Nod-Like Receptor (NLR) is the largest family of Pathogen Recognition Receptors (PRRs) that patrols the cytosolic environment. NLR engagement drives caspase-1 activation that cleaves pro-IL-1B which then gets secreted. Released IL-1B recruits immune cells to the site of infection/injury. Caspase-1 also cleaves Gasdermin-D (GSDM-D) that forms pores within the plasma membrane driving inflammatory cell death called pyroptosis. NLRP3 is the most extensively studied NLR. The NLRP3 gene is encoded by 9 exons, where exon 1 codes for pyrin domain, exon 3 codes for NACHT domain, and Leucine Rich Repeat (LRR) domain is coded by exon 4-9. Exon 2 codes for a highly disorganized loop that connects the rest of the protein to the pyrin domain and may be involved in NLRP3 regulation. The NLRP3 inflammasome is activated by many structurally divergent agonists of microbial, environmental, and host origin. Activated NLRP3 interacts with an adaptor protein, ASC, that bridges it to pro-Caspase-1 forming a multi-protein complex called inflammasome. Dysregulation of NLRP3 inflammasome activity is a hallmark of pathogenesis in several human diseases, indicating its highly significant clinical relevance. In this review, we summarize the existing knowledge about the mechanism of activation of NLRP3 and its regulation during activation by infectious and sterile triggers.

Nod-Like Receptor (NLR) is the largest family of Pathogen Recognition Receptors (PRRs) that patrols the cytosolic environment. NLR engagement drives caspase-1 activation that cleaves pro-IL-1B which then gets secreted. Released IL-1B recruits immune cells to the site of infection/injury. Caspase-1 also cleaves Gasdermin-D (GSDM-D) that forms pores within the plasma membrane driving inflammatory cell death called pyroptosis. NLRP3 is the most extensively studied NLR. The NLRP3 gene is encoded by 9 exons, where exon 1 codes for pyrin domain, exon 3 codes for NACHT domain, and Leucine Rich Repeat (LRR) domain is coded by exon 4-9. Exon 2 codes for a highly disorganized loop that connects the rest of the protein to the pyrin domain and may be involved in NLRP3 regulation. The NLRP3 inflammasome is activated by many structurally divergent agonists of microbial, environmental, and host origin. Activated NLRP3 interacts with an adaptor protein, ASC, that bridges it to pro-Caspase-1 forming a multi-protein complex called inflammasome. Dysregulation of NLRP3 inflammasome activity is a hallmark of pathogenesis in several human diseases, indicating its highly significant clinical relevance. In this review, we summarize the existing knowledge about the mechanism of activation of NLRP3 and its regulation during activation by infectious and sterile triggers.

Non-human primate models will expedite therapeutics and vaccines for coronavirus disease 2019 (COVID-19) to clinical trials. Here, we compare acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old rhesus macaques, baboons and old marmosets. Macaques had clinical signs of viral infection, mild to moderate pneumonitis and extra-pulmonary pathologies, and both age groups recovered in two weeks. Baboons had prolonged viral RNA shedding and substantially more lung inflammation compared with macaques. Inflammation in bronchoalveolar lavage was increased in old versus young baboons. Using techniques including computed tomography imaging, immunophenotyping, and alveolar/peripheral cytokine response and immunohistochemical analyses, we delineated cellular immune responses to SARS-CoV-2 infection in macaque and baboon lungs, including innate and adaptive immune cells and a prominent type-I interferon response. Macaques developed T-cell memory phenotypes/responses and bystander cytokine production. Old macaques had lower titres of SARS-CoV-2-specific IgG antibody levels compared with young macaques. Acute respiratory distress in macaques and baboons recapitulates the progression of COVID-19 in humans, making them suitable as models to test vaccines and therapies. Acute respiratory distress in macaques and baboons recapitulates COVID-19 disease progression in humans, making them suitable as models to test vaccines and therapies.

