Mtb Growth Research Articles

Identification of Mycobacterium tuberculosis CtpF as a target for designing new antituberculous compounds

The emergence of tuberculosis (TB) produced by multi-drug resistance (MDR) and extensively-drug resistance (XDR) Mycobacterium tuberculosis (Mtb), encourages the development of new antituberculous compounds, as well as the identification of novel drug targets. In this regard, plasma membrane P-type ATPases are interesting targets because they play a crucial role in ion homeostasis and mycobacterial survival. We focused on Mtb CtpF, a calcium P-type ATPase that responds to a broad number of intraphagosomal conditions, as a novel target. In this study, we evaluated the capacity of cyclopiazonic acid (CPA), a well-known inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), to inhibit the ATPase activity of CtpF and the Mtb growth demonstrating that CtpF is a druggable target. A homology modeling of CtpF was generated for molecular docking studies of CtpF with CPA and key pharmacophoric features were identified, which were used to perform a pharmacophore-based virtual screening of the ZINC database, and to identify CtpF inhibitor candidates. Molecular docking-based virtual screening and MM-BGSA calculations of candidates allowed identifying six compounds with the best binding energies. The compounds displayed in vitro minimum inhibitory concentrations (MIC) ranging from 50 to 100 μg/mL, growth inhibitions from 29.5 to 64.0% on Mtb, and inhibitions of Ca2+-dependent ATPase activity in Mtb membrane vesicles (IC50) ranging from 4.1 to 35.8 μM. The compound ZINC63908257 was the best candidate by displaying a MIC of 50 μg/mL and a Ca2+ P-type ATPase inhibition of 45% with IC50 = 4.4 μM. Overall, the results indicate that CtpF is a druggable target for designing new antituberculous compounds.

Proteomic characterization of Mycobacterium tuberculosis reveals potential targets of bostrycin

Tuberculosis (TB) is caused by bacterial pathogen Mycobacterium tuberculosis (Mtb) and remains a major health problem worldwide. The increasing prevalence of drug-resistant Mtb strains and the extended duration of anti-TB regimens have created an urgent need for new anti-tuberculosis antibiotics with novel targets or inhibitory strategies. Anthracenedione compound bostrycin has been shown to inhibit the growth of Mtb in vitro and inhibit the activity of the effector protein tyrosine phosphatase (MptpB) secreted by Mtb. In this study, we characterized the proteomic profile of the Mtb strain H37Ra exposed to 1 mg/L and 25 mg/L of bostrycin for 24 h. Bioinformatic analysis of the differential abundant proteins indicated that bostrycin treatment may induce oxidative stress and interfere with essential processes such as synthesis of NAD(+) and the tricarboxylic acid cycle in mycobacteria. Then, the molecular docking of bostrycin and 15 candidates of targeted proteins showed that Rv3684 and Rv1908c got higher scores compared to MptpB, suggesting the direct interaction of bostrycin and these two proteins. Further docking of potential targeted proteins with the functional group-removal derivatives of bostrycin revealed possible key functional groups of bostrycin and provides direction for the modification of bostrycin in future. Biological significanceIt is a challenging work to determine the potential target(s) of an antibiotic accurately and quickly. In this study, we conducted a proteomic analysis of Mtb responding to the treatment of bostrycin, and provided insight into the inhibiting mechanism of this anti-Mtb compound. The proper interaction of bostrycin and targeted proteins, as well as the interacting residues of targets, and functional groups of bostrycin were also identified within the docking surface, providing a direction for further modification of bostrycin. Our study also suggests a reference for the interaction analysis between mycobacteria and antibiotics, and provides potential targets information for other active anthraquinones.

