The current state of animal models and genomic approaches towards identifying and validating molecular determinants of Mycobacterium tuberculosis infection and tuberculosis disease

Animal models are important in understanding both the pathogenesis of and immunity to tuberculosis (TB). Unfortunately, we are beginning to understand that no animal model perfectly recapitulates the human TB syndrome, which encompasses numerous different stages. Furthermore, Mycobacterium tuberculosis infection is a very heterogeneous event at both the levels of pathogenesis and immunity. This review seeks to establish the current understanding of TB pathogenesis and immunity, as validated in the animal models of TB in active use today. We especially focus on the use of modern genomic approaches in these models to determine the mechanism and the role of specific molecular pathways. Animal models have significantly enhanced our understanding of TB. Incorporation of contemporary technologies such as single cell transcriptomics, high-parameter flow cytometric immune profiling, proteomics, proteomic flow cytometry and immunocytometry into the animal models in use will further enhance our understanding of TB and facilitate the development of treatment and vaccination strategies

Development of transgenics in Indian oilseed mustard (Brassica juncea) resistant to herbicide phosphinothricin

Transgenic lines resistant to herbicide phosphinothricin (PPT) were developed in mustard (Brassica juncea), a major oilseed crop grown in more than 6 million hectares of land in North India. Seedling-derived hypocotyl explants were transformed with a disarmed Agrobacterium tumefaciens strain GV3101. The developed constructs contained the bar gene encoding the enzyme phosphinothricin-acetyl-transferase (PAT) which inactivates phosphinothricin (PPT) by acetylating it. The expression of the bar gene was controlled either by the double enhancer version of CaMV35S promoter (35Sdebar) or a CaMV35S promoter with a leader sequence from RNA4 of alfalfa mosaic virus introduced at the 5' end of the bar gene (35SAMVLbar) or without (35Sbar) it. Plant viral leader sequences have been shown to be translational enhancers. In vitro selections for transformed plants were carried out on a medium containing PPT. Transgenic shoots were recovered at a frequency of 23% with 35Sdebar gene construct and at a frequency of 16% with 35SAMVLbar containing construct. Transformation frequencies were low with 35Sbar construct. Individual transgenics with 35Sdebar and 35SAMVLbar constructs were tested for copy number on both the right and left border flanks of T-DNA by Southern hybridization. Single copy transgenic lines were further analysed for transcript levels of the bar gene by Northern blotting and for protein levels by PAT assays. Wide variation in expression levels were observed, particularly amongst the transgenics containing the 35Sdebar construct. Single copy transgenics were selfed to develop homozygous lines which could be used for the study of resistance to herbicide PPT at the field level and to correlate this protection with expression levels observed through molecular analysis. Herbicide- tolerant lines could be used for testing the possibility of low-till or no-till cultivation of mustard in the rain-fed areas where it is extensively grown

Myeloid-derived suppressor cells mediate T cell dysfunction in nonhuman primate TB granulomas

Myeloid-derived suppressor cells (MDSCs) represent an innate immune cell population comprised of immature myeloid cells and myeloid progenitors with very potent immunosuppressive potential. MDSCs are reported to be abundant in the lungs of active tuberculosis (TB) patients. We sought to perform an in-depth study of MDSCs during latent TB infection (LTBI) and active TB (ATB) using the nonhuman primate (NHP) model of pulmonary TB. We found a higher proportion of granulocytic, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in the lungs of ATB animals compared to those with LTBI or naive control animals. Active disease in the lung, but not LTBI, was furthermore associated with higher proliferation, expansion, and immunosuppressive capabilities of PMN-MDSCs, as shown by enhanced expression of Ki67, indoleamine 2,3-dioxygenase (IDO1), interleukin-10 (IL-10), matrix metallopeptidase 9 (MMP-9), inducible nitric oxide synthase (iNOS), and programmed death-ligand 1 (PD-L1). These immunosuppressive PMN-MDSCs specifically localized to the lymphocytic cuff at the periphery of the granulomas in animals with ATB. Conversely, these cells were scarcely distributed in interstitial lung tissue and the inner core of granulomas. This spatial regulation suggests an important immunomodulatory role of PMN-MDSCs by restricting T cell access to the TB granuloma core and can potentially explain dysfunctional anti-TB responses in active granuloma. Our results raise the possibility that the presence of MDSCs can serve as a biomarker for ATB, while their disappearance can indicate successful therapy. Furthermore, MDSCs may serve as a potential target cell for adjunctive TB therapy

Crystal Structure of the 6-Hydroxymethyl-7,8-Dihydropterin Pyrophosphokinase•Dihydropteroate Synthase Bifunctional Enzyme from Francisella tularensis

