CXCL1: A new diagnostic biomarker for human tuberculosis discovered using Diversity Outbred mice

More humans have died of tuberculosis (TB) than any other infectious disease and millions still die each year. Experts advocate for blood-based, serum protein biomarkers to help diagnose TB, which afflicts millions of people in high-burden countries. However, the protein biomarker pipeline is small. Here, we used the Diversity Outbred (DO) mouse population to address this gap, identifying five protein biomarker candidates. One protein biomarker, serum CXCL1, met the World Health Organization’s Targeted Product Profile for a triage test to diagnose active TB from latent M.tb infection (LTBI), non-TB lung disease, and normal sera in HIV-negative, adults from South Africa and Vietnam. To find the biomarker candidates, we quantified seven immune cytokines and four inflammatory proteins corresponding to highly expressed genes unique to progressor DO mice. Next, we applied statistical and machine learning methods to the data, i.e., 11 proteins in lungs from 453 infected and 29 non-infected mice. After searching all combinations of five algorithms and 239 protein subsets, validating, and testing the findings on independent data, two combinations accurately diagnosed progressor DO mice: Logistic Regression using MMP8; and Gradient Tree Boosting using a panel of 4: CXCL1, CXCL2, TNF, IL-10. Of those five protein biomarker candidates, two (MMP8 and CXCL1) were crucial for classifying DO mice; were above the limit of detection in most human serum samples; and had not been widely assessed for diagnostic performance in humans before. In patient sera, CXCL1 exceeded the triage diagnostic test criteria (>90% sensitivity; >70% specificity), while MMP8 did not. Using Area Under the Curve analyses, CXCL1 averaged 94.5% sensitivity and 88.8% specificity for active pulmonary TB (ATB) vs LTBI; 90.9% sensitivity and 71.4% specificity for ATB vs non-TB; and 100.0% sensitivity and 98.4% specificity for ATB vs normal sera. Our findings overall show that the DO mouse population can discover diagnostic-quality, serum protein biomarkers of human TB.     

Mycobacterium Tuberculosis-Specific TNF-α Is a Potential Biomarker for the Rapid Diagnosis of Active Tuberculosis Disease in Chinese Population

Interferon-gamma release assays (IGRAs) have proven to be useful to accurately detect Mycobacterium tuberculosis (Mtb) infection, but they cannot reliably discriminate between active tuberculosis (TB) and latent tuberculosis infection (LTBI). This study aims to test whether Mtb-specific tumor necrosis factor-alpha (TNF-α) could be used as a new tool for the rapid diagnosis of active TB disease. The secretion of TNF-α by Mtb-specific antigen-stimulated peripheral blood mononuclear cells (PBMCs) of sixty seven participants was investigated in the study. Our results showed that the total measurement of TNF-α secretion by Mtb-specific antigen-stimulated PBMCs is not a good biomarker for active TB diagnosis. However, we found that calculation of Mtb-specific TNF-α not only distinguish between active and latent TB infection, but also can differentiate active TB from non-TB patients. Using the cutoff value of 136.9 pg/ml for Mtb-specific TNF-α, we were able to differentiate active TB from LTBI. Sensitivity and specificity were 72% and 90.91%. These data suggest that Mtb-specific TNF-α could be a potential biomarker for the diagnosis of active TB disease.     

Potential of Mycobacterium tuberculosis resuscitation-promoting factors as antigens in novel tuberculosis sub-unit vaccines

Novel vaccines are needed to control tuberculosis (TB), the bacterial infectious disease that together with malaria and HIV is worldwide responsible for high levels of morbidity and mortality. TB can result from the reactivation of an initially controlled latent infection by Mycobacterium tuberculosis (Mtb). Mtb proteins for which a possible role in this reactivation process has been hypothesized are the five homologs of the resuscitation-promoting factor of Micrococcus luteus, namely Mtb Rv0867c (rpfA), Rv1009 (rpfB), Rv1884c (rpfC), Rv2389c (rpfD) and Rv2450c (rpfE). Analysis of the immune recognition of these 5 proteins following Mtb infection or Mycobacterium bovis BCG vaccination of mice showed that Rv1009 (rpfB) and Rv2389c (rpfD) are the most antigenic in the tested models. We therefore selected rpfB and rpfD for testing their vaccine potential as plasmid DNA vaccines. Elevated cellular immune responses and modest but significant protection against intra-tracheal Mtb challenge were induced by immunization with the rpfB encoding DNA vaccine. The results indicate that rpfB is the most promising candidate of the five rpf-like proteins of Mtb in terms of its immunogenicity and protective efficacy and warrants further analysis for inclusion as an antigen in novel TB vaccines.     

