PmtA Regulates Pyocyanin Expression and Biofilm Formation in Pseudomonas aeruginosa

Abstract

The opportunistic pathogen Pseudomonas aeruginosa expresses a small molecular weight, cysteine-rich protein (PmtA), identified as a metallothionein (MT) protein family member. The MT family proteins have been well-characterized in eukaryotes as essential for zinc and copper homeostasis, protection against oxidative stress, and the ability to modify a variety of immune activities. Bacterial MTs share sequence homology, antioxidant chemistry, and heavy metal-binding capacity with eukaryotic MTs, however, the impact of bacterial MTs on virulence and infection have not been well-studied. In the present study, we investigated the role of PmtA in P. aeruginosa PAO1 using a PmtA-deficient strain (ΔpmtA). Here we demonstrated the virulence factor, pyocyanin, relies on the expression of PmtA. We showed that PmtA may be protective against oxidative stress, as an alternative antioxidant, glutathione, can rescue pyocyanin expression. Furthermore, the expression of phzM, which encodes a pyocyanin precursor enzyme, was decreased in the ΔpmtA mutant during early stationary phase. Upregulated pmtA expression was previously detected in confluent biofilms, which are essential for chronic infection, and we observed that the ΔpmtA mutant was disrupted for biofilm formation. As biofilms also modulate antibiotic susceptibility, we examined the ΔpmtA mutant susceptibility to antibiotics and found that the ΔpmtA mutant is more susceptible to cefepime and ciprofloxacin than the wild-type strain. Finally, we observed that the deletion of pmtA results in decreased virulence in a waxworm model. Taken together, our results support the conclusion that PmtA is necessary for the full virulence of P. aeruginosa and may represent a potential target for therapeutic intervention.

CRISPRi-Mediated Gene Suppression Reveals Putative Reverse Transcriptase Gene PA0715 to Be a Global Regulator of Pseudomonas aeruginosa

“…Flagella-mediated swimming, type IV pili-mediated rubbing, pyocyanin, and oxidative phosphorylation are known to be involved in biofilm formation. 33 , 50 Therefore, we assumed that biofilm formation by this strain was also reduced. To verify this hypothesis, we investigated the role of PA0715 in biofilm formation.…”

“…Biofilm formation capacity was assessed using a previously described method with slight modification. 32 , 33 Briefly, overnight cultures of PA01 were diluted 1:100 with LB broth and grown for an additional 3 h. Ten microliters of PAO1 bacterial suspension (adjusted to an OD 600 of 0.6) and 190 μL LB medium were transferred to 96-well flat-bottom microplates to make a final volume of 200 μL, and incubated at 37 °C for 72 h. Following incubation, the culture medium was removed. To exclude planktonic cells that might interfere with the interpretation of results, the bottom of the well plate was washed thrice with sterile 1× PBS buffer.…”

EF-hand protein, EfhP, specifically binds Ca2+ and mediates Ca2+ regulation of virulence in a human pathogen Pseudomonas aeruginosa

“…Pyocyanin is a redox-active pigment that imposes oxidative stress on airway epithelial cells and mediates tissue damage and necrosis during lung infections 74 . Pyocyanin was also shown to cause death in G. mellonella infection model 75 , 76 . We have previously reported that the deletion of efhP completely abolished pyocyanin production in biofilm cells of FRD1, the alginate-producing strain of P. aeruginosa, grown at elevated Ca 2+ 17 .…”

Assessment of Phage-Mediated Inhibition and Removal of Multidrug-Resistant Pseudomonas aeruginosa Biofilm on Medical Implants

“… 32 Besides drug resistance, PA biofilm formation has been reported to be associated in particular with pyocyanin (blue-green lipid-soluble pigment) expression. 33 , 34 On the contrary, no correlation between biofilm formation and drug resistance–or other virulence factors–has been reported, 34 suggestive of PA as a notorious biofilm producer and its continuous involvement in persistent chronic infections. Variation in the source of the isolates highlights the enormous burden of PA in the clinical settings, particularly in device-associated infections, which must be dealt with.…”