Host Protection Research Articles

Z-DNA binding protein 1 mediates necroptotic and apoptotic cell death pathways in murine astrocytes following herpes simplex virus-1 infection

BackgroundThe mechanisms by which glia respond to viral central nervous system (CNS) pathogens are now becoming apparent with the demonstration that microglia and astrocytes express an array of pattern recognition receptors that include intracellular RNA and DNA sensors. We have previously demonstrated that glia express Z-DNA binding protein 1 (ZBP1) and showed that this cytosolic nucleic acid sensor contributes to the inflammatory/neurotoxic responses of these cells to herpes simplex virus-1 (HSV-1). However, the relative contribution made by ZBP1- to HSV-1-mediated cell death in glia has not been determined.MethodsWe have investigated the relative contribution made by ZBP1- to HSV-1-mediated cell death in primary astrocytes derived from mice genetically deficient in this sensor. We have used capture ELISAs and immunoblot analysis to assess inflammatory cytokine production and ZBP1 and phosphorylated mixed lineage kinase domain-like protein (MLKL) expression levels, respectively, following HSV-1 challenge. Furthermore, we have used a commercially available cell viability assay to determine the proportion and rate of cell death in cells following infection with laboratory and neuroinvasive clinical strains of HSV-1, and pharmacological inhibitors of necroptotic and apoptotic pathway components to assess the relative role of each.ResultsWe show that the loss of ZBP1 in astrocytes results in an increase in the number of viral particles released following HSV-1 infection. Importantly, we have confirmed that HSV-1 induces necroptosis in astrocytes and have established the ability of ZBP1 to mediate this cell death pathway. Interestingly, while ZBP1 is best known for its role in necroptotic signaling, our findings indicate that this sensor can also contribute to virally induced apoptosis in these glia.ConclusionsOur findings indicate that ZBP1 serves as a restriction factor for HSV-1 infection and is associated with the induction of both necroptotic and apoptotic cell death pathways in primary murine astrocytes. While it remains to be seen whether ZBP1-mediated activation of cell death in astrocytes contributes significantly to host protection or, rather, exacerbates HSV-1 encephalitis pathology, the identification of such a role in resident CNS cells may represent a novel target for therapeutic intervention to reduce HSV encephalitis-associated morbidity and mortality.

Live Vaccination with Blood-Stage Plasmodium yoelii 17XNL Prevents the Development of Experimental Cerebral Malaria.

In our work, we aim to develop a malaria vaccine with cross-strain (-species) protection. C57BL/6 mice infected with the P. berghei ANKA strain (PbA) develop experimental cerebral malaria (ECM). In contrast, ECM development is inhibited in infected mice depleted of T cells. The clinical applications of immune-cell depletion are limited due to the benefits of host defense against infectious diseases. Therefore, in the present study we attempted to develop a new method for preventing ECM without immune cell depletion. We demonstrated that mice inoculated with a heterologous live-vaccine of P. yoelii 17XNL were able to prevent both ECM and lung pathology and survived longer than control mice when challenged with PbA. Live vaccination protected blood–organ barriers from PbA infection. Meanwhile, live vaccination conferred sterile protection against homologous challenge with the P. yoelii 17XL virulent strain for the long-term. Analysis of the immune response induced by live vaccination showed that cross-reactive antibodies against PbA antigens were generated. IL-10, which has an immunosuppressive effect, was strongly induced in mice challenged with PbA, unlike the pro-inflammatory cytokine IFNγ. These results suggest that the protective effect of heterologous live vaccination against ECM development results from IL-10-mediated host protection.

Three Models of Vaccination Strategies Against Cryptococcosis in Immunocompromised Hosts Using Heat-Killed Cryptococcus neoformans Δsgl1.