Discovery of a unique Mycobacterium tuberculosis protein through proteomic analysis of urine from patients with active tuberculosis

Identification of pathogen-specific biomarkers present in patients' serum or urine samples can be a useful diagnostic approach. In efforts to discover Mycobacterium tuberculosis (Mtb) biomarkers we identified by mass spectroscopy a unique 21-mer Mtb peptide sequence (VVLGLTVPGGVELLPGVALPR) present in the urines of TB patients from Zimbabwe. This peptide has 100% sequence homology with the protein TBCG_03312 from the C strain of Mtb (a clinical isolate identified in New York, NY, USA) and 95% sequence homology with Mtb oxidoreductase (MRGA423_21210) from the clinical isolate MTB423 (identified in Kerala, India). Alignment of the genes coding for these proteins show an insertion point mutation relative to Rv3368c of the reference H37Rv strain, which generated a unique C-terminus with no sequence homology with any other described protein. Phylogenetic analysis utilizing public sequence data shows that the insertion mutation is apparently a rare event. However, sera from TB patients from distinct geographical areas of the world (Peru, Vietnam, and South Africa) contain antibodies that recognize a purified recombinant C-terminus of the protein, thus suggesting a wider distribution of isolates that produce this protein.     

Serum antibody profiles in individuals with latent Mycobacterium tuberculosis infection

One‐third of the world's humans has latent tuberculosis infection (LTBI), representing a large pool of potentially active TB. Recent LTBI carries a higher risk of disease progression than remote LTBI. Recent studies suggest important roles of antibodies in TB pathology, prompting us to investigate serum antibody profiles in a cohort with LTBI. In this single‐center prospective observational study, we analyzed IgG‐antibody concentrations against five major Mycobacterium tuberculosis (Mtb) antigens (including 6 kDa early secretory antigenic target (ESAT6), CFP10, and antigen 85A, which are expressed mainly in the growth phase; and mycobacterial DNA‐binding protein 1 (MDP1) and alpha‐crystallin like protein (Acr), which are expressed in the dormant phases) in individuals with recent (n=13) or remote (n=12) LTBI, no Mtb infection (n=19), or active TB (n=15). Antibody titers against ESAT6 and MDP1 were significantly higher in individuals with recent LTBI than in those with no Mtb infection or remote LTBI. All pairwise antibody titers against these five major antigens were significantly correlated throughout the stages of Mtb infection. Five individuals with recent LTBI had significantly higher antibody titers against ESAT6 (P = 0.03), Ag85A (P = 0.048), Acr (P = 0.057), and MDP1 (P = 0.0001) than in individuals with remote LTBI; they were also outside the normal range (+2 SDs). One of these individuals was diagnosed with active pulmonary TB at 18‐month follow‐up examination. These findings indicated that concentrations of antibodies against both multiplying and dormant Mtb are higher in recent LTBI and that individuals with markedly higher antibody titers may be appropriate candidates for prophylactic therapy.     