While the advent of combination antiretroviral therapy (cART, ART) has significantly improved survival, tuberculosis (TB) still accounts for one-third of deaths in Human Immunodeficiency Virus (HIV)-infected populations, suggesting incomplete restoration of immune function by ART.Mtb/Simian Immunodeficiency Virus (SIV) co-infected macaques have been used to model Mtb/HIV co-infection in humans. To identify the components of TB immunity that remain impaired following HIV-infection and ART treatment, macaques with asymptomatic M. tuberculosis (Mtb) infection were subsequently co-infected with SIV. Animals were then randomly assigned to ART or control groups and studied longitudinally.While ART significantly reduced viral loads and increased CD4+T cell counts in whole blood and BAL samples, it did not reduce the relative risk of SIV- induced TB reactivation (RR 1·23, 95% CI 0·97-1·5) in ART treated macaques. CD4+T cells were poorly restored specifically in the lung interstitial region, despite their significant restoration in the alveolar compartment of the lung as well as in the periphery. IDO1 induction on myeloid cells in the iBALT likely contributes to dysregulated T cell homing and impaired lung immunity.Thus, while ART is indispensable for controlling viral replication, CD4+T cells restoration and in preventing opportunistic infection, it is however inadequate in reversing Mtb-specific T cell impairment. This warrants modeling concurrent treatment of TB and HIV to potentially reduce the risk of reactivation of TB due to HIV. The current and future studies like this have the potential to inform treatment strategies in patients with Mtb/HIV co-infection. The study was funded through grants from the National Institute of Health (NIH).Funding Statement: This work was primarily supported by NIH awards to D.K. and J.R. (R01AI111943, R01AI123047) with additional support from NIH awards R01AI111914, R01AI134240, R01AI135726, U19AI111211 and institutional awards P51OD111033 and P51OD111004.Declaration of Interests: The authors declare that no conflict of interest exists.Ethics Approval Statement: All the animals were housed in the Animal Biosafety level III (ABSL3) at Tulane National Primate Research Center where they were treated as per the standards recommended by Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International and the NIH Guide for the Care and Use of Laboratory Animals. The study was approved by the IACUC of the Tulane National Primate Research Center (protocols P0247R, P0324, P0295R).

SummaryThere are no known cures or vaccines for COVID-19, the defining pandemic of this era. Animal models are essential to fast track new interventions and nonhuman primate (NHP) models of other infectious diseases have proven extremely valuable. Here we compare SARS-CoV-2 infection in three species of experimentally infected NHPs (rhesus macaques, baboons, and marmosets). During the first 3 days, macaques developed clinical signatures of viral infection and systemic inflammation, coupled with early evidence of viral replication and mild-to-moderate interstitial and alveolar pneumonitis, as well as extra-pulmonary pathologies. Cone-beam CT scans showed evidence of moderate pneumonia, which progressed over 3 days. Longitudinal studies showed that while both young and old macaques developed early signs of COVID-19, both groups recovered within a two-week period. Recovery was characterized by low-levels of viral persistence in the lung, suggesting mechanisms by which individuals with compromised immune systems may be susceptible to prolonged and progressive COVID-19. The lung compartment contained a complex early inflammatory milieu with an influx of innate and adaptive immune cells, particularly interstitial macrophages, neutrophils and plasmacytoid dendritic cells, and a prominent Type I-interferon response. While macaques developed moderate disease, baboons exhibited prolonged shedding of virus and extensive pathology following infection; and marmosets demonstrated a milder form of infection. These results showcase in critical detail, the robust early cellular immune responses to SARS-CoV-2 infection, which are not sterilizing and likely impact development of antibody responses. Thus, various NHP genera recapitulate heterogeneous progression of COVID-19. Rhesus macaques and baboons develop different, quantifiable disease attributes making them immediately available essential models to test new vaccines and therapies.

