PatA Regulates Isoniazid Resistance by Mediating Mycolic Acid Synthesis and Controls Biofilm Formation by Affecting Lipid Synthesis in Mycobacteria.

Lipids are prominent components of the mycobacterial cell wall, and they play critical roles not only in maintaining biofilm formation but also in resisting environmental stress, including drug resistance. However, information regarding the mechanism mediating mycobacterial lipid synthesis remains limited. PatA is a membrane-associated acyltransferase and synthesizes phosphatidyl-myo-inositol mannosides (PIMs) in mycobacteria. Here, we found that PatA could regulate the synthesis of lipids (except mycolic acids) to maintain biofilm formation and environmental stress resistance in Mycolicibacterium smegmatis. Interestingly, the deletion of patA significantly enhanced isoniazid (INH) resistance in M. smegmatis, although it reduced bacterial biofilm formation. This might be due to the fact that the patA deletion promoted the synthesis of mycolic acids through an unknown synthesis pathway other than the reported fatty acid synthase (FAS) pathway, which could effectively counteract the inhibition by INH of mycolic acid synthesis in mycobacteria. Furthermore, the amino acid sequences and physiological functions of PatA were highly conserved in mycobacteria. Therefore, we found a mycolic acid synthesis pathway regulated by PatA in mycobacteria. In addition, PatA also affected biofilm formation and environmental stress resistance by regulating the synthesis of lipids (except mycolic acids) in mycobacteria. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, leads to a large number of human deaths every year. This is so serious, which is due mainly to the drug resistance of mycobacteria. INH kills M. tuberculosis by inhibiting the synthesis of mycolic acids, which are synthesized by the FAS pathway. However, whether there is another mycolic acid synthesis pathway is unknown. In this study, we found a PatA-mediated mycolic acid synthesis pathway that led to INH resistance of in patA-deleted mutant. In addition, we first report the regulatory effect of PatA on mycobacterial biofilm formation, which could affect the bacterial response to environmental stress. Our findings represent a new model for regulating biofilm formation by mycobacteria. More importantly, the discovery of the PatA-mediated mycolic acid synthesis pathway indicates that the study of mycobacterial lipids has entered a new stage, and the enzymes might be new targets of antituberculosis drugs.

In Vitro and In Vivo Efficacy of NITD-916 against Mycobacterium fortuitum.

Mycobacterium fortuitum represents one of the most clinically relevant rapid-growing mycobacterial species. Treatments are complex due to antibiotic resistance and to severe side effects of effective drugs, prolonged time of treatment, and co-infection with other pathogens. Herein, we explored the activity of NITD-916, a direct inhibitor of the enoyl-ACP reductase InhA of the type II fatty acid synthase in Mycobacterium tuberculosis. We found that this compound displayed very low MIC values against a panel of M. fortuitum clinical strains and exerted potent antimicrobial activity against M. fortuitum in macrophages. Remarkably, the compound was also highly efficacious in a zebrafish model of infection. Short duration treatments were sufficient to significantly protect the infected larvae from M. fortuitum-induced killing, which correlated with reduced bacterial burdens and abscesses. Biochemical analyses demonstrated an inhibition of de novo synthesis of mycolic acids. Resolving the crystal structure of the InhAMFO in complex with NAD and NITD-916 confirmed that NITD-916 is a direct inhibitor of InhAMFO. Importantly, single nucleotide polymorphism leading to a G96S substitution in InhAMFO conferred high resistance levels to NITD-916, thus resolving its target in M. fortuitum. Overall, these findings indicate that NITD-916 is highly active against M. fortuitum both in vitro and in vivo and should be considered in future preclinical evaluations for the treatment of M. fortuitum pulmonary diseases.

NSC19723, a Thiacetazone-Like Benzaldehyde Thiosemicarbazone Improves the Efficacy of TB Drugs In Vitro and In Vivo.