A Novel Tuberculosis Antigen Identified from Human Tuberculosis Granulomas

Tuberculosis is a global infectious disease caused by Mycobacterium tuberculosis (Mtb). Although novel Mtb biomarkers from both the pathogen and host have been studied, more breakthroughs are still needed to meet different clinic requirements. In an effort to identify Mtb antigens, chaperone-peptide complexes were purified from TB infected lungs using free-solution isoelectric focusing combined with high resolution LTQ Orbitrap Velos mass spectrometry. Antigen specific cellular immune responses in vitro were then examined. Those efforts led to the identification of six Mtb peptides only identified in Tuberculosis lung samples and that were not found in the control samples. Additionally, antigen-specific IFN-γ secretion, T-cell proliferation, cytokine expression, and a cytotoxic assay were also evaluated. Among the peptides isolated, we identified a 34 amino acid peptide named PKA_p belonging to a serine/threonine–protein kinase, as being able to generate Mtb-specific cellular immune responses as noted by elevated antigen-specific cytokine secretion levels, increased CD8⁺ T-cell proliferation and a strong cytotoxic lymphocyte (CTL) response. Moreover, the immune stimulating abilities of PKA_p were further validated in vivo, with target peptide immunized mice showing an increased cellular IFN-γ in both the lungs and spleen without causing immunopathogenesis. In conclusion, we identified novel functional Mtb antigens directly from the granulomatous lesions of Tuberculosis patients, inducing not only significant antigen-specific IFN-γ secretion but also a marked cytotoxic lymphocyte functional response. These findings indicated that PKA_p has potential as a novel antigen biomarker for vaccine development.Mycobacterium tuberculosis (Mtb),¹ the infectious agent that causes tuberculosis, is associated with an estimated 1.4 million deaths per year and remains a major global health concern (1). Current research and diagnostics have focused on antigen screening and biomarker discovery, with most antigen screening methods focused on the bacterial pathogen itself, with less focus on the Mtb infected host (2). The pathogenic progression of TB occurs in the lungs, making the characterization of any functional antigens existing in the lungs during infection potentially useful for immunotherapy or vaccine development. The immune response to an Mtb infection results in the formation of a granuloma that initially contains bacterial expansion, but may fail to eliminate the pathogen (3, 4). This immune response brings with the possibility of identifying Mtb functional antigens in the lung tissue and to gain a clearer understanding of the immune mechanisms (5, 6). Although it has been well studied that a T-cell mediated adaptive immune response plays a central role during Mtb infection and is crucial in both protection and pathogenesis, a better understanding of the antigen induced immune response and correlations to pathogenicity is necessary (2, 7).It has been reported that heat shock proteins (such as the HSP70 family members) and others chaperones such as Gp96 can specifically bind many hydrophobic sequences, enabling them to bind foreign peptides associated with intracellular bacterial or viral challenge (8), such as Gp96 associating with a HBV-specific peptide (9). Previous studies have shown that chaperone-peptide complexes can induce a disease-specific immune response (10–12), with the gp96-peptide complex from H37Rv infected cells able to induce a protective antigen specific immune response (13). Currently, no Mtb chaperone-associated peptides have been isolated directly from patients, thus the present study explores the possible existence of these complexes in TB lung tissue.To achieve this objective, the free-solution isoelectric focusing (FS-IEF) technique, which has been reported to enrich chaperones in cell lysates or tissue samples, was combined with Linear Trap Quadrupole (LTQ) OrbitrapVelos mass spectrometry, which was used to identify the associated Mtb peptides. Using these techniques, we obtained chaperone-rich cell lysates from the granulomatous lung lesions of active TB patients and identified six Mtb-associated peptides not noted in the control samples. Among them, a peptide (PKA_p) derived from Mtb Protein Kinase A not only contributed to significant antigen-specific IFN-γ secretion, but also contributed to CTL function and T-cell proliferation. Importantly, murine immunization with PKA_p derived peptides elicited an antigen-specific cellular activation without the occurrence of immune pathogenesis.     

Portrait of a pathogen: the Mycobacterium tuberculosis proteome in vivo

Background

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a facultative intracellular pathogen that can persist within the host. The bacteria are thought to be in a state of reduced replication and metabolism as part of the chronic lung infection. Many in vitro studies have dissected the hypothesized environment within the infected lung, defining the bacterial response to pH, starvation and hypoxia. While these experiments have afforded great insight, the picture remains incomplete. The only way to study the combined effects of these environmental factors and the mycobacterial response is to study the bacterial response in vivo.

Methodology/Principal Findings

We used the guinea pig model of tuberculosis to examine the bacterial proteome during the early and chronic stages of disease. Lungs were harvested thirty and ninety days after aerosol challenge with Mtb, and analyzed by liquid chromatography-mass spectrometry. To date, in vivo proteomics of the tubercle bacillus has not been described and this work has generated the first large-scale shotgun proteomic data set, comprising over 500 unique protein identifications. Cell wall and cell wall processes, and intermediary metabolism and respiration were the two major functional classes of proteins represented in the infected lung. These classes of proteins displayed the greatest heterogeneity indicating important biological processes for establishment of a productive bacterial infection and its persistence. Proteins necessary for adaptation throughout infection, such as nitrate/nitrite reduction were found at both time points. The PE-PPE protein class, while not well characterized, represented the third most abundant category and showed the most consistent expression during the infection.

Conclusions/Significance

Cumulatively, the results of this work may provide the basis for rational drug design – identifying numerous Mtb proteins, from essential kinases to products involved in metal regulation and cell wall remodeling, all present throughout the course of infection.     

