XopQ induced stromule formation in Nicotiana benthamiana is causally linked to ETI signaling and depends on ADR1 and NRG1

In Nicotiana benthamiana, expression of the Xanthomonas effector XopQ triggers ROQ1-dependent ETI responses and in parallel accumulation of plastids around the nucleus and the formation of stromules. Both processes were proposed to contribute to ETI-related hypersensitive cell death and thereby to plant immunity. Whether these reactions are directly connected to ETI signaling events has not been tested. Here we utilized transient expression experiments to determine whether XopQ-mediated plastid reactions are a result of XopQ perception by ROQ1 or a consequence of XopQ virulence activity. We find that N. benthamiana mutants lacking ROQ1, both RNLs (NRG1 and ADR1) or EDS1, fail to elicit XopQ-dependent host cell death and stromule formation. Mutants lacking only NRG1 lost XopQ-dependent cell death but retained some stromule induction that was abolished in the RNL double mutant. This analysis aligns XopQ-induced stromules with the ETI signaling cascade but not to host programmed cell death. Furthermore, data reveal that XopQ-triggered plastid clustering is not strictly linked to stromule formation during ETI. Our data suggest that stromule formation, in contrast to chloroplast peri-nuclear dynamics, is an integral part of the N. benthamiana ETI response and that both RNL sub-types play a role in this ETI response.

Gasdermin D mediates host cell death but not interleukin-1β secretion in Mycobacterium tuberculosis-infected macrophages

Necrotic cell death represents a major pathogenic mechanism of Mycobacterium tuberculosis (Mtb) infection. It is increasingly evident that Mtb induces several types of regulated necrosis but how these are interconnected and linked to the release of pro-inflammatory cytokines remains unknown. Exploiting a clinical cohort of tuberculosis patients, we show here that the number and size of necrotic lesions correlates with IL-1β plasma levels as a strong indicator of inflammasome activation. Our mechanistic studies reveal that Mtb triggers mitochondrial permeability transition (mPT) and subsequently extensive macrophage necrosis, which requires activation of the NLRP3 inflammasome. NLRP3-driven mitochondrial damage is dependent on proteolytic activation of the pore-forming effector protein gasdermin D (GSDMD), which links two distinct cell death machineries. Intriguingly, GSDMD, but not the membranolytic mycobacterial ESX-1 secretion system, is dispensable for IL-1β secretion from Mtb-infected macrophages. Thus, our study dissects a novel mechanism of pathogen-induced regulated necrosis by identifying mitochondria as central regulatory hubs capable of delineating cytokine secretion and lytic cell death.

The Conserved Colletotrichum spp. Effector Candidate CEC3 Induces Nuclear Expansion and Cell Death in Plants

Plant pathogens secrete proteins, known as effectors, that promote infection by manipulating host cells. Members of the phytopathogenic fungal genus Colletotrichum collectively have a broad host range and generally adopt a hemibiotrophic lifestyle that includes an initial biotrophic phase and a later necrotrophic phase. We hypothesized that Colletotrichum fungi use a set of conserved effectors during infection to support the two phases of their hemibiotrophic lifestyle. This study aimed to examine this hypothesis by identifying and characterizing conserved effectors among Colletotrichum fungi. Comparative genomic analyses using genomes of ascomycete fungi with different lifestyles identified seven effector candidates that are conserved across the genus Colletotrichum. Transient expression assays showed that one of these putative conserved effectors, CEC3, induces nuclear expansion and cell death in Nicotiana benthamiana, suggesting that CEC3 is involved in promoting host cell death during infection. Nuclear expansion and cell death induction were commonly observed in CEC3 homologs from four different Colletotrichum species that vary in host specificity. Thus, CEC3 proteins could represent a novel class of core effectors with functional conservation in the genus Colletotrichum.

Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells

Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection.

A hierarchical GBP network promotes cytosolic LPS recognition and sepsis

Bacterial lipopolysaccharide (LPS) is one of the most bioactive substances known. Trace amounts trigger robust immunity to infection but also life-threatening sepsis causing millions of deaths each year. LPS contamination of the cytosol elicits a caspase-dependent inflammasome pathway promoting cytokine release and host cell death. Here, we report an immune GTPase network controls multiple steps in this pathway by genome-engineering mice to lack 7 different guanylate-binding proteins (GBPs). Gbp2-/- and Gbp3-/- mice had severe caspase-11-driven defects that protected them from septic shock. Gbp2 recruited caspase-11 for LPS recognition whereas Gbp3 assembled and trafficked the pyroptotic pore-forming protein, gasdermin D, after caspase-11 cleavage. Together, our results identify a new functional hierarchy wherein different GBPs choreograph sequential steps in the non-canonical inflammasome pathway to control Gram-negative sepsis.

