BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei

While the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are thought to primarily depend on glycolysis for ATP synthesis, recent studies have shown that they can fully catabolize glucose in a canonical TCA cycle. However, these parasites lack a mitochondrial isoform of pyruvate dehydrogenase and the identity of the enzyme that catalyses the conversion of pyruvate to acetyl-CoA remains enigmatic. Here we demonstrate that the mitochondrial branched chain ketoacid dehydrogenase (BCKDH) complex is the missing link, functionally replacing mitochondrial PDH in both T. gondii and P. berghei. Deletion of the E1a subunit of T. gondii and P. berghei BCKDH significantly impacted on intracellular growth and virulence of both parasites. Interestingly, disruption of the P. berghei E1a restricted parasite development to reticulocytes only and completely prevented maturation of oocysts during mosquito transmission. Overall this study highlights the importance of the molecular adaptation of BCKDH in this important class of pathogens

Erinnerung an W. Thielmann und Willingshausen

While the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are thought to primarily depend on glycolysis for ATP synthesis, recent studies have shown that they can fully catabolize glucose in a canonical TCA cycle. However, these parasites lack a mitochondrial isoform of pyruvate dehydrogenase and the identity of the enzyme that catalyses the conversion of pyruvate to acetyl-CoA remains enigmatic. Here we demonstrate that the mitochondrial branched chain ketoacid dehydrogenase (BCKDH) complex is the missing link, functionally replacing mitochondrial PDH in both T. gondii and P. berghei. Deletion of the E1a subunit of T. gondii and P. berghei BCKDH significantly impacted on intracellular growth and virulence of both parasites. Interestingly, disruption of the P. berghei E1a restricted parasite development to reticulocytes only and completely prevented maturation of oocysts during mosquito transmission. Overall this study highlights the importance of the molecular adaptation of BCKDH in this important class of pathogens

Epithelial endoplasmic reticulum stress orchestrates a protective IgA response.

Immunoglobulin A (IgA) is the major secretory immunoglobulin isotype found at mucosal surfaces, where it regulates microbial commensalism and excludes luminal factors from contacting intestinal epithelial cells (IECs). IgA is induced by both T cell-dependent and -independent (TI) pathways. However, little is known about TI regulation. We report that IEC endoplasmic reticulum (ER) stress induces a polyreactive IgA response, which is protective against enteric inflammation. IEC ER stress causes TI and microbiota-independent expansion and activation of peritoneal B1b cells, which culminates in increased lamina propria and luminal IgA. Increased numbers of IgA-producing plasma cells were observed in healthy humans with defective autophagy, who are known to exhibit IEC ER stress. Upon ER stress, IECs communicate signals to the peritoneum that induce a barrier-protective TI IgA response.Wellcome Trust Senior Investigator Award 106260/Z/14/Z HORIZON2020/European Research Council Consolidator Grant 64888

The 2-methylcitrate cycle is implicated in the detoxification of propionate in Toxoplasma gondii.

Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa phylum. The Coccidia are obligate intracellular pathogens that establish infection in their mammalian host via the enteric route. These parasites lack a mitochondrial pyruvate dehydrogenase complex but have preserved the degradation of branched-chain amino acids (BCAA) as a possible pathway to generate acetyl-CoA. Importantly, degradation of leucine, isoleucine and valine could lead to concomitant accumulation of propionyl-CoA, a toxic metabolite that inhibits cell growth. Like fungi and bacteria, the Coccidia possess the complete set of enzymes necessary to metabolize and detoxify propionate by oxidation to pyruvate via the 2-methylcitrate cycle (2-MCC). Phylogenetic analysis provides evidence that the 2-MCC was acquired via horizontal gene transfer. In T. gondii tachyzoites, this pathway is split between the cytosol and the mitochondrion. Although the rate-limiting enzyme 2-methylisocitrate lyase is dispensable for parasite survival, its substrates accumulate in parasites deficient in the enzyme and its absence confers increased sensitivity to propionic acid. BCAA is also dispensable in tachyzoites, leaving unresolved the source of mitochondrial acetyl-CoA

Author Correction: Uncoupling of invasive bacterial mucosal immunogenicity from pathogenicity.

ISSN:2041-172

Uncoupling of invasive bacterial mucosal immunogenicity from pathogenicity.

