Mycobacterium tuberculosis-Induced Polarization of Human Macrophage Orchestrates the Formation and Development of Tuberculous Granulomas In Vitro

The tuberculous granuloma is an elaborately organized structure and one of the main histological hallmarks of tuberculosis. Macrophages, which are important immunologic effector and antigen-presenting cells, are the main cell type found in the tuberculous granuloma and have high plasticity. Macrophage polarization during bacterial infection has been elucidated in numerous recent studies; however, macrophage polarization during tuberculous granuloma formation and development has rarely been reported. It remains to be clarified whether differences in the activation status of macrophages affect granuloma formation. In this study, the variation in macrophage polarization during the formation and development of tuberculous granulomas was investigated in both sections of lung tissues from tuberculosis patients and an in vitro tuberculous granuloma model. The roles of macrophage polarization in this process were also investigated. Mycobacterium tuberculosis (M. tuberculosis) infection was found to induce monocyte-derived macrophage polarization. In the in vitro tuberculous granuloma model, macrophage transformation from M1 to M2 was observed over time following M. tuberculosis infection. M2 macrophages were found to predominate in both necrotic and non-necrotic granulomas from tuberculosis patients, while both M1 and M2 polarized macrophages were found in the non-granulomatous lung tissues. Furthermore, it was found that M1 macrophages promote granuloma formation and macrophage bactericidal activity in vitro, while M2 macrophages inhibit these effects. The findings of this study provide insights into the mechanism by which M. tuberculosis circumvents the host immune system as well as a theoretical foundation for the development of novel tuberculosis therapies based on reprogramming macrophage polarization.     

Attenuated Mycobacterium tuberculosis SO2 Vaccine Candidate Is Unable to Induce Cell Death

It has been proposed that Mycobacterium tuberculosis virulent strains inhibit apoptosis and trigger cell death by necrosis of host macrophages to evade innate immunity, while non-virulent strains induce typical apoptosis activating a protective host response. As part of the characterization of a novel tuberculosis vaccine candidate, the M. tuberculosis phoP mutant SO2, we sought to evaluate its potential to induce host cell death. The parental M. tuberculosis MT103 strain and the current vaccine against tuberculosis Bacillus Calmette-Guérin (BCG) were used as comparators in mouse models in vitro and in vivo. Our data reveal that attenuated SO2 was unable to induce apoptotic events neither in mouse macrophages in vitro nor during lung infection in vivo. In contrast, virulent MT103 triggers typical apoptotic events with phosphatidylserine exposure, caspase-3 activation and nuclear condensation and fragmentation. BCG strain behaved like SO2 and did not induce apoptosis. A clonogenic survival assay confirmed that viability of BCG- or SO2-infected macrophages was unaffected. Our results discard apoptosis as the protective mechanism induced by SO2 vaccine and provide evidence for positive correlation between classical apoptosis induction and virulent strains, suggesting apoptosis as a possible virulence determinant during M. tuberculosis infection.     

The Prevalence and Drug Sensitivity of Tuberculosis among Patients Dying in Hospital in KwaZulu-Natal,South Africa: A Postmortem Study

Background

Tuberculosis is the leading cause of death in South Africa by death notification, but accurate diagnosis of tuberculosis is challenging in this setting of high HIV prevalence. We conducted limited autopsies on young adults dying in a single public hospital in the province of KwaZulu-Natal between October 2008 and August 2009 in order to estimate the magnitude of deaths attributable to tuberculosis.

Methods and Findings

We studied a representative sample of 240 adult inpatients (aged 20–45 years) dying after admission to Edendale Hospital. Limited autopsies included collection of respiratory tract secretions and tissue by needle core biopsies of lung, liver, and spleen. Specimens were examined by fluorescent microscopy for acid-fast bacilli and cultured in liquid media; cultures positive for M. tuberculosis were tested for drug susceptibility to first- and second-line antibiotics. Ninety-four percent of our study cohort was HIV seropositive and 50% of decedents had culture-positive tuberculosis at the time of death. Fifty percent of the participants were on treatment for tuberculosis at the time of death and 58% of these treated individuals remained culture positive at the time of death. Of the 50% not receiving tuberculosis treatment, 42% were culture positive. Seventeen percent of all positive cultures were resistant to both isoniazid and rifampin (i.e., multidrug resistant); 16% of patients dying during the initiation phase of their first ever course of tuberculosis treatment were infected with multidrug-resistant bacilli.