The complexity and duration of tuberculosis (TB) treatment contributes to the emergence of drug resistant tuberculosis (DR-TB) and drug-associated side effects. Alternate chemotherapeutic agents are needed to shorten the time and improve efficacy of current treatment. In this study, we have assessed the antitubercular activity of NSC19723, a benzaldehyde thiosemicarbazone molecule. NSC19723 is structurally similar to thiacetazone (TAC), a second-line anti-TB drug used to treat individuals with DR-TB. NSC19723 displayed better MIC values than TAC against Mycobacterium tuberculosis and Mycobacterium bovis BCG. In our checkerboard experiments, NSC19723 displayed better profiles than TAC in combination with known first-line and recently approved drugs. Mechanistic studies revealed that NSC19723 inhibits mycolic acid biosynthesis by targeting the HadABC complex. Computational studies revealed that the binding pocket of HadAB is similarly occupied by NSC19723 and TAC. NSC19723 also improved the efficacy of isoniazid in macrophages and mouse models of infection. Cumulatively, we have identified a benzaldehyde thiosemicarbazone scaffold that improved the activity of TB drugs in liquid cultures, macrophages, and mice. IMPORTANCE Mycobacterium tuberculosis, the causative agent of TB is among the leading causes of death among infectious diseases in humans. This situation has worsened due to the failure of BCG vaccines and the increased number of cases with HIV-TB coinfections and drug-resistant strains. Another challenge in the field is the lengthy duration of therapy for drug-sensitive and -resistant TB. Here, we have deciphered the mechanism of action of NSC19723, benzaldehyde thiosemicarbazone. We show that NSC19723 targets HadABC complex and inhibits mycolic acid biosynthesis. We also show that NSC19723 enhances the activity of known drugs in liquid cultures, macrophages, and mice. We have also performed molecular docking studies to identify the interacting residues of HadAB with NSC19723. Taken together, we demonstrate that NSC19723, a benzaldehyde thiosemicarbazone, has better antitubercular activity than thiacetazone.

Trehalose Recycling Promotes Energy-Efficient Biosynthesis of the Mycobacterial Cell Envelope.

The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune, and antibiotic insults. There is considerable evidence of mycomembrane plasticity during infection and in response to host-mimicking stresses. Since mycobacteria are resource and energy limited under these conditions, it is likely that remodeling has distinct requirements from those of the well-characterized biosynthetic program that operates during unrestricted growth. Unexpectedly, we found that mycomembrane remodeling in nutrient-starved, nonreplicating mycobacteria includes synthesis in addition to turnover. Mycomembrane synthesis under these conditions occurs along the cell periphery, in contrast to the polar assembly of actively growing cells, and both liberates and relies on the nonmammalian disaccharide trehalose. In the absence of trehalose recycling, de novo trehalose synthesis fuels mycomembrane remodeling. However, mycobacteria experience ATP depletion, enhanced respiration, and redox stress, hallmarks of futile cycling and the collateral dysfunction elicited by some bactericidal antibiotics. Inefficient energy metabolism compromises the survival of trehalose recycling mutants in macrophages. Our data suggest that trehalose recycling alleviates the energetic burden of mycomembrane remodeling under stress. Cell envelope recycling pathways are emerging targets for sensitizing resource-limited bacterial pathogens to host and antibiotic pressure.IMPORTANCE The glucose-based disaccharide trehalose is a stress protectant and carbon source in many nonmammalian cells. Mycobacteria are relatively unique in that they use trehalose for an additional, extracytoplasmic purpose: to build their outer "myco" membrane. In these organisms, trehalose connects mycomembrane biosynthesis and turnover to central carbon metabolism. Key to this connection is the retrograde transporter LpqY-SugABC. Unexpectedly, we found that nongrowing mycobacteria synthesize mycomembrane under carbon limitation but do not require LpqY-SugABC. In the absence of trehalose recycling, compensatory anabolism allows mycomembrane biosynthesis to continue. However, this workaround comes at a cost, namely, ATP consumption, increased respiration, and oxidative stress. Strikingly, these phenotypes resemble those elicited by futile cycles and some bactericidal antibiotics. We demonstrate that inefficient energy metabolism attenuates trehalose recycling mutant Mycobacterium tuberculosis in macrophages. Energy-expensive macromolecule biosynthesis triggered in the absence of recycling may be a new paradigm for boosting host activity against bacterial pathogens.

N-acetylcysteine (NAC) Attenuating Apoptosis and Autophagy in RAW264.7 Cells in Response to Incubation with Mycolic Acid from Bovine Mycobacterium tuberculosis Complex.

