Gene expression and physiological role of Pseudomonas aeruginosa methionine sulfoxide reductases during oxidative stress

Abstract

Pseudomonas aeruginosa PAO1 has two differentially expressed methionine sulfoxide reductase genes: msrA (PA5018) and msrB (PA2827). The msrA gene is expressed constitutively at a high level throughout all growth phases, whereas msrB expression is highly induced by oxidative stress, such as sodium hypochlorite (NaOCl) treatment. Inactivation of either msrA or msrB or both genes (msrA msrB mutant) rendered the mutants less resistant than the parental PAO1 strain to oxidants such as NaOCl and H2O2. Unexpectedly, msr mutants have disparate resistance patterns when exposed to paraquat, a superoxide generator. The msrA mutant had a higher paraquat resistance level than the msrB mutant, which had a lower paraquat resistance level than the PAO1 strain. The expression levels of msrA showed an inverse correlation with the paraquat resistance level, and this atypical paraquat resistance pattern was not observed with msrB. Virulence testing using a Drosophila melanogaster model revealed that the msrA, msrB, and, to a greater extent, msrA msrB double mutants had an attenuated virulence phenotype. The data indicate that msrA and msrB are essential genes for oxidative stress protection and bacterial virulence. The pattern of expression and mutant phenotypes of P. aeruginosa msrA and msrB differ from previously characterized msr genes from other bacteria. Thus, as highly conserved genes, the msrA and msrB have diverse expression patterns and physiological roles that depend on the environmental niche where the bacteria thrive.

Fig 1

Genetic organization and multiple amino acid sequence alignments of P. aeruginosa MsrA and MsrB. Genetic organization and alignments of MsrA (A) and MsrB (B) from P. aeruginosa with other characterized enzymes from various organisms were performed using the CLUSTALW program. The arrow direction indicates the transcription orientation. Dark gray boxes indicate the MsrA or MsrB signature motif. The asterisk, colon, and period symbols indicate identical residues, conserved substitutions, and semiconserved substitutions, respectively. Values at right are percent identities of the aligned proteins with the P. aeruginosa protein. gpx, glutathione peroxidase homolog (PA2826); and ospR, oxidative stress response and pigment production regulator.

Fig 2

Expression analysis of msrA and msrB during normal growth and in response to oxidants. (A) Autoradiograms of Northern blots of uninduced and 0.2% NaOCl-induced samples probed with either radioactively labeled msrA or msrB. Equal amounts of purified total RNA from uninduced (UN) and 0.2% NaOCl induced (IN) samples were loaded into each lane (10 μg for msrA and 50 μg for msrB). The number below each band represents the fold change in band intensity relative to the level of the uninduced culture, determined using densitometric analysis. (B) Real-time RT-PCR analysis of msrA and msrB expression. Total RNA was isolated from uninduced exponential-phase cultures. Real-time RT-PCR and subsequent analysis were performed on equal amounts of cDNA (10 ng) prepared from total RNA samples as described in Materials and Methods. Data shown are fold change of expression levels using msrB as the reference sample. (C) Real-time RT-PCR analysis of the expression levels of P. aeruginosa msrA and msrB in cultures induced with either 1 mM H₂O₂, 1 mM PQ, or 0.2% NaOCl. Oxidant induction, RNA isolation, real-time RT-PCR, and subsequent analyses were performed as described in Materials and Methods. Data presented are means ± standard deviations of three independent experiments. The asterisk indicates statistically significant difference (P < 0.01) compared with the uninduced condition.

Fig 3

Determination of H₂O₂ and NaOCl resistance levels. The resistance levels of P. aeruginosa strains against 100 mM H₂O₂ or 0.3% NaOCl were determined using a viability test performed as described in Materials and Methods. The percent survival was defined as the percentage of the CFU with treatment divided by the percentage of CFU without treatment. Data presented are means ± standard deviations of three independent experiments. The asterisk indicates a statistically significant difference (P < 0.01) compared with the PAO1 strain.

Fig 4

Determination of paraquat resistance levels. The resistance levels of P. aeruginosa strains against 0.175 mM paraquat were determined using a plate sensitivity assay as described in Materials and Methods and are expressed as percent survival. The percent survival was defined as the percentage of CFU on plates containing paraquat divided by the percentage of CFU on plates without paraquat. Data presented are means ± standard deviations of three independent experiments. The asterisk indicates a statistically significant difference (P < 0.01) compared with the PAO1 harboring pBBR1MCS-4 or the pUFR047 vector control.

Fig 5

Determination of paraquat resistance levels under aerobic and anaerobic conditions. A plate sensitivity assay was performed using LB medium with and without supplementation of 1% KNO₃ and incubated under aerobic and anaerobic conditions. The concentrations of paraquat were 0.175 mM for aerobic experiments and 0.5 mM for anaerobic experiments. The resistance levels of P. aeruginosa strains against paraquat were determined using a plate sensitivity assay and are expressed as percent survival as defined in the legend of Fig. 4. Data presented are means ± standard deviations of three independent experiments. The asterisk indicates a statistically significant difference (P < 0.01) compared with PAO1.

Fig 6

Virulence assay of P. aeruginosa strains. The virulence of P. aeruginosa strains was determined using the Drosophila melanogaster feeding method (30, 31). The flies that survived were counted after 15 h of incubation, and results are expressed as the percent viability. The percent viability was defined as the percentage of the surviving flies after 15 h of incubation with P. aeruginosa cell cultures. Data presented are means ± standard deviations of three independent experiments. The asterisk indicates a statistically significant difference (P < 0.01) compared with PAO1.