Phenazine production enhances extracellular DNA release via hydrogen peroxide generation in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa eDNA is a crucial component essential for biofilm formation and stability. In this study we report that release of eDNA is influenced by the production of phenazine in P. aeruginosa. A ∆phzA-G mutant of P. aeruginosa PA14 deficient in phenazine production generated significantly less eDNA in comparison with the phenazine producing strains. The relationship between eDNA release and phenazine production is bridged via hydrogen peroxide (H2O2) generation and subsequent H2O2 mediated cell lysis and ultimately release of chromosomal DNA into the extracellular environment as eDNA.

Introduction

Pseudomonas aeruginosa is an opportunistic human pathogenic bacterium well known for causing chronic lung infections in cystic fibrosis patients. Similar to many other bacterial species, biofilm formation of P. aeruginosa is facilitated by self-produced extracellular polymeric substances primarily composed of polysaccharides, proteins, lipids and extracellular DNA (eDNA)., eDNA is crucial in various bacterial species for adhesion, cell-to-cell interaction/aggregation, biofilm formation and stability and protection of biofilms against antibiotics and detergents.- In Gram positive bacteria release of eDNA involves lysis of a small population of bacterial cells, mediated through various autolysin proteins such as AtlE in S. epidermidis and gelatinase and serine protease by Enterococcus faecalis. Additionally phage and hydrogen peroxide (H2O2) mediated eDNA release at the cost of cell lysis have also been reported in diverse Streptococcus species.,

In P. aeruginosa, eDNA release is mediated through quorum sensing (QS) molecules such as N-acyl-L-homoserine lactones and the Pseudomonas quinolone signal by inducing phage production. eDNA release via non-QS pathways has also been documented in P. aeruginosa biofilms involving flagella and type IV pili mediated cell lysis., Recently Das and Manefield exposed that eDNA release in P. aeruginosa also happens through production of the phenazine pyocyanin. Phenazine production is mediated via QS controlled expression of phzA-G operons resulting in production of the primary phenazine phenazine-1-carboxylic acid (PCA). PCA is then modified to produce a variety of secondary phenazine molecules such as pyocyanin (PYO) through action of the phzM gene, phenazine-1-carboxamide (PCN), encoded by phzH and 1-hydroxy phenazine (1-OHPHZ) encoded by phzS as shown in Figure 1., In relation to our recently published paper, where we showed pyocyanin enhances eDNA release, in this addendum we demonstrated that like pyocyanin other phenazine molecules also promote eDNA production in P. aeruginosa.

Figure 1. Schematic represents quorum sensing controlled expression of genes for phenazine production. P. aeruginosa synthesizes acylated homoserine lactones (AHLs) and Pseudomonas quinone signaling (PQS) as their primary and secondary quorum sensing signaling molecules. PQS regulates the synthesis of phenazine-1-carboxylic acid (PCA) through a set of primary phenazine producing genes phzA1-G1 and phzA2-G2. PCA then converts into various kinds of specific phenazine encoded by specific genes.

Materials and Methods

Bacterial species, culture conditions and quantification of eDNA

All P. aeruginosa strains used in this study are listed in Table 1. Strains were plated onto LB agar plates and incubated overnight under aerobic conditions at 37°C. Single colonies from the agar plates were used to inoculate 20 ml cultures in LB medium (ph 7) for 0, 1, 3 and 5 d at 30°C, 150 rpm. After growth, the P. aeruginosa strains were harvested and pelleted out by centrifugation at 6,500 rpm (4,912 × g) for 5 min at 10°C. After centrifugation, supernatants were separated from bacterial pellets. In order to remove remaining bacteria, supernatants were again filtered using 0.22 µm Millipore filter units (Millipore). To further ensure the filtered supernatants were free of bacterial cells, 50 µl of filtered supernatant was used to inoculate LB agar plates and incubated aerobically for 48 h at 37°C. No bacterial colony formation on LB agar plates was observed (Fig. 2A). The concentration of eDNA present in the filtered supernatant of various P. aeruginosa strains at various growth days was quantified by using Qubit 2.0 Fluorometer (Invitrogen, Life Technologies).