Bovine tuberculosis is an airborne infectious disease caused by organisms of the Mycobacterium tuberculosis (MTB) complex. Mycolic acid (MA) is the main lipid component of the cell membrane of MTB. It is non-enzymatically reduced by NAD(P)H and further produces reactive oxygen species (ROS), which can cause oxidative stress in human cells. N-acetylcysteine (NAC) is a synthetic precursor of glutathione (GSH) and exhibits anti-ROS activity. However, the underlying mechanisms of its protective properties remain uncertain. Herein, after pre-incubation of RAW264.7 cells with NAC, the factors associated with apoptosis and autophagy were measured. Mechanistically, NAC could reduce MA-induced expression of pro-apoptotic and pro-autophagy proteins. At the mRNA level, NAC can inhibit AMPK and activate mTOR expression. The results indicate that NAC might regulate autophagy in RAW264.7 cells through the AMPK/mTOR pathway. To further prove the effect of NAC on MA, ICR mice were used to evaluate the lung injury. Hematoxylin-eosin (HE) staining was performed on the lung. The results show that NAC could reduce cell injury induced by MA. In conclusion, our research showed that NAC attenuates apoptosis and autophagy in response to incubation with mycolic acid.Bovine tuberculosis is an airborne infectious disease caused by organisms of the Mycobacterium tuberculosis (MTB) complex. Mycolic acid (MA) is the main lipid component of the cell membrane of MTB. It is non-enzymatically reduced by NAD(P)H and further produces reactive oxygen species (ROS), which can cause oxidative stress in human cells. N-acetylcysteine (NAC) is a synthetic precursor of glutathione (GSH) and exhibits anti-ROS activity. However, the underlying mechanisms of its protective properties remain uncertain. Herein, after pre-incubation of RAW264.7 cells with NAC, the factors associated with apoptosis and autophagy were measured. Mechanistically, NAC could reduce MA-induced expression of pro-apoptotic and pro-autophagy proteins. At the mRNA level, NAC can inhibit AMPK and activate mTOR expression. The results indicate that NAC might regulate autophagy in RAW264.7 cells through the AMPK/mTOR pathway. To further prove the effect of NAC on MA, ICR mice were used to evaluate the lung injury. Hematoxylin-eosin (HE) staining was performed on the lung. The results show that NAC could reduce cell injury induced by MA. In conclusion, our research showed that NAC attenuates apoptosis and autophagy in response to incubation with mycolic acid.

Induction of tumor-specific CD8+ cytotoxic T lymphocytes from naïve human T cells by using Mycobacterium-derived mycolic acid and lipoarabinomannan-stimulated dendritic cells.

The main effectors in tumor control are the class I MHC molecule-restricted CD8+ cytotoxic T lymphocytes (CTLs). Tumor-specific CTL induction can be regulated by dendritic cells (DCs) expressing both tumor-derived epitopes and co-stimulatory molecules. Immunosuppressive tolerogenic DCs, having down-regulated co-stimulatory molecules, are seen within the tumor mass and can suppress tumor-specific CTL induction. The tolerogenic DCs expressing down-regulated XCR1+CD141+ appear to be induced by tumor-derived soluble factors or dexamethasone, while the immunogenic DCs usually express XCR1+CD141+ molecules with a cross-presentation function in humans. Thus, if tolerogenic DCs can be reactivated into immunogenic DCs with sufficient co-stimulatory molecules, tumor-specific CD8+ CTLs can be primed and activated in vivo. In the present study, we converted human tolerogenic CD141+ DCs with enhanced co-stimulatory molecule expression of CD40, CD80, and CD86 through stimulation with non-toxic mycobacterial lipids such as mycolic acid (MA) and lipoarabinomannan (LAM), which synergistically enhanced both co-stimulatory molecule expression and interleukin (IL)-12 secretion by XCR1+CD141+ DCs. Moreover, MA and LAM-stimulated DCs captured tumor antigens and presented tumor epitope(s) in association with class I MHCs and sufficient upregulated co-stimulatory molecules to prime naïve CD3+ T cells to become CD8+ tumor-specific CTLs. Repeat CD141+ DC stimulation with MA and LAM augmented the secretion of IL-12. These findings provide us a new method for altering the tumor environment by converting tolerogenic DCs to immunogenic DCs with MA and LAM from Mycobacterium tuberculosis.