Itaconate brings metalloenzyme to a halt

Radical Enzymes Controlled radicals enable unusual enzymatic transformations, but radical generation and management require dedicated systems. Ruetz et al. investigated how the immunometabolite itaconate might undermine these intricate systems to inhibit propionate metabolism, a crucial metabolic pathway in pathogenic Mycobacterium tuberculosis (Mtb) (see the Perspective by Boal). They found that the coenzyme A (CoA) derivative of itaconate can irreversibly inhibit the enzyme methylmalonyl-CoA mutase (MCM), which uses the radical-generating cofactor adenosylcobalamin, or coenzyme B12. Itaconyl-CoA derails the normal radical reaction catalyzed by MCM, forming a long-lived, biradical species, which is incapable of completing the catalytic cycle and cannot be recycled by the endogenous coenzyme B12 regeneration machinery. Itaconate blocks Mtb growth on propionate, and this inhibition mechanism may be relevant to how macrophages resist Mtb infection. Science , this issue p. [589][1]; see also p. [574][2] [1]: /lookup/doi/10.1126/science.aay0934 [2]: /lookup/doi/10.1126/science.aaz4540

Mitofusin 2-Deficiency Suppresses Mycobacterium tuberculosis Survival in Macrophages.

Apoptosis is an important host defense mechanism against mycobacterial infection. However, the molecular mechanisms regulating apoptosis during mycobacterial infection are not well known. Recent reports suggest that bacterial infection regulates mitochondrial fusion and fission in various ways. Here, we investigated the role of mitochondria in Mycobacterium tuberculosis (Mtb)-infected macrophages. Mtb H37Rv (Rv) infection induced mitofusin 2 (MFN2) degradation, leading to mitochondrial fission. Interestingly, Mtb H37Ra (Ra) infection induced significantly greater mitochondrial fragmentation than Rv infection. Mtb-mediated Parkin, an E3 ubiquitin ligase, contributed to the degradation of MFN2. To evaluate the role of endoplasmic reticulum stress in the production of Parkin during Mtb infection, we analyzed Parkin production in 4-phenylbutyric acid (4-PBA)-pretreated macrophages. Pretreatment with 4-PBA reduced Parkin production in Mtb-infected macrophages. In contrast, the level of MFN2 production recovered to a level similar to that of the unstimulated control. In addition, Ra-infected macrophages had reduced mitochondrial membrane potential (MMP) compared to those infected with Rv. Interestingly, intracellular survival of mycobacteria was decreased in siMFN2-transfected macrophages; in contrast, overexpression of MFN2 in macrophages increased Mtb growth compared with the control.

Adjunctive Host-Directed Therapy With Statins Improves Tuberculosis-Related Outcomes in Mice.

Tuberculosis (TB) treatment is lengthy and complicated and patients often develop chronic lung disease. Recent attention has focused on host-directed therapies aimed at optimizing immune responses to Mycobacterium tuberculosis (Mtb), as adjunctive treatment given with antitubercular drugs. In addition to their cholesterol-lowering properties, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have broad anti-inflammatory and immunomodulatory activities. In the current study, we screened 8 commercially available statins for cytotoxic effect, anti-TB activity, synergy with first-line drugs in macrophages, pharmacokinetics and adjunctive bactericidal activity, and, in 2 different mouse models, as adjunctive therapy to first-line TB drugs. Pravastatin showed the least toxicity in THP-1 and Vero cells. At nontoxic doses, atorvastatin and mevastatin were unable to inhibit Mtb growth in THP-1 cells. Simvastatin, fluvastatin, and pravastatin showed the most favorable therapeutic index and enhanced the antitubercular activity of the first-line drugs isoniazid, rifampin, and pyrazinamide in THP-1 cells. Pravastatin modulated phagosomal maturation characteristics in macrophages, phenocopying macrophage activation, and exhibited potent adjunctive activity in the standard mouse model of TB chemotherapy and in a mouse model of human-like necrotic TB lung granulomas. These data provide compelling evidence for clinical evaluation of pravastatin as adjunctive, host-directed therapy for TB.

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112.

Summary Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi‐drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H‐NS‐like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H‐NS, Lsr2 binds DNA in sequence‐dependent and non‐specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA‐binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP‐sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.