Vaccines are one of the greatest medical accomplishments to date, yet no fungal vaccines are currently available in humans mainly because opportunistic mycoses generally occur during immunodeficiencies necessary for vaccine protection. In previous studies, a live, attenuated Cryptococcus neoformans Δsgl1 mutant accumulating sterylglucosides was found to be avirulent and protected mice from a subsequent lethal infection even in absence of CD4+ T cells, a condition most associated with cryptococcosis (e.g., HIV). Here, we tested three strategies of vaccination against cryptococcosis. First, in our preventative model, protection was achieved even after a 3-fold increase of the vaccination window. Second, because live C. neoformans Δsgl1-vaccinated mice challenged more than once with WT strain had a significant decrease in lung fungal burden, we tested C. neoformans Δsgl1 as an immunotherapeutic. We found that therapeutic administrations of HK C. neoformans Δsgl1 post WT challenge significantly improves the lung fungal burden. Similarly, therapeutic administration of HK C. neoformans Δsgl1 post WT challenge resulted in 100% or 70% survival depending on the time of vaccine administration, suggesting that HK Δsgl1 is a robust immunotherapeutic option. Third, we investigated a novel model of vaccination in preventing reactivation from lung granuloma using C. neoformans Δgcs1. Remarkably, we show that administration of HK Δsgl1 prevents mice from reactivating Δgcs1 upon inducing immunosuppression with corticosteroids or by depleting CD4+ T cells. Our results suggest that HK Δsgl1 represents a clinically relevant, efficacious vaccine that confers robust host protection in three models of vaccination against cryptococcosis even during CD4-deficiency.

Vaccinomics to Design a Multi-Epitopes Vaccine for Acinetobacter baumannii.

Antibiotic resistance (AR) is the result of microbes’ natural evolution to withstand the action of antibiotics used against them. AR is rising to a high level across the globe, and novel resistant strains are emerging and spreading very fast. Acinetobacter baumannii is a multidrug resistant Gram-negative bacteria, responsible for causing severe nosocomial infections that are treated with several broad spectrum antibiotics: carbapenems, β-lactam, aminoglycosides, tetracycline, gentamicin, impanel, piperacillin, and amikacin. The A. baumannii genome is superplastic to acquire new resistant mechanisms and, as there is no vaccine in the development process for this pathogen, the situation is more worrisome. This study was conducted to identify protective antigens from the core genome of the pathogen. Genomic data of fully sequenced strains of A. baumannii were retrieved from the national center for biotechnological information (NCBI) database and subjected to various genomics, immunoinformatics, proteomics, and biophysical analyses to identify potential vaccine antigens against A. baumannii. By doing so, four outer membrane proteins were prioritized: TonB-dependent siderphore receptor, OmpA family protein, type IV pilus biogenesis stability protein, and OprD family outer membrane porin. Immuoinformatics predicted B-cell and T-cell epitopes from all four proteins. The antigenic epitopes were linked to design a multi-epitopes vaccine construct using GPGPG linkers and adjuvant cholera toxin B subunit to boost the immune responses. A 3D model of the vaccine construct was built, loop refined, and considered for extensive error examination. Disulfide engineering was performed for the stability of the vaccine construct. Blind docking of the vaccine was conducted with host MHC-I, MHC-II, and toll-like receptors 4 (TLR-4) molecules. Molecular dynamic simulation was carried out to understand the vaccine-receptors dynamics and binding stability, as well as to evaluate the presentation of epitopes to the host immune system. Binding energies estimation was achieved to understand intermolecular interaction energies and validate docking and simulation studies. The results suggested that the designed vaccine construct has high potential to induce protective host immune responses and can be a good vaccine candidate for experimental in vivo and in vitro studies.