Induction of Mycobacterium Tuberculosis Lipid-Specific T Cell Responses by Pulmonary Delivery of Mycolic Acid-Loaded Polymeric Micellar Nanocarriers.

Mycolic acid (MA), a major lipid component of Mycobacterium tuberculosis (Mtb) cell wall, can be presented by the non-polymorphic antigen presenting molecule CD1b to T cells isolated from Mtb-infected individuals. These MA-specific CD1b-restricted T cells are cytotoxic, produce Th1 cytokines, and form memory populations, suggesting that MA can be explored as a potential subunit vaccine candidate for TB. However, the controlled elicitation of MA-specific T cell responses has been challenging due to difficulties in the targeted delivery of lipid antigens and a lack of suitable animal models. In this study, we generated MA-loaded micellar nanocarriers (MA-Mc) comprised of self-assembled poly(ethylene glycol)-bl-poly(propylene sulfide; PEG-PPS) copolymers conjugated to an acid sensitive fluorophore to enhance intracellular delivery of MA to phagocytic immune cells. Using humanized CD1 transgenic (hCD1Tg) mice, we found these nanobiomaterials to be endocytosed by bone marrow-derived dendritic cells (DCs) and localized to lysosomal compartments. Additionally, MA-Mc demonstrated superior efficacy over free MA in activating MA-specific TCR transgenic (DN1) T cells in vitro. Following intranasal immunization, MA-Mc were primarily taken up by alveolar macrophages and DCs in the lung and induced activation and proliferation of adoptively transferred DN1 T cells. Furthermore, intranasal immunization with MA-Mc induced MA-specific T cell responses in the lungs of hCD1Tg mice. Collectively, our data demonstrates that pulmonary delivery of MA via PEG-PPS micelles to DCs can elicit potent CD1b-restricted T cell responses both in vitro and in vivo and MA-Mc could be explored as subunit vaccines against Mtb infection.

HC2091 Kills Mycobacterium tuberculosis by Targeting the MmpL3 Mycolic Acid Transporter.

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is a deadly disease that requires a long course of treatment. The emergence of drug-resistant strains has driven efforts to discover new small molecules that can kill the bacterium. Here, we report characterizations of the compound HC2091, which kills M. tuberculosis in a time- and dose-dependent manner in vitro and inhibits M. tuberculosis growth in macrophages. Whole-genome sequencing of spontaneous HC2091-resistant mutants identified single-nucleotide variants in the mmpL3 mycolic acid transporter gene. HC2091-resistant mutants do not exhibit cross-resistance with the well-characterized Mycobacterium membrane protein large 3 (MmpL3) inhibitor SQ109, suggesting a distinct mechanism of interaction with MmpL3. Additionally, HC2091 does not modulate bacterial membrane potential or kill nonreplicating M. tuberculosis, thus acting differently from other known MmpL3 inhibitors. RNA sequencing (RNA-seq) transcriptional profiling and lipid profiling of M. tuberculosis treated with HC2091 or SQ109 show that the two compounds target a similar pathway. HC2091 has a chemical structure dissimilar to those of previously described MmpL3 inhibitors, supporting the notion that HC2091 is a new class of MmpL3 inhibitor.

Responses of haptoglobin and serum amyloid A in goats inoculated intradermally with C. pseudotuberculosis and mycolic acid extract immunogen.