Induction of autophagy through CLEC4E in combination with TLR4: an innovative strategy to restrict the survival of Mycobacterium tuberculosis

ABSTRACT Host-directed therapies are gaining considerable impetus because of the emergence of drug-resistant strains of pathogens due to antibiotic therapy. Therefore, there is an urgent need to exploit alternative and novel strategies directed at host molecules to successfully restrict infections. The C-type lectin receptor CLEC4E and Toll-like receptor TLR4 expressed by host cells are among the first line of defense in encountering pathogens. Therefore, we exploited signaling of macrophages through CLEC4E in association with TLR4 agonists (C4.T4) to control the growth of Mycobacterium tuberculosis (Mtb). We observed significant improvement in host immunity and reduced bacterial load in the lungs of Mtb-infected mice and guinea pigs treated with C4.T4 agonists. Further, intracellular killing of Mtb was achieved with a 10-fold lower dose of isoniazid or rifampicin in conjunction with C4.T4 than the drugs alone. C4.T4 activated MYD88, PtdIns3K, STAT1 and RELA/NFKB, increased lysosome biogenesis, decreased Il10 and Il4 gene expression and enhanced macroautophagy/autophagy. Macrophages from autophagy-deficient (atg5 knockout or Becn1 knockdown) mice showed elevated survival of Mtb. The present findings also unveiled the novel role of CLEC4E in inducing autophagy through MYD88, which is required for control of Mtb growth. This study suggests a unique immunotherapeutic approach involving CLEC4E in conjunction with TLR4 to restrict the survival of Mtb through autophagy. Abbreviations 3MA: 3 methyladenine; AO: acridine orange; Atg5: autophagy related 5; AVOs: acidic vesicular organelles; BECN1: beclin 1, autophagy related; BMDMs: bone marrow derived macrophages; bw: body weight; C4.T4: agonists of CLEC4E (C4/TDB) and TLR4 (T4/ultra-pure-LPS); CFU: colony forming unit; CLEC4E/Mincle: C-type lectin domain family 4, member e; CLR: c-type lectin receptor; INH: isoniazid; LAMP1: lysosomal-associated membrane protein 1; MφC4.T4: Mtb-infected C4.T4 stimulated macrophages; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MDC: monodansylcadaverine; MTOR: mechanistic target of rapamycin kinase; MYD88: myeloid differentiation primary response 88; NFKB: nuclear factor of kappa light polypeptide gene enhance in B cells; NLR: NOD (nucleotide-binding oligomerization domain)-like receptors; PFA: paraformaldehyde; PPD: purified protein derivative; PtdIns3K: class III phosphatidylinositol 3-kinase; RELA: v-rel reticuloendotheliosis viral oncogene homolog A (avian); RIF: rifampicin; RLR: retinoic acid-inducible gene-I-like receptors; TDB: trehalose-6,6´-dibehenate; TLR4: toll-like receptor 4; Ultra-pure-LPS: ultra-pure lipopolysaccharide-EK; V-ATPase: vacuolar-type H+ ATPase.

Design of high-order antibiotic combinations against M. tuberculosis by ranking and exclusion

Combinations of more than two drugs are routinely used for the treatment of pathogens and tumors. High-order combinations may be chosen due to their non-overlapping resistance mechanisms or for favorable drug interactions. Synergistic/antagonistic interactions occur when the combination has a higher/lower effect than the sum of individual drug effects. The standard treatment of Mycobacterium tuberculosis (Mtb) is an additive cocktail of three drugs which have different targets. Herein, we experimentally measured all 190 pairwise interactions among 20 antibiotics against Mtb growth. We used the pairwise interaction data to rank all possible high-order combinations by strength of synergy/antagonism. We used drug interaction profile correlation as a proxy for drug similarity to establish exclusion criteria for ideal combination therapies. Using this ranking and exclusion design (R/ED) framework, we modeled ways to improve the standard 3-drug combination with the addition of new drugs. We applied this framework to find the best 4-drug combinations against drug-resistant Mtb by adding new exclusion criteria to R/ED. Finally, we modeled alternating 2-order combinations as a cycling treatment and found optimized regimens significantly reduced the overall effective dose. R/ED provides an adaptable framework for the design of high-order drug combinations against any pathogen or tumor.