The cell envelope glycoconjugates of Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) remains the second most common cause of death due to a single infectious agent. The cell envelope of Mycobacterium tuberculosis (Mtb), the causative agent of the disease in humans, is a source of unique glycoconjugates and the most distinctive feature of the biology of this organism. It is the basis of much of Mtb pathogenesis and one of the major causes of its intrinsic resistance to chemotherapeutic agents. At the same time, the unique structures of Mtb cell envelope glycoconjugates, their antigenicity and essentiality for mycobacterial growth provide opportunities for drug, vaccine, diagnostic and biomarker development, as clearly illustrated by recent advances in all of these translational aspects. This review focuses on our current understanding of the structure and biogenesis of Mtb glycoconjugates with particular emphasis on one of the most intriguing and least understood aspect of the physiology of mycobacteria: the translocation of these complex macromolecules across the different layers of the cell envelope. It further reviews the rather impressive progress made in the last 10 years in the discovery and development of novel inhibitors targeting their biogenesis.     

Reengineering Redox Sensitive GFP to Measure Mycothiol Redox Potential of Mycobacterium tuberculosis during Infection

Mycobacterium tuberculosis (Mtb) survives under oxidatively hostile environments encountered inside host phagocytes. To protect itself from oxidative stress, Mtb produces millimolar concentrations of mycothiol (MSH), which functions as a major cytoplasmic redox buffer. Here, we introduce a novel system for real-time imaging of mycothiol redox potential (E_MSH) within Mtb cells during infection. We demonstrate that coupling of Mtb MSH-dependent oxidoreductase (mycoredoxin-1; Mrx1) to redox-sensitive GFP (roGFP2; Mrx1-roGFP2) allowed measurement of dynamic changes in intramycobacterial E_MSH with unprecedented sensitivity and specificity. Using Mrx1-roGFP2, we report the first quantitative measurements of E_MSH in diverse mycobacterial species, genetic mutants, and drug-resistant patient isolates. These cellular studies reveal, for the first time, that the environment inside macrophages and sub-vacuolar compartments induces heterogeneity in E_MSH of the Mtb population. Further application of this new biosensor demonstrates that treatment of Mtb infected macrophage with anti-tuberculosis (TB) drugs induces oxidative shift in E_MSH, suggesting that the intramacrophage milieu and antibiotics cooperatively disrupt the MSH homeostasis to exert efficient Mtb killing. Lastly, we analyze the membrane integrity of Mtb cells with varied E_MSH during infection and show that subpopulation with higher E_MSH are susceptible to clinically relevant antibiotics, whereas lower E_MSH promotes antibiotic tolerance. Together, these data suggest the importance of MSH redox signaling in modulating mycobacterial survival following treatment with anti-TB drugs. We anticipate that Mrx1-roGFP2 will be a major contributor to our understanding of redox biology of Mtb and will lead to novel strategies to target redox metabolism for controlling Mtb persistence.     

Structure-Function Analysis of the Acyl Carrier Protein Synthase (AcpS) from Mycobacterium tuberculosis

We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 Å resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs.     

CD27 expression of T-cells discriminates IGRA-negative TB patients from healthy contacts in Ghana

IFN-γ release assays (IGRAs) have suboptimal sensitivity for detection of Mycobacterium tuberculosis (Mtb) infection and cannot discriminate between tuberculosis (TB) patients and healthy -potentially Mtb infected- contacts (HCs). In a case-control study, we determined T-cell phenotypes of IGRAs in TB patients (n = 20) and HCs (n = 20) from Ghana. CD27 expression of T-cells was significantly lower in TB patients as compared to HCs independent from Mtb-specificity. CD27 expression discriminated both study groups - including TB patients with low or indeterminate IGRA results - effectively. We conclude that CD27 is a promising biomarker for diagnosis of TB patients with inconclusive IGRA results.     

Multiple cytokines are released when blood from patients with tuberculosis is stimulated with Mycobacterium tuberculosis antigens

Background

Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection.

Methods

Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens.

Results

Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1β for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines.

Conclusions

Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone.     