Haptoglobin (Hp) and Serum Amyloid A (SAA) are a group of blood proteins whose concentrations in animals can be influenced by infection, inflammation, surgical trauma or stress. Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA), and Mycolic acid is a virulent factor extracted from C. pseudotuberculosis. There is a dearth of sufficient evidence on the clinical implication of MAs on the responses of Hp and SAA in goats. Therefore, this study was conducted to evaluate the potential effects of Mycolic acid (MAs) and C. pseudotuberculosis on the responses of Hp and SAA in female goats. A total of 12 healthy female goats was divided into three groups; A, B and C each comprising of 4 goats and managed for a period of three months. Group (A) was inoculated with 2 mL of sterile phosphate buffered saline (as a negative control group) intradermally, while group (B) and (C) were inoculated intradermally with 2 ml each of mycolic acid and 1  × 109 cfu of active C. pseudotuberculosis respectively. The result of the study showed that the Hp concentration in goats inoculated with C. pseudotuberculosis was significantly increased up to 7-fold (1.17 ±â€¯0.17 ng/L) while MAs showed a 3-fold increased (0.83 ±â€¯0.01 ng/L) compared with the control. Whereas SAA concentration in C. pseudotuberculosis and MAs groups showed a significant 3-fold (17.85 ±â€¯0.91 pg/mL) and 2-fold (10.97 ±â€¯0.71 pg/mL) increased compared with the control. This study concludes that inoculation of C. pseudotuberculosis and MAs have significant effects on Hp and SAA levels, which indicates that MAs could have a role in the pathogenesis of caseous lymphadenitis.

[Involvement of coronin-1 in foam cell formation of macrophages induced by mycolic acid and its mechanism].

OBJECTIVE: To study the correlation of coronin-1 and mycolic acid (MA)-induced foam cell formation of macrophages and explore its possible mechanism. METHODS: According to the difference of coronin-1 expression level, the experiment included three types of cells: RAW264.7-Cor.Plus, RAW264.7 and RAW264.7-Cor.Minus. After the cells were treated with the polystyrene microspheres coated with 100 µg/mL MA for 24 hours, total proteins were extracted and the level of coronin-1 in each group was detected by Western blotting. With the microspheres coated with 0, 25, 50, 75 and 100 µg/mL MA as phagocytic particles, the cells were swallowed for 24, 48, 72 hours, 5 and 8 days before and after the treatment with 2 µmol/L cytochalasin D (ctyD), and the levels of total cholesterol (TC) and free cholesterol (FC) were tested by TC enzyme kit and FC enzyme kit, respectively; then the cholesterol ester (CE) and the CE/TC ratio were used to quantitatively evaluate the level of foam cell formation. The ctyD-treated cells (RAW264.7-ctyD, RAW264.7-ctyD-MA) and their control groups were stained with FITC-phalloidin, and then the percentage of F-actin rearrangement was calculated. RESULTS: After MA induction, the coronin-1 level of the three experimental groups were significantly higher than that of the corresponding control groups, and there was also obvious difference between the three experimental groups (RAW264.7-Cor.Plus>RAW264.7>RAW264.7-Cor.Minus). The level of foam cell formation of macrophages in each group with different coronin-1 level was positively correlated with the MA coating concentration and its phagocytic time. The highest coronin-1 expression group (RAW264.7-Cor.Plus) had the highest foam cell formation level, and the lowest coronin-1 expression group (RAW264.7-Cor.Minus) had the lowest foam cell formation level. The inhibition of F-actin by ctyD significantly decreased the foam cell formation induced by MA, but the inhibition of F-actin had no significant impact on the positive correlation between coronin-1 and the foam cell formation induced by MA. After phalloidin staining, the F-actin rearrangement rate of MA-treated cells was significantly higher than that of non-MA control cells. CONCLUSION: MA could induce the expression of coronin-1 of macrophage, and the coronin-1 level was positively correlated with the foam cell formation induced by MA. F-actin was involved in the process of lipid accumulation in MA-treated macrophages, but the F-actin was not the key or the only way in coronin-1 regulating the foam cell formation induced by MA.

MmpL3 Inhibitors: Diverse Chemical Scaffolds Inhibit the Same Target.

MmpL3 belongs to the Resistance, Nodulation and Division (RND) superfamily whose role in mycobacteria is the formation of the outer membrane. Indeed, it has been shown that MmpL3 is associated with the export of mycolic acids in the form of trehalose monomycolates (TMM) to the periplasmic space or the outer membrane. In the last few years several whole cell-based screenings of compound libraries brought by a number of diverse chemical scaffolds active against M. tuberculosis (Mtb) that surprisingly share MmpL3 as target. The diverse identified pharmacophores owe important differences among each other, in fact while some of them display inhibitory activity against pathogens that are devoid of mycolic acids and are active against non-replicating Mtb bacilli, some others specifically target mycobacteria and do not kill non-replicating bacilli. The scope of this review is to provide the recent advances in MmpL3 inhibitor discovery with a special focus on structure activity relationship (SAR) studies in order to provide information that could help in developing novel membrane-active anti- TB agents. Moreover, this review will provide the most recent insights into the modes of action of the MmpL3 inhibitors.