While the advent of combination antiretroviral therapy (ART) has significantly improved survival, tuberculosis (TB) remains the leading cause of death in the HIV-infected population. We employed Mtb/Simian Immunodeficiency Virus (SIV) co-infected macaques to model Mtb/HIV co-infection and study the impact of ART on TB reactivation due to HIV-infection. While ART significantly reduced viral loads and increased CD4+ T cell counts in whole blood and BAL samples, it did not reduce the relative risk of SIV- induced TB reactivation in ART treated macaques in the early phase of treatment. CD4+ T cells were poorly restored specifically in the lung interstitium, despite their significant restoration in the alveolar compartment of the lung as well as in the periphery. IDO1 induction on myeloid cells in the iBALT likely contributed to dysregulated T cell homing and impaired lung immunity. Thus, while ART is indispensable in controlling viral replication, CD4+ T cells restoration and preventing opportunistic infection, it appears inadequate in reversing clinical signs of TB reactivation during the relatively short duration of ART and follow-up during this study. This warrants modeling concurrent treatment of TB and HIV to potentially reduce the risk of reactivation of TB due to HIV. The current and future studies like this have the potential to inform treatment strategies in patients with Mtb/HIV co-infection.

The resuscitation-promoting factors of Mycobacterium tuberculosis are hydrolytic enzymes, which are required for resuscitation of dormant cells. RpfB, a peptidoglycan remodelling enzyme similar to the lytic transglycosylase of Escherichia coli, is required for reactivation of M. tuberculosis from chronic infection in vivo, underscoring the need to understand its transcriptional regulation. Here, we identified the transcriptional and translational start points of rpfB, and suggested from rpf promoter-driven GFP expression and in vitro transcription assays that its transcription possibly occurs in a SigB-dependent manner. We further demonstrated that rpfB transcription is regulated by MtrA - the response regulator of the essential two-component system MtrAB. Association of MtrA with the rpfB promoter region in vivo was confirmed by chromatin immunoprecipitation analysis. Electrophoretic mobility shift assays (EMSAs) revealed a loose direct repeat sequence associated with MtrA binding. Binding of MtrA was enhanced upon phosphorylation. MtrA could be pulled down from lysates of M. tuberculosis using a biotinylated DNA fragment encompassing the MtrA-binding site on the rpfB promoter, confirming that MtrA binds to the rpfB promoter. Enhanced GFP fluorescence driven by the rpfB promoter, upon deletion of the MtrA-binding site, and repression of rpfB expression, upon overexpression of MtrA, suggested that MtrA functions as a repressor of rpfB transcription. This was corroborated by EMSAs showing diminished association of RNA polymerase (RNAP) with the rpfB promoter in the presence of MtrA. In vitro transcription assays confirmed that MtrA inhibits RNAP-driven rpfB transcription.

While the advent of combination antiretroviral therapy (ART) has significantly improved survival, tuberculosis (TB) remains the leading cause of death in the HIV-infected population. We used Mycobacterium tuberculosis/simian immunodeficiency virus-coinfected (M. tuberculosis/SIV-coinfected) macaques to model M. tuberculosis/HIV coinfection and study the impact of ART on TB reactivation due to HIV infection. Although ART significantly reduced viral loads and increased CD4(+) T cell counts in blood and bronchoalveolar lavage (BAL) samples, it did not reduce the relative risk of SIV-induced TB reactivation in ART-treated macaques in the early phase of treatment. CD4(+) T cells were poorly restored specifically in the lung interstitium, despite their significant restoration in the alveolar compartment of the lung as well as in the periphery. IDO1 induction in myeloid cells in the inducible bronchus-associated lymphoid tissue (iBALT) likely contributed to dysregulated T cell homing and impaired lung immunity. Thus, although ART was indispensable for controlling viral replication, restoring CD4(+) T cells, and preventing opportunistic infection, it appeared inadequate in reversing the clinical signs of TB reactivation during the relatively short duration of ART administered in this study. This finding warrants the modeling of concurrent treatment of TB and HIV to potentially reduce the risk of reactivation of TB due to HIV to inform treatment strategies in patients with M. tuberculosis/HIV coinfection.

