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Initiation of DNA replication is required for cell viability and passage of genetic information to the next generation. Studies in Escherichia coli and Bacillus subtilis have established A TPases a ssociated with diverse cellular a ctivities (AAA+) as essential proteins required for loading of the replicative helicase at replication origins.

Asthma affects nearly 260 million people worldwide, where severe asthma cases represent the most difficult to treat due to corticosteroid insensitivity. Severe asthma is associated with higher levels of TH17 and TH1 responses, accompanied by neutrophil dominated inflammation. Better understanding of the immune responses to airway allergens that promote or protect against severe asthma is critical for identifying ways to treat these patients. Single nucleotide polymorphisms in theATG5gene, which encodes for a protein required for the cellular recycling process of autophagy, has been associated with higher risk for developing severe asthma. However, the exact role for ATG5 during allergic inflammation and whether other autophagy proteins are involved remains elusive. Using genetic tools to dissect the roles for ATG5 in innate immune cells in house dust mite (HDM)-challenged mice, we have identified a specific role for ATG5 in CD11c+lung macrophages and dendritic cells for suppressing TH17 responses and neutrophil accumulation. We found that this role for ATG5 in CD11c+cells to regulate neutrophil accumulation during allergic airway inflammation also required other autophagy proteins but did not involve regulation of inflammasome, despite higher levels of IL-1β and Caspase 1 in the lungs of mice lacking autophagy in innate immune cells. Our data support a role for autophagy in CD11c+lung macrophages and dendritic cells to promote an immune response to airway allergens that is associated with less severe asthma by suppressing TH17 responses and neutrophil accumulation in the lung.

SUMMARYInflammation is critical for controlling infections, but when left unchecked can cause tissue damage and disease. For tuberculosis, the leading cause of death due to infection1, host inflammation is responsible for the clinical symptoms2, morbidity2, and mortality3,4. Specifically, neutrophil-dominated inflammation is associated with tuberculosis disease progression3,5,6. Therefore, understanding how neutrophil functions are regulated during infection is important for developing ways to prevent disease.Atg5was the first gene shown to specifically function within neutrophils to promote control ofMycobacterium tuberculosis7, the causative agent of tuberculosis. ATG5 is best studied for its role in autophagy8–11, however, the protective activity of ATG5 in neutrophils was unexpectedly independent of other autophagy proteins and remained elusive7. We report here that ATG5, but not other autophagy proteins, is required in neutrophils to suppress neutrophil NETosis and swarming that occur due to elevated type I interferon levels duringM. tuberculosisinfection. The elevated level of NETosis that results from loss of ATG5 expression contributes to the early susceptibility ofAtg5fl/fl-LysM-Cremice duringM. tuberculosisinfection. NETosis is associated with poor disease outcomes in tuberculosis12,13and COVID-19 patients14,15, as well as during other inflammatory diseases in humans16,17. Our studies identify an essential regulator of NETosis and elucidate previously unappreciated roles for ATG5 during infection, which may inform the design of host-directed therapeutics modulating these pathways.

Bacterial pathogens likeMycobacterium tuberculosis(Mtb) employ transcription factors to adapt their physiology to the diverse environments within their host. CarD is a conserved bacterial transcription factor that is essential for viability inMtb. Unlike classical transcription factors that recognize promoters by binding to specific DNA sequence motifs, CarD binds directly to the RNA polymerase (RNAP) to stabilize the open complex intermediate (RPo) during transcription initiation. We previously showed using RNA-sequencing that CarD is capable of both activating and repressing transcriptionin vivo. However, it is unknown how CarD achieves promoter specific regulatory outcomes inMtbdespite binding indiscriminate of DNA sequence. We propose a model where CarD’s regulatory outcome depends on the promoter’s basal RPostability and test this model usingin vitrotranscription from a panel of promoters with varying levels of RPostability. We show that CarD directly activates full-length transcript production from theMtbribosomal RNA promoterrrnAP3 (AP3) and that the degree of transcription activation by CarD is negatively correlated with RPostability. Using targeted mutations in the extended −10 and discriminator region of AP3, we show that CarD directly represses transcription from promoters that form relatively stable RPo. DNA supercoiling also influenced RPostability and affected the direction of CarD regulation, indicating that the outcome of CarD activity can be regulated by factors beyond promoter sequence. Our results provide experimental evidence for how RNAP-binding transcription factors like CarD can exert specific regulatory outcomes based on the kinetic properties of a promoter.

