No abstract

SummaryBacterial nutrition is an essential aspect of host–pathogen interaction. For the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, fatty acids derived from lipid droplets are considered the major carbon source. However, many other soluble nutrients are available inside host cells and may be used as alternative carbon sources. Lactate and pyruvate are abundant in human cells and fluids, particularly during inflammation. In this work, we study Mtb metabolism of lactate and pyruvate combining classic microbial physiology with a ‘multi‐omics’ approach consisting of transposon‐directed insertion site sequencing (TraDIS), RNA‐seq transcriptomics, proteomics and stable isotopic labelling coupled with mass spectrometry‐based metabolomics. We discovered that Mtb is well adapted to use both lactate and pyruvate and that their metabolism requires gluconeogenesis, valine metabolism, the Krebs cycle, the GABA shunt, the glyoxylate shunt and the methylcitrate cycle. The last two pathways are traditionally associated with fatty acid metabolism and, unexpectedly, we found that in Mtb the methylcitrate cycle operates in reverse, to allow optimal metabolism of lactate and pyruvate. Our findings reveal a novel function for the methylcitrate cycle as a direct route for the biosynthesis of propionyl‐CoA, the essential precursor for the biosynthesis of the odd‐chain fatty acids.

The eukaryotic organism Dictyostelium discoideum has become one of the organisms of choice for the overexpression of recombinant myosins and myosin fragments. Here, we describe a protocol that facilitates the screening of cells that have been transformed with myosin expression constructs and allows the rapid purification of recombinant myosins. Depletion of cellular ATP is used to recruit most of the endogenous and recombinant myosin into a rigor-like complex with actin. Following cell lysis the insoluble actomyosin complex is precipitated by centrifugation, washed, and Mg(2+)-ATP is added to extract the recombinant protein from the pellet. More than 90% of the protein in the resulting supernatant corresponds to actin, myosin, and the recombinant myosin fragments. Therefore, it is easy to detect any differences in expression level between individual myosin constructs on SDS-polyacrylamide gels. Additionally, the dependence of expression on external factors, such as cell density, can be readily determined. Furthermore, the presence of a band corresponding to the recombinant protein indicates that the overexpressed protein has at least some of the functional properties that are characteristic for a myosin motor. This rapid and selective extraction protocol can also be utilized to facilitate the purification of recombinant myosin motors on a preparative scale and has proved particularly useful in the purification of myosin head fragments, that are tagged with histidine residues, by Ni(2+)-chelate affinity chromatography.

CD4+ T cells are typically considered as ‘helper’ or ‘regulatory’ populations that support and orchestrate the responses of other lymphocytes. However, they can also develop potent granzyme (Gzm)-mediated cytotoxic activity and CD4+ cytotoxic T cells (CTLs) have been amply documented both in humans and in mice, particularly in the context of human chronic infection and cancer. Despite the established description of CD4+ CTLs, as well as of the critical cytotoxic activity they exert against MHC class II-expressing targets, their developmental and memory maintenance requirements remain elusive. This is at least in part owing to the lack of a murine experimental system where CD4+ CTLs are stably induced. Here, we show that viral and bacterial vectors encoding the same epitope induce distinct CD4+ CTL responses in challenged mice, all of which are nevertheless transient in nature and lack recall properties. Consistent with prior reports, CD4+ CTL differentiation is accompanied by loss of TCF-1 expression, a transcription factor considered essential for memory T cell survival. Using genetic ablation of Tcf7, which encodes TCF-1, at the time of CD4+ T cell activation, we further show that, contrary to observations in CD8+ T cells, continued expression of TCF-1 is not required for CD4+ T cell memory survival. Whilst Tcf7-deficient CD4+ T cells persisted normally following retroviral infection, the CD4+ CTL subset still declined, precluding conclusive determination of the requirement for TCF-1 for murine CD4+ CTL survival. Using xenotransplantation of human CD4+ T cells into murine recipients, we demonstrate that human CD4+ CTLs develop and persist in the same experimental conditions where murine CD4+ CTLs fail to persist. These observations uncover a species-specific defect in murine CD4+ CTL persistence with implications for their use as a model system.

The vast number of poorly characterised enzymes in Mycobacterium tuberculosis (Mtb) is one of the key barriers precluding a better understanding of the biology that underpins pathogenesis. Here, we investigated the Mtb orphan enzyme Rv2498c to delineate its physiological role. Our results from in vitro enzymatic assays, phylogenetic analysis, X-ray crystallography and in vivo Mtb experiments, de-orphan Rv2498c as a multi-functional βhydroxyacyl-CoA lyase/thioesterase (β-HAClyase/thioesterase) that participates in three different metabolic pathways: L-leucine catabolism, itaconate dissimilation, and glyoxylate shunt. Moreover, the deletion of the rv2498c gene from the Mtb genome resulted in attenuation in the mouse model compared to infection with the parent strain. To the best of our knowledge, this is the first report of an (R)-3-hydroxyl-3-methylglutaryl-CoA for leucine catabolism and an itaconate-specific resistance mechanism in Mtb.