Mtb Viability Research Articles

Steroid immune responsive gene regulation in Mycobacterium tuberculosis infection in vitro

Tuberculosis (TB) is an infectious disease displaying a multifactorial pathology. The immunomodulatory role attributed to steroid hormones, such as vitamin D3 (VD3) and 17β-estradiol (E2), highlighted the importance of these hormones against Mycobacterium tuberculosis (Mtb) infection. In order to understand their influence upon gene expression of immune and inflammatory responsive genes against Mtb we tested it in vitro using peripheral blood mononuclear cells (PBMCs). Cells were pretreated with VD3 (50 ng/mL) or E2 (100 nM/mL) and co-cultured with H37Rv Mtb or stimulated with lipopolysaccharide from Escherichia coli (LPS). After 24 h and 72 h of co-culture the Mtb viability in macrophages test was performed, as well the total RNA isolation for gene expression analysis by RT-qPCR of the following target genes: NLRP3, DC-SIGN, IL-1β, and IL-10. We also measured IL-10, TNF, IFN-γ, IL-4, IL-6, and IL-2 supernatant levels. As the main results, we found that VD3 and E2 downregulated the expression of inflammatory genes NLRP3, IL-1β, and IL-10 expression in Mtb co-cultured cells. Finally, VD3 treatment increased the release of the cytokine IFN-γ in Mtb-infected cells, while E2 treatment inhibited the release of IL-10, TNF, IFN-γ, and IL-6. Therefore, we report an immunogenetic influence of VD3 and E2 upon Mtb co-culture.

Acquiring of photosensitivity by Mycobacterium tuberculosis in vitro and inside infected macrophages is associated with accumulation of endogenous Zn–porphyrins

Mycobacterium tuberculosis (Mtb) is able to transition into a dormant state, causing the latent state of tuberculosis. Dormant mycobacteria acquire resistance to all known antibacterial drugs and can survive in the human body for decades before becoming active. In the dormant forms of M. tuberculosis, the synthesis of porphyrins and its Zn-complexes significantly increased when 5-aminolevulinic acid (ALA) was added to the growth medium. Transcriptome analysis revealed an activation of 8 genes involved in the metabolism of tetrapyrroles during the Mtb transition into a dormant state, which may lead to the observed accumulation of free porphyrins. Dormant Mtb viability was reduced by more than 99.99% under illumination for 30 min (300 J/cm2) with 565 nm light that correspond for Zn–porphyrin and coproporphyrin absorptions. We did not observe any PDI effect in vitro using active bacteria grown without ALA. However, after accumulation of active cells in lung macrophages and their persistence within macrophages for several days in the presence of ALA, a significant sensitivity of active Mtb cells (ca. 99.99%) to light exposure was developed. These findings create a perspective for the treatment of latent and multidrug-resistant tuberculosis by the eradication of the pathogen in order to prevent recurrence of this disease.

Transcriptional regulation of suppressors of cytokine signaling during infection with Mycobacterium tuberculosis in human THP-1-derived macrophages and in mice

Mycobacterium tuberculosis (Mtb) infection leads to upregulation of Suppressors of Cytokine signaling (SOCS) expression in host macrophages (Mϕ). SOCS proteins inhibit cytokine signaling by negatively regulating JAK/STAT. We investigated this host-pathogen dialectic at the level of transcription. We used phorbol-differentiated THP-1 Mϕ infected with Mtb to investigate preferential upregulation of some SOCS isoforms that are known to inhibit signaling by IFN-γ, IL-12, and IL-6. We examined time kinetics of likely transcription factors and signaling molecules upstream of SOCS transcription, and survival of intracellular Mtb following SOCS upregulation. Our results suggest a plausible mechanism that involves PGE2 secretion during infection to induce the PKA/CREB axis, culminating in nuclear translocation of C/EBPβ to induce expression of SOCS1. Mtb-infected Mϕ secreted IL-10, suggesting a mechanism of induction of STAT3, which may subsequently induce SOCS3. We provide evidence of temporal variation in SOCS isoform exspression and decay. Small-interfering RNA-mediated knockdown of SOCS1 and SOCS3 restored the pro-inflammatory milieu and reduced Mtb viability. In mice infected with Mtb, SOCS isoforms persisted across Days 28–85 post infection. Our results suggest that differential temporal regulation of SOCS isoforms by Mtb drives the host immune response towards a phenotype that facilitates the pathogen's survival.

