Pentachlorophenol Induction of the Pseudomonas aeruginosa mexAB-oprM Efflux Operon: Involvement of Repressors NalC and MexR and the Antirepressor ArmR

Pentachlorophenol (PCP) induced expression of the NalC repressor-regulated PA3720-armR operon and the MexR repressor-controlled mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa. PCP's induction of PA3720-armR resulted from its direct modulation of NalC, the repressor's binding to PA3720-armR promoter-containing DNA as seen in electromobility shift assays (EMSAs) being obviated in the presence of this agent. The NalC binding site was localized to an inverted repeat (IR) sequence upstream of PA3720-armR and overlapping a promoter region whose transcription start site was mapped. While modulation of MexR by the ArmR anti-repressor explains the upregulation of mexAB-oprM in nalC mutants hyperexpressing PA3720-armR, the induction of mexAB-oprM expression by PCP is not wholly explainable by PCP induction of PA3720-armR and subsequent ArmR modulation of MexR, inasmuch as armR deletion mutants still showed PCP-inducible mexAB-oprM expression. PCP failed, however, to induce mexAB-oprM in a mexR deletion strain, indicating that MexR was required for this, although PCP did not modulate MexR binding to mexAB-oprM promoter-containing DNA in vitro. One possibility is that MexR responds to PCP-generated in vivo effector molecules in controlling mexAB-oprM expression in response to PCP. PCP is an unlikely effector and substrate for NalC and MexAB-OprM - its impact on NalC binding to the PA3720-armR promoter DNA occurred only at high µM levels - suggesting that it mimics an intended phenolic effector/substrate(s). In this regard, plants are an abundant source of phenolic antimicrobial compounds and, so, MexAB-OprM may function to protect P. aeruginosa from plant antimicrobials that it encounters in nature

Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design. © 2013 Nascimento et al

Aminoglycoside-inducible expression of the mexAB-oprM multidrug efflux operon in Pseudomonas aeruginosa: Involvement of the envelope stress-responsive AmgRS two-component system.

Exposure of P. aeruginosa to the aminoglycoside (AG) paromomycin (PAR) induced expression of the PA3720-armR locus and the mexAB-oprM multidrug efflux operon that AmgR controls, although PAR induction of mexAB-oprM was independent of armR. Multiple AGs promoted mexAB-oprM expression and this was lost in the absence of the amgRS locus encoding an aminoglycoside-activated envelope stress-responsive 2-component system (TCS). Purified AmgR bound to the mexAB-oprM promoter region consistent with this response regulator directly regulating expression of the efflux operon. The thiol-active reagent, diamide, which, like AGs, promotes protein aggregation and cytoplasmic membrane damage also promoted AmgRS-dependent mexAB-oprM expression, a clear indication that the MexAB-OprM efflux system is recruited in response to membrane perturbation and/or circumstances that lead to this. Despite the AG and diamide induction of mexAB-oprM, however, MexAB-OprM does not appear to contribute to resistance to these agents

Metagenome meta-analysis reveals an increase in the abundance of some multidrug efflux pumps and mobile genetic elements in chemically polluted environments

International audienceABSTRACT Many human activities contaminate terrestrial and aquatic environments with numerous chemical pollutants that not only directly alter the environment but also affect microbial communities in ways that are potentially concerning to human health, such as selecting for the spread of antibiotic-resistance genes (ARGs) through horizontal gene transfer. In the present study, metagenomes available in the public domain from polluted (with antibiotics, with petroleum, with metal mining, or with coal-mining effluents) and unpolluted terrestrial and aquatic environments were compared to examine whether pollution has influenced the abundance and composition of ARGs and mobile elements, with specific focus on IS26 and class 1 integrons ( intI 1). When aggregated together, polluted environments had a greater relative abundance of ARGs than unpolluted environments and a greater relative abundance of IS26 and intI 1. In general, chemical pollution, notably with petroleum, was associated with an increase in the prevalence of ARGs linked to multidrug efflux pumps. Included in the suite of efflux pumps were mexK , mexB , mexF , and mexW that are polyspecific and whose substrate ranges include multiple classes of critically important antibiotics. Also, in some instances, β-lactam resistance (TEM181 and OXA-541) genes increased, and genes associated with rifampicin resistance (RNA polymerases subunits rpoB and rpoB2 ) decreased in relative abundance. This meta-analysis suggests that different types of chemical pollution can enrich populations that carry efflux pump systems associated with resistance to multiple classes of medically critical antibiotics. IMPORTANCE The United Nations has identified chemical pollution as being one of the three greatest threats to environmental health, through which the evolution of antimicrobial resistance, a seminally important public health challenge, may be favored. While this is a very plausible outcome of continued chemical pollution, there is little evidence or research evaluating this risk. The objective of the present study was to examine existing metagenomes from chemically polluted environments and evaluate whether there is evidence that pollution increases the relative abundance of genes and mobile genetic elements that are associated with antibiotic resistance. The key finding is that for some types of pollution, particularly in environments exposed to petroleum, efflux pumps are enriched, and these efflux pumps can confer resistance to multiple classes of medically important antibiotics that are typically associated with Pseudomonas spp. or other Gram-negative bacteria. This finding makes clear the need for more investigation on the impact of chemical pollution on the environmental reservoir of ARGs and their association with mobile genetic elements that can contribute to horizontal gene transfer events