[Establishment and characterization of macrophage-derived foam cell model induced by mycolic acid].

OBJECTIVE: To explore the formation conditions and characteristics of murine macrophage-derived foam cells induced by the polystyrene microspheres coated with mycolic acid (MA). METHODS: RAW264.7 cells were divided into blank control group and polystyrene microsphere experimental groups where polystyrene microspheres were coated with 0, 25, 50, 75 and 100 µg/mL mycolic acid, respectively. The mixture of RAW264.7 cells and polystyrene microspheres was incubated for 24, 48, 72 hours, 5 and 8 days, respectively. After oil red O staining, the cell morphology of each group was observed under a microscope. The level of intracellular cholesterol ester of each group was measured with total cholesterol and free cholesterol kits, and the cell proliferation was detected with MTT assay. RESULTS: When the concentration of MA was 75 µg/mL and the phagocytosis time was 24 hours, the typical foam cells formed, and its intracellular cholesterol ester content was (60.98±1.32)%. With the phagocytosis time went by, the lower MA concentration was needed for the formation of foam cells. With the gradual accumulation of lipids in foam cells, the cell proliferation activity was gradually weakened. CONCLUSION: MA-coated polystyrene microspheres can quickly induce macrophages to form foam cells. MA concentration and the formation of foam cells were positively correlated, and it is time-dependent.

Mycobacterium tuberculosis keto-mycolic acid and macrophage nuclear receptor TR4 modulate foamy biogenesis in granulomas: a case of a heterologous and noncanonical ligand-receptor pair.

The cell wall of Mycobacterium tuberculosis is configured of bioactive lipid classes that are essential for virulence and potentially involved in the formation of foamy macrophages (FMs) and granulomas. Our recent work established crosstalk between M. tuberculosis cell wall lipids and the host lipid-sensing nuclear receptor TR4. In this study, we have characterized, identified, and adopted a heterologous ligand keto-mycolic acid from among M. tuberculosis lipid repertoire for the host orphan NR TR4. Crosstalk between cell wall lipids and TR4 was analyzed by transactivation and promoter reporter assays. Mycolic acid (MA) was found to transactivate TR4 significantly compared with other cell wall lipids. Among the MA, the oxygenated form, keto-MA, was responsible for transactivation, and the identity was validated by TR4 binding assays followed by TLC and nuclear magnetic resonance. Isothermal titration calorimetry revealed that keto-MA binding to TR4 is energetically favorable. This keto-MA-TR4 axis seems to be essential to this oxygenated MA induction of FMs and granuloma formation as evaluated by in vitro and in vivo model of granuloma formation. TR4 binding with keto-MA features a unique association of host nuclear receptor with a bacterial lipid and adds to the presently known ligand repertoire beyond dietary lipids. Pharmacologic modulation of this heterologous axis may hold promise as an adjunct therapy to frontline tuberculosis drugs.

Structural basis of the binding of fatty acids to peptidoglycan recognition protein, PGRP-S through second binding site.

Short peptidoglycan recognition protein (PGRP-S) is a member of the mammalian innate immune system. PGRP-S from Camelus dromedarius (CPGRP-S) has been shown to bind to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Its structure consists of four molecules A, B, C and D with ligand binding clefts situated at A-B and C-D contacts. It has been shown that LPS, LTA and PGN bind to CPGRP-S at C-D contact. The cleft at the A-B contact indicated features that suggested a possible binding of fatty acids including mycolic acid of Mycobacterium tuberculosis. Therefore, binding studies of CPGRP-S were carried out with fatty acids, butyric acid, lauric acid, myristic acid, stearic acid and mycolic acid which showed affinities in the range of 10(-5) to 10(-8) M. Structure determinations of the complexes of CPGRP-S with above fatty acids showed that they bound to CPGRP-S in the cleft at the A-B contact. The flow cytometric studies showed that mycolic acid induced the production of pro-inflammatory cytokines, TNF-α and IFN-γ by CD3+ T cells. The concentrations of cytokines increased considerably with increasing concentrations of mycolic acid. However, their levels decreased substantially on adding CPGRP-S.

