Drug resistance prediction for Mycobacterium tuberculosis with reference graphs.

Tuberculosis is a global pandemic disease with a rising burden of antimicrobial resistance. As a result, the World Health Organization (WHO) has a goal of enabling universal access to drug susceptibility testing (DST). Given the slowness of and infrastructure requirements for phenotypic DST, whole-genome sequencing, followed by genotype-based prediction of DST, now provides a route to achieving this. Since a central component of genotypic DST is to detect the presence of any known resistance-causing mutations, a natural approach is to use a reference graph that allows encoding of known variation. We have developed DrPRG (Drug resistance Prediction with Reference Graphs) using the bacterial reference graph method Pandora. First, we outline the construction of a Mycobacterium tuberculosis drug resistance reference graph. The graph is built from a global dataset of isolates with varying drug susceptibility profiles, thus capturing common and rare resistance- and susceptible-associated haplotypes. We benchmark DrPRG against the existing graph-based tool Mykrobe and the haplotype-based approach of TBProfiler using 44 709 and 138 publicly available Illumina and Nanopore samples with associated phenotypes. We find that DrPRG has significantly improved sensitivity and specificity for some drugs compared to these tools, with no significant decreases. It uses significantly less computational memory than both tools, and provides significantly faster runtimes, except when runtime is compared to Mykrobe with Nanopore data. We discover and discuss novel insights into resistance-conferring variation for M. tuberculosis - including deletion of genes katG and pncA - and suggest mutations that may warrant reclassification as associated with resistance.

Genomic epidemiology sheds light on the emergence and spread of Mycobacterium bovis Eu2 Clonal Complex in Portugal.

ABSTRACTAnimal tuberculosis (TB) remains a serious concern for animal and human health. Mycobacterium bovis circulates in multi-host systems, dominated by the European 2 clonal complex (Eu2) in Iberia. In this work, we use genomic epidemiology to infer the emergence, spread, and spatiotemporal patterns of Eu2 in the official epidemiological risk area of animal TB in Portugal. Phylogenetic analysis of 144 M. bovis whole-genome sequences from cattle, wild boar, and red deer, representing the 2002-2021 period, distinguished three Eu2 clades that evolved independently. The major Eu2 clade underwent phylodynamic inferences to estimate the time and location of outbreaks, host transitions, and spatial diffusion as well. The origin of this Eu2 clade was attributed to the red deer population in the Castelo Branco district, near the border with Spain. Most host transitions were intraspecific (80%), while interspecific transmissions between wildlife species (wild boar-red deer), and between wild boar and cattle, were highly supported. Phylogeographic reconstruction evidenced that most transitions (82%) occur within municipalities, highlighting local transmission corridors.Our study indicates that M. bovis continues to spread at the cattle-wildlife interface within the animal TB hotspot area, possibly driven by the foraging behaviour of wild boar near agricultural lands. Red deer seems to be an important driver of TB within wildlife hosts, while the wild boar links the multi-host wildlife community and livestock. This work highlights the value of combining genomic epidemiology with phylodynamic inference to resolve host jumps and spatial patterns of M. bovis, providing real-time clues about points of intervention.

Ophthalmic manifestations of tuberculosis.

PURPOSE OF REVIEW: To evaluate the epidemiology, microbiology, and pathology of Mycobacterium Tuberculosis (MTB). Utilizing these basic science concepts, the reader will discover how MTB can cause disease in any part of ophthalmic system. This article will aid clinicians in establishing the difficult diagnosis and management strategies for ophthalmic tuberculosis (OTB). RECENT FINDINGS: Recently, expert panels have reached a consensus on naming conventions and treatment strategies for the variety of ocular tuberculosis (TB). This consensus helps individual clinicians decide when to recommend full anti-TB treatment. SUMMARY: Globally, TB is nearly ubiquitous in the human population. It is most recognized for its pulmonary disease, but pathology of nearly every structure of the ophthalmic system has been identified. This heterogeneity makes establishing a diagnosis difficult, but recent improvements in expert panel naming consensus and nucleic acid amplification tests are improving diagnostic abilities. Clinicians are now feeling more confident with prescribing anti-TB regimens, but ongoing questions regarding the use of oral steroids and risk of medication-induced ocular toxicity remain.

Transcriptional signatures measured in whole blood correlate with protection against tuberculosis in inbred and outbred mice.