Anti-Tuberculosis Potential of OJT008 against Active and Multi-Drug-Resistant Mycobacterium Tuberculosis: In Silico and In Vitro Inhibition of Methionine Aminopeptidase.

Despite the recent progress in the diagnosis of tuberculosis (TB), the chemotherapeutic management of TB continues to be challenging. Mycobacterium tuberculosis (Mtb), the etiological agent of TB, is classified as the 13th leading cause of death globally. In addition, 450,000 people were reported to develop multi-drug-resistant TB globally. The current project focuses on targeting methionine aminopeptidase (MetAP), an essential protein for the viability of Mtb. MetAP is a metalloprotease that catalyzes the excision of the N-terminal methionine (NME) during protein synthesis, allowing the enzyme to be an auspicious target for the development of novel therapeutic agents for the treatment of TB. Mtb possesses two MetAP1 isoforms, MtMetAP1a and MtMetAP1c, which are vital for Mtb viability and, hence, a promising chemotherapeutic target for Mtb therapy. In this study, we cloned and overexpressed recombinant MtMetAP1c. We investigated the in vitro inhibitory effect of the novel MetAP inhibitor, OJT008, on the cobalt ion- and nickel ion-activated MtMetAP1c, and the mechanism of action was elucidated through an in silico approach. The compound's potency against replicating and multi-drug-resistant (MDR) Mtb strains was also investigated. The induction of the overexpressed recombinant MtMetAP1c was optimized at 8 h with a final concentration of 1 mM Isopropyl β-D-1-thiogalactopyranoside. The average yield from 1 L of Escherichia coli culture for MtMetAP1c was 4.65 mg. A preliminary MtMetAP1c metal dependency screen showed optimum activation with nickel and cobalt ions occurred at 100 µM. The half-maximal inhibitory concentration (IC50) values of OJT008 against MtMetAP1c activated with CoCl2 and NiCl2 were 11 µM and 40 µM, respectively. The in silico study showed OJT008 strongly binds to both metal-activated MtMetAP1c, as evidenced by strong molecular interactions and a higher binding score, thereby corroborating our result. This in silico study validated the pharmacophore's metal specificity. The potency of OJT008 against both active and MDR Mtb was <0.063 µg/mL. Our study reports OJT008 as an inhibitor of MtMetAP1c, which is potent at low micromolar concentrations against both active susceptible and MDR Mtb. These results suggest OJT008 is a potential lead compound for the development of novel small molecules for the therapeutic management of TB.

Metabolically distinct roles of NAD synthetase and NAD kinase define the essentiality of NAD and NADP in Mycobacterium tuberculosis.

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated derivative (NADP) are essential cofactors that participate in hundreds of biochemical reactions and have emerged as therapeutic targets in cancer, metabolic disorders, neurodegenerative diseases, and infections, including tuberculosis. The biological basis for the essentiality of NAD(P) in most settings, however, remains experimentally unexplained. Here, we report that inactivation of the terminal enzyme of NAD synthesis, NAD synthetase (NadE), elicits markedly different metabolic and microbiologic effects than those of the terminal enzyme of NADP biosynthesis, NAD kinase (PpnK), in Mycobacterium tuberculosis (Mtb). Inactivation of NadE led to parallel reductions of both NAD and NADP pools and Mtb viability, while inactivation of PpnK selectively depleted NADP pools but only arrested growth. Inactivation of each enzyme was accompanied by metabolic changes that were specific for the affected enzyme and associated microbiological phenotype. Bacteriostatic levels of NAD depletion caused a compensatory remodeling of NAD-dependent metabolic pathways in the absence of an impact on NADH/NAD ratios, while bactericidal levels of NAD depletion resulted in a disruption of NADH/NAD ratios and inhibition of oxygen respiration. These findings reveal a previously unrecognized physiologic specificity associated with the essentiality of two evolutionarily ubiquitous cofactors. IMPORTANCE The current course for cure of Mycobacterium tuberculosis (Mtb)-the etiologic agent of tuberculosis (TB)-infections is lengthy and requires multiple antibiotics. The development of shorter, simpler treatment regimens is, therefore, critical to the goal of eradicating TB. NadE, an enzyme required for the synthesis of the ubiquitous cofactor NAD, is essential for survival of Mtb and regarded as a promising drug target. However, the basis of this essentiality was not clear due to its role in the synthesis of both NAD and NADP. Here, we resolve this ambiguity through a combination of gene silencing and metabolomics. We specifically show that NADP deficiency is bacteriostatic, while NAD deficiency is bactericidal due to its role in Mtb's respiratory capacity. These results argue for a prioritization of NAD biosynthesis inhibitors in anti-TB drug development.