RAMP3 unexplored relevance for innate-T cell immunity

Abstract RAMP3 is a chaperon protein that aids in the transport, activity modulation and pharmacological switch of G-protein coupled receptors (GPCR). Chemokine receptors involved in immune cell recruitment and activation are GPCRs, but the role of RAMP3 in the function of the immune system has not been explored. Our lab found that Ramp3 gene is highly expressed in innate-like T cells in the lungs of mice, particularly for Mucosal-associated invariant T (MAIT) cells. Ramp3 KO mice have an increased absolute number of MAIT cells, while other innate T cells are not affected. These differences are even higher after vaccinations with a live attenuated Salmonella strain containing MAIT-cell antigens. Both at steady-state and after vaccination, Ramp3 KO mice MAIT cells have increased levels of CXCR6, a chemokine receptor that has been associated to lung homing and T cell maintenance in the periphery. Ramp3 KO mice also have a decreased proportion of IL17A-producing MAIT cells in the lung; IL-17-producing innate cells have been shown to be protective, for example following Streptococcus pneumoniae infection. Consistent with the MAIT17 decrease, there was increased bacterial burden after lung infections. While Ramp3 has been reported in several gene signatures related to T cell regulation, its role for T cell immunity has not been explored. This is the first evidence of the relevance of Ramp3 for immune cell function and host protection, influencing innate-like T cell mediated immune function and protection against lung bacterial infections. Supported by grants from NIH 5R01AI137230-04

Indispensable roles of TNF signals in protective immunity against Orientia tsutsugamushi infection

Abstract Scrub typhus is an emerging tropical disease caused by obligate intracellular bacteria Orientia tsutsugamushi, with over one million cases reported yearly. Patients may develop acute lung injury and multi-organ failure; the worldwide average mortality rate is 6%; however, little is known regarding key immune mediators during infection. Using murine scrub typhus models, we demonstrated the essential role of TNF signals in bacterial control and host protection. Mice lacking both TNF receptors (TNFR1/R2−/−) or receiving anti-TNF-α treatment were highly susceptible to infection with significantly increased tissue bacterial loads, inflammatory responses, and mortality. Their lung- and spleen-derived leukocytes displayed profound defects in total cell numbers and activation status for both innate immunity (NK cells, neutrophils, and macrophages) and adaptive immunity (CD4+ and CD8+ T cells). Infection of TNFR1−/− or TNFR2−/− mice confirmed the requirement of both receptors in efficient control of disease progression, as lacking either one receptor greatly reduced activated CD4+ and CD8+ T effector cells. Together, this study demonstrates a critical and indispensable role of TNF signals in host protective immunity against O. tsutsugamushi infection and severe scrub typhus. Supported by grants from NIH (R01 AI132674, R21 AI156536 and R21 AI153586).

The autophagy gene Epg5 promotes susceptibility to enteric viral infection

Abstract Mutations in the autophagy gene EPG5 are responsible for Vici syndrome, a multisystem human genetic disease with a combined immunodeficiency component. However, the mechanism(s) by which EPG5 regulates immune signaling are incompletely understood. Previously, we found that Epg5−/− mice are resistant to influenza and exhibit hyperinflammation in the lungs including elevated IL1B/IL-1β and TNF/TNFa. Here, we show that the disruption of Epg5 results in protection against multiple enteric viruses including norovirus and rotavirus. RNA sequencing revealed that IFN-λ responses are highly upregulated in the intestines of Epg5−/− mice. Further, mice lacking Epg5 exhibit substantial alterations of the intestinal microbiota. Surprisingly, germ-free Epg5−/− mice showed persistent inflammation of both the intestine and lung, suggesting a microbiota independent mechanism. Epg5−/−Ifnlr1−/− mice, which lack the receptor for IFN-λ, regained susceptibility to viral infection but maintained microbial dysbiosis, indicating that IFN-λ signaling is the primary mediator of resistance to enteric viruses but is dispensable for microbial dysbiosis in Epg5−/− mice. Intriguingly, epg-5-mutant Caenorhabditis elegans animals are also resistant to Orsay virus, which is an intestinal cell-tropic virus and the only known virus that naturally infects C. elegans. This study unveils an important role for autophagy gene Epg5 in protection of mammalian hosts by modulating intestinal IFN-λ responses, which is unexpectedly independent of the microbiota, and raises the possibility of a conserved viral resistance mechanism that is shared between C. elegans and mammals.