Self-clearance of Mycobacterium tuberculosis infection: implications for lifetime risk and population at-risk of tuberculosis disease

Background: it is widely assumed that individuals with Mycobacterium tuberculosis (Mtb) infection remain at lifelong risk of tuberculosis (TB) disease. However, there is substantial evidence that self-clearance of Mtb infection can occur. We infer a curve of self-clearance by time since infection and explore its implications for TB epidemiology. Methods and findings: data for self-clearance were inferred using post-mortem and tuberculin-skin-test reversion studies. A cohort model allowing for self-clearance was fitted in a Bayesian framework before estimating the lifetime risk of TB disease and the population infected with Mtb in India, China and Japan in 2019. We estimated that 24.4% (17.8–32.6%, 95% uncertainty interval (UI)) of individuals self-clear within 10 years of infection, and 73.1% (64.6–81.7%) over a lifetime. The lifetime risk of TB disease was 17.0% (10.9–22.5%), compared to 12.6% (10.1–15.0%) assuming lifelong infection. The population at risk of TB disease in India, China and Japan was 35–80% (95% UI) smaller in the self-clearance scenario. Conclusions: the population with a viable Mtb infection may be markedly smaller than generally assumed, with such individuals at greater risk of TB disease. The ability to identify these individuals could dramatically improve the targeting of preventive programmes and inform TB vaccine development, bringing TB elimination within reach of feasibility.     

Novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis

Tuberculosis is a major cause of mortality and morbidity due to infectious disease. However, current clinical diagnostic methodologies such as PCR, sputum culture, or smear microscopy are not ideal. Antibody-based assays are a suitable alternative but require specific antibodies against a suitable biomarker. Mycolic acid, which has been found in patient sputum samples and comprises a large portion of the mycobacterial cell wall, is an ideal target. However, generating anti-lipid antibodies using traditional hybridoma methodologies is challenging and has limited the exploitation of this lipid as a diagnostic marker. We describe here the isolation and characterization of four anti-mycolic acid antibodies from a nonimmune antibody phage display library that can detect mycolic acids down to a limit of 4.5ng. All antibodies were specific for the methoxy subclass of mycolic acid with weak binding for α mycolic acid and did not show any binding to closely related lipids or other Mycobacterium tuberculosis (Mtb) derived lipids. We also determined the clinical utility of these antibodies based on their limit of detection for mycobacteria colony forming units (CFU). In combination with an optimized alkaline hydrolysis method for rapid lipid extraction, these antibodies can detect 10⁵ CFU of Mycobacterium bovis BCG, a close relative of Mtb and therefore represent a novel approach for the development of diagnostic assays for lipid biomarkers.     

Increase of CD4+CD25highFoxP3+ cells impairs in vitro human microbicidal activity against Mycobacterium tuberculosis during latent and acute pulmonary tuberculosis

BackgroundRegulatory T cells (Tregs) play a critical role during Mycobacterium tuberculosis (Mtb) infection, modulating host responses while neutralizing excessive inflammation. However, their impact on regulating host protective immunity is not completely understood. Here, we demonstrate that Treg cells abrogate the in vitro microbicidal activity against Mtb.MethodsWe evaluated the in vitro microbicidal activity of peripheral blood mononuclear cells (PBMCs) from patients with active tuberculosis (TB), individuals with latent tuberculosis infection (LTBI, TST+/IGRA+) and healthy control (HC, TST-/IGRA-) volunteers. PBMCs, depleted or not of CD4⁺CD25⁺ T-cells, were analyzed to determine frequency and influence on microbicidal activity during in vitro Mtb infection with four clinical isolates (S1, S5, R3, and R6) and one reference strain (H37Rv).ResultsThe frequency of CD4⁺CD25^highFoxP3⁺ cells were significantly higher in Mtb infected whole blood cultures from both TB patients and LTBI individuals when compared to HC. Data from CD4⁺CD25⁺ T-cells depletion demonstrate that increase of CD4⁺CD25^highFoxP3⁺ is associated with an impairment of Th-1 responses and a diminished in vitro microbicidal activity of LTBI and TB groups.ConclusionsTregs restrict host anti-mycobacterial immunity during active disease and latent infection and thereby may contribute to both disease progression and pathogen persistence.     

Mycobacterium tuberculosis Proteins Involved in Mycolic Acid Synthesis and Transport Localize Dynamically to the Old Growing Pole and Septum

Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid - Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles'' heel of the bacillus at all its growing stages.     