The 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) enzymes catalyze sequential metabolic reactions in the folate biosynthetic pathway of bacteria and lower eukaryotes. Both enzymes represent validated targets for the development of novel anti-microbial therapies. We report herein that the genes which encode FtHPPK and FtDHPS from the biowarfare agent Francisella tularensis are fused into a single polypeptide. The potential of simultaneously targeting both modules with pterin binding inhibitors prompted us to characterize the molecular details of the multifunctional complex. Our high resolution crystallographic analyses reveal the structural organization between FtHPPK and FtDHPS which are tethered together by a short linker. Additional structural analyses of substrate complexes reveal that the active sites of each module are virtually indistinguishable from those of the monofunctional enzymes. The fused bifunctional enzyme therefore represents an excellent vehicle for finding inhibitors that engage the pterin binding pockets of both modules that have entirely different architectures. To demonstrate that this approach has the potential of producing novel two-hit inhibitors of the folate pathway, we identify and structurally characterize a fragment-like molecule that simultaneously engages both active sites. Our study provides a molecular framework to study the enzyme mechanisms of HPPK and DHPS, and to design novel and much needed therapeutic compounds to treat infectious diseases

Myeloid cell interferon responses correlate with clearance of SARS-CoV-2

Emergence of mutant SARS-CoV-2 strains associated with an increased risk of COVID-19-related death necessitates better understanding of the early viral dynamics, host responses and immunopathology. Single cell RNAseq (scRNAseq) allows for the study of individual cells, uncovering heterogeneous and variable responses to environment, infection and inflammation. While studies have reported immune profiling using scRNAseq in terminal human COVID-19 patients, performing longitudinal immune cell dynamics in humans is challenging. Macaques are a suitable model of SARS-CoV-2 infection. Our longitudinal scRNAseq of bronchoalveolar lavage (BAL) cell suspensions from young rhesus macaques infected with SARS-CoV-2 (n = 6) demonstrates dynamic changes in transcriptional landscape 3 days post- SARS-CoV-2-infection (3dpi; peak viremia), relative to 14-17dpi (recovery phase) and pre-infection (baseline) showing accumulation of distinct populations of both macrophages and T-lymphocytes expressing strong interferon-driven inflammatory gene signature at 3dpi. Type I interferon response is induced in the plasmacytoid dendritic cells with appearance of a distinct HLAD

Mechanisms of reactivation of latent tuberculosis infection due to SIV 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

Impact of SIV infection on mycobacterial lipid-reactive T cell responses in Bacillus Calmette-Guérin (BCG) inoculated macaques

BackgroundAlthough BCG vaccine protects infants from tuberculosis (TB), it has limited efficacy in adults against pulmonary TB. Further, HIV coinfection significantly increases the risk of developing active TB. In the lack of defined correlates of protection in TB disease, it is essential to explore immune responses beyond conventional CD4 T cells to gain a better understanding of the mechanisms of TB immunity.MethodsHere, we evaluated unconventional lipid-reactive T cell responses in cynomolgus macaques following aerosol BCG inoculation and examined the impact of subsequent SIV infection on these responses. Immune responses to cellular lipids of M. bovis and M. tuberculosis were examined ex vivo in peripheral blood and bronchioalveolar lavage (BAL).ResultsPrior to BCG inoculation, innate-like IFN-γ responses to mycobacterial lipids were observed in T cells. Aerosol BCG exposure induced an early increase in frequencies of BAL γδT cells, a dominant subset of lipid-reactive T cells, along with enhanced IL-7R and CXCR3 expression. Further, BCG exposure stimulated greater IFN-γ responses to mycobacterial lipids in peripheral blood and BAL, suggesting the induction of systemic and local Th1-type response in lipid-reactive T cells. Subsequent SIV infection resulted in a significant loss of IL-7R expression on blood and BAL γδT cells. Additionally, IFN-γ responses of mycobacterial lipid-reactive T cells in BAL fluid were significantly lower in SIV-infected macaques, while perforin production was maintained through chronic SIV infection.ConclusionsOverall, these data suggest that despite SIV-induced decline in IL-7R expression and IFN-γ production by mycobacterial lipid-reactive T cells, their cytolytic potential is maintained. A deeper understanding of anti-mycobacterial lipid-reactive T cell functions may inform novel approaches to enhance TB control in individuals with or without HIV infection

The Stress-Response Factor SigH Modulates the Interaction between Mycobacterium tuberculosis and Host Phagocytes

