B American Society for Mass Spectrometry, 2015
J. Am. Soc. Mass Spectrom. (2015)
FOCUS: IMAGING MASS SPECTROMETRY: RESEARCH ARTICLE
Mass Spectrometry Analysis of Pseudomonas aeruginosa
Treated with Azithromycin
Vanessa V. Phelan, Jinshu Fang, Pieter C. Dorrestein
Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences University of
California, San Diego, La Jolla, CA 92093, USA
Abstract. In microbiology, changes in specialized metabolite production (cellto-cell signaling metabolites, virulence factors, and natural products) are measured using phenotypic assays. However, advances in mass spectrometry-based techniques including imaging mass spectrometry (IMS) now allow researchers to directly visualize the production of specialized metabolites from microbial colony biofilms. In this study, a combination of
IMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to visualize the effect of the macrolide antibiotic azithromycin (AZM) on colony biofilms of Pseudomonas aeruginosa. Although previous research suggested that AZM may inhibit cell-to-cell signaling of P. aeruginosa and thereby reduce pathogenicity, we observed no clear decrease in specialized metabolite production.
Keywords: Pseudomonas aeruginosa, Imaging mass spectrometry, Specialized metabolites
Received: 15 December 2014/Revised: 9 February 2015/Accepted: 9 February 2015
Pseudomonas aeruginosa is one of the most common andpersistent opportunistic pathogens affecting patients with cystic fibrosis (CF). Chronic P. aeruginosa infections lead to progressive deterioration of patient lung function, causing morbidity and mortality in CF patients . P. aeruginosa is able to survive and thrive in these patients by growing as biofilms, which provide tolerance to both inflammatory defense mechanisms of the host and antibiotic therapies [2, 3]. Several clinical studies have shown that long-term treatment of CF patients with chronic P. aeruginosa infections with the macrolide antibiotic azithromycin (AZM) leads to improved lung function and increased body weight [4–7]. The median concentration of AZM in the sputum of patients receiving highdose therapy (250 mg AZM per day) is 9.5 μg/mL, well below the minimum inhibitory concentration (MIC) for P. aeruginosa (128 to 512 μg/mL) [4, 6].
It has been suggested that one way AZM treatment improves patient lung function is by inhibiting
P. aeruginosa exchange of molecules involved in cellto-cell interactions [8, 9]. This results in a reduction of exoproducts and pathogenicity. These exoproducts include specialized metabolites (quorum sensors, virulence factors, and natural products) which are key components in the interactions between P. aeruginosa and the host. P. aeruginosa utilizes a hierarchical signaling pathway to control specialized metabolite production where the transcriptional factor pair RhlI-RhlR is subordinate to the LasI-LasR pair [10, 11]. LasI produces N-3oxo-dodecanoyl-L-homoserine lactone (3-oxo-C12-HSL), whereas RhlI produces N-butanoyl-L-homoserine lactone (C4-HSL). These HSLs bind to the transcriptional activators LasR and RhlR, respectively, and activate target promoters. Both las and rhl have been implicated in regulating the production of a third signaling metabolite, 2-heptyl-3-hydroxy-4-quinolone [Pseudomonas quinolone signal (PQS)] [12–14]. The HSLs and PQS have been shown to regulate the expression of genes required for specialized metabolite production, including those for the siderophores pyochelin and pyoverdine, as well as the phenazine, quinolone, and rhamnolipid molecular families . Herein, we describe the application of agar-based microbial matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the effect of AZM on specialized metabolite production of three strains of
P. aeruginosa: FLR01, a non-mucoid clinical isolate, and two common laboratory strains, PAO1 and PA14.Correspondence to: Vanessa Phelan; e-mail: firstname.lastname@example.org
P. aeruginosa strain PA14 was provided by the D. Hung lab (Harvard Medical School, MA, USA) and originated in the lab of F. M. Ausubel (Massachusetts General Hospital, MA, USA) . Strain PAO1 was provided by the S. Noble lab (University of California, San Francisco, CA, USA) and originated in the lab of C. Manoil (University of Washington, Seattle, WA,
USA) . Non-mucoid clinical isolate FLR01 was provided by the F. Rohwer lab (San Diego State University, CA, USA).
All chemicals used for LB and ISP2 media were purchased from Sigma-Aldrich (St. Louis, MO, USA). Azithromycin was purchased from Sigma-Aldrich. LC-MS grade organic solvents were purchased from J.T. Baker (Center Valley, PA, USA).
P. aeruginosa Culture
P. aeruginosa strain PA14, PAO1, and FLR01were cultured to stationary phase (OD600=1.2) overnight in LB liquid medium from a single colony. The cultures were diluted to a 20% glycerol/water stock and stored at –80°C (~2.0× 108 CFU/mL). P. aeruginosa (1 μL) was inoculated directly from glycerol stocks on ISP2 agar medium (10 mL) containing 0, 2, 4, 6, or 8 μg/mL azithromycin in 100 o.d. × 25 mm Petri dishes (Fisherbrand). Cultures were incubated at 30°C for 48 h.
MALDI IMS samples were prepared as previously described [18, 19]. Briefly, for each sample a region of agar including the colony biofilmwas excised from the culture and laid on top of a
Bruker MALDI MSP 96 anchor plate. To measure qualitative concentration differences in metabolite production between samples, cultures of the same strain cultured under different
AZM concentration were analyzed on the same MALDI plate.
A photograph was taken of the agar sections transferred to the
MALDI plates. Universal matrix (Sigma-Aldrich) was applied manually using a 53 μm molecular sieve. Samples were dried at 37°C overnight. All colonies were subjected to MALDITOF IMS in positive reflectron mode using 500 μm spatial resolution in both X and Y dimensions by a Bruker Daltonics (Billerica, MA, USA) Microflex.