Fragment merging strategies with known pyrimidine scaffolds targeting dihydrofolate reductase from Mycobacterium tuberculosis.

Dihydrofolate reductase (DHFR) is a key enzyme involved in the folate pathway that has been heavily targeted for the development of therapeutics against cancer and bacterial and protozoa infections amongst others. Despite being an essential enzyme for Mycobacterium tuberculosis (Mtb) viability, DHFR remains an underexploited target for tuberculosis (TB) treatment. Herein, we report the preparation and evaluation of a series of compounds against Mtb DHFR (MtbDHFR). The compounds have been designed using a merging strategy of traditional pyrimidine-based antifolates with a previously discovered unique fragment hit against MtbDHFR. In this series, four compounds displayed a high affinity against MtbDHFR, with sub-micromolar affinities. Additionally, we determined the binding mode of six of the best compounds using protein crystallography, which revealed occupation of an underutilised region of the active site.

Mycobacterium tuberculosis induces delayed lipid droplet accumulation in dendritic cells depending on bacterial viability and virulence.

Tuberculosis remains a global health threat with high morbidity. Dendritic cells (DCs) participate in the acute and chronic inflammatory responses to Mycobacterium tuberculosis (Mtb) by directing the adaptive immune response and are present in lung granulomas. In macrophages, the interaction of lipid droplets (LDs) with mycobacteria-containing phagosomes is central to host-pathogen interactions. However, the data available for DCs are still a matter of debate. Here, we reported that bone marrow-derived DCs (BMDCs) were susceptible to Mtb infection and replication at similar rate to macrophages. Unlike macrophages, the analysis of gene expression showed that Mtb infection induced a delayed increase in lipid droplet-related genes and proinflammatory response. Hence, LD accumulation has been observed by high-content imaging in late periods. Infection of BMDCs with killed H37Rv demonstrated that LD accumulation depends on Mtb viability. Moreover, infection with the attenuated strains H37Ra and Mycobacterium bovis-BCG induced only an early transient increase in LDs, whereas virulent Mtb also induced delayed LD accumulation. In addition, infection with the BCG strain with the reintroduced virulence RD1 locus induced higher LD accumulation and bacterial replication when compared to parental BCG. Collectively, our data suggest that delayed LD accumulation in DCs is dependent on mycobacterial viability and virulence.

Application of combined CRISPR screening for genetic and chemical-genetic interaction profiling in Mycobacterium tuberculosis.

CRISPR screening, including CRISPR interference (CRISPRi) and CRISPR-knockout (CRISPR-KO) screening, has become a powerful technology in the genetic screening of eukaryotes. In contrast with eukaryotes, CRISPR-KO screening has not yet been applied to functional genomics studies in bacteria. Here, we constructed genome-scale CRISPR-KO and also CRISPRi libraries in Mycobacterium tuberculosis (Mtb). We first examined these libraries to identify genes essential for Mtb viability. Subsequent screening identified dozens of genes associated with resistance/susceptibility to the antitubercular drug bedaquiline (BDQ). Genetic and chemical validation of the screening results suggested that it provided a valuable resource to investigate mechanisms of action underlying the effects of BDQ and to identify chemical-genetic synergies that can be used to optimize tuberculosis therapy. In summary, our results demonstrate the potential for efficient genome-wide CRISPR-KO screening in bacteria and establish a combined CRISPR screening approach for high-throughput investigation of genetic and chemical-genetic interactions in Mtb.

Antibacterial peptide CyclomarinA creates toxicity by deregulating the Mycobacterium tuberculosis ClpC1–ClpP1P2 protease

The ring-forming AAA+ hexamer ClpC1 associates with the peptidase ClpP1P2 to form a central ATP-driven protease in Mycobacterium tuberculosis (Mtb). ClpC1 is essential for Mtb viability and has been identified as the target of antibacterial peptides like CyclomarinA (CymA) that exhibit strong toxicity toward Mtb. The mechanistic actions of these drugs are poorly understood. Here, we dissected how ClpC1 activity is controlled and how this control is deregulated by CymA. We show that ClpC1 exists in diverse activity states correlating with its assembly. The basal activity of ClpC1 is low, as it predominantly exists in an inactive nonhexameric resting state. We show that CymA stimulates ClpC1 activity by promoting formation of supercomplexes composed of multiple ClpC1 hexameric rings, enhancing ClpC1–ClpP1P2 degradation activity toward various substrates. Both the ClpC1 resting state and the CymA-induced alternative assembly state rely on interactions between the ClpC1 coiled-coil middle domains (MDs). Accordingly, we found that mutation of the conserved aromatic F444 residue located at the MD tip blocks MD interactions and prevents assembly into higher order complexes, thereby leading to constitutive ClpC1 hexamer formation. We demonstrate that this assembly state exhibits the highest ATPase and proteolytic activities, yet its heterologous expression in Escherichia coli is toxic, indicating that the formation of such a state must be tightly controlled. Taken together, these findings define the basis of control of ClpC1 activity and show how ClpC1 overactivation by an antibacterial drug generates toxicity.