Of the approximately 10 million cases ofMycobacterium tuberculosis(Mtb) infections each year, over 10% are resistant to the frontline antibiotic isoniazid (INH). INH resistance is predominantly caused by mutations that decrease the activity of the bacterial enzyme KatG, which mediates conversion of the pro-drug INH to its active form INH-NAD. We previously discovered an inhibitor ofMtbrespiration, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a collection of INH-resistant mutants to INH through an unknown mechanism. To investigate the mechanism of action of C10, we exploited the toxicity of high concentrations of C10 to select for resistant mutants. We discovered two mutations that confer resistance to the disruption of energy metabolism and allow for growth ofMtbin high C10 concentrations, indicating that growth inhibition by C10 is associated with inhibition of respiration. Using these mutants as well as direct inhibitors of theMtbelectron transport chain, we provide evidence that inhibition of energy metabolism by C10 is neither sufficient nor necessary to potentiate killing by INH. Instead, we find that C10 acts downstream of INH-NAD synthesis, causingMtbto become particularly sensitive to inhibition of the INH-NAD target, InhA, without changing the concentration of INH-NAD or the activity of InhA, the two predominant mechanisms of potentiating INH. Our studies revealed that there exists a vulnerability inMtbthat can be exploited to renderMtbsensitive to otherwise subinhibitory concentrations of InhA inhibitor.SignificanceIsoniazid (INH) is a critical frontline antibiotic to treatMycobacterium tuberculosis(Mtb) infections. INH efficacy is limited by its suboptimal penetration of theMtb-containing lesion and by the prevalence of clinical INH-resistance. We previously discovered a compound, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a set of INH-resistant mutants to INH. Resistance is typically mediated bykatGmutations that decrease the activation of INH, which is required for INH to inhibit the essential enzyme InhA. Our current work demonstrates that C10 re-sensitizes INH-resistantkatG-hypomorphs without enhancing the activation of INH. We furthermore show that C10 causesMtbto become particularly vulnerable to InhA inhibition without compromising InhA activity on its own. Therefore, C10 represents a novel strategy to curtail the development of INH resistance and to sensitizeMtbto sub-lethal doses of INH, such as those achieved at the infection site.

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homologue (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomesin vivoremains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in non-starved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12-stalk base, suggesting that the aminoacylation status of A-site tRNA is surveyed during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle.SignificanceBacteria persist under nutrient starvation by activating RelA/SpoT homologue (Rsh), which synthesizes a growth regulating alarmone, ppGpp. Rsh is activated specifically upon recognizing a translation elongation complex with deacylated tRNA at the A-site. It is however unclear how Rsh identifies such a complex in vivo. We show here that conditions inducing ribosome hibernation in mycobacteria cause loss of intracellular Rsh, implying that association with translating ribosomes is necessary for intracellular stability of Rsh. Using structural analysis of Rsh-bound 70S translation initiation complex, we propose here that mycobacterial Rsh identifies a Rsh-activating ribosomal complex by constitutively surveying the ribosome entering the translation cycle at the early elongation stage.

SUMMARYNeutrophils are the most abundant cell type in airways of tuberculosis patients. Recent investigations reported induction of neutrophil extracellular traps (NETs) duringMycobacterium tuberculosis(Mtb) infection, however, the molecular regulation and impact of NETosis onMtbpathogenesis is unknown. We find that in response toMtbinfection in neutrophils, PAD4 citrullinates histones to decondense chromatin that gets packaged into vesicles for release as NETs in a manner that can maintain neutrophil viability and promoteMtbreplication. Type I interferon, which has been associated with NETosis in numerous contexts but without a known mechanism, promotes formation of chromatin-containing vesicles and NET release. Analysis of nonhuman primate granulomas supports a model where neutrophils are exposed to type I interferon from macrophages as they migrate into the granuloma, where they release NETs that contribute to necrosis and caseation. Our data reveals NETosis as a promising target to inhibitMtbreplication and granuloma caseation.

