Biology subjects

Boon, E.

Publications and source records attributed to Boon, E..

4 recordsLinked to original sources

bioRxiv2025

Pseudomonas aeruginosa uses kinases NahK and RetS to control the motile-biofilm switch

The multidrug-resistant bacterium Pseudomonas aeruginosa (Pa) poses a significant threat to public health. This Gram-negative bacterium establishes pathogenicity through formation of multicellular communities, known as biofilms, that result in significant resistance to antibiotics and host immune systems. In Pa, the motile-to-biofilm transition is regulated through an interconnected signaling network known as the Gac Multikinase Network (Gac-MKN). This network comprises two regulatory branches: the HptB signaling network and GacS/A signaling network. In the Gac-MKN, several histidine kinases converge to regulate the activity of the post-transcriptional regulator protein, RsmA. Although previous studies have assessed the role of individual kinases in this network, the role of each Gac-MKN kinase in regulating RsmA activity has not been quantitatively characterized and compared in side-by-side experiments in the same reference strain, which is presented here. In this study, we show that kinases NahK and RetS are the predominant regulators of the Gac-MKN. Through controlled testing of RsmA-dependent phenotypes, we demonstrate loss of nahK or retS leads to complete inactivation of RsmA, triggering biofilm formation. Our results support previous findings that RetS regulates RsmA through the GacS/A network but present the new finding that NahK is the central kinase involved in HptB phosphorylation; previous studies have attributed HptB phosphorylation to PA1611 and SagS. Our findings demonstrate that NahK signaling controls RsmA activity to rapidly transition between the motile and biofilm states. We anticipate the results of this study will facilitate the use of targeting the Gac-MKN to trigger biofilm dispersal for improved antibiotic treatment.

microbiology↗

bioRxiv2025

The histidine kinase NahK in Pseudomonas aeruginosa is essential for nitric oxide-stress resistance in nutrient starved media

Biofilms are communities of bacteria growing within a matrix composed of polymeric substances that act as a barrier to antimicrobials, making bacteria within a biofilm recalcitrant to conventional antibiotic treatments. This lifestyle of bacteria is especially relevant during its pathogenesis, as observed in the case of Pseudomonas aeruginosa infections of immunocompromised patients. However, pico- to nanomolar concentrations of nitric oxide (NO) have been shown to be efficient in triggering P. aeruginosa to disperse from biofilms, suggesting that a combination of NO exposure and antibiotic treatment may help in mitigating infections. In P. aeruginosa, the NosP-NahK two component system, which comprises a NO sensing hemoprotein, NosP, and its associated hybrid histidine kinase, NahK, has been proven to be necessary for NO mediated biofilm dispersal. NahK also has additional roles in biofilm formation, motility, denitrification and virulence, due to its regulation of a global post-transcriptional regulator RsmA. Here, we uncover a novel role of NahK in enhancing the resistance of P. aeruginosa to signaling concentrations of NO. Deletion of nahK sensitizes the strain to nanomolar levels of NO, resulting in DNA damage. Consequently, the SOS stress response pathway is induced, which causes phenotypes such as cell filamentation and cell clustering due to lysis-mediated release of extracellular DNA. Our data also indicate that increased susceptibility of the {Delta}nahK strain to NO is due to the antagonization of RsmA, and is restricted to amino acid starved media, suggesting that NahK may also have previously unappreciated roles in modulating amino acid metabolism. ImportanceP. aeruginosa infections of cystic fibrosis patients can result in increased risk of fatality, necessitating the development of better therapeutic strategies to treat its infections. Here, we present data suggesting that nanomolar concentrations of NO, that are normally used to disperse P. aeruginosa from biofilms, can also sensitize the bacterium to NO mediated DNA damage upon loss of function of the histidine kinase NahK. The resulting SOS stress response may benefit the bacterium through favorable mutations but also poses a risk due to SOS-associated pyocin production through cell lysis. Hence, understanding the molecular mechanisms underlying NahK mediated resistance to NO is important to leverage this regulation and identify novel targets to treat P. aeruginosa infections.

microbiology↗

bioRxiv2024

The Histidine Kinase NahK Regulates Denitrification and Nitric Oxide Accumulation through RsmA inPseudomonas aeruginosa

Pseudomonas aeruginosa have a versatile metabolism; they can adapt to many stressors, including limited oxygen and nutrient availability. This versatility is especially important within a biofilm where multiple microenvironments are present. As a facultative anaerobe, P. aeruginosa can survive under anaerobic conditions utilizing denitrification. This process produces nitric oxide (NO) which has been shown to result in cell elongation. However, the molecular mechanism underlying this phenotype is poorly understood. Our laboratory has previously shown that NosP is a NO-sensitive hemoprotein that works with the histidine kinase NahK to regulate biofilm in P. aeruginosa. In this study, we identify NahK as a novel regulator of denitrification under anaerobic conditions. Under anaerobic conditions, deletion of nahK leads to a reduction of growth coupled with reduced transcriptional expression and activity of the denitrification reductases. Further, during stationary phase under anaerobic conditions, {Delta}nahK does not exhibit cell elongation, which is characteristic of P. aeruginosa. We determine the loss of cell elongation is due to changes in NO accumulation in{Delta} nahK. We further provide evidence that NahK may regulate denitrification through modification of RsmA activity. ImportanceP. aeruginosa is an opportunistic multi-drug resistance pathogen that is associated with hospital acquired infections. P. aeruginosa is highly virulent, in part due to its versatile metabolism and ability to form biofilms. Therefore, better understanding of the molecular mechanisms that regulate these processes should lead to new therapeutics to treat P. aeruginosa infections. The histidine kinase NahK has been previously shown to be involved in both NO signaling and quorum sensing through RsmA. The data presented here demonstrate that NahK is responsive to NO produced during denitrification to regulate cell morphology. Understanding NahKs role in metabolism under anaerobic conditions has larger implications in determining Nahks role in a heterogeneous metabolic environment such as a biofilm.

microbiology↗

bioRxiv2023

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 (P. 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 led to a four-fold 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 systems, with a large upregulation of the Pseudomonas quinolone signal (PQS) 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, this data contributes to understanding how the GacS MKN modulates QS and virulence. ImportancePseudomonas 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 demonstrates 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.

microbiology↗