Although BCG has been used for almost 100 years to immunize against Mycobacterium tuberculosis, TB remains a global public health threat. Numerous clinical trials are underway studying novel vaccine candidates and strategies to improve or replace BCG, but vaccine development still lacks a well-defined set of immune correlates to predict vaccine-induced protection against tuberculosis. This study aimed to address this gap by examining transcriptional responses to BCG vaccination in C57BL/6 inbred mice, coupled with protection studies using Diversity Outbred mice. We evaluated relative gene expression in blood obtained from vaccinated mice, because blood is easily accessible, and data can be translated to human studies. We first determined that the average peak time after vaccination is 14 days for gene expression of a small subset of immune-related genes in inbred mice. We then performed global transcriptomic analyses using whole blood samples obtained two weeks after mice were vaccinated with BCG. Using comparative bioinformatic analyses and qRT-PCR validation, we developed a working correlate panel of 18 genes that were highly correlated with administration of BCG but not heat-killed BCG. We then tested this gene panel using BCG-vaccinated Diversity Outbred mice and revealed associations between the expression of a subset of genes and disease outcomes after aerosol challenge with M. tuberculosis. These data therefore demonstrate that blood-based transcriptional immune correlates measured within a few weeks after vaccination can be derived to predict protection against M. tuberculosis, even in outbred populations.

[Effects of macrophage glycolytic reprogramming on tuberculosis granuloma formation].

The formation of granulomatous lesions is a typical pathological feature of tuberculosis, and infection with Mycobacterium tuberculosis is the main cause. Although the mechanism underlying granuloma formation remains unclear, increasing evidence suggests that immune metabolism plays an important role. In this review, we summarized the latest advances in macrophage glycolytic reprogramming in tuberculosis granuloma formation to discover new methods for early diagnosis and provided new ideas for tuberculosis therapeutics based on the regulation of immune metabolism.

The role of Mycobacterium tuberculosis acetyltransferase and protein acetylation modifications in tuberculosis.

Tuberculosis (TB) is a widespread infectious disease caused by Mycobacterium tuberculosis (M. tb), which has been a significant burden for a long time. Post-translational modifications (PTMs) are essential for protein function in both eukaryotic and prokaryotic cells. This review focuses on the contribution of protein acetylation to the function of M. tb and its infected macrophages. The acetylation of M. tb proteins plays a critical role in virulence, drug resistance, regulation of metabolism, and host anti-TB immune response. Similarly, the PTMs of host proteins induced by M. tb are crucial for the development, treatment, and prevention of diseases. Host protein acetylation induced by M. tb is significant in regulating host immunity against TB, which substantially affects the disease's development. The review summarizes the functions and mechanisms of M. tb acetyltransferase in virulence and drug resistance. It also discusses the role and mechanism of M. tb in regulating host protein acetylation and immune response regulation. Furthermore, the current scenario of isoniazid usage in M. tb therapy treatment is examined. Overall, this review provides valuable information that can serve as a preliminary basis for studying pathogenic research, developing new drugs, exploring in-depth drug resistance mechanisms, and providing precise treatment for TB.

Macrophage targeted polymeric curcumin nanoparticles limit intracellular survival of Mycobacterium tuberculosis through induction of autophagy and augment anti-TB activity of isoniazid in RAW 264.7 macrophages.

Rapid emergence of antibiotic resistance in tuberculosis has left us with limited resources to treat and manage multi drug resistant (MDR) cases of tuberculosis, prompting the development of novel therapeutics. Mycobacterium tuberculosis (MTB) perturbs the host protective pathways for its survival, therefore host directed therapeutic (HDT) interventions offer an attractive alternative strategy. Curcumin (CMN), the principle curcuminoid from Curcuma longa is known to have anti-TB activity against MDR strains of MTB in macrophages. We discovered that treatment of CMN induced autophagy in uninfected and MTB infected macrophages which was evident by conversion of LC3-I to LC3-II and degradation of p62. Inhibition of autophagy by a pharmacological inhibitor 3-MA resulted in significant inhibition of intracellular killing activity of CMN, suggesting the involvement of autophagy in intracellular clearance of MTB. Moreover, annexin v-FITC/PI staining data suggested induction of apoptosis in uninfected and MTB infected macrophages post CMN treatment. This finding was further corroborated by up-regulated expression of pro-apoptotic proteins, Bax, cleaved caspase-3 and PARP and diminished expression of anti-apoptotic protein Bcl-2 as evaluated by immunoblotting. Using GFP-MTB H37Rv and Lysotracker Red staining we demonstrated co-localization of GFP-MTB H37Rv containing phagosome to lysosome after CMN treatment, indicating enhanced phagosome lysosome fusion. Due to poor bioavailability of CMN, its clinical use is limited, therefore to overcome this issue, CMN was encapsulated in Poly(lactic-co-glycolic) acid (PLGA) shell, resulting in polymeric CMN nano particles (ISCurNP). Flow cytometric evaluation suggested >99% uptake of ISCurNP after 3h of treatment. In BALB/c mice, oral dose of ISCurNP resulted in 6.7-fold increase in the bioavailability compared to free CMN. Moreover, ISCurNP treatment resulted in significant decrease in the intracellular survival of MTB H37Rv through induction of autophagy. Adjunct action of ISCurNP and CMN in combination with isoniazid (INH) revealed >99% decrease in intracellular survival of MTB in macrophage as compared to ISCurNP, CMN or INH alone. In conclusion, our findings suggest the role of ISCurNP as novel host directed formulation to combat both sensitive and MDR strains of MTB by induction of autophagy.