Molecular structure of the Mycobacterium tuberculosis virulence factor, mycolic acid, determines the elicited inflammatory pattern.

Mycolic acids (MAs) occur in the cell wall of Mycobacterium tuberculosis as variable mixtures of different classes and chain lengths. Here, we address the relationship between the structure and its inflammatory function of this virulence factor using single synthetic MA isomers, differing in oxygenation class and cis- versus α-methyl-trans proximal cyclopropane orientation. Analysis of bronchoalveolar inflammation, lung histopathology and alveolar macrophage transcription revealed a strong dependence on these meromycolic chemistries of mouse pulmonary inflammation in response to intratracheal treatments with MAs. Whereas α-MA was inert, oxygenated methoxy- and keto-MA with cis-cyclopropane stereochemistry elicited solid to mild inflammatory responses respectively. In trans-cyclopropane orientation, methoxy-MA partially lost its inflammatory activity and keto-MA exerted anti-inflammatory alternative activation of alveolar macrophages and counteracted cis-methoxy-MA induced airway inflammation. The differential innate immune activities of MAs demonstrated here, dependent on oxygenation class and cis versus α-methyl-trans cyclopropane chemistry, identify a novel means for M. tuberculosis to steer host immune responses during infection.

Effect of mycolic acid on surface activity of binary surfactant lipid monolayers.

In pulmonary tuberculosis, Mycobacterium tuberculosis lies in close physical proximity to alveolar surfactant. Cell walls of the mycobacteria contain loosely bound, detachable surface-active lipids. In this study, the effect of mycolic acid (MA), the most abundant mycobacterial cell wall lipid, on the surface activity of phospholipid mixtures from lung surfactant was investigated using Langmuir monolayers and atomic force microscopy (AFM). In the presence of mycolic acid, all the surfactant lipid mixtures attained high minimum surface tensions (between 20 and 40 mN/m) and decreased surface compressibility moduli <50 mN/m. AFM images showed that the smooth surface topography of surfactant lipid monolayers was altered with addition of MA. Aggregates with diverse heights of at least two layer thicknesses were found in the presence of mycolic acid. Mycolic acids could aggregate within surfactant lipid monolayers and result in disturbed monolayer surface activity. The extent of the effect of mycolic acid depended on the initial state of the monolayer, with fluid films of DPPC-POPC and DPPC-CHOL being least affected. The results imply inhibitory effects of mycolic acid toward lung surfactant lipids and could be a mechanism of lung surfactant dysfunction in pulmonary tuberculosis.

Effect of mycobacterial lipids on surface properties of Curosurf: implications for lung surfactant dysfunction in tuberculosis.

In this study, the effect of mycobacterial lipids on the surface activity of lung surfactant was evaluated. Mycolic acid and cord factor, the most abundant surface active lipids of the mycobacterial cell wall when combined with Curosurf led to alteration of its surface properties. Addition of graded amounts of mycolic acid increased the minimum surface tension of Curosurf monolayers from <10 mN/m to approximately 20-27 mN/m. Presence of mycolic acid also slowed the rate of Curosurf adsorption. Similarly, presence of cord factor increased the minimum surface tension achieved by Curosurf to approximately 16-27 mN/m. AFM imaging revealed presence of aggregates on addition of mycobacterial lipids to Curosurf monolayers. Results of this study show that mycolic acid and cord factor can biophysically inactivate porcine lung surfactant extract (Curosurf). This suggests that biophysical inhibition of lung surfactant is possible in vivo in pulmonary tuberculosis which could aggravate areas of alveolar atelectasis.

Macrophage reprogramming by mycolic acid promotes a tolerogenic response in experimental asthma.