Conclusions

Our findings reveal the immense toll of tuberculosis among HIV-positive individuals in KwaZulu-Natal. The majority of decedents who remained culture positive despite receiving tuberculosis treatment were infected with pan-susceptible M. tuberculosis, suggesting that the diagnosis of tuberculosis was made too late to alter the fatal course of the infection. There is also a significant burden of undetected multidrug-resistant tuberculosis among HIV-coinfected individuals dying in this setting. New public health approaches that improve early diagnosis of tuberculosis and accelerate the initiation of treatment are urgently needed in this setting. Please see later in the article for the Editors'' Summary     

Evaluation of the BD MGIT TBc Identification Test (TBc ID), a rapid chromatographic immunoassay for the detection of Mycobacterium tuberculosis complex from liquid culture

The BACTEC MGIT 960 system is increasingly used to culture Mycobacterium tuberculosis. We evaluated the performance of the new immunochromatographic assay BD MGIT TBc Identification Test (TBc ID) for the rapid identification of M. tuberculosis complex in clinical samples when performed directly from BACTEC MGIT 960 culture positive for acid-fast bacilli (AFB).Of 92 cultures evaluated, the sensitivity and specificity of the TBc ID test was 98.5% and 100%, respectively compared to sequencing of the 16S rRNA gene. One culture that was TBc ID test negative but that was identified as M. tuberculosis by 16S rRNA sequencing was confirmed to have a mutation in the mpt64 gene.The TBc ID test is an easy and sensitive method for the identification of M. tuberculosis complex in liquid culture medium, does not require a high level of skills, neither any additional specific equipment and gives results in 15 min, which provide a good alternative for the rapid identification of M. tuberculosis complex in liquid medium.     

Intracellular replication of attenuated Mycobacterium tuberculosis phoP mutant in the absence of host cell cytotoxicity

Intracellular pathogen Mycobacterium tuberculosis survives and replicates in macrophages but limited information is available on its replication into non-phagocytic cells. Here we study the role of the M. tuberculosis virulence gene phoP in the intracellular growth with rat and human lung fibroblasts. In contrast to macrophages, attenuated M. tuberculosis phoP mutant was able to multiply intracellularly in fibroblasts at the same level as the virulent M. tuberculosis. However, when M. tuberculosis virulence was studied using human foetal lung fibroblasts, MRC-5 cell line, the virulent strain caused a significant damage in cells compared with attenuated strains BCG and M. tuberculosis phoP mutant. We analysed the effect of cytoskeleton inhibitors in NRK-49F fibroblasts. M. tuberculosis invasion was not inhibited, suggesting that mycobacterial uptake was microtubule and microfilament independent. Our results suggest that PhoP in M. tuberculosis does not regulate intracellular replication in fibroblasts, contrary to what happens in macrophages. The ability of M. tuberculosis phoP mutant to replicate within non-phagocytic cells, such as fibroblasts, without causing damage, could be a potential advantage for a live attenuated vaccine against tuberculosis.     

