Identification of Genes Involved in Pseudomonas aeruginosa Biofilm-Specific Resistance to Antibiotics

Pseudomonas aeruginosa is a key opportunistic pathogen characterized by its biofilm formation ability and high-level multiple antibiotic resistance. By screening a library of random transposon insertion mutants with an increased biofilm-specifc antibiotic suscepti bility, we previously ident ified 3 genes or operons of P. aeruginosa UCBPP-PA14 ( ndvB , PA1875–1877 and tssC1 ) that do not affect biofilm formation but are involved in biofilm-specific antibiotic resistance. In this study, we demonstrate that PA0756–0757 (encoding a putative two-component regulatory system), PA2070 and PA5033 (encoding hypothetical proteins of unknown function) display increased expression in biofilm cells and also have a role in biofilm-specific antibiotic resistance. Furthermore, deletion of each of PA0756, PA2070 and PA5033 resulted in a significant reduction of lethality in Caenorhabditis elegans, indicating a role for these genes in both biofilm-specific antibiotic resistance and persistence in vivo. Together, these data suggest that these genes are potential targets for antimicrobial agents

A Farnesyltransferase Acts to Inhibit Ectopic Neurite Formation in C. elegans.

Genetic pathways that regulate nascent neurite formation play a critical role in neuronal morphogenesis. The core planar cell polarity components VANG-1/Van Gogh and PRKL-1/Prickle are involved in blocking inappropriate neurite formation in a subset of motor neurons in C. elegans. A genetic screen for mutants that display supernumerary neurites was performed to identify additional factors involved in this process. This screen identified mutations in fntb-1, the β subunit of farnesyltransferase. We show that fntb-1 is expressed in neurons and acts cell-autonomously to regulate neurite formation. Prickle proteins are known to be post-translationally modified by farnesylation at their C-terminal CAAX motifs. We show that PRKL-1 can be recruited to the plasma membrane in both a CAAX-dependent and CAAX-independent manner but that PRKL-1 can only inhibit neurite formation in a CAAX-dependent manner

Minimal bactericidal concentrations of antibiotics for PA14 deletion mutants tested with planktonic cells (MBC-P) and biofilm cells (MBC-B) (µg/ml)^a.

a<p>the values represent the mode of at least 6 biological replicates.</p

Identification of Genes Involved in <i>Pseudomonas aeruginosa</i> Biofilm-Specific Resistance to Antibiotics

<div><p><i>Pseudomonas aeruginosa</i> is a key opportunistic pathogen characterized by its biofilm formation ability and high-level multiple antibiotic resistance. By screening a library of random transposon insertion mutants with an increased biofilm-specifc antibiotic susceptibility, we previously identified 3 genes or operons of <i>P. aeruginosa</i> UCBPP-PA14 (<i>ndvB</i>, PA1875–1877 and <i>tssC1</i>) that do not affect biofilm formation but are involved in biofilm-specific antibiotic resistance. In this study, we demonstrate that PA0756–0757 (encoding a putative two-component regulatory system), PA2070 and PA5033 (encoding hypothetical proteins of unknown function) display increased expression in biofilm cells and also have a role in biofilm-specific antibiotic resistance. Furthermore, deletion of each of PA0756, PA2070 and PA5033 resulted in a significant reduction of lethality in <i>Caenorhabditis elegans</i>, indicating a role for these genes in both biofilm-specific antibiotic resistance and persistence <i>in vivo</i>. Together, these data suggest that these genes are potential targets for antimicrobial agents.</p></div

Antibiotic susceptibility of PA14 deletion mutants containing pJB866-based plasmids and assayed in LB.

<p>Antibiotic susceptibility of PA14 deletion mutants containing pJB866-based plasmids and assayed in LB.</p

<i>P. aeruginosa</i> strains and plasmids used in this study.

<p><i>P. aeruginosa</i> strains and plasmids used in this study.</p

qPCR analysis of the expression of PA0756, PA2070 and PA5033 in planktonic and biofilm cells.

<p>For each condition, three biological replicate samples were tested in triplicate qPCRs.</p

Primers used in this study.

*<p>Locations of restriction sites are underlined and in bold.</p

Inactivation of PA0756-0757, PA2070 or PA5033 does not affect biofilm formation.

<p>A. Air-liquid interface assay of biofilm formation of <i>P. aeruginosa</i> PA14 wild type and its deletion mutants, ΔPA0765-0757, ΔPA2070 and ΔPA5033. The images of biofilms at 200 X magnification were taken by phase-contrast microscopy after 24 h of growth at 37°C in M63-arginine medium. B. Quantification of biofilm formation by measurement of the biofilm-associated crystal violet (OD₅₅₀). The data shown are the mean ± standard deviation from two separate experiments (each with quadruplicate samples) using the microtitre plate biofilm assay and display no significant difference (<i>p</i>>0.05) among the four groups as determined by one-way ANOVA.</p

ΔPA0756-0757, ΔPA2070 and ΔPA5033 are attenuated in a <i>C. elegans</i> slow-killing model.

<p>Slow-killing conditions were used for each strain and death of <i>C. elegans</i> was measured every 24 h for a total of 72 h. Exposure to bacterial lawn represents when L4 or young adult hermaphrodite <i>C. elegans</i> were added to pathogenic plates. Values represent the results from at least three biological replicates. Error bars represent standard deviation, and lack of error bars means a standard deviation of zero.</p