Interkingdom metabolic transformations captured by microbial imaging mass spectrometry

Abstract

In polymicrobial infections, microbes can interact with both the host immune system and one another through direct contact or the secretion of metabolites, affecting disease progression and treatment options. The thick mucus in the lungs of patients with cystic fibrosis is highly susceptible to polymicrobial infections by opportunistic pathogens, including the bacterium Pseudomonas aeruginosa and the fungus Aspergillus fumigatus. Unravelling the hidden molecular interactions within such polymicrobial communities and their metabolic exchange processes will require effective enabling technologies applied to model systems. In the present study, MALDI-TOF and MALDI-FT-ICR imaging mass spectrometry (MALDI-IMS) combined with MS/MS networking were used to provide insight into the interkingdom interaction between P. aeruginosa and A. fumigatus at the molecular level. The combination of these technologies enabled the visualization and identification of metabolites secreted by these microorganisms grown on agar. A complex molecular interplay was revealed involving suppression, increased production, and biotransformation of a range of metabolites. Of particular interest is the observation that P. aeruginosa phenazine metabolites were converted by A. fumigatus into other chemical entities with alternative properties, including enhanced toxicities and the ability to induce fungal siderophores. This work highlights the capabilities of MALDI-IMS and MS/MS network analysis to study interkingdom interactions and provides insight into the complex nature of polymicrobial metabolic exchange and biotransformations. M icrobes that colonize mammalian hosts can form polymicrobial communities, such as biofilms, where they establish commensual, mutualistic, competitive, or antagonistic interactions with one another and with the host. In microbial disease, this complex interplay can affect the outcome of antimicrobial therapy (1). Therefore, it is important to understand polymicrobial populations and their interactions at the molecular level.In persons with cystic fibrosis (CF), the lungs are lined with a viscous mucus layer susceptible to polymicrobial infections (2). Pseudomonas aeruginosa, a Gram-negative bacterial opportunistic pathogen, is the most prevalent and persistent microorganism (3) isolated from the sputum of CF lungs and leading cause of mortality in CF patients (4). Within the CF lung, P. aeruginosa exists in biofilm-like macrocolonies (5) and is refractory to antimicrobial agents and the host immune response (6). Aspergillus fumigatus, an opportunistic fungal pathogen, is the second-most persistent microbe in the CF lung, with a 10-57% prevalence rate (3), and is capable of causing allergic bronchopulmonary aspergillosis (7).Superinfection with both P. aeruginosa and A. fumigatus in CF patients leads to decreased pulmonary function compared with monoinfection with either microbe (8). Interestingly, however, in a pulmonary mouse model, mice coinfected with P. aeruginosa and A. fumigatus had a higher survival rate than mice...

Impact of a Transposon Insertion in phzF2 on the Specialized Metabolite Production and Interkingdom Interactions of Pseudomonas aeruginosa

“…PAO1, the phzF2 mutant, and the complemented PhzF2 pJA09-bearing strain were grown from a single colony in LB media overnight to stationary phase (optical density [OD] ϭ 1.2), diluted to a 1.6 ϫ 10 8 -CFU/ml, 20% glycerol stock, and stored at Ϫ80°C in 50-l aliquots. Af293 spore stock was prepared as previously described (7.2 ϫ 10 6 -CFU/ml, 20% glycerol stock) and stored at Ϫ80°C in 50-l aliquots (11). These frozen stocks were used directly for inoculations.…”

“…Pyocyanin (PYO) (Ն98% by high-performance liquid chromatography [HPLC]), 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) (Ն96% by HPLC), and 3-oxo-C 12 -homoserine lactone (C 12 -HSL) were purchased from Sigma-Aldrich. Phenazine-1-carboxamide (PCN) (95% by nuclear magnetic resonance [NMR]) and phenazine-1-carboxylic acid (PCA) (95% by NMR) were purchased from Princeton Biomolecular Research, Inc. 1-HP, PCN, and PCA were purified by HPLC as previously described, as they contained multiple phenazines (11).…”

“…In vitro studies show that metabolites secreted by P. aeruginosa inhibit A. fumigatus filamentation and biofilm formation (10). In 2012, we detailed the metabolic interaction between these two microbes, with an emphasis on the A. fumigatus Af293-mediated biotransformation of the phenazine class of specialized metabolites produced by P. aeruginosa PA14 (11). The phenazines have a number of roles in both intra-and interspecies interactions, including antibacterial and antifungal activities, electron shuttling through their redox capabilities, generation of reactive oxidative species, and cell signaling (12,13).…”

OpenMSI: A High-Performance Web-Based Platform for Mass Spectrometry Imaging

“…All tests are performed using a 100×100×100,000 test dataset. We, evaluate the performance of k×k×l layouts with k= [1,2,4,8,16, 32] and l= [128,256,512,1024,2048,4096,8192]. We omitted 32×32×128, 32×32×256, 32×32×512 due to the poor spectrum-at-a-time write performance of these data layouts.…”

Development of an Imaging Mass Spectrometry Technique for Visualizing Localized Cellular Signaling Mediators in Tissues

“…1,2) e emergence of IMS technology coupled with MALDI, 3,4) secondary ion mass spectrometry (SIMS), 5,6) desorption electrospray ionization (DESI), 7,8) and other desorption/ionization techniques [9][10][11] has added another attractive metabolite imaging technique to the established molecular imaging methods. In addition to its capability for simultaneous visualization of multiple metabolites, there are several other unique advantages of IMS, including its label-free principle, which has led to the emergence of a high number of IMS studies in biological [12][13][14][15] and medical research [16][17][18] in recent years focused on the analysis of small molecules.…”