Convergent evolution of bacterial ceramide synthesis

Bacteria synthesize numerous types of sphingolipids with various physiological functions. Despite their roles in mediating host inflammation, cellular differentiation, and protection from environmental stress, their biosynthetic pathway remains undefined since several essential eukaryotic ceramide synthesis enzymes have no bacterial homologue. Using genetic and biochemical approaches, we identified the complete pathway for bacterial ceramide synthesis. Bioinformatic and phylogenetic analyses revealed the presence of these genes in a broad range of bacterial taxa and led to our discovery of the first Gram‐positive species to produce ceramides. Biochemical experiments with purified proteins support a model in which the bacterial pathway operates in a different order than in eukaryotes. Furthermore, phylogenetic analyses are consistent with the independent evolution of the bacterial and eukaryotic ceramide pathways. Current work is being done to elucidate the specific subcellular localization of the synthetic enzymes and identify additional proteins required for the transport of sphingolipids to the outer membrane of Gram‐negative bacteria.

Goblet cells regulate expansion of colonic iNKT cells in CD1d-dependent manner

Abstract Invariant natural killer T (iNKT) cells are innate-like T cells that secrete a wide array of cytokines and immune mediators. These cells recognize self or microbial ligands presented by cells expressing CD1d, and can contribute to host protection or/and pathogenesis during intestinal inflammation. The colonic iNKT cell population is established in early life under the influence of the microbiota and current understanding is that this iNKT cells are not manipulable in later life. Previous studies have identified that goblet cell associated passages (GAPs) play a role in luminal antigen delivery and the induction and maintenance of peripherally induced T regulatory cells in the steady state. Colonic GAPs are largely absent in adult mice due to goblet cell (GC) microbial sensing, which inhibits GAP formation. We hypothesized, that when present, colonic GAPs may deliver glycolipids to stimulate colonic iNKT cells. We found that the glycolipids can be delivered through GAPs and that inducing colonic GAPs in adult mice using pharmacological inhibitors or transgenic mouse models resulted in significant iNKT cell expansion. Further deletion of CD1d on GCs inhibited iNKT cell expansion suggesting a role for colonic GCs in presenting glycolipids to iNKT cells. Single cell RNA sequencing of sorted colonic iNKT cells showed significantly expanded iNKT2 and iNKT1 subsets after colonic GAP induction. Furthermore, the iNKT cells expanding after opening colonic GAPs were protective in DSS-induced colitis. Our findings suggest that the GAP function and CD1d expression by GCs plays a role in modulating colonic iNKT cell subsets in adulthood and can be protective in some colitis models.

The extracellular matrix maintained by hypodermal macrophages via IGF1 is a niche for Staphylococcus aureus infection

Abstract Macrophages (MΦ) play a vital role in tissue homeostasis, but their functions in skin hypodermis, a common site of Staphylococcus aureus (S. aureus) infection, remains largely unexplored. Using flow-cytometry, bulk RNA-seq and single-cell RNA-seq, two MΦ subsets with distinct transcriptomes were uncovered. Both MΦ subsets relied on colony stimulating factor 1 (CSF1) receptor signaling for their survival. Single-cell RNA-seq of non-immune cells identified fibroblasts (FB) as a major source of dermal and hypodermal CSF1. Analysis of dermal and hypodermal stromal cells transcriptomes revealed Tek (encoding Tie2) in hypodermal FB as a potential driver for skin layer-specific ablation of CSF1. Tek-cre x Csf1-floxed mice (Csf1ΔTek) displayed a striking post-natal ablation of hypodermal MΦ associated with an alteration of the collagen network and increased accumulation of hyaluronic acid (HA). Generation of a ligand-receptor map identified candidate genes involved in hypodermal MΦ-FB crosstalk. Among which, depletion of insulin-like growth factor 1 (IGF1) from MΦ in Csf1r-cre x Igf1-floxed (Igf1ΔCsf1r) recapitulated the alteration of the ECM observed in absence of hypodermal MΦ. Interestingly, upon injecting S. aureus into the hypodermis, Csf1ΔTek and Igf1ΔCsf1r mice displayed striking reduction of the cellulitis phenotype as compared to WT mice. Pre-injection of HA attenuated cellulitis in WT mice, suggesting that deposition of HA in the absence of MΦ or MΦ-derived IGF1 conferred host protection against S. aureus. Thus, while hypodermal MΦ critically maintained ECM homeostasis, this provided a niche for S. aureus infection, highlighting implications for novel therapeutic strategies in S. aureus soft-tissue infections.

