Characterization of Alanine Catabolism inPseudomonas aeruginosaand Its Importance for Proliferation In Vivo

Abstract

The opportunistic pathogen Pseudomonas aeruginosa causes a variety of infections in immunocompromised individuals, including individuals with the heritable disease cystic fibrosis. Like the carbon sources metabolized by many disease-causing bacteria, the carbon sources metabolized by P. aeruginosa at the host infection site are unknown. We recently reported that L-alanine is a preferred carbon source for P. aeruginosa and that two genes potentially involved in alanine catabolism (dadA and dadX) are induced during in vivo growth in the rat peritoneum and during in vitro growth in sputum (mucus) collected from the lungs of individuals with cystic fibrosis. The goals of this study were to characterize factors required for alanine catabolism in P. aeruginosa and to assess the importance of these factors for in vivo growth. Our results reveal that dadA and dadX are arranged in an operon and are required for catabolism of L-alanine. The dad operon is inducible by L-alanine, D-alanine, and L-valine, and induction is dependent on the transcriptional regulator Lrp. Finally, we show that a mutant unable to catabolize DL-alanine displays decreased competitiveness in a rat lung model of infection.A hallmark of successful bacterial pathogens is their ability to replicate within their hosts, where they not only must acquire nutrients for growth but also often compete with commensal microorganisms. Although this basic tenet of bacterial pathogenesis, which was espoused originally by Louis Pasteur in the late nineteenth century (21) and more recently by E. D. Garber and other workers (3, 6), has been recognized for some time, it has generally been overlooked, and the metabolic pathways critical for proliferation in most infection sites are unknown. Basic knowledge regarding metabolic processes utilized by infecting bacteria is of fundamental importance for understanding bacterial pathogenesis and may offer opportunities for development of novel therapeutics. Indeed, interfering with bacterial metabolism in vivo has been efficacious for inhibiting the pathogenesis of several bacterial pathogens. For example, mutants of the poultry pathogen Campylobacter jejuni that are unable to catabolize L-serine display markedly reduced colonization of the chick gut (32). Likewise, loss of threonine utilization by the pulmonary pathogen Legionella pneumophila prevents replication in alveolar macrophages (26).The gram-negative opportunistic pathogen Pseudomonas aeruginosa is a leading cause of ophthalmic, burn wound, and nosocomial infections and causes chronic pulmonary infections in individuals with cystic fibrosis (CF) (9). In addition, P. aeruginosa colonizes numerous environments outside the host, and its ability to catabolize a wide array of carbon sources likely allows proliferation in these diverse environments. From a host-pathogen perspective, the carbon sources available for growth significantly affect production of extracellular virulence factors and biofilm formation in P. aeruginosa (19,20,29). Despite these findings, we have lit...

Pseudomonas aeruginosa Enhances Production of an Antimicrobial in Response to N -Acetylglucosamine and Peptidoglycan

“…To map the transcriptional start site, a nonradioactive primer extension assay, previously used to identify Helicobacter pylori and P. aeruginosa transcriptional start sites (4,21), was employed. Briefly, a fluorescently labeled primer antisense to the coding strand of PA14_15830 was used to generate cDNA from GlcNAc-grown P. aeruginosa RNA.…”

“…Primer extension was performed using fluorescently labeled 6-carboxyfluorescein (FAM) primers as previously described (4,21,31). Briefly, 1 l of a 0.2 M 5Ј-FAM-labeled primer was added to 20 g total RNA in a final volume of 20 l and incubated at 70°C for 10 min.…”

Regulation and Characterization of the dadRAX Locus for d -Amino Acid Catabolism in Pseudomonas aeruginosa PAO1

“…(B) The nucleotide sequence of the dadA regulatory region in P. aeruginosa PAO1. The Ϫ10 element and the transcriptional initiation site (ϩ1) of the dadA promoter as described by Boulette et al (1) are labeled, and the ribosome binding site in front of the ATG initiation codon of dadA is underlined. The 5Ј ends of probes QA1 to -6 are labeled accordingly, the common 3Ј end of these probes is highlighted and double underlined, and four putative DadR binding sites are numbered and underlined.…”

“…Expression of dadAX in P. aeruginosa requires DadR (1), a transcriptional activator of the Lrp family (3,7). The dadAX operon was also described by Boulette et al for its importance in P. aeruginosa proliferation and infection in rat (1). The gene organization in the dadRAX locus is highly conserved among Pseudomonas bacteria (Fig.…”

Homeostasis and Catabolism of Choline and Glycine Betaine: Lessons from Pseudomonas aeruginosa

“…However, during liquid broth enrichment of cultures from soil and aquatic environments, P. aeruginosa and many of its nonpathogenic relatives such as P. putida, P. fluorescens, and P. stutzeri are capable of rapid growth and often outcompete numerically abundant microbes from the original system, leading them to be categorized as optimal exploiters of nutrient pulses (12,13). For P. aeruguinosa, the ability to utilize diverse carbon and nitrogen sources and its high growth rate under benign conditions likely contribute to its broad distribution and infectious potential (1,14,15).…”