HIV is a major driver of tuberculosis (TB) reactivation. Depletion of CD4+ T cells is assumed to be the basis behind TB reactivation in individuals with latent tuberculosis infection (LTBI) coinfected with HIV. Nonhuman primates (NHPs) coinfected with a mutant simian immunodeficiency virus (SIVΔGY) that does not cause depletion of tissue CD4+ T cells during infection failed to reactivate TB. To investigate the contribution of CD4+ T cell depletion relative to other mechanisms of SIV-induced reactivation of LTBI, we used CD4R1 antibody to deplete CD4+ T cells in animals with LTBI without lentiviral infection. The mere depletion of CD4+ T cells during LTBI was insufficient in generating reactivation of LTBI. Instead, direct cytopathic effects of SIV resulting in chronic immune activation, along with the altered effector T cell phenotypes and dysregulated T cell homeostasis, were likely mediators of reactivation of LTBI. These results revealed important implications for TB control in HIV-coinfected individuals.

Coordinated bacterial cell septation and cell wall biosynthesis require formation of protein complexes at the sites of division and elongation, in a temporally controlled manner. The protein players in these complexes remain incompletely understood in mycobacteria. Using in vitro and in vivo assays, we showed that Rv2147c (or SepF) of Mycobacterium tuberculosis interacts with the principal driver of cytokinesis, FtsZ. SepF also interacts with itself both in vitro and in vivo. Amino acid residues 189A, 190K and 215F are required for FtsZ-SepF interaction, and are conserved across Gram-positive bacteria. Using Mycobacterium smegmatis as a surrogate system, we confirmed that sepFMSMEG is essential. Knockdown of SepF led to cell elongation, defective growth and failure of FtsZ to localize to the site of division, suggesting that SepF assists FtsZ localization at the site of division. Furthermore, SepF interacted with MurG, a peptidoglycan-synthesizing enzyme, both in vitro and in vivo, suggesting that SepF could serve as a link between cell division and peptidoglycan synthesis. SepF emerges as a newly identified essential component of the cell division complex in mycobacteria.

Mycobacterium tuberculosis employs two-component systems (TCSs) for survival within its host. The TCS MtrAB is conserved among mycobacteria. The response regulator MtrA is essential in M. tuberculosis. The genome-wide chromatin immunoprecipitation (ChIP) sequencing performed in this study suggested that MtrA binds upstream of at least 45 genes of M. tuberculosis, including those involved in cell wall remodelling, stress responses, persistence and regulation of transcription. It binds to the promoter regions and regulates the peptidoglycan hydrolases rpfA and rpfC, which are required for resuscitation from dormancy. It also regulates the expression of whiB4, a critical regulator of the oxidative stress response, and relF, one-half of the toxin-antitoxin locus relFG. We have identified a new consensus 9 bp loose motif for MtrA binding. Mutational changes in the consensus sequence greatly reduced the binding of MtrA to its newly identified targets. Importantly, we observed that overexpression of a gain-of-function mutant, MtrAY102C, enhanced expression of the aforesaid genes in M. tuberculosis isolated from macrophages, whereas expression of each of these targets was lower in M. tuberculosis overexpressing a phosphorylation-defective mutant, MtrAD56N. This result suggests that phosphorylated MtrA (MtrA-P) is required for the expression of its targets in macrophages. Our data have uncovered new MtrA targets that suggest that MtrA is required for a transcriptional response that likely enables M. tuberculosis to persist within its host and emerge out of dormancy when the conditions are favourable.

The genome of M. tuberculosis (Mtb) encodes eleven paired two component systems (TCSs) consisting of a sensor kinase (SK) and a response regulator (RR). The SKs sense environmental signals triggering RR-dependent gene expression pathways that enable the bacterium to adapt in the host milieu. We demonstrate that a conserved motif present in the C-terminal domain regulates the DNA binding functions of the OmpR family of Mtb RRs. Molecular docking studies against this motif helped to identify two molecules with a thiazolidine scaffold capable of targeting multiple RRs, and modulating their regulons to attenuate bacterial replication in macrophages. The changes in the bacterial transcriptome extended to an altered immune response with increased autophagy and NO production, leading to compromised survival of Mtb in macrophages. Our findings underscore the promise of targeting multiple RRs as a novel yet unexplored approach for development of new anti-mycobacterial agents particularly against drug-resistant Mtb.