The histidine kinase NahK regulates pyocyanin production through the PQS system.

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.

Negative regulation of biofilm formation by nitric oxide sensing proteins.

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.

Self-Patterned Nanoscale Topography of Thin Copolymer Films Prepared by Evaporative Assembly-Resist Early-Stage Bacterial Adhesion.

Biofilm formation on the surfaces of indwelling medical devices has become a growing health threat due to the development of antimicrobial resistance to infection-causing bacteria. For example, ventilator-associated pneumonia caused by Pseudomonas and Staphylococci species has become a significant concern in treatment of patients during COVID-19 pandemic. Nanostructured surfaces with antifouling activity are of interest as a promising strategy to prevent bacterial adhesion without triggering drug resistance. In this study, we report a facile evaporative approach to prepare block copolymer film coatings with nanoscale topography that resist bacterial adhesion. The initial attachment of the target bacterium Pseudomonas aeruginosa PAO1 to copolymer films as well as homopolymer films was evaluated by fluorescence microscopy. Significant reduction in bacterial adhesion (93-99% less) and area coverage (>92% less) on the copolymer films was observed compared with that on the control and homopolymer films [poly(methacrylic acid) (PMAA)─only 40 and 23% less, respectively]. The surfaces of poly(styrene)-PMAA copolymer films with patterned nanoscale topography that contains sharp peaks ranging from 20 to 80 nm spaced at 30-50 nm were confirmed by atomic force microscopy and the corresponding surface morphology analysis. Investigation of the surface wettability and surface potential of polymer films assists in understanding the effect of surface properties on the bacterial attachment. Comparison of bacterial growth studies in polymer solutions with the growth studies on coatings highlights the importance of physical nanostructure in resisting bacterial adhesion, as opposed to chemical characteristics of the copolymers. Such self-patterned antifouling surface coatings, produced with a straightforward and energy-efficient approach, could provide a convenient and effective method to resist bacterial fouling on the surface of medical devices and reduce device-associated infections.