Tuberculosis (TB) results in 1.5 million deaths every year. The rise in multi-drug resistant TB underscores the urgent need to develop new antibacterials, particularly those with new chemical entities and/or novel mechanisms of action that can be used in combination therapy with existing drugs to prevent the rapid emergence of resistance. Herein, we report the discovery and synthesis of a new series of compounds containing a 3-thio-1,2,4-triazole moiety that show inhibition ofMycobacterium tuberculosis(Mtb) growth and survival. Structure-activity relationship studies led us to identify potent analogs displaying nanomolar inhibitor activity, specifically againstMtb. These potent analogs exhibit a promising ADME/pharmacokinetic profile and no cytotoxicity in mammalian cells at over 100 times the effective dose inMtb. Our preliminary investigations into the mechanism of action suggest this series is not engaging promiscuous targets and, thereby, could be acting on a novel target.Abstract Figure

SummaryHost autophagy had been associated with the control ofMycobacterium tuberculosis(Mtb) infection due to its ability to sequesters microorganisms through a process termed “xenophagy”1–4. Xenophagy purportedly limits Mtb replication within infected macrophages1–4. However, studies in mice using a standard low-dose infection model demonstrated that xenophagy in infected phagocytes is not required to control Mtb pathogenesis5,6. Instead, an autophagy-independent function of ATG5 in myeloid cells controls low-dose Mtb infection through limiting neutrophilic inflammation5. Hitherto, anin vivorole for autophagy during Mtb infection remained to be elucidated. We report herein that autophagy in myeloid cells mediates protection against high-dose Mtb infection, providing the first evidence for a role for autophagy in myeloid cells during Mtb infectionin vivo. With the exception of ATG5, the autophagy proteins required to control high-dose Mtb infection are dispensable for host defense against a standard low-dose Mtb infection. Specifically, autophagy is required in CD11c+cells, but is dispensable in neutrophils, to control a high-dose Mtb infection in the lung. The role for autophagy is not to directly degrade Mtb in macrophages through xenophagy, but mainly to limit myeloid-derived suppressor cell accumulation and to promote sustained protective T cell responses. Together, our data highlight a novel role for autophagy in controlling Mtb infection, distinct from that of Atg5 during low-dose Mtb infection, or any previously reported roles for autophagy. In addition, our finding that the result of a pathogen-plus-susceptibility gene interaction is dependent on pathogen burden has important implications on our understanding of how Mtb infection in humans can lead to a spectrum of outcomes, the variables that contribute to autophagy gene function during infection and inflammation, and the potential use of autophagy modulators in clinical medicine.

