Pseudomonas aeruginosa gshA Mutant Is Defective in Biofilm Formation, Swarming, and Pyocyanin Production

Abstract

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that can cause severe opportunistic infections. The principal redox buffer employed by this organism is glutathione (GSH). To assess the role of GSH in the virulence of P. aeruginosa, a number of analyses were performed using a mutant strain deficient in gshA, which does not produce GSH. The mutant strain exhibited a growth delay in minimal medium compared to the wild-type strain. Furthermore, the gshA mutant was defective in biofilm and persister cell formation and in swimming and swarming motility and produced reduced levels of pyocyanin, a key virulence factor. Finally, the gshA mutant strain demonstrated increased sensitivity to methyl viologen (a redox cycling agent) as well as the thiol-reactive antibiotics fosfomycin and rifampin. Taken together, these data suggest a key role for GSH in the virulence of P. aeruginosaIMPORTANCEPseudomonas aeruginosa is a ubiquitous bacterium that can cause severe opportunistic infections, including many hospital-acquired infections. It is also a major cause of infections in patients with cystic fibrosis. P. aeruginosa is intrinsically resistant to a number of drugs and is capable of forming biofilms that are difficult to eradicate with antibiotics. The number of drug-resistant strains is also increasing, making treatment of P. aeruginosa infections very difficult. Thus, there is an urgent need to understand how P. aeruginosa causes disease in order to find novel ways to treat infections. We show that the principal redox buffer, glutathione (GSH), is involved in intrinsic resistance to the fosfomycin and rifampin antibiotics. We further demonstrate that GSH plays a role in P. aeruginosa disease and infection, since a mutant lacking GSH has less biofilm formation, is less able to swarm, and produces less pyocyanin, a pigment associated with infection.

Keywords: Pseudomonas aeruginosa; biofilms; glutathione; pyocyanin; thiols; virulence.

FIG 1

The gshA transposon mutants, strains PW4072 and PW9759, produce little to no detected glutathione. Glutathione (A) and cysteine (B) levels were quantified by HPLC. (A) The levels of glutathione were significantly decreased in the two mutant strains compared to the level in the wild-type strain (MPAO1). Additionally, there was a significant increase in glutathione production in the cis-complemented strain, BH01. (B) There was no significant difference in cysteine production among the strains tested. Values are means plus standard deviations (error bars) from four independent experiments. Values that are significantly different (P < 0.001) are indicated by three asterisks.

FIG 2

The gshA transposon mutant (PW4072) has a decreased growth rate compared to the wild-type (MPAO1) and complemented (TJB10) strains. Overnight cultures of each strain were grown in tryptic soy broth (TSB) supplemented with the appropriate antibiotics (tetracycline [60 µg/ml] and chloramphenicol [10 µg/ml] for strain PW4072; spectinomycin [200 µg/ml] for strain TJB10). A 250-µl portion of the overnight culture was used to inoculate 5 ml of fresh M9 minimal medium. Once the culture was grown to an OD₆₀₀ of 0.5, each culture was diluted to an OD₆₀₀ of 0.05 and inoculated into the wells of a 96-well plate. The cultures were grown with shaking at 37°C, and the OD₆₀₀ was measured every hour for 18 h. Each strain was tested in triplicate.

FIG 3

The gshA transposon mutant is defective for biofilm formation compared to the wild-type and complemented strains. Biofilms were grown in 96-well plates for 24 h in M9 medium (A) and TSB (B). Biofilms were rinsed three times with PBS, treated with 0.4% crystal violet for 15 min, rinsed again three times with PBS, and resuspended in 33% acetic acid. Biomass was read using a spectrophotometer at 550 nm. Values are means plus standard deviations (error bars) from four independent experiments. Values that are significantly different (P < 0.05) are indicated by an asterisk.

FIG 4

A lack of glutathione contributes to decreased swarming and swimming motility but has no effect on twitching motility in gshA mutant strains (PW4072 and PW9759). (A and B) Swarming and swimming motility was assessed for each strain by inoculating 3 µl of a 16-h overnight culture onto appropriate plates and incubating for 24 to 48 h at 30°C. (C) Twitching motility was assessed by stabbing an overnight culture through twitching agar and incubating for 24 to 48 h at 30°C. Data show decreased swarming and swimming motility of the mutant strains based on measured diameter. Values are means plus standard deviations (error bars) from four independent experiments. Values that are significantly different from the value for the wild-type MPAO1 strain are indicated by asterisks as follows: **, P < 0.01; ***, P < 0.001.

FIG 5

The gshA transposon mutants (PW4072 and PW9759) have different pigments than the wild-type strain (MPAO1).

FIG 6

The gshA transposon mutants (PW4072 and PW9759) are deficient in pyocyanin production. Spent medium supernatants of cultures of each strain grown overnight in TSB were extracted with chloroform and HCl, and absorbance readings were taken at 520 nm for pyocyanin quantification. There was significantly less pyocyanin production in the gshA transposon mutant strain compared to the wild-type (MPAO1), complemented (TJB10 and BH01), and control (PW5404) strains. Values are means plus standard deviations from five independent experiments. Values that are significantly different from the value for the wild-type MPAO1 strain are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01.

FIG 7

Glutathione disruption does not inhibit quorum sensing (QS) mediated by acyl homoserine lactones (AHLs). QS mediated by AHL was assayed using a cross-feeding assay. The P. aeruginosa strain of interest was streaked 0.75 cm from an E. coli reporter strain on MacConkey agar. The plates were incubated at 37°C for 24 h. In each image, the E. coli strain is on the left, while the P. aeruginosa strain is on the right. Strain PW3597 is defective for QS and serves as a negative control. All strains except PW3597 were positive for AHL-mediated QS as indicated by the pinkish color of the E. coli after 24 h of incubation.

FIG 8

Persister cell formation is defective in gshA transposon mutants (PW4072 and PW9759). Persister cells were formed by exposing one stationary-phase culture to 50 µg/ml ofloxacin for 3.5 h. The number of CFU of untreated cultures was compared to that of ofloxacin-treated cultures to calculate the percentage of persister cells formed. The two mutant strains (PW4072 and PW9759) produced few to no persister cells, while the cis-complemented strain (BH01) produced significantly more persister cells than the wild-type strain (MPAO1). Values are means plus standard deviations from four independent experiments. Values that are significantly different from the value for the wild-type MPAO1 strain are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01.

FIG 9

The gshA transposon mutant (PW4072) has increased sensitivity to methyl viologen. Strains were grown to exponential phase and plated on tryptic soy agar (TSA) alone or supplemented with 1.0 mM methyl viologen. The surviving percentage was calculated by dividing the number of CFU from plates containing TSA plus methyl viologen by the number of CFU from plates containing TSA only. Wild-type (MPAO1) and complemented (TJB10 and BH01) strains had between 10- and 14-fold increased survival on methyl viologen compared to the transposon mutant (PW4072). Values are means plus standard deviations from five independent experiments. Values that are significantly different (P < 0.05) from the value for the wild-type strain are indicated by an asterisk.