apparatus that directly injects bacterial toxins into infected host cells—are inversely regulated with biofilm determinants, such as the production of the exopolysaccharides Psl and Pel [6]. Some recently published data challenges this idea [7]–[9].…”

Section: Introductionmentioning

confidence: 99%

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The Pseudomonas aeruginosa Type III Translocon Is Required for Biofilm Formation at the Epithelial Barrier

et al. 2014

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Clinical infections by Pseudomonas aeruginosa, a deadly Gram-negative, opportunistic pathogen of immunocompromised hosts, often involve the formation of antibiotic-resistant biofilms. Although biofilm formation has been extensively studied in vitro on glass or plastic surfaces, much less is known about biofilm formation at the epithelial barrier. We have previously shown that when added to the apical surface of polarized epithelial cells, P. aeruginosa rapidly forms cell-associated aggregates within 60 minutes of infection. By confocal microscopy we now show that cell-associated aggregates exhibit key characteristics of biofilms, including the presence of extracellular matrix and increased resistance to antibiotics compared to planktonic bacteria. Using isogenic mutants in the type III secretion system, we found that the translocon, but not the effectors themselves, were required for cell-associated aggregation on the surface of polarized epithelial cells and at early time points in a murine model of acute pneumonia. In contrast, the translocon was not required for aggregation on abiotic surfaces, suggesting a novel function for the type III secretion system during cell-associated aggregation. Supernatants from epithelial cells infected with wild-type bacteria or from cells treated with the pore-forming toxin streptolysin O could rescue aggregate formation in a type III secretion mutant, indicating that cell-associated aggregation requires one or more host cell factors. Our results suggest a previously unappreciated function for the type III translocon in the formation of P. aeruginosa biofilms at the epithelial barrier and demonstrate that biofilms may form at early time points of infection.

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“…We compiled lists from six transcriptomic (2,(83)(84)(85)(86)(87) and five proteomic (88-92) investigations of differential expression between biofilm and planktonic cells. This expands on previous analyses (93)(94)(95).…”

Section: Resultsmentioning

confidence: 99%

Contribution of Stress Responses to Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms

Stewart

Franklin

Williamson

et al. 2015

Antimicrob Agents Chemother

125

118

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bEnhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoSregulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4-to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin.

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Biofilms and type III secretion are not mutually exclusive in Pseudomonas aeruginosa

Cited by 41 publications

References 69 publications

The Cyclic AMP-Vfr Signaling Pathway in Pseudomonas aeruginosa Is Inhibited by Cyclic Di-GMP

The Cyclic AMP-Vfr Signaling Pathway in Pseudomonas aeruginosa Is Inhibited by Cyclic Di-GMP

The Pseudomonas aeruginosa Type III Translocon Is Required for Biofilm Formation at the Epithelial Barrier

Contribution of Stress Responses to Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms

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