Discovery of SARS-CoV-2-E channel inhibitors as antiviral candidates

Lack of efficiency has been a major problem shared by all currently developed anti-SARS-CoV-2 therapies. Our previous study shows that SARS-CoV-2 structural envelope (2-E) protein forms a type of cation channel, and heterogeneously expression of 2-E channels causes host cell death. In this study we developed a cell-based high throughput screening (HTS) assay and used it to discover inhibitors against 2-E channels. Among 4376 compounds tested, 34 hits with cell protection activity were found. Followed by an anti-viral analysis, 15 compounds which could inhibit SARS-CoV-2 replication were identified. In electrophysiological experiments, three representatives showing inhibitory effect on 2-E channels were chosen for further characterization. Among them, proanthocyanidins directly bound to 2-E channel with binding affinity (KD) of 22.14 μM in surface plasmon resonance assay. Molecular modeling and docking analysis revealed that proanthocyanidins inserted into the pore of 2-E N-terminal vestibule acting as a channel blocker. Consistently, mutations of Glu 8 and Asn 15, two residues lining the proposed binding pocket, abolished the inhibitory effects of proanthocyanidins. The natural product proanthocyanidins are widely used as cosmetic, suggesting a potential of proanthocyanidins as disinfectant for external use. This study further demonstrates that 2-E channel is an effective antiviral drug target and provides a potential antiviral candidate against SARS-CoV-2.

Management of cell death in parasitic infections

For a long time, host cell death during parasitic infection has been considered a reflection of tissue damage, and often associated with disease pathogenesis. However, during their evolution, protozoan and helminth parasites have developed strategies to interfere with cell death so as to spread and survive in the infected host, thereby ascribing a more intriguing role to infection-associated cell death. In this review, we examine the mechanisms used by intracellular and extracellular parasites to respectively inhibit or trigger programmed cell death. We further dissect the role of the prototypical “eat-me signal” phosphatidylserine (PtdSer) which, by being exposed on the cell surface of damaged host cells as well as on some viable parasites via a process of apoptotic mimicry, leads to their recognition and up-take by the neighboring phagocytes. Although barely dissected so far, the engagement of different PtdSer receptors on macrophages, by shaping the host immune response, affects the overall infection outcome in models of both protozoan and helminth infections. In this scenario, further understanding of the molecular and cellular regulation of the PtdSer exposing cell-macrophage interaction might allow the identification of new therapeutic targets for the management of parasitic infection.

Zinc oxide nanoparticles conjugated with clinically-approved medicines as potential antibacterial molecules

At present, antibiotic resistance is one of the most pressing issues in healthcare globally. The development of new medicine for clinical applications is significantly less than the emergence of multiple drug-resistant bacteria, thus modification of existing medicines is a useful avenue. Among several approaches, nanomedicine is considered of potential therapeutic value. Herein, we have synthesized Zinc oxide nanoparticles (ZnO-NPs) conjugated with clinically-approved drugs (Quercetin, Ceftriaxone, Ampicillin, Naringin and Amphotericin B) with the aim to evaluate their antibacterial activity against several Gram-positive (Methicillin resistant Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes) and Gram-negative (Escherichia coli K1, Serratia marcescens and Pseudomonas aeruginosa) bacteria. The nanoparticles and their drug conjugates were characterized using UV-visible spectrophotometry, dynamic light scattering, Fourier transform infrared spectroscopy and atomic force microscopy. Antibacterial activity was performed by dilution colony forming unit method and finally 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to determine their cytotoxic effects against human cell lines. ZnO-NPs revealed maxima surface plasmon resonance band at 374 and after conjugation with beta-cyclodextrin at 379 nm, polydispersity with size in range of 25–45 nm with pointed shaped morphology. When conjugated with ZnO-NPs, drug efficacy against MDR bacteria was enhanced significantly. In particular, Ceftriaxone- and Ampicillin-conjugated ZnO-NPs exhibited potent antibacterial effects. Conversely, ZnO-NPs and drugs conjugated NPs showed negligible cytotoxicity against human cell lines except Amphotericin B (57% host cell death) and Amphotericin B-conjugated with ZnO-NPs (37% host cell death). In conclusion, the results revealed that drugs loaded on ZnO-NPs offer a promising approach to combat increasingly resistant bacterial infections.