The atypical ubiquitin E2 conjugase UBE2L3 is an indirect caspase-1 target and controls IL-1beta secretion by inflammasomes

Caspase-1 activation by inflammasome signalling scaffolds initiates inflammation and antimicrobial responses. Caspase-1 proteolytically converts newly induced pro-IL-1α into its mature form and directs its secretion, triggers pyroptosis and the release of non-substrate alarmins such as IL-1α and HMGB1. While some caspase-1 substrates involved in these events are known, the identities and roles of non-proteolytic targets remain unknown. Here we report using unbiased proteomics that the UBE2L3 ubiquitin conjugase is an indirect target of caspase-1. Caspase-1, but not caspase-4, control led pyroptosis-and ubiquitin-independent proteasomal degradation of UBE2L3 upon canonical and non-canonical inflammasome activation by sterile danger signals and bacterial infection. Mechanistically, UBE2L3 acted post-translationally to promote K48-ubiquitylation and turnover of pro-IL-1β and dampen mature-IL-1β production. UBE2L3 depletion increased pro-IL-1β levels and mature-IL-1β secretion by inflammasomes. These findings on UBE2L3 as a molecular rheostat have implications for IL-1-driven pathology in hereditary fever syndromes, and autoinflammatory conditions associated with UBE2L3 polymorphisms

Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis

The guanylate binding protein (GBP) family of interferon-inducible GTPases promotes antimicrobial immunity and cell death. During bacterial infection, multiple mouse Gbps, human GBP2, and GBP5 support the activation of caspase-1-containing inflammasome complexes or caspase-4 which trigger pyroptosis. Whether GBPs regulate other forms of cell death is not known. The apicomplexan parasite Toxoplasma gondii causes macrophage death through unidentified mechanisms. Here we report that Toxoplasma-induced death of human macrophages requires GBP1 and its ability to target Toxoplasma parasitophorous vacuoles through its GTPase activity and prenylation. Mechanistically, GBP1 promoted Toxoplasma detection by AIM2, which induced GSDMD-independent, ASC-, and caspase-8-dependent apoptosis. Identical molecular determinants targeted GBP1 to Salmonella-containing vacuoles. GBP1 facilitated caspase-4 recruitment to Salmonella leading to its enhanced activation and pyroptosis. Notably, GBP1 could be bypassed by the delivery of Toxoplasma DNA or bacterial LPS into the cytosol, pointing to its role in liberating microbial molecules. GBP1 thus acts as a gatekeeper of cell death pathways, which respond specifically to infecting microbes. Our findings expand the immune roles of human GBPs in regulating not only pyroptosis, but also apoptosis

Differential spatiotemporal targeting of Toxoplasma and Salmonella by GBP1 assembles caspase signalling platforms

Human guanylate binding proteins (GBPs), a family of IFNγ-inducible GTPases, promote cell-intrinsic defence against pathogens and host cell death. We previously identified GBP1 as a mediator of cell death of human macrophages infected with Toxoplasma gondii (Tg) or Salmonella Typhimurium (STm). How GBP1 targets microbes for AIM2 activation during Tg infection and caspase-4 during STm infection remains unclear. Here, using correlative light and electron microscopy and EdU labelling of Tg-DNA, we reveal that GBP1-decorated parasitophorous vacuoles (PVs) lose membrane integrity and release Tg-DNA for detection by AIM2-ASC-CASP8. In contrast, differential staining of cytosolic and vacuolar STm revealed that GBP1 does not contribute to STm escape into the cytosol but decorates almost all cytosolic STm leading to the recruitment of caspase-4. Caspase-5, which can bind LPS and whose expression is upregulated by IFNγ, does not target STm pointing to a key role for caspase-4 in pyroptosis. We also uncover a regulatory mechanism involving the inactivation of GBP1 by its cleavage at Asp192 by caspase-1. Cells expressing non-cleavable GBP1D192E therefore undergo higher caspase-4-driven pyroptosis during STm infection. Taken together, our comparative studies elucidate microbe-specific spatiotemporal roles of GBP1 in inducing cell death by leading to assembly and regulation of divergent caspase signalling platforms

Mycobacterium tuberculosis Rv3586 (DacA) Is a Diadenylate Cyclase That Converts ATP or ADP into c-di-AMP

Cyclic diguanosine monophosphate (c-di-GMP) and cyclic diadenosine monophosphate (c-di-AMP) are recently identified signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA), which is an ortholog of Bacillus subtilis DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-performance liquid chromatography (HPLC), mass spectrometry (MS) and thin layer chromatography (TLC). Our results showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA, c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional dimerization. Both the N-terminal and the C-terminal domains were essential for the DacA's enzymatically active conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg2+, Mn2+ or Co2+. DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for interacting with ATP, and mutation of this motif to AAA completely abolished DacA's diadenylate cyclase activity. These results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the biological function of this enzyme in M. tuberculosis

