Microbial metabolomics in open microscale platforms

Abstract

The microbial secondary metabolome encompasses great synthetic diversity, empowering microbes to tune their chemical responses to changing microenvironments. Traditional metabolomics methods are ill-equipped to probe a wide variety of environments or environmental dynamics. Here we introduce a class of microscale culture platforms to analyse chemical diversity of fungal and bacterial secondary metabolomes. By leveraging stable biphasic interfaces to integrate microculture with small molecule isolation via liquid-liquid extraction, we enable metabolomics-scale analysis using mass spectrometry. This platform facilitates exploration of culture microenvironments (including rare media typically inaccessible using established methods), unusual organic solvents for metabolite isolation and microbial mutants. Utilizing Aspergillus, a fungal genus known for its rich secondary metabolism, we characterize the effects of culture geometry and growth matrix on secondary metabolism, highlighting the potential use of microscale systems to unlock unknown or cryptic secondary metabolites for natural products discovery. Finally, we demonstrate the potential for this class of microfluidic systems to study interkingdom communication between fungi and bacteria.

Figure 1. The micrometabolomics platform workflow is simpler, faster, and takes up less space than traditional metabolite extraction.

Traditional fungal metabolomics workflows (top) require serial inoculation of cultures, collection and homogenization of the sample, extraction of metabolites with solvent, and finally evaporation of that solvent prior to analysis. The microscale workflow (bottom) allows for arrayed inoculation and on-chip metabolite extraction without the need for culture collection and homogenization. Besides the streamlined process, the micrometabolomics platform also uses ∼1000 × less solvent, which cuts down on evaporation time and makes the workflow faster.

Figure 2. Extraction at microscale.

(a) The micrometabolomics device is comprised of a central culture well with an overlying pipet-accessible solvent channel. (b) The device is operated in three simple steps. (c) Simulations of fluid flow in the platform demonstrate that solvent removal does not disturb the liquid culture underneath. (d) The devices are arrayable and compatible with a multichannel pipette. (e) The extraction module can also be integrated with a platform for pooled extraction. (f) Sporulating culture of A. fumigatus overlayed on solid GMM agar grown in the micrometabolomics platform for 2 days. Scale bar, 250 μm.

Figure 3. Solvent selection impacts extraction of secondary metabolites.

(a) Experimental design and culture photo. PeOH is 1-pentanol, CHCl₃ is chloroform and γ-Capro is γ-caprolactone. (b) Principal component analysis of A. nidulans cultured in the micrometabolomics platform. Only features that could be annotated as known secondary metabolites were used for clustering (Table 1). Each dot represents one of five independent cultures per condition from one experiment and the shaded ellipses represent 95% confidence intervals. Variance explained refers to the amount of total variation observed between samples that can be attributed to segregation along that principal component. (c) Loadings plot of individual features for the PCA in b. (d–f) Peak areas (integrated peak intensities, arbitrary units (a.u.)) of three of the features numbered in c. Error bars represent s.d. of the five replicates and statistics were performed using the Kruskal–Wallis test as described in the methods; ^**P value <0.01. Peak areas for features 14–16 were summed as they are adducts of the same compound. Structures are of the putative annotation for each peak.

Figure 4. Global metabolite profiles segregate by well diameter but not well depth.

(a) Panel of devices with varying diameters and depths. (b) Principal component analysis of A. nidulans grown on GMM agar in wells of varying diameter and depth and extracted with PeOH. Legend values of diameter and depth are given in microns. ‘Macroscale' refers to core samples of fungal growth on a 10-cm petri dish while ‘Media control' refers to core samples of agar alone. Each dot represents one of five independent cultures per condition from one experiment and the shaded ellipses represent 95% confidence intervals for each of the different diameters or control conditions. (c) Loadings plot of individual features for the PCA in b. Each dot represents one feature. Features in pink were putatively annotated as A. nidulans secondary metabolites based on exact mass <10 p.p.m. error (Supplementary Table 3).

Figure 5. A. fumigatus metabolite production varies when grown on blood as compared to GMM.

(a) Experimental conditions tested in the micrometabolomics platform in triplicate. Data for all three independent cultures from one experiment are shown in b–e. (b) Overlap of global metabolite profiles extracted with PeOH from blood, A. fumigatus grown on blood, and A. fumigatus grown on GMM. Peaks were extracted and aligned using XCMS online. Numbers represent peaks unique in m/z value and retention time. (c) Putative annotation of peaks based on exact mass of known A. fumigatus secondary metabolites. Error given in p.p.m. Adducts are compatible with the observed spectrum and colour corresponds to peak location in b. (d) Peak areas for three putative secondary metabolites produced by A. fumigatus grown on either blood or GMM. The peak areas for both TAF adducts were summed. The dotted line is the peak threshold of 60,000 below which is considered noise. Error bars represent s.d. of three microchannels. P values were calculated using unpaired Student's t-tests: *P value <0.05, ^** P value <0.005. (e) LC-MS/MS of the three peaks with putative IDs in d. Replicates were pooled prior to LC-MS/MS. TAF and gliotoxin were analysed in positive mode, endocrocin in negative mode.

Figure 6. A microscale coculture platform that enables segregated analysis of interkingdom communication.

(a) The coculture platform is comprised of two micrometabolomics devices in diffusion contact via four pores in the floor of each culture well. (b) Diffusion of a 25-μM solution of fluorescent dye, Alexa 488 hydrazide, through the agar culture pads into the upper solvent well is time dependent. Error bars represent s.d. of three devices. (c) Photos of bacterial and fungal growth within the coculture devices. (d) Monoculture and coculture of P. aeruginosa and A. fumigatus after 3 days at 30 °C shows that coculture prevents A. fumigatus growth. (e) Monoculture and coculture of R. solanacearum and A. flavus for 3 days at 30 °C shows that coculture causes A. flavus to generate chlamydospores. Images were taken at × 4 ((d,e) i–iv), scale bar, 250 μm, and a subset of wells were stained with calcafluor white and imaged at × 10 ((d,e) v–vi), scale bar, 25 μm. Images are representative of three culture wells.