The CD40/CD40 ligand system in linking acute neuroinflammation with chronic progressive demyelination

Abstract CD4+ T cells play critical roles in mediating adaptive immunity and developing protective host immunity by eliminating infectious viruses. CD40 Ligand expressed on CD4+ T cells known to interact with its cognate receptor CD40 expressed on monocyte/macrophage/microglia to induce several immune functions during inflammation and host immunity. Our study aimed to delineate a potential CD4+ T cell-microglia nexus at the cellular and molecular level using CD4−/− and CD40L−/− mice. Results showed that the absence of CD40L renders mice highly susceptible to murine-coronavirus-MHV-A59/RSA59 infection due to reduced microglia/macrophage activation and significantly dampened effector CD4+ T recruitment to the CNS at the acute-adaptive bridging phase of neuroinflammation. CD40L deficiency significantly impaired the priming, expansion, and activation of lymphoid cells in the deep cervical lymph nodes. Additionally, both CD4−/− and CD40L−/− mice presented with severe chronic phase demyelination and axonal pathology in the brain and spinal cord which was correlated with prolonged virus persistence. Results from this study provide exciting information about a critical role of CD40L in mediating RSA59 induced activation of CD4+ T cells in the CLN and their consequent infiltration into CNS in addition to affecting the numbers and activation of monocyte/macrophage/microglia during the acute-adaptive transition phase. CD40-CD40L interaction in linking the innate-acute and chronic-adaptive immune responses may suggest a novel target in designing prophylaxis for virus-induced demyelination and axonal degeneration, in contrast to autoimmune suppression, which holds only for autoimmune mechanisms of inflammatory demyelination. This work was supported by a Department of Biotechnology, India, research grant (BT/PR 20922/MED/122/37/2016). We thank the Council of Scientific and Industrial Research (CSIR) India for providing fellowships to F.S. the Ministry of Human Resource Development (MHRD), India for providing fellowship to D.C; and the University Grants Commission (UGC), India, for providing fellowship to M.K.

T-Cell Aging-Associated Phenotypes in Autoimmune Disease.

The aging process causes profound restructuring of the host immune system, typically associated with declining host protection against cancer and infection. In the case of T cells, aging leads to the accumulation of a diverse set of T-cell aging-associated phenotypes (TASP), some of which have been implicated in driving tissue inflammation in autoimmune diseases. T cell aging as a risk determinant for autoimmunity is exemplified in two classical autoimmune conditions: rheumatoid arthritis (RA), a disease predominantly affecting postmenopausal women, and giant cell arteritis (GCA), an inflammatory vasculopathy exclusively occurring during the 6th–9th decade of life. Pathogenic T cells in RA emerge as a consequence of premature immune aging. They have shortening and fragility of telomeric DNA ends and instability of mitochondrial DNA. As a result, they produce a distinct profile of metabolites, disproportionally expand their endoplasmic reticulum (ER) membranes and release excess amounts of pro-inflammatory effector cytokines. Characteristically, they are tissue invasive, activate the inflammasome and die a pyroptotic death. Patients with GCA expand pathogenic CD4+ T cells due to aberrant expression of the co-stimulatory receptor NOTCH1 and the failure of the PD-1/PD-L1 immune checkpoint. In addition, GCA patients lose anti-inflammatory Treg cells, promoting tissue-destructive granulomatous vasculitis. In summary, emerging data identify T cell aging as a risk factor for autoimmune disease and directly link TASPs to the breakdown of T cell tolerance and T-cell-induced tissue inflammation.