Effect of Iron Supplementation on the Outcome of Non-Progressive Pulmonary Mycobacterium tuberculosis Infection.

The human response to Mycobacterium tuberculosis (Mtb) infection is affected by the availability of iron (Fe), which is necessary for proper immune cell function and is essential for the growth and virulence of bacteria. Increase in host Fe levels promotes Mtb growth and tuberculosis (TB) pathogenesis, while Fe-supplementation to latently infected, asymptomatic individuals is a significant risk factor for disease reactivation. However, the effect of Fe-supplementation on the host immunity during latent Mtb infection remains unclear, due partly to the paucity in availability of animal models that recapitulate key pathophysiological features seen in humans. We have demonstrated that rabbits can develop non-progressive latency similar to infected humans. In this study, using this model we have evaluated the effect of Fe-supplementation on the bacterial growth, disease pathology, and immune response. Systemic and lung Fe parameters, gene expression profile, lung bacterial burden, and disease pathology were determined in the Mtb-infected/Fe- or placebo-supplemented rabbits. Results show that Fe-supplementation to Mtb-infected rabbits did not significantly change the hematocrit and Hb levels, although it elevated total Fe in the lungs. Expression of selected host iron- and immune-response genes in the blood and lungs was perturbed in Mtb-infected/Fe-supplemented rabbits. Iron-supplementation during acute or chronic stages of Mtb infection did not significantly affect the bacterial burden or disease pathology in the lungs. Data presented in this study is of significant relevance for current public health policies on Fe-supplementation therapy given to anemic patients with latent Mtb infection.

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

Tuberculosis and malaria walk side by side in the Brazilian Amazon: an ecological approach.

To assess the spatial distribution of TB and malaria incidence, as well as their spatial association with each other, regardless of environmental and socio-economic factors commonly reported as determinants of both disease rates among the municipalities of Amazonas State, Brazil between 2012 and 2015. Through an ecological approach considering municipalities of Amazonas, Brazil, as unit of analysis, a negative binomial regression model was used to assess association between malaria and TB rates, in which the dependent variable was the average municipal tuberculosis incidence rate. Positive associations of overall malaria (β=0.100 [CI=0.032, 0.168], P=0.004), P.vivax malaria (β=0.115 [CI=0.036, 0.195], P=0.005), and P.falciparum malaria (β=0.389 [CI=-0.0124, 0.791], P=0.057) with TB rates were found, regardless of the sociodemographic factors included in the study. In the Brazilian Amazon, TB and malaria are spatially associated. Therefore, it is very likely that co-infections also occur in this region, regardless of the HIV status.

Curdlan Limits Mycobacterium tuberculosis Survival Through STAT-1 Regulated Nitric Oxide Production

Host-directed therapies have emerged as an innovative and promising approach in tuberculosis (TB) treatment due to the observed limitations of current TB regimen such as lengthy duration and emergence of drug resistance. Thus, we explored the role of curdlan (beta glucan polysaccharide) as a novel strategy to activate macrophages against Mycobacterium tuberculosis (Mtb). The aim of the study was to investigate the role of curdlan in restricting the Mtb growth both in vitro and in vivo. Further, the immunomodulatory potential of curdlan against Mtb and the underlying mechanism is largely unknown. We found that curdlan treatment enhanced the antigen presentation, pro-inflammatory cytokines, Mtb uptake and killing activity of macrophages. In vivo studies showed that curdlan therapy significantly reduced the Mtb burden in lung and spleen of mice. Administration of curdlan triggered the protective Th1 and Th17 immunity while boosting the central and effector memory response in Mtb infected mice. Curdlan mediated anti-Mtb activity is through signal transducer and activator of transcription-1 (STAT-1), which regulates nitric oxide (NO) production through inducible NO synthase (iNOS) induction; along with this activation of nuclear factor kappa B (NF-κB) was also evident in Mtb infected macrophages. Thus, we demonstrate that curdlan exerts effective anti-tuberculous activity anti-tuberculous activity. It can be used as a potential host-directed therapy against Mtb.

