Mycobacterial Lipids Research Articles

A Proteins of ANL Family FACLs and FAALs, Next Anti TB Drug Targets.

The adenylating enzymes (AEs) of the ANL family contribute to the lipid synthesis process in Mycobacterium tuberculosis (MTB). FAALs (Fatty Acyl-AMP Ligase) and FACLs (Fatty Acyl-CoA Ligase) are members of the ANL enzyme family and are closely associated with the metabolism of mycobacterial lipids responsible for successful infections. Therefore, given their role in both biosynthesis and breakdown processes within fatty acid metabolism, these two groups of proteins might be potential drug targets. The growing abundance of structural, biochemical, and genetic information on mycobacterial FAAL and FACL enzymes could provide a solid foundation for the creation of next-generation anti-tuberculosis drugs, via rational drug designing.

Quantification of mycolic acids in different mycobacterial species by standard addition method through liquid chromatography mass spectrometry

Mycobacteria possess unique and robust lipid profile responsible for their pathogenesis and drug resistance. Mycolic acid (MA) represents an attractive diagnostic biomarker being absent in humans, inert and known to modulate host-pathogen interaction. Accurate measurement of MA is significant to design efficient therapeutics. Despite considerable advances in Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) based approaches, quantification of mycobacterial lipids including MA is still challenging mainly because of ion suppression effects due to complex matrix and non-availability of suitable internal standards for MA. The current study demonstrates the use of standard addition method (SAM) to circumvent this problem and provides a reliable and exhaustive analytical method to quantify mycobacterial MA based on reversed-phase ultra-high-performance liquid chromatography- mass spectrometry data acquisition. In this method, multiple reaction monitoring (MRM) has been applied, wherein 16 MRM channels or transitions have been chosen for quantification of alpha-, methoxy- and keto-MAs with C-24 and C-26 hydrocarbon chains that are actually best suited for TB diagnostics. We found that the overall methodological limit of detection and limit of quantification were in the range 0.05–0.71 ng/µl and 0.16–2.16 ng/µl. Taken together, SAM quantitative technique could serve as promising alternative for relative concentration determination of MA to aid medical research.

Derivatives of Pyrimidine Nucleosides Affect Artificial Membranes Enriched with Mycobacterial Lipids.

The mechanisms of action of pyrimidine nucleoside derivatives on model lipid membranes of various compositions were studied. A systematic analysis of the tested agents' effects on the membrane physicochemical properties was performed. Differential scanning microcalorimetry data indicated that the ability of nucleoside derivatives to disorder membrane lipids depended on the types of nucleoside bases and membrane-forming lipids. The 5'-norcarbocyclic uracil derivatives were found to be ineffective, while N4-alkylcytidines demonstrated the most pronounced effects, significantly decreasing the dipalmitoylphosphocholine melting temperature and cooperativity of phase transition. The elongation of hydrophobic acyl radicals potentiated the disordering action of N4-alkylcytidines, while an increase in hydrophilicity due to replacing deoxyribose with ribose inhibited this effect. The ability of compounds to form transmembrane pores was also tested. It was found that 5-alkyluridines produced single, ion-permeable pores in phosphatidylglycerol membranes, and that methoxy-mycolic acid and trehalose monooleate potentiated the pore-forming activity of alkyloxymethyldeoxyuridines. The results obtained open up perspectives for the development of innovative highly selective anti-tuberculosis agents, which may be characterized by a low risk of developing drug resistance due to the direct action on the membranes of the pathogen.

N - acetyl-transferases required for iron uptake and aminoglycoside resistance promote virulence lipid production in M. marinum.

Acetylation is a modification of protein N-termini, lysine residues, antibiotics and lipids. Many of the enzymes that promote acetylation belong to the GNAT family of proteins. M. marinum is a well-established as a model to understand how M. tuberculosis causes tuberculosis. In this study we sought to identify conserved GNAT proteins required for early stages of mycobacterial infection. Using M. marinum, we determined that several GNAT proteins are required for the lytic activity of M. marinum. We uncovered previously unknown connections between acetyl-transferases required for iron uptake and antimicrobial resistance, and the production of the unique mycobacterial lipids, PDIM and PGLOur data support that acetyl-transferases from the GNAT family are interconnected, and have activities beyond those previously reported.