Targeting polyketide synthase 13 for the treatment of tuberculosis.

Tuberculosis (TB) is one of the most threatening diseases for humans, however, the drug treatment strategy for TB has been stagnant and inadequate, which could not meet current treatment needs. TB is caused by Mycobacterial tuberculosis, which has a unique cell wall that plays a crucial role in its growth, virulence, and drug resistance. Polyketide synthase 13 (Pks13) is an essential enzyme that catalyzes the biosynthesis of the cell wall and its critical role is only found in Mycobacteria. Therefore, Pks13 is a promising target for developing novel anti-TB drugs. In this review, we first introduced the mechanism of targeting Pks13 for TB treatment. Subsequently, we focused on summarizing the recent advance of Pks13 inhibitors, including the challenges encountered during their discovery and the rational design strategies employed to overcome these obstacles, which could be helpful for the development of novel Pks13 inhibitors in the future.

An acidic microenvironment in Tuberculosis increases extracellular matrix degradation by regulating macrophage inflammatory responses.

Mycobacterium tuberculosis (M.tb) infection causes marked tissue inflammation leading to lung destruction and morbidity. The inflammatory extracellular microenvironment is acidic, however the effect of this acidosis on the immune response to M.tb is unknown. Using RNA-seq we show that acidosis produces system level transcriptional change in M.tb infected human macrophages regulating almost 4000 genes. Acidosis specifically upregulated extracellular matrix (ECM) degradation pathways with increased expression of Matrix metalloproteinases (MMPs) which mediate lung destruction in Tuberculosis. Macrophage MMP-1 and -3 secretion was increased by acidosis in a cellular model. Acidosis markedly suppresses several cytokines central to control of M.tb infection including TNF-α and IFN-γ. Murine studies demonstrated expression of known acidosis signaling G-protein coupled receptors OGR-1 and TDAG-8 in Tuberculosis which are shown to mediate the immune effects of decreased pH. Receptors were then demonstrated to be expressed in patients with TB lymphadenitis. Collectively, our findings show that an acidic microenvironment modulates immune function to reduce protective inflammatory responses and increase extracellular matrix degradation in Tuberculosis. Acidosis receptors are therefore potential targets for host directed therapy in patients.

Comparison of in silico predicted Mycobacterium tuberculosis spoligotypes and lineages from whole genome sequencing data.

Bacterial strain-types in the Mycobacterium tuberculosis complex underlie tuberculosis disease, and have been associated with drug resistance, transmissibility, virulence, and host-pathogen interactions. Spoligotyping was developed as a molecular genotyping technique used to determine strain-types, though recent advances in whole genome sequencing (WGS) technology have led to their characterization using SNP-based sub-lineage nomenclature. Notwithstanding, spoligotyping remains an important tool and there is a need to study the congruence between spoligotyping-based and SNP-based sub-lineage assignation. To achieve this, an in silico spoligotype prediction method ("Spolpred2") was developed and integrated into TB-Profiler. Lineage and spoligotype predictions were generated for > 28 k isolates and the overlap between strain-types was characterized. Major spoligotype families detected were Beijing (25.6%), T (18.6%), LAM (13.1%), CAS (9.4%), and EAI (8.3%), and these broadly followed known geographic distributions. Most spoligotypes were perfectly correlated with the main MTBC lineages (L1-L7, plus animal). Conversely, at lower levels of the sub-lineage system, the relationship breaks down, with only 65% of spoligotypes being perfectly associated with a sub-lineage at the second or subsequent levels of the hierarchy. Our work supports the use of spoligotyping (membrane or WGS-based) for low-resolution surveillance, and WGS or SNP-based systems for higher-resolution studies.

Target Identification in Anti-Tuberculosis Drug Discovery.

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB), a disease that, although preventable and curable, remains a global epidemic due to the emergence of resistance and a latent form responsible for a long period of treatment. Drug discovery in TB is a challenging task due to the heterogeneity of the disease, the emergence of resistance, and uncomplete knowledge of the pathophysiology of the disease. The limited permeability of the cell wall and the presence of multiple efflux pumps remain a major barrier to achieve effective intracellular drug accumulation. While the complete genome sequence of Mtb has been determined and several potential protein targets have been validated, the lack of adequate models for in vitro and in vivo studies is a limiting factor in TB drug discovery programs. In current therapeutic regimens, less than 0.5% of bacterial proteins are targeted during the biosynthesis of the cell wall and the energetic metabolism of two of the most important processes exploited for TB chemotherapeutics. This review provides an overview on the current challenges in TB drug discovery and emerging Mtb druggable proteins, and explains how chemical probes for protein profiling enabled the identification of new targets and biomarkers, paving the way to disruptive therapeutic regimens and diagnostic tools.

Structure of an endogenous mycobacterial MCE lipid transporter.

To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4-7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.

Deciphering the emerging role of phytocompounds: Implications in the management of drug-resistant tuberculosis and ATDs-induced hepatic damage.

Tuberculosis is a disease of poverty, discrimination, and socioeconomic burden. Epidemiological studies suggest that the mortality and incidence of tuberculosis are unacceptably higher worldwide. Genomic mutations in embCAB, embR, katG, inhA, ahpC, rpoB, pncA, rrs, rpsL, gyrA, gyrB, and ethR contribute to drug resistance reducing the susceptibility of Mycobacterium tuberculosis to many antibiotics. Additionally, treating tuberculosis with antibiotics also poses a serious risk of hepatotoxicity in the patient's body. Emerging data on drug-induced liver injury showed that anti-tuberculosis drugs remarkably altered levels of hepatotoxicity biomarkers. The review is an attempt to explore the anti-mycobacterial potential of selected, commonly available, and well-known phytocompounds and extracts of medicinal plants against strains of Mycobacterium tuberculosis. Many studies have demonstrated that phytocompounds such as flavonoids, alkaloids, terpenoids, and phenolic compounds have antibacterial action against Mycobacterium species, inhibiting the bacteria's growth and replication, and sometimes, causing cell death. Phytocompounds act by disrupting bacterial cell walls and membranes, reducing enzyme activity, and interfering with essential metabolic processes. The combination of these processes reduces the overall survivability of the bacteria. Moreover, several phytochemicals have synergistic effects with antibiotics routinely used to treat TB, improving their efficacy and decreasing the risk of resistance development. Interestingly, phytocompounds have been presented to reduce isoniazid- and ethambutol-induced hepatotoxicity by reversing serum levels of AST, ALP, ALT, bilirubin, MDA, urea, creatinine, and albumin to their normal range, leading to attenuation of inflammation and hepatic necrosis. As a result, phytochemicals represent a promising field of research for the development of new TB medicines.

Is there a need to optimise pyrazinamide doses in patients with tuberculosis? A systematic review.

Pyrazinamide (PZA) is a first-line antituberculosis drug with potent sterilising activity. Variability in drug exposure may translate into suboptimal treatment responses. This systematic review, conducted according to PRISMA guidelines, aimed to evaluate the concentration-effect relationship. In vitro/in vivo studies had to contain information on the infection model, PZA dose and concentration, and microbiological outcome. Human studies had to present information on PZA dose, measures of drug exposure and maximum concentration, and microbiological response parameter or overall treatment outcome. A total of 34 studies were assessed, including in vitro (n = 2), in vivo (n = 3) and clinical studies (n = 29). Intracellular and extracellular models demonstrated a direct correlation between PZA dose of 15-50 mg/kg/day and reduction in bacterial count between 0.50-27.7 log10 CFU/mL. Consistent with this, higher PZA doses (>150 mg/kg) were associated with a greater reduction in bacterial burden in BALB/c mice models. Human pharmacokinetic studies displayed a linear positive correlation between PZA dose (i.e. 21.4-35.7 mg/kg/day) and drug exposure (AUC range 220.6-514.5 mg·h/L). Additionally, human studies confirmed a dose-effect relationship, with an increased 2-month sputum culture conversion rate at AUC/MIC targets of 8.4-11.3 with higher exposure/susceptibility ratios leading to greater efficacy. A 5-fold variability in AUC was observed at PZA dose of 25 mg/kg. A direct concentration-effect relationship and increased treatment efficacy with higher PZA exposure to susceptibility ratios was observed. Taking into account variability in drug exposure and treatment response, further studies on dose optimisation are justified.

