The role of NosP in Pseudomonas aeruginosa biofilm development

Bacterial biofilms are a public health threat because they cause chronic and hospital-acquired infections but are
resistant to antibiotics. Failure to characterize the biochemical machinery that drives biofilm dispersal risks
missing key targets for treatment of infectious disease. Although nitric oxide (NO)-triggered biofilm dispersal in
Pseudomonas aeruginosa (PA), a principal pathogen in cystic fibrosis and hospital-acquired infections, is well
documented, the underlying biochemical processes responsible are not understood. To bridge this knowledge
gap, a long-term goal of the PI is to determine the mechanism of NO signaling in bacteria and to use this
knowledge to develop therapeutic strategies to disperse biofilms. In her previous RO1 funding period, the PI
established the NosP (NO sensing protein) family of hemoproteins, which were discovered in her laboratory, as
NO sensors that regulate biofilm in many bacteria. Based on studies from the previous funding period, the
objective of the proposed work is to characterize the NosP-associated histidine kinase (NahK) and determine
the molecular mechanism underlying NO/NosP/NahK regulation of biofilm and virulence in PA. It is hypothesized
that NO produced downstream of RsmA binds NosP to trigger biofilm dispersal through NahK and the GacS/Rsm
MKN. The GacS/Rsm MKN integrates signals from many sensor kinases to control RsmA, the master regulator
of motility/acute infection v. biofilm/chronic infection in PA. The proposal is innovative because it challenges the
established GacS/Rsm MKN and forges new logical connections between anaerobic respiration, NO, biofilm,
and virulence. This proposal is significant because elucidation of the basis for NO signaling in PA will open new
therapeutic opportunities for controlling infection caused by this important human pathogen. The hypothesis will
be tested by pursuing three specific aims: (1) to identify NosP/NahK interactions within the GacS/Rsm MKN; (2)
to delineate the role of NosP/NahK in modulation of RsmA-controlled phenotypes; and (3) to establish the link
between RsmA-regulated denitrification and NosP/NahK signaling. Under aim 1, NahK protein-protein and
phosphotransfer interactions with GacS/Rsm MKN members will be characterized as a function of NO/NosP
regulation. Under aim 2, the effect of NO/NosP/NahK on virulence, quorum sensing, and cyclic-di-GMP pathways
controlled by the RsmA will be quantified. Under aim 3, the effect of NO/NosP/NahK on RsmA-controlled
denitrification will be quantified and investigated as a regulatory feedback loop. The PI has significant experience
with the proposed assays. Upon completion of these aims, NO/NosP/NahK is expected to be established in
controlling the GacS/Rsm motility/virulence switch. This would represent a fundamentally important discovery,
defining a new signaling pathway and novel antibiotic targets, for which there is a pressing need, especially
considering the increased antibiotic resistance typically seen in biofilming organisms. NO-triggered biofilm
dispersal has been widely observed in bacteria, so clinical interventions based on NO signaling have the potential
for widespread application, furthering the significance of this project.

Grant Number: 5R01GM118894-08
NIH Institute/Center: NIH
Principal Investigator: ELIZABETH BOON