Transcriptional profiling of macrophages reveals distinct parasite stage-driven signatures during early infection by Leishmania donovani

Macrophages undergo swift changes in mRNA abundance upon pathogen invasion. Herein we describe early remodelling of the macrophage transcriptome during infection by amastigotes or promastigotes of Leishmania donovani. Approximately 10–16% of host mRNAs were differentially modulated in L. donovani-infected macrophages when compared to uninfected controls. This response was partially stage-specific as a third of changes in mRNA abundance were either exclusively driven by one of the parasite forms or significantly different between them. Gene ontology analyses identified categories associated with immune functions (e.g. antigen presentation and leukocyte activation) among significantly downregulated mRNAs during amastigote infection while cytoprotective-related categories (e.g. DNA repair and apoptosis inhibition) were enriched in upregulated transcripts. Interestingly a combination of upregulated (e.g. cellular response to IFNβ) and repressed (e.g. leukocyte activation, chemotaxis) immune-related transcripts were overrepresented in the promastigote-infected dataset. In addition, Ingenuity Pathway Analysis (IPA) associated specific mRNA subsets with a number of upstream transcriptional regulators predicted to be modulated in macrophages infected with L. donovani amastigotes (e.g. STAT1 inhibition) or promastigotes (e.g. NRF2, IRF3, and IRF7 activation). Overall, our results indicate that early parasite stage-driven transcriptional remodelling in macrophages contributes to orchestrate both protective and deleterious host cell responses during L. donovani infection.

Neonatal Immune Responses to Respiratory Viruses.

Respiratory tract infections are a leading cause of morbidity and mortality in newborns, infants, and young children. These early life infections present a formidable immunologic challenge with a number of possibly conflicting goals: simultaneously eliminate the acute pathogen, preserve the primary gas-exchange function of the lung parenchyma in a developing lung, and limit long-term sequelae of both the infection and the inflammatory response. The latter has been most well studied in the context of childhood asthma, where multiple epidemiologic studies have linked early life viral infection with subsequent bronchospasm. This review will focus on the clinical relevance of respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and rhinovirus (RV) and examine the protective and pathogenic host responses within the neonate.

Neutrophils: Amoeboid Migration and Swarming Dynamics in Tissues.

Neutrophils are key cells of our innate immune response with essential roles for eliminating bacteria and fungi from tissues. They are also the prototype of an amoeboid migrating leukocyte. As one of the first blood-recruited immune cell types during inflammation and infection, these cells can invade almost any tissue compartment. Once in the tissue, neutrophils undergo rapid shape changes and migrate at speeds higher than most other immune cells. They move in a substrate-independent manner in interstitial spaces and do not follow predetermined tissue paths. Instead, neutrophil navigation is largely shaped by the chemokine and chemoattractant milieu around them. This highlights the decisive role of attractant-sensing G-protein coupled receptors (GPCRs) and downstream molecular pathways for controlling amoeboid neutrophil movement in tissues. A diverse repertoire of cell-surface expressed GPCRs makes neutrophils the perfect sentinel cell type to sense and detect danger-associated signals released from wounds, inflamed interstitium, dying cells, complement factors or directly from tissue-invading microbes. Moreover, neutrophils release attractants themselves, which allows communication and coordination between individual cells of a neutrophil population. GPCR-mediated positive feedback mechanisms were shown to underlie neutrophil swarming, a population response that amplifies the recruitment of amoeboid migrating neutrophils to sites of tissue injury and infection. Here we discuss recent findings and current concepts that counteract excessive neutrophil accumulation and swarm formation. In particular, we will focus on negative feedback control mechanisms that terminate neutrophil swarming to maintain the delicate balance between tissue surveillance, host protection and tissue destruction.

Filamentous prophage capsid proteins contribute to superinfection exclusion and phage defence in Pseudomonas aeruginosa.

Filamentous prophages in Pseudomonas aeruginosa PAO1 are converted to superinfective phage virions during biofilm development. Superinfection exclusion is necessary for the development of resistance against superinfective phage virions in host cells. However, the molecular mechanisms underlying the exclusion of superinfective Pf phages are unknown. In this study, we found that filamentous prophage-encoded structural proteins allow exclusion of superinfective Pf phages by interfering with type IV pilus (T4P) function. Specifically, the phage minor capsid protein pVII inhibits Pf phage adsorption by interacting with PilC and PilJ of T4P, and overproduction of pVII completely abrogates twitching motility. The minor capsid protein pIII provides partial superinfection exclusion and interacts with the PilJ and TolR/TolA proteins. Furthermore, pVII provides full host protection against infection by pilus-dependent lytic phages, and pIII provides partial protection against infection by pilus-independent lytic phages. Considering that filamentous prophages are common in clinical Pseudomonas isolates and their induction is often activated during biofilm formation, this study suggests the need to rethink the strategy of using lytic phages to treat P. aeruginosa biofilm-related infections.