Table 1.P. aeruginosa strains used in this study and their relevant phenazine producing characteristics

P.aeruginosa strains	Phenazine production					Source
	PCA	PCN	5-MCA	1-OHPHZ	PYO
PAO1 Wildtype	+	+	+	+	+	15
∆phzS	+	+	+	-	-	18
∆phzH	+	-	+	+	+	18
∆phzM	+	+	-	+	-	18
phzSH	+	-	-	-	++	15
PA14 Wildtype	+	+	+	+	+	15
PA14 ∆phzM	+	+	-	+	-	D.K. Newman lab
PA14 ∆phzA-G	-	-	-	-	-	19

Note: +, produce basal level of phenazine; ++, produce elevated amount of phenazine.

Figure 2. Different kinds of phenazine production and its influence in eDNA release. Comparison of bacterial colony formation on LB agar plate after 48 h incubation with non-filtered (upper half) and filtered (bottom half) bacterial supernatant (A). Production of various kinds of phenazine molecules by P. aeruginosa PAO1 strains (B). Quantification of eDNA release in supernatants of various kinds of phenazine producing P. aeruginosa PAO1 (C) and PA14 (D) strains. Error bars represents standard deviations from the mean (n = 3). Asterisks indicate statistically significant (p < 0.05) differences in eDNA concentration in comparison to the PAO1 wildtype (C) and mutant strain ∆phzA-G (D). Schematic represents the relationship between eDNA release and phenazine production is bridged via H2O2 generation and subsequent H2O2 mediated cell lysis (E).

Statistical analysis

The amount of eDNA release by various phenazine producing P. aeruginosa strains was analyzed using a two-tailed Student’s t-test. Differences were considered significant if p < 0.05.

Results and Discussion

By disrupting the activity of specific phenazine genes in wildtype PA01 the ratio of production of the various phenazine molecules is altered as indicated by the change in bacterial cell free supernatant color (Fig. 2B). For instance the mutant ∆phzS appears red because it lacks the gene responsible for 1-OHPHZ production and also responsible for conversion of 5-methylphenazine-1-carboxylic acid (5-MCA), an immediate precursor of PYO (which is red colored compound), into PYO. The ∆phzH mutant appears light green because it produces 1-OHPHZ and PYO but lacks production of PCN. The double mutant ∆phzSH is unable to produce PCN or 1-OHPHZ and consequently overproduces PYO giving the supernatant a dark green appearance.

Figure 2C demonstrates that mutant strains over-producing specific phenazines released significantly more eDNA than the PAO1 wildtype strain especially after 3 and 5 d of batch culture growth. In support of this observation it has been reported previously that altering the proportion of specific phenazine production, via activating or deleting specific phz genes, has significant impacts on Pseudomonas adhesion, biofilm formation and biocontrol activity.- Similar observations were made using a ∆phzA-G mutant of P. aeruginosa strain PA14 incapable of producing phenazines. In this case, significantly lower eDNA production was observed in comparison to PA14 wildtype that predominantly produces pyocyanin and a PA14 ∆phzM mutant that is deficient in pyocyanin production but producing PCN and 1-OHPHZ (Fig. 2D).

The relationship between eDNA release and phenazine production is bridged via H2O2 generation as an intermediate agent. H2O2 generation occurs when phenazines, which are electrochemically active, accept electrons from NADH in the biofilm or cell culture and subsequently transfers that electrons it to molecular oxygen. Phenazines are thus involved in reduction of molecular oxygen to form reactive oxygen species like O2-, H2O2.1 H2O2 can react with metals to produce highly reactive hydroxyl radicals that damage bacterial cell walls resulting in lysis of cells and ultimately release of chromosomal DNA forming eDNA (Fig. 2E). Linking the results from the previous and current study, we propose that phenazines may have significant ecological impact not only on P. aeruginosa biofilm formation but also on other bacterial species that persist in mixed biofilms along with P. aeruginosa. Phenazine influenced H2O2 generation and lysis of competing bacterial cells in mixed biofilms and subsequent eDNA release may give it a competitive edge.

Moreover, large quantities of pyocyanin as well as DNA are known to exist in the sputum of cystic fibrosis (CF) patients, and these factors could promote biofilm formation and subsequently enhance mortality in CF patients through deterioration of the host immune system. Host immune cells called neutrophils kill infecting microbes by trapping them in the neutrophil extracellular matrix which mainly consists of DNA and antimicrobial peptides. However, killing of microbes may further encourage release of microbial eDNA and excess of DNA is responsible for failure of host immune systems by inciting autoimmune diseases (systemic lupus erythematosus). This makes the biological significance of eDNA of greater interest for further research since it not only encourages biofilm formation but also damages host immune systems.