Syndecans promote mycobacterial internalization by lung epithelial cells

Pulmonary tuberculosis (TB) is an airborne disease caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). Alveolar epithelial cells and macrophages are the first point of contact for Mtb in the respiratory tract. However, the mechanisms of mycobacterial attachment to, and internalization by, nonprofessional phagocytes, such as epithelial cells, remain incompletely understood. We identified syndecan 4 (Sdc4) as mycobacterial attachment receptor on alveolar epithelial cells. Sdc4 mRNA expression was increased in human and mouse alveolar epithelial cells after mycobacterial infection. Sdc4 knockdown in alveolar epithelial cells or blocking with anti‐Sdc4 antibody reduced mycobacterial attachment and internalization. At the molecular level, interactions between epithelial cells and mycobacteria involved host Sdc and the mycobacterial heparin‐binding hemagglutinin adhesin. In vivo, Sdc1/Sdc4 double‐knockout mice were more resistant to Mtb colonization of the lung. Our work reveals a role for distinct Sdcs in promoting mycobacterial entry into alveolar epithelial cells with impact on outcome of TB disease.     

Mycobacterium tuberculosis has diminished capacity to counteract redox stress induced by elevated levels of endogenous superoxide

Mycobacterium tuberculosis (Mtb) has evolved protective and detoxification mechanisms to maintain cytoplasmic redox balance in response to exogenous oxidative stress encountered inside host phagocytes. In contrast, little is known about the dynamic response of this pathogen to endogenous oxidative stress generated within Mtb. Using a noninvasive and specific biosensor of cytoplasmic redox state of Mtb, we for first time discovered a surprisingly high sensitivity of this pathogen to perturbation in redox homeostasis induced by elevated endogenous reactive oxygen species (ROS). We synthesized a series of hydroquinone-based small molecule ROS generators and found that ATD-3169 permeated mycobacteria to reliably enhance endogenous ROS including superoxide radicals. When Mtb strains including multidrug-resistant (MDR) and extensively drug-resistant (XDR) patient isolates were exposed to this compound, a dose-dependent, long-lasting, and irreversible oxidative shift in intramycobacterial redox potential was detected. Dynamic redox potential measurements revealed that Mtb had diminished capacity to restore cytoplasmic redox balance in comparison with Mycobacterium smegmatis (Msm), a fast growing nonpathogenic mycobacterial species. Accordingly, Mtb strains were extremely susceptible to inhibition by ATD-3169 but not Msm, suggesting a functional linkage between dynamic redox changes and survival. Microarray analysis showed major realignment of pathways involved in redox homeostasis, central metabolism, DNA repair, and cell wall lipid biosynthesis in response to ATD-3169, all consistent with enhanced endogenous ROS contributing to lethality induced by this compound. This work provides empirical evidence that the cytoplasmic redox poise of Mtb is uniquely sensitive to manipulation in steady-state endogenous ROS levels, thus revealing the importance of targeting intramycobacterial redox metabolism for controlling TB infection.     

Mycobacterium tuberculosis: the honey badger of pathogens

Mycobacterium tuberculosis is a fascinating object of study: it is one of the deadliest pathogens of humankind, able to fend off persistent attacks by the immune system or drugs Subject Categories: Immunology, Microbiology, Virology & Host Pathogen Interaction, Chemical Biology

