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Effect of temperature on Burkholderia pseudomallei growth, proteomic changes, motility and resistance to stress environments.

ABSTRACT: Burkholderia pseudomallei is a flagellated, gram-negative environmental bacterium that causes melioidosis, a severe infectious disease of humans and animals in tropical areas. We hypothesised that B. pseudomallei may undergo phenotypic adaptation in response to an increase in growth temperature. We analysed the growth curves of B. pseudomallei strain 153 cultured in Luria-Bertani broth at five different temperatures (25?°C-42?°C) and compared the proteomes of bacteria cultured at 37?°C and 42?°C. B. pseudomallei exhibited the highest growth rate at 37?°C with modest reductions at 30?°C, 40?°C and 42?°C but a more marked delay at 25?°C. Proteome analysis revealed 34 differentially expressed protein spots between bacterial cultures at 42?°C versus 37?°C. These were identified as chaperones (7 spots), metabolic enzymes (12 spots), antioxidants (10 spots), motility proteins (2 spots), structural proteins (2 spots) and hypothetical proteins (1 spot). Of the 22 down-regulated proteins at 42?°C, redundancy in motility and antioxidant proteins was observed. qRT-PCR confirmed decreased expression of fliC and katE. Experiments on three B. pseudomallei strains demonstrated that these had the highest motility, greatest resistance to H2O2 and greatest tolerance to salt stress at 37?°C. Our data suggest that temperature affects B. pseudomallei motility and resistance to stress.

SUBMITTER: Paksanont S

PROVIDER: S-EPMC6004011 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Effect of temperature on Burkholderia pseudomallei growth, proteomic changes, motility and resistance to stress environments.

Paksanont Suporn S Sintiprungrat Kitisak K Yimthin Thatcha T Pumirat Pornpan P Peacock Sharon J SJ Chantratita Narisara N

Scientific reports 20180615 1

Burkholderia pseudomallei is a flagellated, gram-negative environmental bacterium that causes melioidosis, a severe infectious disease of humans and animals in tropical areas. We hypothesised that B. pseudomallei may undergo phenotypic adaptation in response to an increase in growth temperature. We analysed the growth curves of B. pseudomallei strain 153 cultured in Luria-Bertani broth at five different temperatures (25 °C-42 °C) and compared the proteomes of bacteria cultured at 37 °C and 42 °C ...[more]

PMID: 29907803

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Project description:Burkholderia pseudomallei is the etiological agent of melioidosis. Because of the bacterium's intrinsic resistance and propensity to establish latent infections, melioidosis therapy is complicated and prolonged. Newer generation ?-lactams, specifically ceftazidime, are used for acute phase therapy, but resistance to this cephalosporin has been observed. The chromosomally encoded penA gene encodes a putative twin arginine translocase (TAT)-secreted ?-lactamase, and penA mutations have been implicated in ceftazidime resistance in clinical isolates. However, the role of PenA in resistance has not yet been systematically studied in isogenetic B. pseudomallei mutant backgrounds. We investigated the effects of penA deletion, point mutations, and up-regulation, as well as tat operon deletion and PenA TAT-signal sequence mutations. These experiments were made possible by employing a B. pseudomallei strain that is excluded from Select Agent regulations. Deletion of penA significantly (>4-fold) reduced the susceptibility to six of the nine ?-lactams tested and ?16-fold for ampicillin, amoxicillin, and carbenicillin. Overexpression of penA by single-copy, chromosomal expression of the gene under control of the inducible P(tac) promoter, increased resistance levels for all ?-lactams tested 2- to 10-fold. Recreation of the C69Y and P167S PenA amino acid substitutions previously observed in resistant clinical isolates increased resistance to ceftazidime by ?85- and 5- to 8-fold, respectively. Similarly, a S72F substitution resulted in a 4-fold increase in resistance to amoxicillin and clavulanic acid. Susceptibility assays with PenA TAT-signal sequence and ?tatABC mutants, as well as Western blot analysis, confirmed that PenA is a TAT secreted enzyme and not periplasmic but associated with the spheroplastic cell fraction. Lastly, we determined that two LysR-family regulators encoded by genes adjacent to penA do not play a role in transcriptional regulation of penA expression.

Project description:BackgroundBurkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins.Principal findingsMicrobiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A) enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F) inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM) suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe), increasing the 'greasiness' of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem). In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive.Conclusion/significanceOur results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue Tyr119 affects the permeability of the BpsOmp38 channel to neutral sugars and antimicrobial agents.

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Effect of temperature on Burkholderia pseudomallei growth, proteomic changes, motility and resistance to stress environments.

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Effect of temperature on Burkholderia pseudomallei growth, proteomic changes, motility and resistance to stress environments.

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