The immune response toMycobacterium tuberculosisinfection determines tuberculosis disease outcomes, yet we have an incomplete understanding of what immune factors contribute to a protective immune response. Neutrophilic inflammation has been associated with poor disease prognosis in humans and in animal models duringM. tuberculosisinfection and, therefore, must be tightly regulated. ATG5 is an essential autophagy protein that is required in innate immune cells to control neutrophil-dominated inflammation and promote survival duringM. tuberculosisinfection, however, the mechanistic basis for how ATG5 regulates neutrophil recruitment is unknown. To interrogate what innate immune cells require ATG5 to control neutrophil recruitment duringM. tuberculosisinfection, we used different mouse strains that conditionally deleteAtg5in specific cell types. We found that ATG5 is required in CD11c+cells (lung macrophages and dendritic cells) to control the production of proinflammatory cytokines and chemokines duringM. tuberculosisinfection, which would otherwise promote neutrophil recruitment. This role for ATG5 is autophagy-dependent, but independent of mitophagy, LC3-associated phagocytosis, and inflammasome activation, which are the most well-characterized ways that autophagy proteins regulate inflammation. In addition to the increase in proinflammatory cytokine production duringM. tuberculosisinfection, loss of ATG5 in innate immune cells also results in an early induction of TH17 responses. Despite prior publishedin vitrocell culture experiments supporting a role for autophagy in controllingM. tuberculosisreplication in macrophages, loss of autophagy does not affectM. tuberculosisburden in macrophagesin vivoand, therefore, the effects of autophagy on inflammatory responses occur without changes in pathogen numbers. These findings reveal new roles for autophagy proteins in lung resident macrophages and dendritic cells that are required to suppress inflammatory responses that are associated with poor control ofM. tuberculosisinfection.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a major health threat listed among the top 10 causes of death worldwide. Treatment of multidrug-resistant Mtb requires use of additional second-line drugs that prolong the treatment process and result in higher death rates. Our team previously identified a 2-pyridone molecule (C10) that blocks tolerance to the first-line drug isoniazid at C10 concentrations that do not inhibit bacterial growth. Here, we discovered that the genes rv3160c and rv3161c are highly induced by C10, which led us to investigate them as potential targets. We show that Rv3160c acts as a TetR-like transcriptional repressor binding to a palindromic sequence located in the rv3161c promoter. We also demonstrate that C10 interacts with Rv3160c, inhibiting its binding to DNA. We deleted the rv3161c gene, coding for a putative oxygenase, to investigate its role in drug and stress sensitivity as well as C10 activity. This Δrv3161c strain was more tolerant to isoniazid and lysozyme than wild type Mtb. However, this tolerance could still be blocked by C10, suggesting that C10 functions independently of Rv3161c to influence isoniazid and lysozyme sensitivity.

Innate and adaptive immune responses that prime myeloid cells, such as macrophages, protect against pathogens1,2. However, if left uncontrolled, these responses may lead to detrimental inflammation3. Macrophages, particularly those resident in tissues, must therefore remain quiescent between infections despite chronic stimulation by commensal microorganisms. The genes required for quiescence of tissue-resident macrophages are not well understood. Autophagy, an evolutionarily conserved cellular process by which cytoplasmic contents are targeted for lysosomal digestion, has homeostatic functions including maintenance of protein and organelle integrity and regulation of metabolism4. Recent research has shown that degradative autophagy, as well as various combinations of autophagy genes, regulate immunity and inflammation5–12. Here, we delineate a function of the autophagy proteins Beclin 1 and FIP200—but not of other essential autophagy components ATG5, ATG16L1 or ATG7—in mediating quiescence of tissue-resident macrophages by limiting the effects of systemic interferon-γ. The perturbation of quiescence in mice that lack Beclin 1 or FIP200 in myeloid cells results in spontaneous immune activation and resistance to Listeria monocytogenes infection. While antibiotic-treated wild-type mice display diminished macrophage responses to inflammatory stimuli, this is not observed in mice that lack Beclin 1 in myeloid cells, establishing the dominance of this gene over effects of the bacterial microbiota. Thus, select autophagy genes, but not all genes essential for degradative autophagy, have a key function in maintaining immune quiescence of tissue-resident macrophages, resulting in genetically programmed susceptibility to bacterial infection. The autophagy proteins Beclin 1 and FIP200, but not other essential autophagy components, such as ATG5, ATG16L1 or ATG7, regulate quiescence of tissue-resident macrophages, thereby modulating immune activation and resistance to Listeria monocytogenes infection.