Inhibiting glycolysis by targeting the enzyme PFKFB3 restricts macrophage anti-mycobacterial activity and neutrophil phagocytosis of Mycobacterium tuberculosis.

Abstract Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB), a leading cause of infectious disease-related mortality around the world. Myeloid cells are important in TB as Mtb host cells and anti-Mtb effector cells but the factors that differentiate these roles are not clearly defined. Immunometabolism is a driver of immune function and may be an important determinant of this interaction and represents a potential target for host-directed therapies against TB. To better understand how immunometabolism relates to TB, we incubated macrophages and neutrophils from Mtb-infected macaques with a panel of metabolic inhibitors and measured how this affected cellular responses to Mtb infection. Specifically, we targeted glycolysis, oxidative phosphorylation, and fatty acid oxidation and measured how this affected macrophage and neutrophil phagocytosis, macrophage control over Mtb viability and replication, and neutrophil production of extracellular traps. We found that targeting glycolysis by inhibiting PFKFB3, a rate-limiting enzyme in glycolysis, reduced macrophage anti-Mtb activity and neutrophil phagocytosis, while inhibition with the glucose analog 2-DG did not. In contrast, incubating cell-free mycobacteria with PFKFB3 inhibitors severely inhibited their growth, suggesting that these bacteria use different metabolic pathways in cells and culture to survive. Inhibiting oxidative phosphorylation with metformin or fatty acid oxidation with etomoxir did not change how macrophages or neutrophils responded to Mtb or affect mycobacterial growth in culture. These results indicate a role for immunometabolism in myeloid responses to Mtb and may inform efforts for development of metabolism-targeted therapies for TB. Supported by grants from NIH (R01AI134183)

Identification of cell wall synthesis inhibitors active against Mycobacterium tuberculosis by competitive activity-based protein profiling

SummaryThe identification and validation of a small molecule’s targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecule’s targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here, we study the effects of 1,2,3-triazole ureas on Mycobacterium tuberculosis (Mtb). After screening ∼200 compounds, we focus on 4 compounds that form a structure-activity series. The compound with negligible activity reveals targets, the inhibition of which is functionally less relevant for Mtb growth and viability, an aspect not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirms that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings show that ABPP identifies the targets most likely relevant to a compound's antibacterial activity.

High-throughput screening to discover inhibitors of the CarD\xb7RNA polymerase protein\u2013protein interaction in Mycobacterium tuberculosis

Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) accounts for 3.7% of new cases of TB annually worldwide and is a major threat to global public health. Due to the prevalence of the MDR-TB and extensively drug resistant tuberculosis (XDR-TB) cases, there is an urgent need for new drugs with novel mechanisms of action. CarD, a global transcription regulator in MTB, binds RNAP and activates transcription by stabilizing the transcription initiation open-promoter complex (RPo). CarD is required for MTB viability and it has highly conserved homologues in many eubacteria. A fluorescence polarization (FP) assay which monitors the association of MTB RNAP, native rRNA promoter DNA and CarD has been developed. Overall, our objective is to identify and characterize small molecule inhibitors which block the CarD/RNAP interaction and to understand the mechanisms by which CarD interacts with the molecules. We expect that the development of a new and improved anti-TB compound with a novel mechanism of action will relieve the burden of resistance. This CarD FP assay is amenable to HTS and is an enabling tool for future novel therapeutic discovery.