The Mycobacterium tuberculosis stress response factor SigH plays a crucial role in modulating the pathogen's response to heat, oxidative-stress, envelope damage and hypoxia. We hypothesized that the lack of this key stress response factor would alter the interaction between the pathogen and its host cells. We compared the interaction of Mtb, Mtb:Δ-sigH and a strain where the mutation had been genetically complemented (Mtb: Δ-sigH:CO) with primary rhesus macaque bone marrow derived macrophages (Rh-BMDMs). The expression of numerous inducible and homeostatic (CCL) β-chemokines and several apoptotic markers was induced to higher levels in the cells infected with Mtb:Δ-sigH, relative to Mtb or the complemented strain. The differential expression of these genes manifested into functional differences in chemotaxis and apoptosis in cells infected with these two strains. The mutant strain also exhibited reduced late-stage survival in Rh-BMDMs. We hypothesize that the product of one or more SigH-dependent genes may modulate the innate interaction of Mtb with host cells, effectively reducing the chemokine-mediated recruitment of immune effector cells, apoptosis of infected monocytes and enhancing the long-term survival and replication of the pathogen in this milieu The significantly higher induction of Prostaglandin Synthetase 2 (PTGS2 or COX2) in Rh-BMDMs infected with Mtb relative to Mtb: Δ-sigH may explain reduced apoptosis in Mtb-infected cells, as PTGS2 is known to inhibit p53-dependent apoptosis.The SigH-regulon modulates the innate interaction of Mtb with host phagocytes, perhaps as part of a strategy to limit its clearance and prolong its survival. The SigH regulon appears to be required to modulate innate immune responses directed against Mtb

Improved Xenobiotic Metabolism and Reduced Susceptibility to Cancer in Gluten-Sensitive Macaques upon Introduction of a Gluten-Free Diet

A non-human primate (NHP) model of gluten sensitivity was employed to study the gene perturbations associated with dietary gluten changes in small intestinal tissues from gluten-sensitive rhesus macaques (Macaca mulatta).Stages of remission and relapse were accomplished in gluten-sensitive animals by administration of gluten-free (GFD) and gluten-containing (GD) diets, as described previously. Pin-head-sized biopsies, obtained non-invasively by pediatric endoscope from duodenum while on GFD or GD, were used for preparation of total RNA and gene profiling, using the commercial Rhesus Macaque Microarray (Agilent Technologies),targeting expression of over 20,000 genes.When compared with normal healthy control, gluten-sensitive macaques showed differential gene expressions induced by GD. While observed gene perturbations were classified into one of 12 overlapping categories--cancer, metabolism, digestive tract function, immune response, cell growth, signal transduction, autoimmunity, detoxification of xenobiotics, apoptosis, actin-collagen deposition, neuronal and unknown function--this study focused on cancer-related gene networks such as cytochrome P450 family (detoxification function) and actin-collagen-matrix metalloproteinases (MMP) genes.A loss of detoxification function paralleled with necessity to metabolize carcinogens was revealed in gluten-sensitive animals while on GD. An increase in cancer-promoting factors and a simultaneous decrease in cancer-preventing factors associated with altered expression of actin-collagen-MMP gene network were noted. In addition, gluten-sensitive macaques showed reduced number of differentially expressed genes including the cancer-associated ones upon withdrawal of dietary gluten. Taken together, these findings indicate potentially expanded utility of gluten-sensitive rhesus macaques in cancer research

Transcriptional Reprogramming in Nonhuman Primate (Rhesus Macaque) Tuberculosis Granulomas

In response to Mtb infection, the host remodels the infection foci into a dense mass of cells known as the granuloma. The key objective of the granuloma is to contain the spread of Mtb into uninfected regions of the lung. However, it appears that Mtb has evolved mechanisms to resist killing in the granuloma. Profiling granuloma transcriptome will identify key immune signaling pathways active during TB infection. Such studies are not possible in human granulomas, due to various confounding factors. Nonhuman Primates (NHPs) infected with Mtb accurately reflect human TB in clinical and pathological contexts.We studied transcriptomics of granuloma lesions in the lungs of NHPs exhibiting active TB, during early and late stages of infection. Early TB lesions were characterized by a highly pro-inflammatory environment, expressing high levels of immune signaling pathways involving IFNgamma, TNFalpha, JAK, STAT and C-C/C-X-C chemokines. Late TB lesions, while morphologically similar to the early ones, exhibited an overwhelming silencing of the inflammatory response. Reprogramming of the granuloma transcriptome was highly significant. The expression of approximately two-thirds of all genes induced in early lesions was later repressed.The transcriptional characteristics of TB granulomas undergo drastic changes during the course of infection. The overwhelming reprogramming of the initial pro-inflammatory surge in late lesions may be a host strategy to limit immunopathology. We propose that these host profiles can predict changes in bacterial replication and physiology, perhaps serving as markers for latency and reactivation