Project description:We used time-resolved metabolic footprinting, an important technical approach used to monitor changes in extracellular compound concentrations during microbial growth, to study the order of substrate utilization (i.e., substrate preferences) and kinetics of a fast-growing soil isolate, Paraburkholderia sp. strain 1N. The growth of Paraburkholderia sp. 1N was monitored under aerobic conditions in a soil-extracted solubilized organic matter medium, representing a realistic diversity of available substrates and gradient of initial concentrations. We combined multiple analytical approaches to track over 150 compounds in the medium and complemented this with bulk carbon and nitrogen measurements, allowing estimates of carbon use efficiency throughout the growth curve. Targeted methods allowed the quantification of common low-molecular-weight substrates: glucose, 20 amino acids, and 9 organic acids. All targeted compounds were depleted from the medium, and depletion followed a sigmoidal curve where sufficient data were available. Substrates were utilized in at least three distinct temporal clusters as Paraburkholderia sp. 1N produced biomass at a cumulative carbon use efficiency of 0.43. The two substrates with highest initial concentrations, glucose and valine, exhibited longer usage windows, at higher biomass-normalized rates, and later in the growth curve. Contrary to hypotheses based on previous studies, we found no clear relationship between substrate nominal oxidation state of carbon (NOSC) or maximal growth rate and the order of substrate depletion. Under soil solution conditions, the growth of Paraburkholderia sp. 1N induced multiauxic substrate depletion patterns that could not be explained by the traditional paradigm of catabolite repression.IMPORTANCE: Exometabolomic footprinting methods have the capability to provide time-resolved observations of the uptake and release of hundreds of compounds during microbial growth. Of particular interest is microbial phenotyping under environmentally relevant soil conditions, consisting of relatively low concentrations and modeling pulse input events. Here, we show that growth of a bacterial soil isolate, Paraburkholderia sp. 1N, on a dilute soil extract resulted in a multiauxic metabolic response, characterized by discrete temporal clusters of substrate depletion and metabolite production. Our data did not support the hypothesis that compounds with lower energy content are used preferentially, as each cluster contained compounds with a range of nominal oxidation states of carbon. These new findings with Paraburkholderia sp. 1N, which belongs to a metabolically diverse genus, provide insights on ecological strategies employed by aerobic heterotrophs competing for low-molecular-weight substrates in soil solution.

Project description:Unraveling the diversity of Th2 effector cells subsets may help us to understand their pathogenic role in Type-2 associated inflammatory disorders, including allergic diseases and helminth infections. To characterize the heterogeneity and function of these effector Th2 cells, we analyzed 47 subjects’ PBMCs [23 filarial-infected (Fil+) with or without coincident HDM sensitization (Fil+HDM+ n=12; Fil+HDM-, n=11) and 24 subjects without filarial infection (Fil-) (Fil-HDM+ n=12; and Fil-HDM-, n=12) using multiparameter flow cytometry and two-level FlowSOM analysis. The frequency of 3 memory CD4+ T cell clusters, including CCR4+CCR6+CRTH2- (subset 1), CCR4+CCR6-CRTH2+ (subset 2), and CCR6+CCR4+CRTH2+ (subset 3) were markedly enriched among Fil+ subjects. The functional characterization indicated subset 2 and 3 as distinct Th2 cytokine producers, which together were responsible for the majority of IL-4, IL-5, or IL-13 produced among the Fil+ subjects. These subsets were sorted and analyzed by multiomic single cell RNA profiling. Indeed, both subset 2 and subset 3 presented features of pathogenic Th2 effector cells based on their single cell molecular signature, including downregulation of cd27 and high expression levels of itga4, il-17rb, hpgds, klrb1, ptgdr2, il-9r, il-4, il5 and il-13 genes when compared with conventional Th2 cells from healthy controls. When the Fil+ subjects were divided based on allergic status, the Fil+HDM+ subjects had an expansion of both subset 2 (3.19% vs 1.28%, p=0.001) and subset 3 (0.39% vs 0.15%, p=0.002) Th2 cells when compared with Fil+HDM-. Gene expression analysis further demonstrated that concomitant HDM sensitization in the presence of filarial infection reshaped the molecular program of these pathogenic effector Th2 subsets by even further upregulating the expression of gata3, il17rb, clrf2, and klrb1. Notably, Fil+HDM+ patients’ cells showed higher responsiveness to filarial or HDM antigenic stimulation, producing even higher levels of IL-4, IL-5 and IL-13 when compared with Fil+HDM- patients. This distinct molecular and functional program of Th2 effector cells subsets sheds new light on the Th2 cell plasticity and their contribution to immune regulation in helminth infection and allergic diseases.

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