Lack of impact of PCP on MexR repressor binding to the <i>mexR-mexA</i> intergenic region.

<p>Mobility shift assay in which purified MexR-His (500 ng) was incubated with 40 ng of a 351-bp DNA fragment encompassing the <i>mexR-mexA</i> intergenic region and increasing amounts of PCP as indicated. Lane 1, DNA only control.</p

Bacterial strains and plasmids.

<p>Ap^r, ampicillin resistant; Km^r, kanamycin resistant; Tc^r, tetracycline resistant.</p

ArmR-/PA3720-independence of PCP induction of <i>mexAB-oprM</i>.

<p>Expression of <i>mexA</i> (as a measure of <i>mexAB-oprM</i>) was assessed in <i>P. aeruginosa</i> strains K767 (wild type; WT), K3145 (Δ<i>armR</i>), K3146 (ΔPA3720) and K3130 (ΔPA3720-<i>armR</i>) exposed or not to PCP (0.75 mM; 1.5 hr) using qRT-PCR. Expression was normalized to <i>rpsL</i> controls and is reported relative (fold change) to untreated <i>P. aeruginosa</i> K767. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032684#s3" target="_blank">Results</a> shown are the mean +/− standard error of one cDNA sample for each, processed in triplicate, and are representative of 2 independent experiments.</p

PCP induction of PA3720-<i>armR</i>.

<p>qRT-PCR results showing impact of PCP and/or a <i>nalC</i> mutation on PA3720 expression (as a measure of PA3720-<i>armR</i> expression). Expression of PA3720 is reported relative to the <i>rpsL</i> internal control for wild type <i>P. aeruginosa</i> K767 and its <i>nalC</i> derivative, K1454 exposed or not to PCP (0.75 mM; 1.5 hr). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032684#s3" target="_blank">Results</a> shown are the mean +/− standard error of one cDNA sample for each, processed in triplicate, and are representative of 2 independent experiments.</p

Mapping the PA3720-<i>armR</i> transcriptional start site.

<p>The <i>nalC</i>/PA3720-<i>armR</i> intergenic region highlighting the RACE-determined transcription start site for PA3720-<i>armR</i> (bolded and italicized), the predicted transcriptional initiation site (marked with an asterisk; assessed using neural network promoter prediction software provided by M. G. Reese at <a href="http://www.fruitfly.org/seq_tools/promoter.html" target="_blank">http://www.fruitfly.org/seq_tools/promoter.html</a>), the <i>nalC</i> and PA3720 start codons (arrows), and putative −10/−35 sites for <i>nalC</i> (overlined) and PA3720-<i>armR</i> (underlined) promoters. The end-points of PCR-generated PA3720-distal (I) and -proximal (II) fragments and oligonucleotides (III and IV) used in EMSAs with purified NalC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032684#pone-0032684-g002" target="_blank">Fig. 2B</a>) are identified by arrowheads above the sequence. The shaded sequence corresponds to an inverted repeat and possible NalC-binding site.</p

PCP modulation of NalC repressor binding to the PA3720-<i>armR</i> upstream region.

<p>A) Mobility shift assay in which 50 ng of a 209-bp DNA fragment encompassing the <i>nalC</i>-PA3720 intergenic region was incubated without (lane 1) or with 200 (lane 2), 300 (lane 3), 400 (lane 4), 500 (lane 5), 800 (lane 6), 1000 (lane 7), 2000 (lane 8) or 3000 (lane 9) ng of purified NalC-His. B) Mobility shift assay in which purified NalC-His (800 ng) was incubated with 50 ng of a 209-bp DNA fragment encompassing the <i>nalC</i>-PA3720 intergenic region and increasing amounts of PCP as indicated. Lane 1, DNA only control.</p