A fundamental requirement for life is the replication of an organism’s DNA. Studies in Escherichia coli and Bacillus subtilis have set the paradigm for DNA replication in bacteria. During replication initiation in E. coli and B. subtilis, the replicative helicase is loaded onto the DNA at the origin of replication by an ATPase helicase loader. However, most bacteria do not encode homologs to the helicase loaders in E. coli and B. subtilis. Recent work has identified the DciA protein as a predicted helicase operator that may perform a function analogous to the helicase loaders in E. coli and B. subtilis. DciA proteins, which are defined by the presence of a DUF721 domain (termed the DciA domain herein), are conserved in most bacteria but have only been studied in mycobacteria and γ-proteobacteria (Pseudomonas aeruginosa and Vibrio cholerae). Sequences outside of the DciA domain in Mycobacterium tuberculosis DciA are essential for protein function but are not conserved in the P. aeruginosa and V. cholerae homologs, raising questions regarding the conservation and evolution of DciA proteins across bacterial phyla. To comprehensively define the DciA protein family, we took a computational evolutionary approach and analyzed domain architectures and sequence properties of DciA-domain containing proteins across the tree of life. These analyses identified lineage-specific domain architectures amongst DciA homologs as well as broadly conserved sequence-structural motifs. The diversity of DciA proteins represents the evolution of helicase operation in bacterial DNA replication and highlights the need for phylum-specific analyses of this fundamental biological process.IMPORTANCEDespite the fundamental importance of DNA replication for life, this process remains understudied in bacteria outside of Escherichia coli and Bacillus subtilis. In particular, most bacteria do not encode the helicase loading proteins that are essential in E. coli and B. subtilis for DNA replication. Instead, most bacteria encode a DciA homolog that likely constitutes the predominant mechanism of helicase operation in bacteria. However, it is still unknown how DciA structure and function compare across diverse phyla that encode DciA proteins. In this study, we perform computational evolutionary analyses to uncover tremendous diversity amongst DciA homologs. These studies provide a significant advance in our understanding of an essential component of the bacterial DNA replication machinery.

Macroautophagy/autophagy regulates inflammation via multiple mechanisms, including lysosomal degradation of specific cellular components. Certain autophagy gene “cassettes” also participate in non-canonical processes to mediate important biological activities. While select autophagy genes in myeloid cells have been implicated in protecting mice in models of cytokine storm syndromes (CSS), a more extensive genetic analysis of the autophagy pathway for this disorder has not been reported to date. We determined that multiple canonical autophagy genes in the myeloid compartment protected against fatal disease from both intravenous TNF and intraperitoneal LPS, with the notable exception that Atg14 was dispensable for the latter. Serum cytokine analyses and genetic crosses further revealed distinct mechanisms contribute to the hypersensitivity of autophagy gene-deficient mice in these CSS models. Surprisingly, TNF was dispensable for the increased mortality of myeloid 5-deficient mice challenged with LPS. Tissue-specific ablation of Atg5 in cells expressing ITGAX/CD11c and LYZ2/LYSM, but not S100A8/MRP8, defined a myeloid subset that protected against TNF, while protection against LPS was conferred by Atg5 in a distinct subset of LYZ2-expressing cells. Together, this study identifies autophagy gene sets and specific cell types that protect against fatal inflammation due to CSS, highlighting important differences in two commonly used murine models of the disorder. ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; ATG16L1: autophagy related 16-like 1 (S. cerevisiae); BECN1: beclin 1, autophagy related; CASP1: caspase 1; CASP4/CASP11: caspase 4, apoptosis-related cysteine peptidase; CIM: conditionally immortalized macrophage; CLP: cecal ligation and puncture; CSS: cytokine storm syndrome; DC: dendritic cell; IFNG/IFNγ: interferon gamma; IFNGR1: interferon gamma receptor 1; ip: intraperitoneal; iv: intravenous; IL12/p70: interleukin 12, p70 heterodimer; IL18: Interleukin 18; ITGAX/CD11c: integrin alpha X; LAP: LC3-associated phagocytosis; LPS: lipopolysaccharide; LYZ2/LYSM: lysozyme 2; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; RB1CC1/FIP200: RB1-inducible coiled-coil 1; S100A8/MRP8: S100 calcium binding protein A8 (calgranulin A); TICAM1/TRIF: TIR domain containing adaptor molecule 1; TLR4: toll-like receptor 4; TNF: tumor necrosis factor.