To be or not to be a pseudogene: a molecular epidemiological approach to the mclx genes and its impact in tuberculosis

Tuberculosis presents a myriad of symptoms, progression routes and propagation patterns not yet fully understood. Whereas for a long time research has focused solely on the patient immunity and overall susceptibility, it is nowadays widely accepted that the genetic diversity of its causative agent, Mycobacterium tuberculosis, plays a key role in this dynamic. This study focuses on a particular family of genes, the mclxs (Mycobacterium cyclase/LuxR-like genes), which codify for a particular and nearly mycobacterial-exclusive combination of protein domains. mclxs genes were found to be pseudogenized by frameshift-causing insertion(s)/deletion(s) in a considerable number of M. tuberculosis complex strains and clinical isolates. To discern the functional implications of the pseudogenization, we have analysed the pattern of frameshift-causing mutations in a group of M. tuberculosis isolates while taking into account their microbial-, patient- and disease-related traits. Our logistic regression-based analyses have revealed disparate effects associated with the transcriptional inactivation of two mclx genes. In fact, mclx2 (Rv1358) pseudogenization appears to be primarily driven by the microbial phylogenetic background, being mainly related to the Euro-American (EAm) lineage; on the other hand, mclx3 (Rv2488c) presents a higher tendency for pseudogenization among isolates from patients born on the Western Pacific area, and from isolates causing extra-pulmonary infections. These results contribute to the overall knowledge on the biology of M. tuberculosis infection, whereas at the same time launch the necessary basis for the functional assessment of these so far overlooked genes.This work was supported by Fundacao para a Ciencia e Tecnologia (FCT), Portugal, and cofunded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), Quadro de Referencia Estrategico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER), and from Projeto Estrategico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013). H.N.-G. received a personal FCT Grant (SFRH/BD/33902/2209). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Very long O-antigen chains of Salmonella Paratyphi A inhibit inflammasome activation and pyroptotic cell death

Salmonella Paratyphi A (SPtA) remains one of the leading causes of enteric (typhoid) fever. Yet, despite the recent increased rate of isolation from patients in Asia, our understanding of its pathogenesis is incomplete. Here we investigated inflammasome activation in human macrophages infected with SPtA. We found that SPtA induces GSDMD‐mediated pyroptosis via activation of caspase‐1, caspase‐4 and caspase‐8. Although we observed no cell death in the absence of a functional Salmonella pathogenicity island‐1 (SPI‐1) injectisome, HilA‐mediated overexpression of the SPI‐1 regulon enhances pyroptosis. SPtA expresses FepE, an LPS O‐antigen length regulator, which induces the production of very long O‐antigen chains. Using a ΔfepE mutant we established that the very long O‐antigen chains interfere with bacterial interactions with epithelial cells and impair inflammasome‐mediated macrophage cell death. Salmonella Typhimurium (STm) serovar has a lower FepE expression than SPtA, and triggers higher pyroptosis, conversely, increasing FepE expression in STm reduced pyroptosis. These results suggest that differential expression of FepE results in serovar‐specific inflammasome modulation, which mirrors the pro‐ and anti‐inflammatory strategies employed by STm and SPtA, respectively. Our studies point towards distinct mechanisms of virulence of SPtA, whereby it attenuates inflammasome‐mediated detection through the elaboration of very long LPS O‐polysaccharides

Identification of Colletotrichum species associated with anthracnose disease of coffee in Vietnam

Colletotrichum gloeosporioides, C. acutatum, C. capsici and C. boninense associated with anthracnose disease on coffee (Coffea spp.) in Vietnam were identified based on morphology and DNA analysis. Phylogenetic analysis of DNA sequences from the internal transcribed spacer region of nuclear rDNA and a portion of mitochondrial small subunit rRNA were concordant and allowed good separation of the taxa. We found several Colletotrichum isolates of unknown species and their taxonomic position remains unresolved. The majority of Vietnamese isolates belonged to C. gloeosporioides and they grouped together with the coffee berry disease (CBD) fungus, C. kahawae. However, C. kahawae could be distinguished from the Vietnamese C. gloeosporioides isolates based on ammonium tartrate utilization, growth rate and pathogenictity. C. gloeosporioides isolates were more pathogenic on detached green berries than isolates of the other species, i.e. C. acutatum, C capsici and C. boninense. Some of the C. gloeosporioides isolates produced slightly sunken lesion on green berries resembling CBD symptoms but it did not destroy the bean. We did not find any evidence of the presence of C. kahawae in Vietnam

Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells.

The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11-IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens