Mechanopathology of biofilm-like Mycobacterium tuberculosis cords.

Mycobacterium tuberculosis (Mtb) cultured axenically without detergent forms biofilm-like cords, a clinical identifier of virulence. In lung-on-chip (LoC) and mouse models, cords in alveolar cells contribute to suppression of innate immune signaling via nuclear compression. Thereafter, extracellular cords cause contact-dependent phagocyte death but grow intercellularly between epithelial cells. The absence of these mechanopathological mechanisms explains the greater proportion of alveolar lesions with increased immune infiltration and dissemination defects in cording-deficient Mtb infections. Compression of Mtb lipid monolayers induces a phase transition that enables mechanical energy storage. Agent-based simulations demonstrate that the increased energy storage capacity is sufficient for the formation of cords that maintain structural integrity despite mechanical perturbation. Bacteria in cords remain translationally active despite antibiotic exposure and regrow rapidly upon cessation of treatment. This study provides a conceptual framework for the biophysics and function in tuberculosis infection and therapy of cord architectures independent of mechanisms ascribed to single bacteria.

Uptake-independent killing of macrophages by extracellular Mycobacterium tuberculosis aggregates.

Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular Mtb replication induces the death of the infected macrophages and the release of bacterial aggregates. Here, we show that these aggregates can evade phagocytosis by killing macrophages in a contact-dependent but uptake-independent manner. We use time-lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytosolic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb ESX-1 secretion system, however, this system alone cannot induce calcium accumulation and macrophage death in the absence of the Mtb surface-exposed lipid phthiocerol dimycocerosate. Unexpectedly, we found that blocking ESX-1-mediated secretion of the EsxA/EsxB virulence factors does not eliminate the uptake-independent killing of macrophages and that the 50-kDa isoform of the ESX-1-secreted protein EspB can mediate killing in the absence of EsxA/EsxB secretion. Treatment with an ESX-1 inhibitor reduces uptake-independent killing of macrophages by Mtb aggregates, suggesting that novel therapies targeting this anti-phagocytic mechanism could prevent the propagation of extracellular bacteria within the lung.

Preexisting variation in DNA damage response predicts the fate of single mycobacteria under stress.

Clonal microbial populations are inherently heterogeneous, and this diversification is often considered as an adaptation strategy. In clinical infections, phenotypic diversity is found to be associated with drug tolerance, which in turn could evolve into genetic resistance. Mycobacterium tuberculosis, which ranks among the top ten causes of mortality with high incidence of drug-resistant infections, exhibits considerable phenotypic diversity. In this study, we quantitatively analyze the cellular dynamics of DNA damage responses in mycobacteria using microfluidics and live-cell fluorescence imaging. We show that individual cells growing under optimal conditions experience sporadic DNA-damaging events manifested by RecA expression pulses. Single-cell responses to these events occur as transient pulses of fluorescence expression, which are dependent on the gene-network structure but are triggered by extrinsic signals. We demonstrate that preexisting subpopulations, with discrete levels of DNA damage response, are associated with differential susceptibility to fluoroquinolones. Our findings reveal that the extent of DNA integrity prior to drug exposure impacts the drug activity against mycobacteria, with conceivable therapeutic implications.

Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-on-chip model.

Severe cases of SARS-CoV-2 infection are characterized by hypercoagulopathies and systemic endotheliitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of an immune cell-mediated cytokine storm remain unknown. Using a vascularized lung-on-chip model, we find that infection of alveolar epithelial cells leads to limited apical release of virions, consistent with reports of monoculture infection. However, viral RNA and proteins are rapidly detected in underlying endothelial cells, which are themselves refractory to apical infection in monocultures. Although endothelial infection is unproductive, it leads to the formation of cell clusters with low CD31 expression, a progressive loss of barrier integrity and a pro-coagulatory microenvironment. Viral RNA persists in individual cells generating an inflammatory response, which is transient in epithelial cells but persistent in endothelial cells and typified by IL-6 secretion even in the absence of immune cells. Inhibition of IL-6 signalling with tocilizumab reduces but does not prevent loss of barrier integrity. SARS-CoV-2-mediated endothelial cell damage thus occurs independently of cytokine storm.

Mycobacterium tuberculosis EspK Has Active but Distinct Roles in the Secretion of EsxA and EspB.

The Mycobacterium tuberculosis type-7 protein secretion system ESX-1 is a major driver of its virulence. While the functions of most ESX-1 components are characterized, many others remain poorly defined. In this study, we examined the role of EspK, an ESX-1-associated protein that is thought to be dispensable for ESX-1 activity in members of the Mycobacterium tuberculosis complex. We show that EspK is needed for the timely and optimal secretion of EsxA and absolutely essential for EspB secretion in M. tuberculosis Erdman. We demonstrate that only the EsxA secretion defect can be alleviated in EspK-deficient M. tuberculosis by culturing it in media containing detergents like Tween 80 or tyloxapol. Subcellular fractionation experiments reveal EspK is exported by M. tuberculosis in an ESX-1-independent manner and localized to its cell wall. We also show a conserved W-X-G motif in EspK is important for its interaction with EspB and enabling its secretion. The same motif, however, is not important for EspK localization in the cell wall. Finally, we show EspB in EspK-deficient M. tuberculosis tends to adopt higher-order oligomeric conformations, more so than EspB in wild-type M. tuberculosis. These results suggest EspK interacts with EspB and prevents it from assembling prematurely into macromolecular complexes that are presumably too large to pass through the membrane-spanning ESX-1 translocon assembly. Collectively, our findings indicate M. tuberculosis EspK has a far more active role in ESX-1-mediated secretion than was previously appreciated and underscores the complex nature of this secretion apparatus. IMPORTANCE Mycobacterium tuberculosis uses its ESX-1 system to secrete EsxA and EspB into a host to cause disease. We show that EspK, a protein whose role in the ESX-1 machinery was thought to be nonessential, is needed by M. tuberculosis for optimal EsxA and EspB secretion. Culturing EspK-deficient M. tuberculosis with detergents alleviates EsxA but not EspB secretion defects. We also show that EspK, which is exported by M. tuberculosis in an ESX-1-independent manner to the cell wall, interacts with and prevents EspB from assembling into large structures inside the M. tuberculosis cell that are nonsecretable. Collectively, our observations demonstrate EspK is an active component of the ESX-1 secretion machinery of the tubercle bacillus.

Identification of aminopyrimidine-sulfonamides as potent modulators of Wag31-mediated cell elongation in mycobacteria.

There is an urgent need to discover new anti-tubercular agents with novel mechanisms of action in order to tackle the scourge of drug-resistant tuberculosis. Here, we report the identification of such a molecule - an AminoPYrimidine-Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis. Mutations in APYS1-resistant M. tuberculosis mapped exclusively to wag31, a gene that encodes a scaffolding protein thought to orchestrate cell elongation. Recombineering confirmed that a Gln201Arg mutation in Wag31 was sufficient to cause resistance to APYS1, however, neither overexpression nor conditional depletion of wag31 impacted M. tuberculosis susceptibility to this compound. In contrast, expression of the wildtype allele of wag31 in APYS1-resistant M. tuberculosis was dominant and restored susceptibility to APYS1 to wildtype levels. Time-lapse imaging and scanning electron microscopy revealed that APYS1 caused gross malformation of the old pole of M. tuberculosis, with eventual lysis. These effects resembled the morphological changes observed following transcriptional silencing of wag31 in M. tuberculosis. These data show that Wag31 is likely not the direct target of APYS1, but the striking phenotypic similarity between APYS1 exposure and genetic depletion of Wag31 in M. tuberculosis suggests that APYS1 might indirectly affect Wag31 through an as yet unknown mechanism.

Elucidating the role of (p)ppGpp in mycobacterial persistence against antibiotics.

Bacterial persistence, the ability of bacteria to survive high concentrations of antibiotics for extended periods of time, is an important contributing factor to therapy failure and development of chronic and recurrent infections. Several recent studies have suggested that this persistence is mediated primarily by (p)ppGpp, through its interactions with toxin-antitoxin modules and polyphosphates. In this study, we address whether these key players play a role in mycobacterial persistence against antibiotics. We targeted these specific pathways in Mycobacterium smegmatis by constructing deletion strains of (p)ppGpp synthetase/hydrolase (relA), polyphosphate kinases (ppk1 and ppk2), exopolyphosphatases (ppx1 and ppx2), and the lon protease. None of these mutant strains exhibited altered levels of persisters against isoniazid and ciprofloxacin, when compared with wild-type strain. Even under conditions in which the stringent response usually gets activated, these strains displayed wild-type persister levels. Interestingly, we also found that unlike Escherichia coli, maintaining M. smegmatis in exponential phase by repeated passaging does not eliminate persisters suggesting that at least against the antibiotics tested, stationary-phase dependent persisters (type I) are not the major contributors. Thus, our data demonstrate that multiple mechanisms of antibiotic persistence exist and that these vary widely among different bacterial species. © 2018 IUBMB Life, 70(9):836-844, 2018.

EspI regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis.

The function of EspI, a 70 kDa protein in Mycobacterium tuberculosis, has remained unclear. Although EspI is encoded by a gene within the esx-1 locus, in this study we clarify previous conflicting results and show that EspI is not essential for ESX-1-mediated secretion or virulence in M. tuberculosis. We also provide evidence that reduction of cellular ATP levels in wild-type M. tuberculosis using the drug bedaquiline completely blocks ESX-1-mediated secretion. Remarkably, M. tuberculosis lacking EspI fails to exhibit this phenotype. Furthermore, mutagenesis of a highly conserved ATP-binding motif in EspI renders M. tuberculosis incapable of shutting down ESX-1-mediated secretion during ATP depletion. Collectively these results show that M. tuberculosis EspI negatively regulates the ESX-1 secretion system in response to low cellular ATP levels and this function requires the ATP-binding motif. In light of our results the potential significance of EspI in ESX-1 function during latent tuberculosis infection and reactivation is also discussed.

Assessing the essentiality of the decaprenyl-phospho-d-arabinofuranose pathway in Mycobacterium tuberculosis using conditional mutants.

In Mycobacterium tuberculosis the decaprenyl-phospho-d-arabinofuranose (DPA) pathway is a validated target for the drugs ethambutol and benzothiazinones. To identify other potential drug targets in the pathway, we generated conditional knock-down mutants of each gene involved using the TET-PIP OFF system. dprE1, dprE2, ubiA, prsA, rv2361c, tkt and rpiB were confirmed to be essential under non-permissive conditions, whereas rv3807c was not required for survival. In the most vulnerable group, DprE1-depleted cells died faster in vitro and intracellularly than those lacking UbiA and PrsA. Downregulation of DprE1 and UbiA resulted in similar phenotypes, namely swelling of the bacteria, cell wall damage and lysis as observed at the single cell level, by real time microscopy and electron microscopy. By contrast, depletion of PrsA led to cell elongation and implosion, which was suggestive of a more pleiotropic effect. Drug sensitivity assays with known DPA-inhibitors supported the use of conditional knock-down strains for target-based whole-cell screens. Together, our work provides strong evidence for the vulnerability of all but one of the enzymes in the DPA pathway and generates valuable tools for the identification of lead compounds targeting the different biosynthetic steps. PrsA, phosphoribosyl-pyrophosphate synthetase, appears to be a particularly attractive new target for drug discovery.

Bioluminescence for assessing drug potency against nonreplicating Mycobacterium tuberculosis.

Targeting dormant Mycobacterium tuberculosis represents a challenge to antituberculosis drug discovery programs. We previously reported and validated the use of the streptomycin (STR)-dependent M. tuberculosis 18b strain as a tool for assessing drug potency against nonreplicating bacteria both in vitro and in vivo. In this study, we generated a luminescent 18b strain, named 18b-Lux, by transforming the bacteria with a vector expressing the luxCDABE operon from Photorhabdus luminescens. Luciferase expression was demonstrated under replicating conditions, and, more importantly, luminescence levels significantly above background were detected following STR removal. The sensitivity of STR-starved 18b-Lux to approved and candidate antituberculosis therapeutic agents was evaluated by means of a luciferase assay in a 96-well format. Results mirrored the data obtained with the standard resazurin reduction microplate assay, and the luminescence readout allowed time course assessments of drug efficacy in vitro. Specifically, we proved that bedaquiline, the rifamycins, and sutezolid displayed time-dependent activity against dormant bacteria, while pyrazinamide and SQ109 showed bactericidal effects at the highest concentrations tested. Overall, we established the optimal conditions for an inexpensive, simple, and very sensitive assay with great potential for future applications.

Rapid cytolysis of Mycobacterium tuberculosis by faropenem, an orally bioavailable ß-lactam antibiotic.

Recent clinical studies indicate that meropenem, a ß-lactam antibiotic, is a promising candidate for therapy of drug-resistant tuberculosis. However, meropenem is chemically unstable, requires frequent intravenous injection, and must be combined with a ß-lactamase inhibitor (clavulanate) for optimal activity. Here, we report that faropenem, a stable and orally bioavailable ß-lactam, efficiently kills Mycobacterium tuberculosis even in the absence of clavulanate. The target enzymes, L,D-transpeptidases, were inactivated 6- to 22-fold more efficiently by faropenem than by meropenem. Using a real-time assay based on quantitative time-lapse microscopy and microfluidics, we demonstrate the superiority of faropenem to the frontline antituberculosis drug isoniazid in its ability to induce the rapid cytolysis of single cells. Faropenem also showed superior activity against a cryptic subpopulation of nongrowing but metabolically active cells, which may correspond to the viable but nonculturable forms believed to be responsible for relapses following prolonged chemotherapy. These results identify faropenem to be a potential candidate for alternative therapy of drug-resistant tuberculosis.

Combinations of ß-Lactam Antibiotics Currently in Clinical Trials Are Efficacious in a DHP-I-Deficient Mouse Model of Tuberculosis Infection.

We report here a dehydropeptidase-deficient murine model of tuberculosis (TB) infection that is able to partially uncover the efficacy of marketed broad-spectrum ß-lactam antibiotics alone and in combination. Reductions of up to 2 log CFU in the lungs of TB-infected mice after 8 days of treatment compared to untreated controls were obtained at blood drug concentrations and time above the MIC (T>MIC) below clinically achievable levels in humans. These findings provide evidence supporting the potential of ß-lactams as safe and mycobactericidal components of new combination regimens against TB with or without resistance to currently used drugs.

The phosphatidyl-myo-inositol mannosyltransferase PimA is essential for Mycobacterium tuberculosis growth in vitro and in vivo.

The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.

Mycobacterium tuberculosis EspB binds phospholipids and mediates EsxA-independent virulence.

The type-VII ESX-1 secretion apparatus, encoded by the esx-1 genetic locus, is essential for the export of EsxA and EsxB, two major virulence factors of Mycobacterium tuberculosis. ESX-1 also requires the products of the unlinked espACD operon for optimal function and these proteins are considered integral parts of the secretion apparatus. Here we show that the espACD operon is not necessary for the secretion of EspB, another ESX-1 substrate, and this unimpeded secretion of EspB is associated with significant residual virulence. Upon further investigation, we found that purified EspB can facilitate M. tb virulence even in the absence of EsxA and EsxB, and may do so by binding the bioactive phospholipids phosphatidic acid and phosphatidylserine, both of which are potent bioactive molecules with prominent roles in eukaryotic cell signalling. Our findings provide new insights into the impact of the espACD operon on the ESX-1 apparatus and reveal a distinct virulence function for EspB with novel implications in M. tb-host interactions.

In vitro and in vivo activities of three oxazolidinones against nonreplicating Mycobacterium tuberculosis.

Oxazolidinones represent a new class of antituberculosis drugs that exert their function by inhibiting protein synthesis. Here, we compared the activities of three oxazolidinones, linezolid, PNU-100480, and AZD5847, against latent tuberculosis using a simple model employing the streptomycin-starved Mycobacterium tuberculosis strain 18b. The in vitro drug susceptibility results showed that the three oxazolidinones had a bacteriostatic effect against actively growing bacilli but potent bactericidal activity against nonreplicating cells. In the murine model of latent infection with M. tuberculosis 18b, the efficacy of the three compounds varied greatly. Indeed, AZD5847 or its prodrug exhibited no activity or only modest activity, respectively, after 2 months of treatment, whereas both linezolid and PNU-100480 were effective against latent bacilli in mice and showed promising outcomes in combination therapy with rifampin. Moreover, the potency of PNU-100480 was significantly greater than that of linezolid, making it an attractive drug candidate in the development of new combination therapies for latent tuberculosis.

Simple and rapid method to determine antimycobacterial potency of compounds by using autoluminescent Mycobacterium tuberculosis.

A major obstacle in the process of discovery of drugs against Mycobacterium tuberculosis is its extremely slow growth rate and long generation time (∼20 to 24 h). Consequently, determination of MICs and minimum bactericidal concentrations (MBCs) of potential drug candidates using current methods requires 7 days (resazurin-based MIC assay [REMA]) and 1 month (CFU enumeration), respectively. We employed a synthetic luciferase operon optimized for expression in high-GC-content bacteria and adapted it for use in mycobacteria. Using luminescence-based readouts, we were able to determine the MICs and bactericidal activities of approved tuberculosis (TB) drugs, which correlated well with currently used methods. Although luminescence-based readouts have been used previously to determine the MICs and bactericidal activities of approved TB drugs, in this study we adapted this assay to carry out a pilot screen using a library of 1,114 compounds belonging to diverse chemical scaffolds. We found that MICs derived from a 3-day luminescence assay matched well with REMA-based MIC values. To determine the bactericidal potencies of compounds, a 1:10 dilution of the cultures from the MIC plate was carried out on day 7, and the bactericidal concentrations determined based on time to positivity in 2 weeks were found to be comparable with MBC values determined by the conventional CFU approach. Thus, the luminescent mycobacterium-based approach not only is very simple and inexpensive but also allowed us to generate the information in half the time required by conventional methods.

Quantification of Mycobacterium tuberculosis Growth in Cell-Based Infection Assays by Time-Lapse Fluorescence Microscopy.

Quantification of Mycobacterium tuberculosis (Mtb) growth dynamics in cell-based in vitro infection models is traditionally carried out by measurement of colony forming units (CFU). However, Mtb being an extremely slow growing organism (16-24 h doubling time), this approach requires at least 3 weeks of incubation to obtain measurable readouts. In this chapter, we describe an alternative approach based on time-lapse microscopy and quantitative image analysis that allows faster quantification of Mtb growth dynamics in host cells. In addition, this approach provides the capability to capture other readouts from the same experimental setup, such as host cell viability, bacterial localization as well as the dynamics of propagation of infection between the host cells.

Establishing Air-Liquid Interface (ALI) Airway Culture Models for Infectious Disease Research.

Air-liquid interface (ALI) airway culture models serve as a powerful tool to emulate the characteristic features of the respiratory tract in vitro. These models are particularly valuable for studying emerging respiratory viral and bacterial infections. Here, we describe an optimized protocol to obtain the ALI airway culture models using normal human bronchial epithelial cells (NHBECs). The protocol outlined below enables the generation of differentiated mucociliary airway epithelial cultures by day 28 following exposure to air.

Increase in acute pancreatitis, especially gallstone related, as the cause for emergency admissions: Temporal trend from Kashmir, India.

BACKGROUND: The incidence of acute pancreatitis is increasing globally. Gallstones (GS) and ascariasis are the major causes for acute pancreatitis in the Kashmiri population. In recent years, we have observed an increase in the admission rate of acute pancreatitis. Many patients who present first time as gallstone pancreatitis have asymptomatic gallstones. We aimed at studying the etiology and yearly admission rate of acute pancreatitis with main focus on gallstone pancreatitis and the contribution of asymptomatic gallstones. METHODS: This was a hospital-based, prospective, observational study from January 2015 to December 2019 for a period of five years. Patients of acute pancreatitis were evaluated for etiology and yearly admission rate. Patients of gallstone pancreatitis were evaluated in terms of clinical profile, risk factors, nature (symptomatic/asymptomatic, known/unknown gallstones), size of stones, treatment and outcome in terms of severity and mortality. The data was analyzed by Statistical Package for the Social Sciences (SPSS) version 20.0, as mean (SD), frequencies and percentages. RESULTS: As many as 702 (8.5%) patients of acute pancreatitis were admitted among 8245 gastrointestinal emergencies in five years. The yearly admission rate of acute pancreatitis was 5.6%, 7.3%, 8.7%, 9.5% and 10.3%, respectively (p = 0.013). Gallstones, Ascariasis, alcohol and idiopathic acute pancreatitis were 47.7%, 6.9%, 1.2% and 33.7%, respectively. Gallstone pancreatitis increased from 31% in 2015 to 52.4% in 2019 (p = 0.045) and ascariasis-related acute pancreatitis declined from 14.4% to 1.6% (p = 0.034). Asymptomatic gallstones constituted 87.7% of cases. Known/unknown asymptomatic gallstones and symptomatic gallstones were 24.4%, 63.2% and 12.2%, respectively. Gallstones < 5 mm and > 5 mm were76.1% and 23.8% respectively (p = 0.027). Cholecystectomy rate in index admission was 4.7%. Mild, moderate and severe gallstone pancreatitis was 60.2%, 18.8% and 20.8%, respectively. Mortality in gallstone pancreatitis was 10.4%. CONCLUSION: The incidence of acute pancreatitis is increasing due to gallstone pancreatitis. Ascariasis-related acute pancreatitis has declined. There is significant contribution of asymptomatic gallstones in patients who present for the first time as acute pancreatitis. Small gallstones < 5 mm are likely to be the risk factors for gallstone pancreatitis.

Phenotypic profiling of Mycobacterium tuberculosis EspA point mutants reveals that blockage of ESAT-6 and CFP-10 secretion in vitro does not always correlate with attenuation of virulence.

The EspA protein of Mycobacterium tuberculosis is essential for the type VII ESX-1 protein secretion apparatus, which delivers the principal virulence factors ESAT-6 and CFP-10. In this study, site-directed mutagenesis of EspA was performed to elucidate its influence on the ESX-1 system. Replacing Trp(55) (W55) or Gly(57) (G57) residues in the putative W-X-G motif of EspA with arginines impaired ESAT-6 and CFP-10 secretion in vitro and attenuated M. tuberculosis. Replacing the Phe(50) (F50) and Lys(62) (K62) residues, which flank the W-X-G motif, with arginine and alanine, respectively, destabilized EspA, abolished ESAT-6 and CFP-10 secretion in vitro, and attenuated M. tuberculosis. Likewise, replacing the Phe(5) (F5) and Lys(41) (K41) residues with arginine and alanine, respectively, also destabilized EspA and blocked ESAT-6 and CFP-10 secretion in vitro. However, these two particular mutations did not attenuate M. tuberculosis in cellular models of infection or during acute infection in mice. We have thus identified amino acid residues in EspA that are important for facilitating ESAT-6 and CFP-10 secretion and virulence. However, our data also indicate for the first time that blockage of M. tuberculosis ESAT-6 and CFP-10 secretion in vitro and attenuation are mutually exclusive.