RNA polymerase (RNAP) binding protein A (RbpA) is essential for mycobacterial viability and regulates transcription initiation by increasing the stability of the RNAP-promoter open complex (RP

Summary Although programmed cell death can control intracellular bacterial replication, the role of cell death during systemic Salmonella infection remained elusive. In this issue of Immunity, Doerflinger et al. discover a critical but overlapping role for cell death associated caspases during Salmonella infection.

The ability to regulate gene expression through transcription initiation underlies the adaptability and survival of all bacteria. Recent work has revealed that the transcription machinery in many bacteria diverges from the paradigm that has been established in Escherichia coli . Mycobacterium tuberculosis ( Mtb ) encodes the RNA polymerase (RNAP)-binding protein CarD, which is absent in E. coli but is required to form stable RNAP-promoter open complexes (RP o ) and is essential for viability in Mtb . The stabilization of RP o by CarD has been proposed to result in activation of gene expression; however, CarD has only been examined on limited promoters that do not represent the typical promoter structure in Mtb . In this study, we investigate the outcome of CarD activity on gene expression from Mtb promoters genome-wide by performing RNA sequencing on a panel of mutants that differentially affect CarD’s ability to stabilize RP o . In all CarD mutants, the majority of Mtb protein encoding transcripts were differentially expressed, demonstrating that CarD had a global effect on gene expression. Contrary to the expected role of CarD as a transcriptional activator, mutation of CarD led to both up- and down-regulation of gene expression, suggesting that CarD can also act as a transcriptional repressor. Furthermore, we present evidence that stabilization of RP o by CarD could lead to transcriptional repression by inhibiting promoter escape, and the outcome of CarD activity is dependent on the intrinsic kinetic properties of a given promoter region. Collectively, our data support CarD’s genome-wide role of regulating diverse transcription outcomes.

The pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, enacts unique transcriptional regulatory mechanisms when subjected to host-derived stresses. Initiation of transcription by the Mycobacterial RNA polymerase (RNAP) has previously been shown to exhibit different open complex kinetics and stabilities relative to Escherichia coli (Eco) RNAP. However, transcription initiation rates also depend on the kinetics following open complex formation such as initial nucleotide incorporation and subsequent promoter escape. Here, using a real-time fluorescence assay, we present the first in-depth kinetic analysis of initial transcription and promoter escape for the Mtb RNAP. We show that in relation to Eco RNAP, Mtb displays slower initial nucleotide incorporation but faster overall promoter escape kinetics on the Mtb rrnAP3 promoter. Furthermore, in the context of the essential transcription factors CarD and RbpA, Mtb promoter escape is slowed via differential effects on initially transcribing complexes. Finally, based on their ability to increase the rate of open complex formation and decrease the rate of promoter escape, we suggest that CarD and RbpA are capable of activation or repression depending on the rate-limiting step of a given promoter's basal initiation kinetics.

Mycobacterium tuberculosis ( Mtb ) killed more people in 2017 than any other single infectious agent. This dangerous pathogen is able to withstand stresses imposed by the immune system and tolerate exposure to antibiotics, resulting in persistent infection. The global tuberculosis (TB) epidemic has been exacerbated by the emergence of mutant strains of Mtb that are resistant to frontline antibiotics. Thus, both phenotypic drug tolerance and genetic drug resistance are major obstacles to successful TB therapy. Using a chemical approach to identify compounds that block stress and drug tolerance, as opposed to traditional screens for compounds that kill Mtb , we identified a small molecule, C10, that blocks tolerance to oxidative stress, acid stress, and the frontline antibiotic isoniazid (INH). In addition, we found that C10 prevents the selection for INH-resistant mutants and restores INH sensitivity in otherwise INH-resistant Mtb strains harboring mutations in the katG gene, which encodes the enzyme that converts the prodrug INH to its active form. Through mechanistic studies, we discovered that C10 inhibits Mtb respiration, revealing a link between respiration homeostasis and INH sensitivity. Therefore, by using C10 to dissect Mtb persistence, we discovered that INH resistance is not absolute and can be reversed.