Heterogeneous GM-CSF signaling in macrophages is associated with control of Mycobacterium tuberculosis

Variability in bacterial sterilization is a key feature of Mycobacterium tuberculosis (Mtb) disease. In a population of human macrophages, there are macrophages that restrict Mtb growth and those that do not. However, the sources of heterogeneity in macrophage state during Mtb infection are poorly understood. Here, we perform RNAseq on restrictive and permissive macrophages and reveal that the expression of genes involved in GM-CSF signaling discriminates between the two subpopulations. We demonstrate that blocking GM-CSF makes macrophages more permissive of Mtb growth while addition of GM-CSF increases bacterial control. In parallel, we find that the loss of bacterial control that occurs in HIV-Mtb coinfected macrophages correlates with reduced GM-CSF secretion. Treatment of coinfected cells with GM-CSF restores bacterial control. Thus, we leverage the natural variation in macrophage control of Mtb to identify a critical cytokine response for regulating Mtb survival and identify components of the antimicrobial response induced by GM-CSF.

Impact of selective immune-cell depletion on growth of Mycobacterium tuberculosis (Mtb) in a whole-blood bactericidal activity (WBA) assay.

We investigated the contribution of host immune cells to bacterial killing in a whole-blood bactericidal activity (WBA) assay, an ex vivo model used to test efficacy of drugs against mycobacterium tuberculosis (Mtb). We performed WBA assays with immuno-magnetic depletion of specific cell types, in the presence or absence of rifampicin. Innate immune cells decreased Mtb growth in absence of drug, but appeared to diminish the cidal activity of rifampicin, possibly attributable to intracellular bacterial sequestration. Adaptive immune cells had no effect with or without drug. The WBA assay may have potential for testing adjunctive host-directed therapies acting on phagocytic cells.

Macrophage heterogeneity correlates with autophagy-dependent anti-mycobacterial activity and ability to present antigen in human macrophages

Abstract Mycobacterium tuberculosis (Mtb) kills around 1.5 million people each year. Albeit, only 10% of humans develop active disease after aerosol infection, the basis for differential susceptibility of humans remains unclear. To determine if human macrophages are heterogeneous in their ability to kill Mtb, cord blood derived macrophages were treated with either IFN-γ or IL-4 to prepare M1 and M2 phenotype respectively in vitro. Macrophages were infected with a 1:1 ratio of Mtb and days 3 and 5 post infection, macrophage lysates were plated for CFU counts. In addition, intracellular autophagy was measured in gfpMtb infected macrophages using cytoID stains to detect autophagosomes and LC3 lipidation through western blot of macrophage lysates. Mtb infected macrophages were overlaid using CD4 T cells specific for the Antigen-85B epitope to measure antigen presentation. M1 macrophages showed a bacteriostatic ability and restricted the growth of Mtb while IL-4 treated M2 macrophages showed nearly 1 log increase in the growth of Mtb. Thus M2 macrophages were permissive for growth of Mtb. Consistent with this observation, gfpMtb co-localized with autophagosomes more frequently in M1 macrophages suggesting a role for autophagy during growth inhibition. M1 macrophages also presented Ag85B to CD4 T cells better than M2 macrophages. Enhanced antigen presentation by M1 macrophages was also found to be dependent on IFITM3, Atg5 and Atg 16 genes as siRNA knock down of these specific genes resulted in reduced antigen presentation by M1 macrophages. These observations suggest that autophagy pathways within macrophage may govern their heterogeneity into M1 or M2 hence their innate ability to kill intracellular Mtb and trigger immune activation.