Decreased Expression of miR-21, miR-26a,miR-29a,and miR-142-3p in CD4+ T Cells and Peripheral Blood from Tuberculosis Patients

The vast majority of Mycobacterium tuberculosis (M. tuberculosis) infected individuals are protected from developing tuberculosis and T cells are centrally involved in this process. MicroRNAs (miRNA) regulate T-cell functions and are biomarker candidates of disease susceptibility and treatment efficacy in M. tuberculosis infection. We determined the expression profile of 29 selected miRNAs in CD4⁺ T cells from tuberculosis patients and contacts with latent M. tuberculosis infection (LTBI). These analyses showed lower expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4⁺ T cells from tuberculosis patients. Whole blood miRNA candidate analyses verified decreased expression of miR-26a, miR-29a, and miR-142-3p in children with tuberculosis as compared to healthy children with LTBI. Despite marked variances between individual donor samples, trends of increased miRNA candidate expression during treatment and recovery were observed. Functional in vitro analysis identified increased miR-21 and decreased miR-26a expression after re-stimulation of T cells. In vitro polarized Interleukin-17 positive T-cell clones showed activation-dependent miR-29a up-regulation. In order to characterize the role of miR-29a (a described suppressor of Interferon-γ in tuberculosis), we analyzed M. tuberculosis specific Interferon-γ expressing T cells in children with tuberculosis and healthy contacts but detected no correlation between miR-29a and Interferon-γ expression. Suppression of miR-29a in primary human T cells by antagomirs indicated no effect on Interferon-γ expression after in vitro activation. Finally, classification of miRNA targets revealed only a moderate overlap between the candidates. This may reflect differential roles of miR-21, miR-26a, miR-29a, and miR-142-3p in T-cell immunity against M. tuberculosis infection and disease.     

The Genetic Requirements for Fast and Slow Growth in Mycobacteria

Mycobacterium tuberculosis infects a third of the world''s population. Primary tuberculosis involving active fast bacterial replication is often followed by asymptomatic latent tuberculosis, which is characterised by slow or non-replicating bacteria. Reactivation of the latent infection involving a switch back to active bacterial replication can lead to post-primary transmissible tuberculosis. Mycobacterial mechanisms involved in slow growth or switching growth rate provide rational targets for the development of new drugs against persistent mycobacterial infection. Using chemostat culture to control growth rate, we screened a transposon mutant library by Transposon site hybridization (TraSH) selection to define the genetic requirements for slow and fast growth of Mycobacterium bovis (BCG) and for the requirements of switching growth rate. We identified 84 genes that are exclusively required for slow growth (69 hours doubling time) and 256 genes required for switching from slow to fast growth. To validate these findings we performed experiments using individual M. tuberculosis and M. bovis BCG knock out mutants. We have demonstrated that growth rate control is a carefully orchestrated process which requires a distinct set of genes encoding several virulence determinants, gene regulators, and metabolic enzymes. The mce1 locus appears to be a component of the switch to slow growth rate, which is consistent with the proposed role in virulence of M. tuberculosis. These results suggest novel perspectives for unravelling the mechanisms involved in the switch between acute and persistent TB infections and provide a means to study aspects of this important phenomenon in vitro.     

Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4–6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.     

Identification of T-Cell Antigens Specific for Latent Mycobacterium Tuberculosis Infection

Background

T-cell responses against dormancy-, resuscitation-, and reactivation-associated antigens of Mycobacterium tuberculosis are candidate biomarkers of latent infection in humans.

Methodology/Principal Findings

We established an assay based on two rounds of in vitro restimulation and intracellular cytokine analysis that detects T-cell responses to antigens expressed during latent M. tuberculosis infection. Comparison between active pulmonary tuberculosis (TB) patients and healthy latently M. tuberculosis-infected donors (LTBI) revealed significantly higher T-cell responses against 7 of 35 tested M. tuberculosis latency-associated antigens in LTBI. Notably, T cells specific for Rv3407 were exclusively detected in LTBI but not in TB patients. The T-cell IFNγ response against Rv3407 in individual donors was the most influential factor in discrimination analysis that classified TB patients and LTBI with 83% accuracy using cross-validation. Rv3407 peptide pool stimulations revealed distinct candidate epitopes in four LTBI.

Conclusions

Our findings further support the hypothesis that the latency-associated antigens can be exploited as biomarkers for LTBI.     