I have always been interested in infectious diseases since I began to study biology. As a graduate student, my pathogen of choice was Salmonella typhimurium, which typically causes diarrhea that can potentially lead to death. Salmonella''s rapid doubling time, and the availability of elegant genetic tools, a wealth of reagents, and a robust animal infection model put this bug at the apex of ideal host–pathogen systems to study. After I finished my PhD studies—and for reasons to be told another day—my career took an unexpected detour into an area of research I never thought I would be interested in: I went from the sublime to the ridiculous, from Salmonella to Mycobacterium tuberculosis (Mtb), an excruciatingly slow‐growing bacillus with few genetic tools, a paucity of reagents, and an animal model in which an experiment can take a year or longer. Having said all of that, I love working on this pathogen.For those of you who do not know much about Mtb, it is the world''s deadliest bacterium that causes the disease tuberculosis (TB). As Mtb is spread in aerosol droplets coughed up by infected individuals, TB is highly contagious, and about one‐third of the world''s population may be infected with Mtb, although this number has been reasonably challenged (Behr et al, 2021). Even if the numbers of latent or asymptomatic infections are debated, there are some back‐of‐the‐envelope estimates that Mtb has killed more than a billion humans over the millennia. Although TB is often treatable with antibiotics and most Mtb‐infected healthy individuals are asymptomatic, the appearance of multi‐drug‐resistant Mtb and HIV/AIDS has further increased the number of deaths caused by this pathogen.How has Mtb become such a successful pathogen? For one, we lack an effective vaccine to prevent infection. Many readers may point out that they have themselves been given a TB vaccine; known as “BCG” for bacille Calmette–Guérin, this is a laboratory‐attenuated strain of a species related to Mtb called Mycobacterium bovis. While BCG does provide some protection for children against TB, BCG is essentially ineffective against pulmonary TB in adults. For this reason, it is not used in the USA and many other countries.Another major challenge to treating TB has been a lack of antimicrobials that can access Mtb bacilli in privileged sites known as granulomas, which are cell‐fortified structures our immune system builds to contain microbial growth. In addition to the granuloma walls, Mtb has a highly complex cell envelope that protects it from many small molecules. I imagine that antimicrobial molecules have the challenging task of reaching an enemy shielded in armor, hiding deep inside a castle keep, and surrounded by a vast moat, and an army of orcs.On top of these therapeutic barriers, most antimicrobials work on metabolically active or growing bacteria. Mtb, however, grows very slowly, with a doubling time under optimal laboratory conditions of about 20 h—compared with 20 min for Salmonella. Moreover, Mtb is believed to enter a “persistent” or “latent” state in its natural host with limited cell divisions. This extremely slow growth makes treatment a long and tedious prospect: 6–12 months of antibiotic treatment are generally required, during which time one cannot drink alcohol due to the potential liver toxicity of the drugs. Believe it or not, there are people who would rather refuse TB treatment than give up alcohol for a few months. Additionally, the perception of “feeling cured” after a few weeks of TB therapy can also lead to a lapse in compliance. The consequence of failing to clear a partially treated infection is the emergence of drug resistance, which has created strains that are extensively resistant to most frontline TB drugs.When thinking about the difficulty of curing Mtb infections, I am reminded of the fierce and fearless honey badger, which came to fame through a viral YouTube video. The narrator points out how honey badgers “don''t care” about battling vicious predators in order to get food: venomous snakes, stinging bees—you name it. I once saw a photo of a honey badger that looked more like a pin cushion, harpooned with numerous porcupine quills. This battle royale of the wilderness is a perfect analogy of Mtb versus the immune system: Like the honey badger, Mtb really don''t care.Vaccines primarily work by coaxing our immune system to make antibodies that neutralize foreign invaders, most typically viruses, but also bacteria, some of which have evolved mechanisms to evade detection by antibodies or otherwise render them useless. In most cases, phagocytes then gobble up and kill invading bacteria. While phagocytes are critical in controlling Mtb infections, it is unclear which of their molecules or “effectors” act as executioners of Mtb. For example, nitric oxide and copper play roles in controlling Mtb in a mouse model, but it is unknown how these molecules exert their host‐protective activity, and whether or not they play a similar role in humans. Furthermore, despite the production of these antibacterial effectors—the “porcupine quills”—Mtb often persists due to intrinsic resistance mechanisms. Thus, while our immune system may have the tools to keep Mtb under control, it falls short of eradicating it from our bodies and, in many cases, fails to prevent the progression of the disease. Perhaps a most worrying observation is that prior infection, which is generally considered the most effective path to immunity for many infectious diseases, does not consistently protect against reinfection with Mtb.The above facts have left the TB field scrambling to identify new ways to fight this disease. Much of this work requires that researchers understand both the fundamental processes of the bacterium and its host. Studies of human populations around the globe have revealed differences in susceptibility to infection, the genetic and immunological bases of which are being investigated (Bellamy et al, 2000; Berry et al, 2010; Möller et al, 2010). These studies have made researchers increasingly aware that how the immune system responds to Mtb may play a critical role in disease control. For example, understanding why some individuals or populations are more or less susceptible to TB may help in the development of better vaccines. Also, the more we understand what makes this pathogen so resilient to the immune system could facilitate the development of new antibacterial drugs or host‐directed therapies. These questions can only be answered once we fully understand how the host combats Mtb infections, and how the bacteria counteract these host defenses. While it is a daunting endeavor, my hope is that the efforts of many laboratories around the world will get a better understanding of the host–Mtb interface and ultimately help to eradicate this disease for good.