Negative regulation of biofilm formation by nitric oxide sensing proteins

Abstract

Biofilm-based infections pose a serious threat to public health. Biofilms are surface-attached communities of microorganisms, most commonly bacteria and yeast, residing in an extracellular polymeric substance (EPS). The EPS is composed of several secreted biomolecules that shield the microorganisms from harsh environmental stressors and promote antibiotic resistance. Due to the increasing prominence of multidrug-resistant microorganisms and a decreased development of bactericidal agents in clinical production, there is an increasing need to discover alternative targets and treatment regimens for biofilm-based infections. One promising strategy to combat antibiotic resistance in biofilm-forming bacteria is to trigger biofilm dispersal, which is a natural part of the bacterial biofilm life cycle. One signal for biofilm dispersal is the diatomic gas nitric oxide (NO). Low intracellular levels of NO have been well documented to rapidly disperse biofilm macrostructures and are sensed by a widely conserved NO-sensory protein, NosP, in many pathogenic bacteria. When bound to heme and ligated to NO, NosP inhibits the autophosphorylation of NosP's associated histidine kinase, NahK, reducing overall biofilm formation. This reduction in biofilm formation is regulated by the decrease in secondary metabolite bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). The NosP/NahK signaling pathway is also associated with other major regulatory systems in the maturation of bacterial biofilms, including virulence and quorum sensing. In this review, we will focus on recent discoveries investigating NosP, NahK and NO-mediated biofilm dispersal in pathogenic bacteria.

Keywords: NosP; biofilm; c-di-GMP; histidine kinase; nitric oxide; two component signaling systems.

Figure 1.. NosP/NahK TCSSs regulate various processes amongst bacterial species.

NosP is a heme-binding, NO-sensing protein that functions to inhibit the kinase activity of NosP’s associated histidine kinase, NahK. It has been reported that disruption of NahK activity disrupts various bacterial processes amongst Gram-negative biofilm-forming pathogens, including c-di-GMP metabolism, biofilm formation and dispersal, QS and virulence. In all figures, the beige parallelogram represents heme and the blue and red circles represent NO.

Figure 2:. NosP is a structurally conserved protein more present in hnoX deficient bacteria.

A. AlphaFold 2.0 Protein Structure Prediction Database predicts the Burkholderia thailandensis NosP to form a globular protein consisting of two distinct subunits, a tri-symmetrical base and a disordered cap. FIST_N (red) composes two of the symmetrical units of the base, while FIST_C composes the third symmetrical unit as well as the disordered cap. B. Using the matchmaker function on ChimeraX to visualize the protein overlay, the calculated structures of NosP from Burkholderia thailandesis, Legionella pneumophila, Pseudomonas aeruginosa, Shewanella oneidensis, and Vibrio cholerae have high structural similarity with calculated RMSDs from 3.789 Å to 5.842 Å across all 366 amino acids. C. Multiple sequence alignment of the amino acid NosP sequences from B. thailandesis, L. pneumophila, P. aeruginosa, S. oneidensis, and V. cholerae highlight several conserved amino acids across the N- and C-terminal FIST domains. D. Sequence Similarity Network (SSN) of Burkholderia thailandensis NosP generated using the EFI - Enzyme Similarity Tool. The SSN was visualized using Cytoscape with a %id edge filter set to 75%.

Figure 3.. NosP signaling pathway in B. thailandensis.

In B. thailandensis, BtNosP bound to heme (yellow parallelogram) inhibits the autophosphorylation activity of its co-cistronic BtNahK, leading to a decreased biofilm formation phenotype, similar to a ΔBtnahK mutant strain.

Figure 4.. NosP signaling in V. cholerae.

A. The NosP domain of CdpA is required for bacterial attachment in V. cholerae. Inhibition of the PDE activity of CdpA contributes to an accumulation of c-di-GMP which binds to MSHA pili. This allows for the pili to extend and promotes bacterial attachment. B. In the presence of NO, the NosP domain will promote PDE activity of CdpA contributing to pili retraction and bacterial detachment. C. The NosP (VpsV) and VpsS signaling cascade in the LuxU QS circuit. When NosP ligates ferrous NO, it inhibits VpsS, preventing VpsS from phosphorylating LuxU. This prevents QS signaling that leads to biofilm formation; overall, inactivation of VpsS promotes biofilm dispersal. Yellow parallelogram - heme, nitrogen - blue, oxygen - red.

Figure 5.. NosP signals in the GacS MKN in P. aeruginosa PA14.

When PaNosP is not ligated to ferrous NO, PaNahK auto-phosphorylates and transfers a phosphoryl group to HptB. Downstream, this phosphorylation of HptB promotes transcriptional activation of sRNA rsmY. rsmY is an inhibitor of post-transcriptional regulator RsmA. rsmZ is another sRNA that inhibits RsmA, but is only known to be transcriptionally regulated by GacA. RsmA activity promotes PQS and PYO production while inhibiting las and rhl production. Yellow parallelogram - heme, nitrogen - blue, oxygen - red.

Figure 6.. NosP signaling pathway in L. pneumophila.

In L. pneumophila, NO (nitrogen – blue, oxygen – red) ligation by the heme (yellow parallelogram) bound NosP activates NahK autophosphorylation, causing an overall decrease in c-di-GMP concentration, due to an increase in PDE activity and decrease in DGC activity of the bifunctional response regulator NarR.

Figure 7.. NosP signaling modulates biofilm formation through regulation of c-di-GMP in Shewanella oneidensis.

A. Both NosP and NahK promote biofilm formation in the absence of NO by inhibiting HnoK. This inhibition prevents the phosphotransfer from HnoK to HnoB, HnoC and HnoD, reducing PDE activity. The accumulation of c-di-GMP will allow for the development of mature biofilm macrostructures. B. NO-bound NosP/NahK complex promotes NO-mediated biofilm dispersal through HnoK. In the presence of NO, NosP will inhibit NahK and HnoK is now in the active state. This allows for the phosphorylation of HnoD and HnoB. This promotes the degradation of c-di-GMP into pGpG and a reduction in biofilm formation.