Interactions between Na?ve and Infected Macrophages Reduce Mycobacterium tuberculosis Viability

A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with naïve macrophages produced an antimicrobial effect, but only if naïve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the naïve macrophages. The antimicrobial effect of naïve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the naïve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of naïve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which promotes nitric oxide-dependent antimicrobial activity against bacilli in autolysosomes of heavily infected macrophages. This cooperative, innate antimicrobial interaction may limit the maximal growth rate of M. tuberculosis prior to the expression of adaptive immunity in pulmonary tuberculosis.     

Homology Modeling, Molecular Docking and DNA Binding Studies of Nucleotide Excision Repair UvrC Protein from M. tuberculosis

Mycobacterium tuberculosis is a Gram positive, acid-fast bacteria belonging to genus Mycobacterium, is the leading causative agent of most cases of tuberculosis. The pathogenicity of the bacteria is enhanced by its developed DNA repair mechanism which consists of machineries such as nucleotide excision repair. Nucleotide excision repair consists of excinuclease protein UvrABC endonuclease, multi-enzymatic complex which carries out repair of damaged DNA in sequential manner. UvrC protein is a part of this complex and thus helps to repair the damaged DNA of M. tuberculosis. Hence, structural bioinformatics study of UvrC protein from M. tuberculosis was carried out using homology modeling and molecular docking techniques. Assessment of the reliability of the homology model was carried out by predicting its secondary structure along with its model validation. The predicted structure was docked with the ATP and the interacting amino acid residues of UvrC protein with the ATP were found to be TRP539, PHE89, GLU536, ILE402 and ARG575. The binding of UvrC protein with the DNA showed two different domains. The residues from domain I of the protein VAL526, THR524 and LEU521 interact with the DNA whereas, amino acids interacting from the domain II of the UvrC protein included ARG597, GLU595, GLY594 and GLY592 residues. This predicted model could be useful to design new inhibitors of UvrC enzyme to prevent pathogenesis of Mycobacterium and so the tuberculosis.     

Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis – actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease – which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.     

Culture Conversion Rate at 2 Months of Treatment According to Diagnostic Methods among Patients with Culture-Positive Pulmonary Tuberculosis

Introduction

The culture-negative conversion rate of sputum after 2 months of treatment in patients with pulmonary tuberculosis (TB) is used as a reliable surrogate marker for relapse after completion of treatment. We hypothesized that culture conversion of sputum at 2 months of anti-TB treatment and the time to culture conversion are different among pulmonary TB patients who are diagnosed using different methods.

Methods

Culture-confirmed pulmonary TB patients who were diagnosed between 1 January, 2011 and 31 December, 2012 were classified into three groups based on the diagnostic method that prompted treatment initiation: positive acid-fast bacilli (AFB) staining of sputum (smear-positive group), negative AFB staining, but Mycobacterium tuberculosis was cultured from sputum (culture-positive group), and positive AFB staining, positive polymerase chain reaction (PCR) for M. tuberculosis, or culture of M. tuberculosis from a bronchoscopic specimen (bronchoscopy group). Rates of negative mycobacterial culture conversion at 2 months of anti-TB treatment and the time to negative culture conversion of sputum were compared among the three groups.

Results

A total of 203 patients with culture-confirmed pulmonary TB were included in the final analysis. TB patients in the culture-positive group (94.1%) and the bronchoscopy group (97.6%) showed a higher culture conversion rate at 2 months of treatment than those in the smear-positive group (78.7%, P = 0.001). Additionally, the time to culture conversion was longer in the smear-positive group (median, 40 days) than in the culture-positive (median, 19 days; P = 0.009) and bronchoscopy groups (median, 29 days; P = 0.004).

Conclusions

The higher culture conversion rate at 2 months and the shorter time to culture conversion among pulmonary TB patients with a negative AFB smear suggests the feasibility of shortening treatment duration and isolation in these patients.     