Natural mutations in the sensor kinase of the PhoPR two-component regulatory system modulate virulence of ancestor-like tuberculosis bacilli.

The molecular factors and genetic adaptations that contributed to the emergence of Mycobacterium tuberculosis (MTB) from an environmental Mycobacterium canettii-like ancestor, remain poorly investigated. In MTB, the PhoPR two-component regulatory system controls production and secretion of proteins and lipid virulence effectors. Here, we describe that several mutations, present in phoR of M. canettii relative to MTB, impact the expression of the PhoP regulon and the pathogenicity of the strains. First, we establish a molecular model of PhoR and show that some substitutions found in PhoR of M. canettii are likely to impact the structure and activity of this protein. Second, we show that STB-K, the most attenuated available M. canettii strain, displays lower expression of PhoP-induced genes than MTB. Third, we demonstrate that genetic swapping of the phoPR allele from STB-K with the ortholog from MTB H37Rv enhances expression of PhoP-controlled functions and the capacities of the recombinant strain to colonize human macrophages, the MTB target cells, as well as to cause disease in several mouse infection models. Fourth, we extended these observations to other M. canettii strains and confirm that PhoP-controlled functions are expressed at lower levels in most M. canettii strains than in M. tuberculosis. Our findings suggest that distinct PhoR variants have been selected during the evolution of tuberculosis bacilli, contributing to higher pathogenicity and persistence of MTB in the mammalian host.

Antimycobacterial pyridine carboxamides: From design to in vivo activity.

Tuberculosis is the number one killer of infectious diseases caused by a single microbe, namely Mycobacterium tuberculosis (Mtb). The success rate of curing this infection is decreasing due to emerging antimicrobial resistance. Therefore, novel treatments are urgently needed. As an attempt to develop new antituberculars effective against both drugs-sensitive and drug-resistant Mtb, we report the synthesis of a novel series inspired by combining fragments from the first-line agents isoniazid and pyrazinamide (series I) and isoniazid with the second-line agent 4-aminosalicylic acid (series II). We identified compound 10c from series II with selective, potent in vitro antimycobacterial activity against both drug-sensitive and drug-resistant Mtb H37Rv strains with no in vitro or in vivo cytotoxicity. In the murine model of tuberculosis, compound 10c caused a statistically significant decrease in colony-forming units (CFU) in spleen. Despite having a 4-aminosalicylic acid fragment in its structure, biochemical studies showed that compound 10c does not directly affect the folate pathway but rather methionine metabolism. In silico simulations indicated the possibility of binding to mycobacterial methionine-tRNA synthetase. Metabolic study in human liver microsomes revealed that compound 10c does not have any known toxic metabolites and has a half-life of 630 min, overcoming the main drawbacks of isoniazid (toxic metabolites) and 4-aminosalicylic acid (short half-life).

[An Infant with Zoonotic Pulmonary Tuberculosis due to Mycobacterium bovis]. / Mycobacterium bovis'e Bagli Zoonotik Pulmoner Tüberküloz Tanili Bir Infant.

Bovine tuberculosis might be seen in low-income countries, especially in children fed with raw milk. The most common transmission route is fecal-oral way, and it is most likely through unpasteurized dairy products. Although clinical and radiological findings are like non-zoonotic tuberculosis, treatment approaches may differ in individuals with zoonotic tuberculosis. Prevention of zoonotic diseases requires multidisciplinary approaches. These approaches include the development of veterinary and surveillance studies for the detection of communicable diseases in farm animals, as well as informing the public about raw milk consumption. In this case report, a patient with zoonotic pulmonary tuberculosis related to Mycobacterium bovis because of consumption of raw milk was presented. A five-month-old male was admitted to the hospital due to a persistent, feverless, non-productive cough since birth. Empirical antibiotic treatment was started with a preliminary diagnosis of pneumonia because of left upper lobe and right pericardial infiltration on chest X-ray. However, after two weeks of antimicrobial therapy, the patient's clinical and laboratory findings did not improve. This led to the referral for a computed tomography imaging, which revealed tracheomalacia, consolidation on the right upper lobe, an indistinguishable mass or consolidation on the left middle lobe of the lung, peribronchial thickening on the basal segment of the lower lobe, and mediastinal lymphadenopathy. Three consecutive days of fasting gastric lavage fluid was sent to the reference laboratory for acid-resistant bacillus examination, polymerase chain reaction (PCR) and culture studies. As the clinical findings were compatible and PCR was positive, the patient was started on quadruple antituberculous therapy. After initiation of anti-tuberculosis drugs, the patient's findings radiologically and clinically were improved. Mycobacterium bovis was grown in the culture. In the meantime, it was discovered that the patient was fed with raw milk. Due to the patient's clinical symptoms and the growth of Mycobacterium bovis in the gastric lavage fluid culture, the diagnosis of bovine tuberculosis was made. The culprit was that the milk of the cow belonging to the patient's family, which was later found to be infected with M.bovis, was milked and given to the patient without boiling. Today, unpasteurized dairy products continue to be consumed, especially in rural areas. One of the most important steps to prevent zoonotic diseases is to raise awareness about not consuming raw milk and undercooked meat. To elucidate the epidemiological link in childhood, taking a good anamnesis, including questioning raw milk consumption, is essential in the diagnosis of tuberculosis.

A strip-based assay for detection of CrfA enzyme activity to differentiate Mycobacterium tuberculosis and non-tuberculous mycobacteria.

There is an unmet need for tools that permit diagnosis of Tuberculosis (TB) that are affordable, low-tech, and can differentiate Mycobacterium tuberculosis (M.tb) from non-tuberculous mycobacteria (NTM). In this study, we have developed a strip-based assay to detect the activity of a unique Carbapenem Resistance Factor A (CrfA) enzyme present only in M.tb. The strip comprises of PVDF (Polyvinylidene fluoride) membrane that has an immobilized anti-CrfA antibody to capture the CrfA enzyme from M.tb lysate. Lysate of mycobacteria is applied to the strip, washed, and incubated in the presence of chromogenic reporter dye which is a substrate for CrfA. A change in the color of the dye that is readily visible to the naked eye is the readout. We evaluated lysates from M.tb and various NTMs namely, M. abscessus, M. chelonae, M. avium, M. obuense, M. paraintracellulare, M. kansasi, including the patient-derived sputum samples. The strip assay selectively identified only those samples containing M.tb. Based on this evidence, this new assay enables the identification and differentiation of M.tb from NTMs in patient sputum samples. As this tool can be simple to use, therefore has the potential to serve the unmet need for diagnosis of TB and NTM infections in resource-limited settings.

Autophagy prevents early proinflammatory responses and neutrophil recruitment during Mycobacterium tuberculosis infection without affecting pathogen burden in macrophages.

The immune response to Mycobacterium tuberculosis infection determines tuberculosis disease outcomes, yet we have an incomplete understanding of what immune factors contribute to a protective immune response. Neutrophilic inflammation has been associated with poor disease prognosis in humans and in animal models during M. tuberculosis infection and, therefore, must be tightly regulated. ATG5 is an essential autophagy protein that is required in innate immune cells to control neutrophil-dominated inflammation and promote survival during M. tuberculosis infection; however, the mechanistic basis for how ATG5 regulates neutrophil recruitment is unknown. To interrogate what innate immune cells require ATG5 to control neutrophil recruitment during M. tuberculosis infection, we used different mouse strains that conditionally delete Atg5 in specific cell types. We found that ATG5 is required in CD11c+ cells (lung macrophages and dendritic cells) to control the production of proinflammatory cytokines and chemokines during M. tuberculosis infection, which would otherwise promote neutrophil recruitment. This role for ATG5 is autophagy dependent, but independent of mitophagy, LC3-associated phagocytosis, and inflammasome activation, which are the most well-characterized ways that autophagy proteins regulate inflammation. In addition to the increased proinflammatory cytokine production from macrophages during M. tuberculosis infection, loss of ATG5 in innate immune cells also results in an early induction of TH17 responses. Despite prior published in vitro cell culture experiments supporting a role for autophagy in controlling M. tuberculosis replication in macrophages, the effects of autophagy on inflammatory responses occur without changes in M. tuberculosis burden in macrophages. These findings reveal new roles for autophagy proteins in lung resident macrophages and dendritic cells that are required to suppress inflammatory responses that are associated with poor control of M. tuberculosis infection.