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Proteome profiling of Pseudomonas aeruginosa PAO1 identifies novel responders to copper stress.

ABSTRACT: BACKGROUND:The opportunistic pathogen, Pseudomonas aeruginosa is well known for its environmental and metabolic versatility, yet many of the functions of its gene-products remain to be fully elucidated. This study's objective was to illuminate the potential functions of under-described gene-products during the medically relevant copper-stress condition. RESULTS:We used data-independent acquisition mass spectrometry to quantitate protein expression changes associated with copper stress in P. aeruginosa PAO1. Approximately 2000 non-redundant proteins were quantified, with 78 proteins altering in abundance by +/- 1.5-fold or more when cultured to mid-log growth in the presence of 50 μM copper sulfate. One-third of those differentially expressed proteins have no prior established functional roles. CONCLUSIONS:This study provides evidence for the functional involvement of some specific proteins in enabling P. aeruginosa to survive under sub-lethal concentrations of copper. This further paves the way for targeted investigations into the specific mechanisms of their activity.

SUBMITTER: Wright BW

PROVIDER: S-EPMC6444534 | biostudies-other | 2019 Apr

REPOSITORIES: biostudies-other

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Proteome profiling of Pseudomonas aeruginosa PAO1 identifies novel responders to copper stress.

Wright Bradley W BW Kamath Karthik S KS Krisp Christoph C Molloy Mark P MP

BMC microbiology 20190401 1

<h4>Background</h4>The opportunistic pathogen, Pseudomonas aeruginosa is well known for its environmental and metabolic versatility, yet many of the functions of its gene-products remain to be fully elucidated. This study's objective was to illuminate the potential functions of under-described gene-products during the medically relevant copper-stress condition.<h4>Results</h4>We used data-independent acquisition mass spectrometry to quantitate protein expression changes associated with copper st ...[more]

PMID: 30935370

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Project description:Purpose: The intensive use of metal-based nanoparticles results in their continuous release into the environment and the subsequent destroying microbial biodiversity and causing occurrence of antibiotic resistant determinants. Although previous studies have indicated that nanoparticles may be toxic to microorganisms, there is a scarcity of data available to assess the underlying molecular mechanisms of inhibitory and biocidal effects of nanoparticles on microorganisms, which is a critical gap in our comprehensive understanding of the impacts of nanoparticles on microbial ecosystems. Methods: By combining the global activated/suppressed gene profiles and detected physiological responses, we detected the microbial response to CuO NPs exposure and revealed potential mechanisms of CuO NPs on this prevalent opportunistic pathogen and environmentally relevant denitrifier. Results:Our results indicate that nanoparticles could react directly with the biological membrane, but also change gene expressions. Transmission electron microscopy (TEM) results indicate that CuO nanoparticles not only damaged the structure of the bacterial membrane but also entered the cells. Planktonic cells of P. aeruginosa are energetically compromised after exposure to a sublethal level (1 and 10 mg/L) of CuO nanoparticles. Respiration was likely inhibited as denitrification activity was severely depleted in terms of decreased transcript levels of most denitrification genes. In addition, and of high concern, bacteriophage genes were activated and it is speculated that phage mediated cell lysis. Meanwhile, CuO NP exposure induced significantly up-regulated expressions of metal resistance gene, resistance-nodulation-division, P-type ATPase efflux and cation diffusion facilitator transporters, which can form an integrated network controlling metals concentrations in the cytoplasm and periplasm P. aeruginosa. Conclusion: Based on our results, it is evident that the response of bacteria under the exposure of nanoparticles is very complex, though a clear and comprehensive understanding of the true mechanisms of inhibition or toxicity is still lacking. Our findings will provide insights on whole picture regarding the fundamental mechanisms of microbial susceptibility, tolerance and resistance to exposure of CuO NPs, as well highlight to re-estimate potential risks of CuO NP to public health and environment.

Project description:Pseudomonas aeruginosa PAO1 produces three polysaccharides, alginate, Psl, and Pel that play distinct roles in attachment and biofilm formation for monospecies biofilms. Considerably less is known about their role in the development of mixed species biofilm communities. This study has investigated the roles of alginate, Psl, and Pel during biofilm formation of P. aeruginosa in a defined and experimentally informative mixed species biofilm community, consisting of P. aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Loss of the Psl polysaccharide had the biggest impact on the integration of P. aeruginosa in the mixed species biofilms, where the percent composition of the psl mutant was significantly lower (0.06%) than its wild-type (WT) parent (2.44%). In contrast, loss of the Pel polysaccharide had no impact on mixed species biofilm development. Loss of alginate or its overproduction resulted in P. aeruginosa representing 8.4 and 18.11%, respectively, of the mixed species biofilm. Dual species biofilms of P. aeruginosa and K. pneumoniae were not affected by loss of alginate, Pel, or Psl, while the mucoid P. aeruginosa strain achieved a greater biomass than its parent strain. When P. aeruginosa was grown with P. protegens, loss of the Pel or alginate polysaccharides resulted in biofilms that were not significantly different from biofilms formed by the WT PAO1. In contrast, overproduction of alginate resulted in biofilms that were comprised of 35-40% of P. aeruginosa, which was significantly higher than the WT (5-20%). Loss of the Psl polysaccharide significantly reduced the percentage composition of P. aeruginosa in dual species biofilms with P. protegens (<1%). Loss of the Psl polysaccharide significantly disrupted the communal stress resistance of the three species biofilms. Thus, the polysaccharide composition of an individual species significantly impacts mixed species biofilm development and the emergent properties of such communities.

Project description:The P. aeruginosa reference strain PAO1 has been used to delineate much of the physiology, metabolism, and fundamental biology of the species. The wild-type parent of PAO1 was lost, and PAO1 carries a regulatory mutation introduced for positive genetic selection that affects antibiotic resistance, virulence, quorum sensing, and other traits. The mutation is a loss-of-function change in an oxidoreductase gene (mexS), which constitutively activates a stress response controlled by a positive regulator (MexT). Fitness defects associated with the constitutive response have led to the inadvertent selection of mexT-minus suppressor mutations, creating genetic heterogeneity in PAO1 sublines studied in different laboratories. To help circumvent complications due to the mexS-minus phenotypes, we created a wild-type version of PAO1 (called LPAO) by "reverting" its mexS to the functional allele likely to have been in its parent. Phenotypic analysis revealed that the mexS-minus allele in PAO1 makes growth sensitive to salt (NaCl) and is lethal when combined with mutations inactivating the major sodium antiporter (ShaABCDEF). The salt sensitivity of PAO1 may underlie some complex mexS-minus phenotypes and help explain the selection of mexT-minus suppressor mutations. To facilitate genetic comparisons of PAO1, LPAO, and other P. aeruginosa strains, we developed a transformation procedure to transfer selectable alleles, such as transposon insertion alleles, between strains. Overall, the study helps explain phenotypic heterogeneity of PAO1-derived strains and provides resources to help recognize and eliminate difficulties due to it. IMPORTANCE The P. aeruginosa reference strain PAO1 carries a regulatory mutation that may affect processes characterized in it. To eliminate complications due to the mutation, we constructed a version of the missing wild-type parent strain and developed methods to transfer mutations between PAO1 and the new strain. The methods are likely to be applicable to other isolates of P. aeruginosa as well.

Dataset Information

Proteome profiling of Pseudomonas aeruginosa PAO1 identifies novel responders to copper stress.

Publications

Proteome profiling of Pseudomonas aeruginosa PAO1 identifies novel responders to copper stress.

Similar Datasets

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