Nearly 40% of Cincinnati VA patients suffer from chronic obstructive pulmonary disease (COPD) that often suffer from airway infection by opportunistic bacteria, the most prevalent of which is Pseudomonas aeruginosa (PA). PA is found at high titers in chronically infected COPD airways and many strains are mucoid, resulting from overproduction of a viscous exopolysaccharide called alginate. The major mechanism of mucoid conversion of PA is via mutations. These mutations occur predominantly (>84-92%) within mucA, encoding an anti-sigma factor. Without MucA, the sigma factor AlgT(U) directs transcription of genes involved in alginate biosynthesis, resulting in mucoidy. In 2006, we published a paper in the Journal of Clinical Investigation, demonstrating that mucoid mucA mutant bacteria are killed during anaerobic exposure to acidified nitrite (A- NO2-). However, inactivation of algT(U) in the mucA background did not relieve sensitivity to acicified nitrite strongly indicating the affects observed were MucA-specific. Provision of mucA in trans restored A-NO2- resistance and subsequent experiments established that nitric oxide (NO) plays a role in cell death. Importantly, no adverse effects were observed when A-NO2- was applied to human airway epithelia. In summary, we have discovered a novel, non-toxic agent that could potentially achieve the translational goal of eradicating mucoid PA from the airways of COPD patients.
Three specific aims are proposed and designed to determine (i) the mechanism(s) underlying A-NO2- sensitivity in mucA mutant bacteria, (ii) the role of MucA and members of the anaerobic respiratory cascade in biofilm sensitivity to A-NO2-, and (iii) to test the hypothesis that mucA and double anaerobic regulator mutants will be even more sensitive to A-NO2- in a tried-and-true mouse model of chronic lung infection.
Aim 1. Identify the molecular basis underlying anaerobic acidified NO2- sensitivity in mucoid mucA mutant PA. Although our discovery in 2006 describes an "Achilles'Heel" of mucoid, mucA mutant bacteria, we still do not know the mechanism of killing of these organisms by A-NO2-. Specifically, the role of MucA, NO3-/NO2- transport, anaerobic regulatory machinery and NO-sensitive sulfhydryl/Fe-containing proteins is very much underappreciated. The molecular basis will be determined by (i) micoarray studies of mucA and wild-type strains grown under aerobic and anaerobic conditions;(ii) determination of the cellular MucA levels that allow nitrite sensitivity, (iii) determining the rates/levels of NO2- and NO3- transport in mucA and WT and mucA double (anaerobic regulatory hierarchy genes) and;(iv) elucidate the status of critical cellular proteins known to be targets of nitrosylation.
Aim 2. Determine the effects of NO2- on viability of wild-type versus mucoid, DmucA mutants in complex, highly organized communities known as biofilms using 3 different established model systems. The biofilm mode of growth is that which has been determined to exist and actually thrive within the thick CF airway mucus. We will use 3 complimentary yet contrasting approaches that include (i) a static biofilm system, representing the stagnant mucus of COPD airways, (ii) a flow-through system that represents a contrasting biofilm mode of growth, and finally (iii) growth is static biofilms in airway surface liquid derived from human primary cells.
Aim 3. Determine the effects of NO2- on viability of wild-type versus DmucA and double mucA anaerobic regulatory mutants in an established murine chronic lung infection model. Proof-of-principle animal studies are required to show the relative efficacy of the aforementioned treatments on not only mucA mutant organisms, but also mucA mutants with selected second site mutations in genes encoding proteins that are S-nitrosylated upon exposure to A-NO2-.
Chronic obstructive pulmonary disease (COPD) is a debilitating lung disorder encompassing chronic bronchitis and/or emphysema, and characterized by nonreversible airflow limitation. In 2005, COPD was the 4th most common discharge diagnosis at VA hospitals and care was estimated at $3 billion in direct healthcare costs in 1999. At the Cincinnati VAMC, the prevalence of COPD exceeds 40% and, in 2008, it accounted for over $20M in expenditures. Exacerbations are a major contributing factor to COPD morbidity/mortality. Episodes of breathlessness mediated by Pseudomonas aeruginosa (PA) infection often precipitate hospitalizations and is associated with increased COPD patient mortality. PA causes two distinct patterns of carriage: (1) short-term colonization with exacerbation followed by clearance and (2) long-term persistence which is associated with mucoid strains. Improved treatment options, in this case the use of sodium nitrite for the management of COPD exacerbations precipitated by PA infection are needed to reduce mortality and morbidity.