Time to Culture Positivity and Sputum Smear Microscopy during Tuberculosis Therapy

Sputum smear microscopy is widely used for tuberculosis diagnosis and treatment monitoring. We evaluated the correlation between smear microscopy and time to liquid culture positivity during early tuberculosis treatment. The study included patients with smear-positive pulmonary tuberculosis hospitalized at a tuberculosis reference centre in Germany between 01/2012 and 05/2013. Patient records were reviewed and clinical, radiological and microbiological data were analysed. Sputum samples were collected before treatment initiation and weekly thereafter. A number of 310 sputum samples from 30 patients were analysed. Time to liquid culture positivity inversely correlated with smear grade (Spearman''s rho −0.439, p<0.001). There was a better correlation within the first two months vs. after two months of therapy (−0.519 vs. −0.416) with a trend to a more rapid increase in time to positivity between baseline and week 2 in patients who culture-converted within the first two months (5.9 days vs. 9.4 days, p = 0.3). In conclusion, the numbers of acid-fast bacilli in sputum smears of patients with pulmonary tuberculosis and time to culture positivity for M. tuberculosis cultures from sputum are correlated before and during tuberculosis treatment. A considerable proportion of patients with culture conversion after two months of therapy continued to have detectable acid-fast bacilli on sputum smears.     

Phenylalanine-Rich Peptides Potently Bind ESAT6, a Virulence Determinant of Mycobacterium tuberculosis,and Concurrently Affect the Pathogen's Growth

Background

The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung.

Methodology/Principal Findings

During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage.

Conclusions/Significance

While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide''s binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen.     

Discovery of Mycobacterium tuberculosis α-1,4-Glucan Branching Enzyme (GlgB) Inhibitors by Structure- and Ligand-based Virtual Screening

GlgB (α-1,4-glucan branching enzyme) is the key enzyme involved in the biosynthesis of α-glucan, which plays a significant role in the virulence and pathogenesis of Mycobacterium tuberculosis. Because α-glucans are implicated in the survival of both replicating and non-replicating bacteria, there exists an exigent need for the identification and development of novel inhibitors for targeting enzymes, such as GlgB, involved in this pathway. We have used the existing structural information of M. tuberculosis GlgB for high throughput virtual screening and molecular docking. A diverse database of 330,000 molecules was used for identifying novel and efficacious therapeutic agents for targeting GlgB. We also used three-dimensional shape as well as two-dimensional similarity matrix methods to identify diverse molecular scaffolds that inhibit M. tuberculosis GlgB activity. Virtual hits were generated after structure and ligand-based screening followed by filters based on interaction with human GlgB and in silico pharmacokinetic parameters. These hits were experimentally evaluated and resulted in the discovery of a number of structurally diverse chemical scaffolds that target M. tuberculosis GlgB. Although a number of inhibitors demonstrated in vitro enzyme inhibition, two compounds in particular showed excellent inhibition of in vivo M. tuberculosis survival and its ability to get phagocytosed. This work shows that in silico docking and three-dimensional chemical similarity could be an important therapeutic approach for developing inhibitors to specifically target the M. tuberculosis GlgB enzyme.     

Mycobacterium tuberculosis TlyA Protein Negatively Regulates T Helper (Th) 1 and Th17 Differentiation and Promotes Tuberculosis Pathogenesis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M. tuberculosis have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor in several bacterial infections and is evolutionarily conserved in many Gram-positive bacteria, but its function in M. tuberculosis pathogenesis has not been elucidated. Here, we report that TlyA significantly contributes to the pathogenesis of M. tuberculosis. We show that a TlyA mutant M. tuberculosis strain induces increased IL-12 and reduced IL-1β and IL-10 cytokine responses, which sharply contrasts with the immune responses induced by wild type M. tuberculosis. Furthermore, compared with wild type M. tuberculosis, TlyA-deficient M. tuberculosis bacteria are more susceptible to autophagy in macrophages. Consequently, animals infected with the TlyA mutant M. tuberculosis organisms exhibited increased host-protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M. tuberculosis. Thus, M. tuberculosis employs TlyA as a host evasion factor, thereby contributing to its virulence.     

Nitazoxanide Stimulates Autophagy and Inhibits mTORC1 Signaling and Intracellular Proliferation of Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.