There is the notion that infection with a virulent intestinal pathogen induces generally stronger mucosal adaptive immunity than the exposure to an avirulent strain. Whether the associated mucosal inflammation is important or redundant for effective induction of immunity is, however, still unclear. Here we use a model of auxotrophic Salmonella infection in germ-free mice to show that live bacterial virulence factor-driven immunogenicity can be uncoupled from inflammatory pathogenicity. Although live auxotrophic Salmonella no longer causes inflammation, its mucosal virulence factors remain the main drivers of protective mucosal immunity; virulence factor-deficient, like killed, bacteria show reduced efficacy. Assessing the involvement of innate pathogen sensing mechanisms, we show MYD88/TRIF, Caspase-1/Caspase-11 inflammasome, and NOD1/NOD2 nodosome signaling to be individually redundant. In colonized animals we show that microbiota metabolite cross-feeding may recover intestinal luminal colonization but not pathogenicity. Consequent immunoglobulin A immunity and microbial niche competition synergistically protect against Salmonella wild-type infection

Economia della conoscenza e creativit\ue0

Questo capitolo si pone l'obiettivo di analizzare le diverse concezioni di creativit\ue0 per comprendere come, passando da una visione individuale di creativit\ue0 ad un'altra pi\uf9 sociale, diventi critico analizzare e capire le caratteristiche del contesto di riferimento e delle interazioni che esistono tra i diversi attori. Per raggiungere tale obiettivo, analizzeremo innanzitutto quei cambiamenti, avvenuti negli ultimi decenni nelle economie dei Paesi pi\uf9 avanzati, che hanno posto all\u2019ordine del giorno l\u2019importanza della creativit\ue0 come leva strategica per lo sviluppo economico delle imprese e dei territori. Successivamente, passeremo in rassegna le diverse concezioni di creativit\ue0 per capire quali fattori possono stimolarla oppure ostacolarla. Infine, approfondiremo la concezione di creativit\ue0 come processo sociale analizzando il ruolo delle dinamiche relazionali

<i>Toxoplasma gondii</i> BCKDH-complex is required for normal growth and virulence.

<p>(A) Schematic representation of pathways to produce acetyl-CoA in the mitochondrion. In green are pathways specific to <i>T. gondii</i> and in red pathways common to <i>T. gondii</i> and <i>Plasmodium</i> spp. (B) Total lysates from extracellular RH<i>ku80_ko</i> (RH) and Tg<i>e1a_ko</i> tachyzoites were analysed by Western blot. Expression of BCKDH-E1a was assessed using polyclonal anti-PfBCKDH-E1a antibodies. Detection of profilin was used as loading control. (C) Plaque assays were performed by inoculating HFF monolayers with RH or Tg<i>e1a_ko</i> parasites for 7 days. Plaques were revealed by Giemsa staining of HFFs. Scale bar represents 1 mm. (D) Intracellular growth of RH (blue) and Tg<i>e1a_ko</i> (red) was assessed after 24 h in complete media, media lacking glutamine, or glucose. Following 24 h of growth in glucose-depleted environment, glucose was added back to the media and rescue of the parasite’s growth was assessed. Data are represented as means ± SD from three independent biological replicates. (E) The apicoplast targeting sequence of TgPDH-E1a (aa 1–225, ABE76506) and mitochondrial targeting sequence of TgBCKDH-E1a (aa 1–73, XP_002366588) were replaced with the mitochondrial transit peptide of the superoxide dismutase 3 (SOD3) and myc-tagged <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004263#ppat.1004263-Pino2" target="_blank">[71]</a> to direct the expression of the fusion protein in the mitochondrion of Tg<i>e1a_ko</i> parasites for complementation (creating pTub8-SOD3mycPDHE1a and pTub8-SOD3mycBCKDHE1a respectively). Immunofluorescence assay shows localization of SOD3mycPDHE1a and SOD3mycBCKDHE1a in the single tubular mitochondrion (anti-myc (in green), anti-GAP45 (pellicle marker in red)). (F) Intracellular growth assay at 32 h post transient transfection of Tg<i>e1a_ko</i> with pTub8-SOD3mycPDHE1a, pTub8-SOD3mycBCKDHE1a and pTub8-mycNtGAP45 (negative control) in complete media or media depleted in glucose. Data are represented as means ± SD from three independent biological replicates. Only vacuoles containing parasites transiently expressing the transgene were taken into account. Over 200 vacuoles were counted per replicate. (G) CD1 mice were infected with RH (in blue) or Tg<i>e1a_ko</i> (in red) tachyzoites (∼15 parasites per mouse) and survival was assessed over 21 days. A challenge with ∼1000 wild-type RH tachyzoites was performed on mice that survived initial infection and survival followed for a further 10 days. Five mice were infected per condition.</p

Epithelial endoplasmic reticulum stress orchestrates a protective IgA response

Immunoglobulin A (IgA) is the major secretory immunoglobulin isotype found at mucosal surfaces, where it regulates microbial commensalism and excludes luminal factors from contacting intestinal epithelial cells (IECs). IgA is induced by both T cell-dependent and -independent (TI) pathways. However, little is known about TI regulation. We report that IEC endoplasmic reticulum (ER) stress induces a polyreactive IgA response, which is protective against enteric inflammation. IEC ER stress causes TI and microbiota-independent expansion and activation of peritoneal B1b cells, which culminates in increased lamina propria and luminal IgA. Increased numbers of IgA-producing plasma cells were observed in healthy humans with defective autophagy, who are known to exhibit IEC ER stress. Upon ER stress, IECs communicate signals to the peritoneum that induce a barrier-protective TI IgA response