Phagosomal signalling of the C-type lectin receptor Dectin-1 is terminated by intramembrane proteolysis

Sensing of pathogens by pattern recognition receptors (PRR) is critical to initiate protective host defence reactions. However, activation of the immune system has to be carefully titrated to avoid tissue damage necessitating mechanisms to control and terminate PRR signalling. Dectin-1 is a PRR for fungal β-glucans on immune cells that is rapidly internalised after ligand-binding. Here, we demonstrate that pathogen recognition by the Dectin-1a isoform results in the formation of a stable receptor fragment devoid of the ligand binding domain. This fragment persists in phagosomal membranes and contributes to signal transduction which is terminated by the intramembrane proteases Signal Peptide Peptidase-like (SPPL) 2a and 2b. Consequently, immune cells lacking SPPL2b demonstrate increased anti-fungal ROS production, killing capacity and cytokine responses. The identified mechanism allows to uncouple the PRR signalling response from delivery of the pathogen to degradative compartments and identifies intramembrane proteases as part of a regulatory circuit to control anti-fungal immune responses.

Reassembling a cannon in the DNA defense arsenal: Genetics of StySA, a BREX phage exclusion system in Salmonella lab strains.

Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA. Recent investigation has uncovered BREX (BacteRiophage EXclusion) systems. These RM-like activities employ host protection by DNA modification, but immediate replication arrest occurs without evident of nuclease action on unmodified phage DNA. Here we show that the historical stySA RM locus of Salmonella enterica sv Typhimurium is a variant BREX system. A laboratory strain disabled for both the restriction and methylation activity of StySA nevertheless has wild type sequence in pglX, the modification gene homolog. Instead, flanking genes pglZ and brxC each carry multiple mutations (μ) in their C-terminal domains. We further investigate this system in situ, replacing the mutated pglZμ and brxCμ genes with the WT counterpart. PglZ-WT supports methylation in the presence of either BrxCμ or BrxC-WT but not in the presence of a deletion/insertion allele, ΔbrxC::cat. Restriction requires both BrxC-WT and PglZ-WT, implicating the BrxC C-terminus specifically in restriction activity. These results suggests that while BrxC, PglZ and PglX are principal components of the BREX modification activity, BrxL is required for restriction only. Furthermore, we show that a partial disruption of brxL disrupts transcription globally.

NLRP1B and NLRP3 Control the Host Response following Colonization with the Commensal Protist Tritrichomonas musculis.

Commensal intestinal protozoa, unlike their pathogenic relatives, are neglected members of the mammalian microbiome. These microbes have a significant impact on the host's intestinal immune homeostasis, typically by elevating anti-microbial host defense. Tritrichomonas musculis, a protozoan gut commensal, strengthens the intestinal host defense against enteric Salmonella infections through Asc- and Il1r1-dependent Th1 and Th17 cell activation. However, the underlying inflammasomes mediating this effect remain unknown. In this study, we report that colonization with T. musculis results in an increase in luminal extracellular ATP that is followed by increased caspase activity, higher cell death, elevated levels of IL-1β, and increased numbers of IL-18 receptor-expressing Th1 and Th17 cells in the colon. Mice deficient in either Nlrp1b or Nlrp3 failed to display these protozoan-driven immune changes and lost resistance to enteric Salmonella infections even in the presence of T. musculis These findings demonstrate that T. musculis-mediated host protection requires sensors of extracellular and intracellular ATP to confer resistance to enteric Salmonella infections.

Interferons in coronavirus pathogenesis: The good, the bad, and the ugly

Interferons (IFNs) are a key component of the innate antiviral immunity and are generally implicated in protective host immune responses. Here, I discuss the central role of IFNs during different coronavirus (CoV) infections, the importance of timing of the IFN response, and how emerging human coronaviruses subvert antiviral IFN response to cause severe disease.