Frances Smith

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix ofPseudomonas aeruginosabiofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening aP.aeruginosatransposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin.P.aeruginosa tatmutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes inpqsAexpression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to thetatmutants did not returnpqsA,rhlAorphzA1expression or pyocyanin production to wild type levels. However, transformation of thetatmutants with the AQ-independentpqseffectorpqsErestoredphzA1expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. ApqsA::luxfusion was introduced into each of 34 validatedP.aeruginosaTat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochromebc₁complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production,rhlAexpression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion ofcytBorcytC₁. Thus, either lack of the Rieske sub-unit or mutation of cytochromebc₁genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA , rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc ₁ complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms. Author Summary: Pseudomonas aeruginosa is a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections. Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate. The biofilm extracellular matrix incorporates extracellular DNA (eDNA). This stabilizes biofilm architecture and helps confer tolerance to antibiotics. Since mechanisms that control eDNA release are not well understood, we screened a P. aeruginosa mutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane. Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling. This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles. Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability of tat mutants to translocate the Rieske subunit of the cytochrome bc ₁ complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa , our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.

Pachyonychia congenita (PC) is a rare autosomal dominant skin disorder characterized predominantly by hypertrophic nail dystrophy, oral leukokeratosis, and painful palmoplantar keratoderma. It is associated with a mutation in one of five keratin genes, KRT6A, KRT6B, KRT6C, KRT16, or KRT17. The International PC Research Registry (IPCRR) confirms that as of January 2014 there have been 547 cases of PC genetically confirmed. It is estimated that there are between 2000 and 10,000 cases of PC in the world. However, the exact prevalence of PC is not yet established. We report a case of PC-K6a, p.Arg164Pro, in a 40-year-old man. Initially he was diagnosed with onychomycosis and was treated with systemic antifungals. This is the first genetically confirmed case of PC in the Czech Republic.

Keratin 7 is expressed in simple epithelia but is expressed at low or undetectable levels in gastrointestinal epithelial cells. In the pancreas, it is present in ductal but not in acinar cells. K7 mRNA is overexpressed in pancreatic cancers. Here we use luciferase reporter assays to analyze the tissue‐specific regulatory elements of murine keratin 7 ( Krt7 ) promoter in vitro and in vivo . All elements required for appropriate cell and tissue specificity in reporter assays are present within the Krt7 —234 bp sequence. This fragment appears more selective to pancreatic ductal cells than the Krt19 promoter. GC‐rich sequences corresponding to putative Sp1,AP‐2 binding sites are essential for in vitro activity. Krt7‐LacZ transgenic mice were generated to analyze in vivo activity. Sequences located 1.5 or 0.25 kb upstream of the transcription initiation site drive reporter expression to ductal, but not acinar, cells in transgenic mice. LacZ mRNA was detected in the pancreas as well as in additional epithelial tissues— such as the intestine and the lung—using both promoter constructs. An AdK7Luc adenovirus was generated to assess targeting selectivity in vivo by intravenous injection to immunocompetent mice and in a xenograft model of pancreatic cancer. The —0.25 kb region showed pancreatic selectivity, high activity in pancreatic cancers, and sustained transgene expression in xenografts. In conclusion, the krt7 promoter is useful to target pancreatic ductal adenocarcinoma cells in vitro and in vivo .— Pujal, J., Huch, M., Jose, A., Abasolo, I., Rodolosse, A., Duch, A., Sanchez‐Palazon, L., Smith, F. J. D., McLean, W. H. I., Fillat, C., Real, F. X. Keratin 7 promoter selectively targets transgene expression to normal and neoplastic pancreatic ductal cells in vitro and in vivo . FASEBJ. 23, 1366–1375 (2009)