Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS) Promotes Biofilm Formation of Pseudomonas aeruginosa by Increasing Iron Availability.

The 2-alkyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase HodC was previously described to cleave the Pseudomonas quinolone signal, PQS, which is exclusively used in the complex quorum sensing (QS) system of Pseudomonas aeruginosa, an opportunistic pathogen employing QS to regulate virulence and biofilm development. Degradation of PQS by exogenous addition of HodC to planktonic cells of P. aeruginosa attenuated production of virulence factors, and reduced virulence in planta. However, proteolytic cleavage reduced the efficacy of HodC. Here, we identified the secreted protease LasB of P. aeruginosa to be responsible for HodC degradation. In static biofilms of the P. aeruginosa PA14 lasB::Tn mutant, the catalytic activity of HodC led to an increase in viable biomass in newly formed but also in established biofilms, and reduced the expression of genes involved in iron metabolism and siderophore production, such as pvdS, pvdL, pvdA, and pvdQ. This is likely due to an increase in the levels of bioavailable iron by degradation of PQS, which is able to sequester iron from the surrounding environment. Thus, HodC, despite its ability to quench the production of virulence factors, is contraindicated for combating P. aeruginosa biofilms.

Knockout of extracytoplasmic function sigma factor ECF-10 affects stress resistance and biofilm formation in Pseudomonas putida KT2440.

Pseudomonas putida is a Gram-negative soil bacterium which is well-known for its versatile lifestyle, controlled by a large repertoire of transcriptional regulators. Besides one- and two-component regulatory systems, the genome of P. putida reveals 19 extracytoplasmic function (ECF) sigma factors involved in the adaptation to changing environmental conditions. In this study, we demonstrate that knockout of extracytoplasmic function sigma factor ECF-10, encoded by open reading frame PP4553, resulted in 2- to 4-fold increased antibiotic resistance to quinolone, ß-lactam, sulfonamide, and chloramphenicol antibiotics. In addition, the ECF-10 mutant exhibited enhanced formation of biofilms after 24 h of incubation. Transcriptome analysis using Illumina sequencing technology resulted in the detection of 12 genes differentially expressed (>2-fold) in the ECF-10 knockout mutant strain compared to their levels of expression in wild-type cells. Among the upregulated genes were ttgA, ttgB, and ttgC, which code for the major multidrug efflux pump TtgABC in P. putida KT2440. Investigation of an ECF-10 and ttgA double-knockout strain and a ttgABC-overexpressing strain demonstrated the involvement of efflux pump TtgABC in the stress resistance and biofilm formation phenotypes of the ECF-10 mutant strain, indicating a new role for this efflux pump beyond simple antibiotic resistance in P. putida KT2440.

Fabrication of highly uniform gel coatings by the conversion of surface-anchored metal-organic frameworks.

We report the fabrication of 3D, highly porous, covalently bound polymer films of homogeneous thickness. These surface-bound gels combine the advantages of metal-organic framework (MOF) materials, namely, the enormous flexibility and the large size of the maximum pore structures and, in particular, the possibility to grow them epitaxially on modified substrates, with those of covalently connected gel materials, namely, the absence of metal ions in the deposited material, a robust framework consisting of covalent bonds, and, most importantly, pronounced stability under biological conditions. The conversion of a SURMOF (surface-mounted MOF) yields a surface-grafted gel. These SURGELs can be loaded with bioactive compounds and applied as bioactive coatings and provide a drug-release platform in in vitro cell culture studies.

TypA is involved in virulence, antimicrobial resistance and biofilm formation in Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is an important opportunistic human pathogen and is extremely difficult to treat due to its high intrinsic and adaptive antibiotic resistance, ability to form biofilms in chronic infections and broad arsenal of virulence factors, which are finely regulated. TypA is a GTPase that has recently been identified to modulate virulence in enteric Gram-negative pathogens. RESULTS: Here, we demonstrate that mutation of typA in P. aeruginosa resulted in reduced virulence in phagocytic amoebae and human macrophage models of infection. In addition, the typA mutant was attenuated in rapid cell attachment to surfaces and biofilm formation, and exhibited reduced antibiotic resistance to ß-lactam, tetracycline and antimicrobial peptide antibiotics. Quantitative RT-PCR revealed the down-regulation, in a typA mutant, of important virulence-related genes such as those involved in regulation and assembly of the Type III secretion system, consistent with the observed phenotypes and role in virulence of P. aeruginosa. CONCLUSIONS: These data suggest that TypA is a newly identified modulator of pathogenesis in P. aeruginosa and is involved in multiple virulence-related characteristics.