Involvement of Sphingosine Kinase (SK1) during autophagosome formation plays a critical role in controlling the growth of Mycobacterium tuberculosis within Human Mesenchymal Stem Cells

Abstract Mycobacterium tuberculosis (Mtb) evades host defense mechanisms by infecting and persisting in Mesenchymal Stem Cells (MSCs). Thus, MSCs contain M. tuberculosis DNA and harbor viable bacilli, and our recent study showed that autophagy plays a critical role in restricting the growth of Mtb in human MSCs. Activation of human MSCs using rapamycin enhanced the bactericidal function of MSCs. Here, we examined the autophagy pathway associated with the control of Mtb growth within MSCs. When compared to human macrophages, MSCs had increased expression of Sphingosine Kinase 1 (SK1) during Mtb infection, which was also found to be associated with overall increase in autophagic flux and LC3B positive puncta. SK1 also more often co-localized with gfpMtb containing autophagosomes within MSCs when compared to human macrophages. Percentage of SK1 and LC3B double positive M. tuberculosis containing autophagosomes were also significantly higher in MSCs compared to macrophages suggesting that Mtb induces SK1 mediated autophagy in MSCs during infection. To validate the role of SK1 mediated autophagy in inhibition of Mtb growth within MSCs, we knocked down SK1 using siRNA and scrambled control and examined the survival of M. tuberculosis. SK1-siRNA treated MSCs showed an enhanced survival of Mtb, and replication compared to control siRNA treated cells over 21 days of incubation. These results together indicate that SK1 mediates autophagy, autophagosome formation and autophagy dependent inhibition of Mtb growth within MSCs.

Type I Interferon confers susceptibility to Mycobacterium tuberculosis infection within granulomas of SIV co-infected non-human primates

Abstract Mycobacterium tuberculosis (Mtb) co-infection with human immunodeficiency virus (HIV) typically results in poor disease outcome for tuberculosis (TB). While the loss of CD4+ T cells due to HIV is known to cause increased TB disease susceptibility, the mechanisms which govern the loss of disease control in co-infected individuals are not well understood particularly within the lung granulomas that keep TB contained. This study aims to profile the transcriptional patterns of lung granulomas from non-human primates (NHP) co-infected with Mtb and simian immunodeficiency virus (SIV) as a model for TB-SIV co-infection in humans. We compared the transcriptional profiles of granulomas from four Mauritian cynomolgus macaques that were infected with SIV for six months and then co-infected with Mtb for eight weeks against animals that were infected with Mtb for eight weeks. Granulomas from co-infected animals were found to have generally elevated transcripts involved in Type I interferon pathways (IFIT1), anti-bacterial effectors (defensin A, cathepsin G) and evidence of increased neutrophilic inflammation compared to Mtb-only control lung granulomas. Pathways that were downregulated in co-infected animals were related to Type II interferon signaling (IRF8). These results suggest that SIV infection alters the host immune response in ways that may be advantageous to Mtb growth by lowering IFNγ responsiveness and promoting myeloid cell-associated inflammation. We hypothesize this leads to a more permissive and less antibacterial environment that promotes bacterial survival, dissemination, and ultimately leads to increased susceptibility of co-infected individuals towards TB disease.

Neutrophils transiently harbor Mycobacterium tuberculosis and represent a metabolically permissive niche for bacterial growth

Abstract Neutrophils are rapidly recruited to sites of mycobacterial infection, where they phagocytose bacilli. At the early stage of infection, Mycobacterium tuberculosis is predominantly present in neutrophils, which are also frequently infected in human TB. However, neutrophils are less well studied than other components of the host response to Mtb and much of the available data are conflicting. To better understand the role of neutrophils in tuberculosis, we focused on the fate of Mtb in neutrophils. We demonstrated that Mtb-infected neutrophils are short-lived and actively engulfed by two ontologically distinct lung macrophages, alveolar and interstitial macrophages in vivo. RNA-seq data also confirms the expression of efferocytosis-related genes in both types of macrophages. Importantly, Mtb remains viable in lung macrophages after being transported from neutrophils, implying a critical step for Mtb spreading between host phagocytes. Furthermore, neutrophils upregulate CD64 and reveal a more activated phenotype. Exploitation of reporter Mtb strains, we observed comparable replication status of Mtb in both CD64+ and CD64− neutrophils, indicating that Mtb replication is independent of the neutrophil activation state. Mechanistically, although CD64+ neutrophils are more glycolytically active than CD64− neutrophils, CD64+ neutrophils highly express CD36, and internalize greater amounts of fatty acid, LDL and oxLDL, the preferred nutrients for Mtb growth. In conclusion, our data support the hypothesis that neutrophils function as a transient carrier that delivers Mtb to macrophages. The metabolic state in neutrophils is distinct from macrophages yet still represents a hospitable environment supporting bacterial growth.