Host transcriptional response to TB preventive therapy differentiates two sub-groups of IGRA-positive individuals

We hypothesised that individuals with immunological sensitisation to Mycobacterium tuberculosis (Mtb), conventionally regarded as evidence of latent tuberculosis infection (LTBI), would demonstrate binary responses to preventive therapy (PT), reflecting the differential immunological consequences of the sterilisation of viable infection in those with active Mtb infection versus no Mtb killing in those who did not harbour viable bacilli.We investigated longitudinal whole blood transcriptional profile responses to PT of Interferon gamma release assay (IGRA)-positive tuberculosis contacts and IGRA-negative, tuberculosis-unexposed controls. Longitudinal unsupervised clustering analysis with a subset of 474 most variable genes in antigen-stimulated blood separated the IGRA-positive participants into two distinct subgroups, one of which clustered with the IGRA-negative controls. 117 probes were differentially expressed over time between the two cluster groups, many of them associated with immunological pathways important in mycobacterial control.We contend that the differential host RNA response reflects lack of Mtb viability in the group that clustered with the IGRA-negative unexposed controls, and Mtb viability in the group (1/3 of IGRA-positives) that clustered away.Gene expression patterns in the blood of IGRA-positive individuals emerging during the course of PT, which reflect Mtb viability, could have major implications in the identification of risk of progression, treatment stratification and biomarker development.

Acyldepsipeptide Antibiotics and a Bioactive Fragment Thereof Differentially Perturb Mycobacterium tuberculosis ClpXP1P2 Activity in Vitro.

Proteolytic complexes in Mycobacterium tuberculosis (Mtb), the deadliest bacterial pathogen, are major foci in tuberculosis drug development programs. The Clp proteases, which are essential for Mtb viability, are high-priority targets. These proteases function through the collaboration of ClpP1P2, a barrel-shaped heteromeric peptidase, with associated ATP-dependent chaperones like ClpX and ClpC1 that recognize and unfold specific substrates in an ATP-dependent fashion. The critical interaction of the peptidase and its unfoldase partners is blocked by the competitive binding of acyldepsipeptide antibiotics (ADEPs) to the interfaces of the ClpP2 subunits. The resulting inhibition of Clp protease activity is lethal to Mtb. Here, we report the surprising discovery that a fragment of the ADEPs retains anti-Mtb activity yet stimulates rather than inhibits the ClpXP1P2-catalyzed degradation of proteins. Our data further suggest that the fragment stabilizes the ClpXP1P2 complex and binds ClpP1P2 in a fashion distinct from that of the intact ADEPs. A structure-activity relationship study of the bioactive fragment defines the pharmacophore and points the way toward the development of new drug leads for the treatment of tuberculosis.

Novel T9 loop conformation of filamenting temperature-sensitive mutant Z from Mycobacterium tuberculosis.

As of 2017, tuberculosis had infected 1.7 billion people (23% of the population of the world) and caused ten million deaths. Mycobacterium tuberculosis (Mtb) is quickly evolving, and new strains are classified as multidrug resistant. Thus, the identification of novel druggable targets is essential to combat the proliferation of these drug-resistant strains. Filamenting temperature-sensitive mutant Z (FtsZ) is a key protein involved in cytokinesis, an important process for Mtb proliferation and viability. FtsZ is required for bacterial cell division because it polymerizes into a structure called the Z-ring, which recruits accessory division proteins to the septum. Here, the crystal structure of the MtbFtsZ protein has been determined to 3.46 Å resolution and is described as a dimer of trimers, with an inter-subunit interface between protomers AB and DE. In this work, a novel conformation of MtbFtsZ is revealed involving the T9 loop and the nucleotide-binding pocket of protomers BC and EF.

Fragment Profiling Approach to Inhibitors of the Orphan M. tuberculosis P450 CYP144A1

Similarity between the ligand binding profiles of enzymes may aid functional characterization and be of greater relevance to inhibitor development than sequence similarity or structural homology. Fragment screening is an efficient approach for characterization of the ligand binding profile of an enzyme and has been applied here to study the family of cytochrome P450 enzymes (P450s) expressed by Mycobacterium tuberculosis (Mtb). The Mtb P450s have important roles in bacterial virulence, survival, and pathogenicity. Comparing the fragment profiles of seven of these enzymes revealed that P450s which share a similar biological function have significantly similar fragment profiles, whereas functionally unrelated or orphan P450s exhibit distinct ligand binding properties, despite overall high structural homology. Chemical structures that exhibit promiscuous binding between enzymes have been identified, as have selective fragments that could provide leads for inhibitor development. The similarity between the fragment binding profiles of the orphan enzyme CYP144A1 and CYP121A1, a characterized enzyme that is important for Mtb viability, provides a case study illustrating the subsequent identification of novel CYP144A1 ligands. The different binding modes of these compounds to CYP144A1 provide insight into structural and dynamic aspects of the enzyme, possible biological function, and provide the opportunity to develop inhibitors. Expanding this fragment profiling approach to include a greater number of functionally characterized and orphan proteins may provide a valuable resource for understanding enzyme-ligand interactions.