Chemical synthesis of conjugate vaccines, consisting of a polysaccharide linked to a protein, can be technically challenging, and in vivo bacterial conjugations (bioconjugations) have emerged as manufacturing alternatives. Bioconjugation relies upon an oligosaccharyltransferase to attach polysaccharides to proteins, but currently employed enzymes are not suitable for the generation of conjugate vaccines when the polysaccharides contain glucose at the reducing end, which is the case for ~75% of Streptococcus pneumoniae capsules. Here, we use an O-linking oligosaccharyltransferase to generate a polyvalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at their reducing end. In addition, we show that different vaccine carrier proteins can be glycosylated using this system. Pneumococcal bioconjugates are immunogenic, protective and rapidly produced within E. coli using recombinant techniques. These proof-of-principle experiments establish a platform to overcome limitations of other conjugating enzymes enabling the development of bioconjugate vaccines for many important human and animal pathogens., Bioconjugation is a promising process to manufacture conjugate vaccines, but currently employed enzymes cannot generate the full spectrum of bacterial glycoproteins. Here, the authors use an O-linking oligosaccharyltransferase to generate a polyvalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at their reducing end.

Significance MM3122 represents an advanced lead candidate for clinical development as a novel antiviral drug for COVID-19. In addition to being novel drugs, these selective TMRSS2 inhibitors can be used as valuable chemical probes to help elucidate mechanisms of viral pathogenesis. Since TMPRSS2 plays a key role as a viral protein processing protease in the pathogenesis of other coronaviruses (SARS-CoV, MERS-CoV) as well as influenza viruses, MM3122 and this class of TMPRSS2 inhibitors hold much promise as new drugs to not only treat SARS-CoV-2 infections but also potentially represent broad-spectrum antivirals., The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

Mycobacterium tuberculosis ( Mtb), the causative agent of tuberculosis (TB), remains a leading cause of death due to infection in humans. To more effectively combat this pandemic, many aspects of TB control must be developed, including better point of care diagnostics, shorter and safer drug regimens, and a protective vaccine. To address all these areas of need, better understanding of the pathogen, host responses, and clinical manifestations of the disease is required. Recently, the application of cutting-edge technologies to the study of Mtb pathogenesis has resulted in significant advances in basic biology, vaccine development, and antibiotic discovery. This leaves us in an exciting era of Mtb research in which our understanding of this deadly infection is improving at a faster rate than ever, and renews hope in our fight to end TB. In this review, we reflect on what is known regarding Mtb pathogenesis, highlighting recent breakthroughs that will provide leverage for the next leaps forward in the field.

Macrophages activated with interferon-γ (IFN-γ) in combination with other proinflammatory stimuli, such as lipopolysaccharide or tumor necrosis factor-α (TNF-α), respond with transcriptional and cellular changes that enhance clearance of intracellular pathogens at the risk of damaging tissues. IFN-γ effects must therefore be carefully balanced with inhibitory mechanisms to prevent immunopathology. We performed a genome-wide CRISPR knockout screen in a macrophage cell line to identify negative regulators of IFN-γ responses. We discovered an unexpected role of the ubiquitin-fold modifier (Ufm1) conjugation system (herein UFMylation) in inhibiting responses to IFN-γ and lipopolysaccharide. Enhanced IFN-γ activation in UFMylation-deficient cells resulted in increased transcriptional responses to IFN-γ in a manner dependent on endoplasmic reticulum stress responses involving Ern1 and Xbp1. Furthermore, UFMylation in myeloid cells is required for resistance to influenza infection in mice, indicating that this pathway modulates in vivo responses to infection. These findings provide a genetic roadmap for the regulation of responses to a key mediator of cellular immunity and identify a molecular link between the UFMylation pathway and immune responses.