Potassium efflux and serum amyloid A3 are involved in IL-1β production by macrophages infected with Mycobacterium tuberculosis

Abstract While interleukin (IL)-1β is essential for the protective immunity against tuberculosis infection, it also exacerbates the pathology of tuberculosis. Although previous studies suggested that early secreted antigenic target of 6-kDa (ESAT-6) of Mycobacterium tuberculosis (Mtb) is a potent activator of NLRP3 inflammasome for IL-1β maturation, the mechanisms remain unclear. Therefore, we studied IL-1β production by C57BL/6 mouse bone marrow-derived macrophages (BMDM) infected with Mtb H37Rv, its esat-6 deletion mutant or esat-6 complemented strain. Our data clearly showed that BMDM infected with esat-6 deletion mutant produced significantly reduced IL-1β compared with the cells infected with H37Rv and esat-6 complemented strain despite having comparable levels of pro-IL-1β, implying that ESAT-6 is critical for the maturation but not synthesis of pro-IL-1β. IL-1β production by Mtb-infected BMDM is inhibited by MCC950, a selective NLRP3 inhibitor. Interestingly blocking K+ efflux by high concentrations of extracellular K+ or quinine, an inhibitor of TWIK2 K+ efflux channel, inhibited IL-1β production, but increased bacilli growth in BMDM. Infection of mice with esat-6 deletion mutant showed defects in lung IL-1β production compared with H37Rv. Recombinant ESAT-6 induced IL-1β production in mouse lung. RNA-seq analysis identified serum amyloid A (Saa) 3 as one of the most highly expressed genes in ESAT-6 inoculated mouse lung. Silencing Saa3 in BMDM led to reduced IL-1β production by H37Rv or ESAT-6. These data together imply that Mtb induces secretion of IL-1β in an ESAT-6 dependent manner with the involvement of NLRP3, Saa3 and K+ efflux channel which is also required for Mtb growth control in macrophages.

γ-herpesvirus latency attenuates Mycobacterium tuberculosis infection in mice

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a bacterial pathogen which is transmitted via aerosol and establishes a chronic lung infection. In naïve hosts, Mtb grows for several weeks without being restricted by IFNγ-producing T cells, which eventually accumulate and limit Mtb dissemination. In this study, we used a mouse model of Mtb/γ-herpesvirus (γHV) coinfection to test the hypothesis that latent γHV infection alters host resistance to Mtb. γHVs are DNA viruses which elicit a polyclonal T cell response and attenuate some acute bacterial pathogens in mice; whether γHVs modulate infection with Mtb is unknown. Here, mice harboring latent mouse gammaherpesvirus 68 (MHV68)—a γHV genetically and biologically related to human Epstein Barr virus (EBV)—were infected via aerosol with a low dose of virulent Mtb. Mtb burdens and IFNγ+ T cell frequencies in mice with latent MHV68 (MHV68POS mice) were subsequently measured and compared to control mice that did not harbor latent MHV68 (MHV68NEG mice). Relative to MHV68NEG controls, MHV68POS mice more effectively limited Mtb growth and dissemination, and had higher frequencies of CD4+IFNγ+ cells in lung-draining lymph nodes. Collectively, our results support a model wherein latent γHV confers moderate protection against subsequent Mtb infection.