Alu repeats as transcriptional regulatory platforms in macrophage responses to M. tuberculosis infection.

To understand the epigenetic regulation of transcriptional response of macrophages during early-stage M. tuberculosis (Mtb) infection, we performed ChIPseq analysis of H3K4 monomethylation (H3K4me1), a marker of poised or active enhancers. De novo H3K4me1 peaks in infected cells were associated with genes implicated in host defenses and apoptosis. Our analysis revealed that 40% of de novo regions contained human/primate-specific Alu transposable elements, enriched in the AluJ and S subtypes. These contained several transcription factor binding sites, including those for members of the MEF2 and ATF families, and LXR and RAR nuclear receptors, all of which have been implicated in macrophage differentiation, survival, and responses to stress and infection. Combining bioinformatics, molecular genetics, and biochemical approaches, we linked genes adjacent to H3K4me1-associated Alu repeats to macrophage metabolic responses against Mtb infection. In particular, we show that LXRα signaling, which reduced Mtb viability 18-fold by altering cholesterol metabolism and enhancing macrophage apoptosis, can be initiated at response elements present in Alu repeats. These studies decipher the mechanism of early macrophage transcriptional responses to Mtb, highlighting the role of Alu element transposition in shaping human transcription programs during innate immunity.

Cigarette smoking impairs human pulmonary immunity to Mycobacterium tuberculosis.

Cigarette smoking is linked to important aspects of tuberculosis, such as susceptibility to infection, disease reactivation, mortality, transmission, and persistent infectiousness. The mechanistic basis for this remains poorly understood. To compare the functional impairment seen in human alveolar macrophages (AM) from nonsmokers, smokers, and ex-smokers after infection with Mycobacterium tuberculosis (Mtb). AM were acquired at bronchoscopy, and number and viability from smoking donors were compared with nonsmoking donors. AM were challenged in vitro with Mtb and intracellular bacterial viability was measured. Cytokine secretion was measured 24 hours postinfection by ELISA. Previously we determined the frequency of CD4(+)FoxP3(+) T cells in the presence or absence of allogeneic AM, and data were reanalyzed to separate the patient subjects according to smoking status. There were significantly more AM from smokers compared with nonsmokers or ex-smokers (P < 0.01). AM from smokers could not control intracellular Mtb growth. Nonsmokers' AM generated significantly more tumor necrosis factor (TNF)-α, IFN-γ, and IL-1β after Mtb infection compared with uninfected AM (P < 0.05). However, Mtb-infected AM from smokers did not secrete significantly more TNF-α, IFN-γ, and IL-1β compared with uninfected smokers' AM. AM taken from ex-smokers also failed to secrete significantly increased TNF-α, IFN-γ, and IL-1β after Mtb infection. Both smokers' and nonsmokers' AM induced FoxP3(+) T regulatory cell phenotype responses in allogeneic admixed T cells (>4.8 fold; P < 0.05). Even after Mtb infection, AM continued to drive this regulatory phenotype. In smokers, the pulmonary compartment has a number of macrophage-specific immune impairments that provide some mechanistic explanations whereby cigarette smoking renders a patient susceptible to tuberculosis infection and disease.

Primary and alternative S18 ribosomal protein in Mycobacterium tuberculosis

Protein synthesis is a tightly regulated process in every living organism and is likely to be a point of control in Mycobacterium tuberculosis (Mtb) during the course of infection. Mtb has four alternative ribosomal proteins organized in a single operon, whose function is not known, but may play an important role in translation regulation. Intriguingly, one of these proteins, S18–2, and its primary ribosomal protein paralog, S18–1, are both phosphorylated in vivo. Measured by their ability to bind another protein in a course of a ribosome biogenesis, their activities are not affected by phosphorylation, but it changes their stability in vitro. This possibly helps the exchange in response to zinc concentration, which regulates (represses) only expression of S18–2. In addition, presence of zinc had a profound and opposed effect on in vitro activities of S18–1 and S18–2 proteins, i.e., zinc activates S18–1 and inhibits S18–2. These data together suggest a model in which S18–1 is able to be a dominant ribosome constituent in high zinc conditions, but that it may be replaced by S18–2 when zinc is deficient. Therefore, S18–2 and other alternative ribosomal proteins may play a role in Mtb viability in necrotizing granulomas, which are thought to be a low zinc environment and are important for TB transmission.