RATIONALE: Mycolic acid (MA) constitutes a major and distinguishing cell wall biolipid from Mycobacterium tuberculosis. MA interferes with the lipid homeostasis of alveolar macrophages, inducing differentiation into foamy macrophages exhibiting increased proinflammatory function. OBJECTIVES: We verified the interference of this altered macrophage function with inhaled antigen-triggered allergic airway inflammation and underlying Th2 lymphocyte reactivity. METHODS: Using ovalbumin (OVA) as model allergen, C57BL/6 or BALB/C mice were sensitized by OVA-alum immunization. Experimental asthma, triggered subsequently by repetitive nebulized OVA inhalation, was assessed, using as readout parameters eosinophilia, peribronchial inflammation, and Th2 cytokine function. MEASUREMENTS AND MAIN RESULTS: A single intratracheal treatment of sensitized mice with MA, inserted into liposomes as carriers, prevented the onset of OVA-triggered allergic airway inflammation and promoted unresponsiveness to a secondary set of allergen exposures. The development of this tolerant condition required an 8-d lapse after MA instillation, coinciding with the appearance of foamy alveolar macrophages. MA-conditioned CD11b(+)F4/80(+) macrophages, transferred to the airways, mimicked the tolerogenic function of instilled MA; however, without the 8-d lapse requirement. Indicative of a macrophage-mediated tolerogenic antigen-presenting function, major histocompatibility complex (MHC)-mismatched donor macrophages failed to promote tolerance. Furthermore, Treg markers were strongly increased and established tolerance was lost after in situ depletion of CD25(+) Treg cells. Contrary to the interleukin-10 dependence of tolerogenic dendritic cells, IFN-gamma deficiency but not interleukin-10 deficiency abrogated the tolerogenic capacity of MA-conditioned macrophages. CONCLUSIONS: These results document an innate-driven Mycobacterium tuberculosis MA-triggered immune regulatory mechanism in control of pulmonary allergic responses by converting macrophages into IFN-gamma-dependent tolerogenic antigen-presenting cells.

Enhanced biodegradation of diesel oil by a newly identified Rhodococcus baikonurensis EN3 in the presence of mycolic acid.

AIMS: The aim of the present study was to isolate and characterize a bacterium, strain EN3, capable of using diesel oil as a major carbon and energy source, and to analyse the enhancement of diesel oil degradation by this organism using synthetic mycolic acid (2-hexyl-3-hydroxyldecanoic acid). METHOD AND RESULTS: An actinomycete with the ability to degrade diesel oil was isolated from oil contaminated soil and characterized. The strain had phenotypic properties consistent with its classification in the genus Rhodococcus showing a 16S rRNA gene similarity of 99.7% with Rhodococcus baikonurensis DSM 44587(T). The ability of the characterized strain to degrade diesel oil at various concentrations (1000, 5000, 10 000 and 20 000 mg l(-1)) was determined. The effect of synthetic mycolic acid on the biodegradation of diesel oil was investigated at the 20 000 mg l(-1) concentration; the surfactant was added to the flask cultures at three different concentrations (10, 50 and 100 mg l(-1)) and degradation followed over 7 days. Enhanced degradation was found at all three concentrations of the surfactant. In addition, the enhancement of diesel oil degradation by other surfactants was observed. CONCLUSIONS: The synthetic mycolic acid has potential for the remediation of petroleum-contaminated sites from both an economic and applied perspective as it can stimulate biodegradation at low concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that the synthesized mycolic acid can be used for potential applications in the bioremediation industries, for example, in oil spill clean-up, diesel fuel remediation and biostimulation.

Planejamento racional de tuberculostáticos com base na estrutura dos ácidos micólicos da parede celular do Mycobacterium tuberculosis / Rational design of tuberculostatic based on the structure of mycolic acids of the cell wall of Mycobacterium tuberculosis

Os ácidos micólicos, principais componentes da parede celular do M. tuberculosis, são alvos específicos para o planejamento de novos agentes potenciais contra a tuberculose (TB) e o estudo de seu processo biossintético é de importância fundamental no desenvolvimento de inibidores mais seletivos. À vista de tal fato, desenvolveu-se estudo utilizando técnicas de modelagem molecular (CADD) com o objetivo de contribuir para o planejamento racional de protótipos de atividade tuberculostática potencial, com base em análogos estruturais da isoniazida (INH), cujo mecanismo de ação está ligado à inibição da síntese de ácidos micólicos...