The histidine kinase NahK regulates pyocyanin production through the PQS system

Abstract

Many bacterial histidine kinases work in two-component systems that combine into larger multi-kinase networks. NahK is one of the kinases in the GacS Multi-Kinase Network (MKN), which is the MKN that controls biofilm regulation in the opportunistic pathogen Pseudomonas aeruginosa. This network has also been associated with regulating many virulence factors P. aeruginosa secretes to cause disease. However, the individual role of each kinase is unknown. In this study, we identify NahK as a novel regulator of the phenazine pyocyanin (PYO). Deletion of nahK leads to a fourfold increase in PYO production, almost exclusively through upregulation of phenazine operon two (phz2). We determined that this upregulation is due to mis-regulation of all P. aeruginosa quorum-sensing (QS) systems, with a large upregulation of the Pseudomonas quinolone signal system and a decrease in production of the acyl-homoserine lactone-producing system, las. In addition, we see differences in expression of quorum-sensing inhibitor proteins that align with these changes. Together, these data contribute to understanding how the GacS MKN modulates QS and virulence and suggest a mechanism for cell density-independent regulation of quorum sensing. IMPORTANCE Pseudomonas aeruginosa is a Gram-negative bacterium that establishes biofilms as part of its pathogenicity. P. aeruginosa infections are associated with nosocomial infections. As the prevalence of multi-drug-resistant P. aeruginosa increases, it is essential to understand underlying virulence molecular mechanisms. Histidine kinase NahK is one of several kinases in P. aeruginosa implicated in biofilm formation and dispersal. Previous work has shown that the nitric oxide sensor, NosP, triggers biofilm dispersal by inhibiting NahK. The data presented here demonstrate that NahK plays additional important roles in the P. aeruginosa lifestyle, including regulating bacterial communication mechanisms such as quorum sensing. These effects have larger implications in infection as they affect toxin production and virulence.

Keywords: NahK; NosP; PQS; nitric oxide; pyocyanin; quorum sensing; signaling; virulence.

Fig 1

Pyocyanin is overproduced in ΔnahK. (A) Representation of NahK in the GacS MKN. All kinases function to regulate the activity of post-transcriptional regulator protein, RsmA. RsmA activity regulates quorum sensing, which in turn regulates PYO production. (B) Chloroform extract of ΔnahK supernatant has a bright blue color. (C) PYO is characterized by a peak at 520 nm in 0.1M HCl (9). ΔnahK is diluted fourfold in 0.1M HCl compared to wild type. (D, E) Beer’s law quantification of pyocyanin production at 520 nm (extinction coefficient 17.072 from 21). (D) Planktonic, liquid cultures (n = 3), and (E) biofilm, agar cultures. n = 3. (F) Overexpression of rsmA in wild type and ΔnahK complements the PYO phenotype, suggesting that absence of nahK represses RsmA activity, which in turn increases PYO production. P-values were calculated using one-way analysis of variance and a Tukey multiple comparisons test. *P ≤ 0.5.

Fig 2

ΔnahK is more virulent than PA14 wild type. Slow-killing kinetics of all strains in the nematode C. elegans. After 5 days of exposure to the bacteria, wild type shows ~20% killing, while ΔnahK shows ~55% killing. Error bars represent the standard deviation of at least four biological replicates, with each replicate consisting of 30–35 worms. P-values were calculated using one-way analysis of variance. *P ≤ 0.5, ****P ≤ 0.0001.

Fig 3

Phz2 is the driver of PYO production. (A) Pphz2-mS is expressed more than Pphz1-mS in the wild type and ΔnahK planktonically. ΔnahK has higher expression of Pphz2-mS than wild type overall. P-values were calculated using one-way analysis of variance and a Tukey multiple comparisons test at final time point. n = 3. (B) Pphz2-mS is expressed more than Pphz1-mS in the wild type and ΔnahK biofilms. P-values were calculated using one-way analysis of variance and a Tukey multiple comparisons test. n = 3. (C) High-performance liquid chromatography (HPLC) quantification of phenazines PCA and PYO in wild type, ∆nahK, ∆nahK∆phz1HMS, and ∆nahK∆phz2HMS. n = 3. (D) Quantitative PCR for phzM and phzS in ∆nahK compared to wild type. Gyrase A was used as a housekeeping gene (30,). P-values were calculated using unpaired, two-tailed t-tests comparing ΔnahK ΔCt values to wild type ΔCt values. n = 3. *P-value <0.05, **P-value <0.01, *** P-value <0.001.

Fig 4

Pyocyanin production may be regulated by the PQS system and rsaL. qPCR for the main quorum-sensing transcriptional regulators in ∆nahK compared to wild type. Pyocyanin production is potentially induced by each of these transcriptional regulators. Gyrase A was used as a housekeeping gene (30). Bars above and below the threshold represent upregulation and downregulation, respectively. P-values were calculated using unpaired, two-tailed t-tests comparing ΔnahK ΔCt values to wild type ΔCt values. n = 3. *P-value <0.05, **P-value <0.01.

Fig 5

PQS precursors are overexpressed in ∆nahK supernatant, while C12-HSL is reduced. Values are a ratio of prevalence in ∆nahK:wild type in positive mode LCMS. Bars above and below the threshold represent upregulation and downregulation, respectively. P-values were calculated using unpaired, two-tailed t-tests (Table S4).

Fig 6

Quorum-sensing inhibitors for the las and rhl systems are upregulated, whereas the pqs inhibitor is downregulated. qPCR for the main QS inhibitors in ∆nahK compared to wild type. Gyrase A was used as a housekeeping gene. Bars above and below the threshold represent upregulation and downregulation, respectively. P-values were calculated using unpaired, two-tailed t-tests comparing ΔnahK ΔCt values to wild type ΔCt values. n = 3. *P-value <0.05, **P-value <0.01.

Fig 7

PQS drives phz2 in ΔnahK. (A) Co-cultured wild type or ΔnahK with ΔpqsABC (planktonic) containing either the phz1 or phz2 mScarlet reporter shows increased activation of phz2 with both donors, with a higher activation in ΔnahK. n = 3. P-values were calculated using one-way analysis of variance and a Tukey multiple comparisons test at final time point. *P ≤ 0.05. (B) 3-day biofilms of co-cultured wild type or ΔnahK with ΔpqsABC containing either the phz1 or phz2 mScarlet reporter show increased activation of phz2 with both donors, with a higher activation in ΔnahK. n = 3. P-values were calculated using one-way analysis of variance and a Tukey multiple comparisons test at final time point. *P ≤ 0.05.

Fig 8

NahK regulates PQS and QS inhibitors by modulating RsmA. Inhibition of NahK leads to decreased RsmA signaling. This signaling activates PQS production and represses PQS inhibitor QslA. It also represses the LasR system through activation of LasR QS inhibitors. Increased RsaL and PQS production increases phz2-mediated PYO production.