This study demonstrates that the transcription factor basic helix-loop-helix family member e40 (Bhlhe40) is essential during Mycobacterium tuberculosis infection to specifically regulate Il10 expression, revealing the importance of strict control of IL-10 production by innate and adaptive immune cells during infection., The cytokine IL-10 antagonizes pathways that control Mycobacterium tuberculosis (Mtb) infection. Nevertheless, the impact of IL-10 during Mtb infection has been difficult to decipher because loss-of-function studies in animal models have yielded only mild phenotypes. We have discovered that the transcription factor basic helix-loop-helix family member e40 (Bhlhe40) is required to repress Il10 expression during Mtb infection. Loss of Bhlhe40 in mice results in higher Il10 expression, higher bacterial burden, and early susceptibility similar to that observed in mice lacking IFN-γ. Deletion of Il10 in Bhlhe40−/− mice reverses these phenotypes. Bhlhe40 deletion in T cells or CD11c+ cells is sufficient to cause susceptibility to Mtb. Bhlhe40 represents the first transcription factor found to be essential during Mtb infection to specifically regulate Il10 expression, revealing the importance of strict control of IL-10 production by innate and adaptive immune cells during infection. Our findings uncover a previously elusive but significant role for IL-10 in Mtb pathogenesis., Graphical Abstract

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

There is a clear link between defects in autophagy and the development of autoimmune and chronic inflammatory diseases, raising interest in better understanding the roles of autophagy within the immune system. In addition, autophagy has been implicated in the immune response to infection by pathogenic microbes. As such, there are efforts currently underway to develop modulators of autophagy as a therapeutic strategy for the treatment of the autoimmune, inflammatory, and infectious diseases. In this review, we discuss the numerous roles for autophagy in immunity and how these activities are linked to disease. We highlight how autophagy affects pathogen clearance, phagocytosis, pattern recognition receptor signaling, inflammation, antigen presentation, cell death, and immune cell development and maintenance. With these diverse and extensive immune-related functions for autophagy in mind, we finish by considering the possible implications of targeting autophagy as a therapeutic strategy.

Nair et al. define a key role for Irg1 in minimizing the pathological immune response associated with Mtb infection. Using Irg1−/− and Irg1fl/fl conditional mice, detailed immune cell analysis, and transcriptional profiling, their data supports a model where Irg1 expression in myeloid cell subsets tempers inflammation and controls the recruitment and infection of neutrophils during Mtb infection., Immune-Responsive Gene 1 (Irg1) is a mitochondrial enzyme that produces itaconate under inflammatory conditions, principally in cells of myeloid lineage. Cell culture studies suggest that itaconate regulates inflammation through its inhibitory effects on cytokine and reactive oxygen species production. To evaluate the functions of Irg1 in vivo, we challenged wild-type (WT) and Irg1−/− mice with Mycobacterium tuberculosis (Mtb) and monitored disease progression. Irg1−/−, but not WT, mice succumbed rapidly to Mtb, and mortality was associated with increased infection, inflammation, and pathology. Infection of LysM-Cre Irg1fl/fl, Mrp8-Cre Irg1fl/fl, and CD11c-Cre Irg1fl/fl conditional knockout mice along with neutrophil depletion experiments revealed a role for Irg1 in LysM+ myeloid cells in preventing neutrophil-mediated immunopathology and disease. RNA sequencing analyses suggest that Irg1 and its production of itaconate temper Mtb-induced inflammatory responses in myeloid cells at the transcriptional level. Thus, an Irg1 regulatory axis modulates inflammation to curtail Mtb-induced lung disease., Graphical Abstract

Research in recent years has focused significantly on the role of selective macroautophagy in targeting intracellular pathogens for lysosomal degradation, a process termed xenophagy. In this review we evaluate the proposed roles for xenophagy in controlling bacterial infection, highlighting the concept that successful pathogens have evolved ways to subvert or exploit this defense, minimizing the actual effectiveness of xenophagy in innate immunity. Instead, studies in animal models have revealed that autophagy-associated proteins often function outside of xenophagy to influence bacterial pathogenesis. In light of current efforts to manipulate autophagy and the development of host-directed therapies to fight bacterial infections, we also discuss the implications stemming from the complicated relationship that exists between autophagy and bacterial pathogens.