Residual Membrane Fluidity in Mycobacterial Cell Envelope Layers under Extreme Conditions Underlines Membrane-Centric Adaptation.

One of the routes for adaptation to extreme environments is via remodeling of cell membrane structure, composition, and biophysical properties rendering a functional membrane. Collective studies suggest some form of membrane feedback in mycobacterial species that harbor complex lipids within the outer and inner cell wall layers. Here, we study the homeostatic membrane landscape of mycobacteria in response to high hydrostatic pressure and temperature triggers using high pressure fluorescence, mass and infrared spectroscopies, NMR, SAXS, and molecular dynamics simulations. Our findings reveal that mycobacterial membrane possesses unique and lipid-specific pressure-induced signatures that attenuate progression to highly ordered phases. Both inner and outer membrane layers exhibit phase coexistence of nearly identical lipid phases keeping residual fluidity over a wide range of temperature and pressure, but with different sensitivities. Lipidomic analysis of bacteria grown under pressure revealed lipidome remodeling in terms of chain length, unsaturation, and specific long-chained characteristic mycobacterial lipids, rendering a fluid bacterial membrane. These findings could help understand how bacteria may adapt to a broad spectrum of harsh environments by modulating their lipidome to select lipids that enable the maintenance of a fluid functional cell envelope.

Mycobacterium tuberculosis Rv2617c is involved in stress response and phage infection resistance

Mycobacterium tuberculosis (M. tuberculosis) is the pathogen of human tuberculosis (TB). Resistance to numerous in vivo stresses, including oxidative stress, is determinant for M. tuberculosis intracellular survival, and understanding associated mechanisms is crucial for developing new therapeutic strategies. M. tuberculosis Rv2617c has been associated with oxidative stress response when interacting with other proteins in M. tuberculosis; however, its functional promiscuity and underlying molecular mechanisms remain elusive. In this study, we investigated the phenotypic changes of Mycobacterium smegmatis (M. smegmatis) expressing Rv2617c (Ms_Rv2617c) and its behavior in the presence of various in vitro stresses and phage infections. We found that Rv2617c conferred resistance to SDS and diamide while sensitizing M. smegmatis to oxidative stress (H2O2) and altered mycobacterial phenotypic properties (single-cell clone and motility), suggestive of reprogrammed mycobacterial cell wall lipid contents exemplified by increased cell wall permeability. Interestingly, we also found that Rv2617c promoted M. smegmatis resistance to infection by phages (SWU1, SWU2, D29, and TM4) and kept phage TM4 from destroying mycobacterial biofilms. Our findings provide new insights into the role of Rv2617c in resistance to oxide and acid stresses and report for the first time on its role in phage resistance in Mycobacterium.

Promising antibacterial efficacy of arenicin peptides against the emerging opportunistic pathogen Mycobacterium abscessus

BackgroundMycobacterium abscessus, a fast-growing non-tuberculous mycobacterium, is an emerging opportunistic pathogen responsible for chronic bronchopulmonary infections in people with respiratory diseases such as cystic fibrosis (CF). Due to its intrinsic polyresistance to a wide range of antibiotics, most treatments for M. abscessus pulmonary infections are poorly effective. In this context, antimicrobial peptides (AMPs) active against bacterial strains and less prompt to cause resistance, represent a good alternative to conventional antibiotics. Herein, we evaluated the effect of three arenicin isoforms, possessing two or four Cysteines involved in one (Ar-1, Ar-2) or two disulfide bonds (Ar-3), on the in vitro growth of M. abscessus.MethodsThe respective disulfide-free AMPs, were built by replacing the Cysteines with alpha-amino-n-butyric acid (Abu) residue. We evaluated the efficiency of the eight arenicin derivatives through their antimicrobial activity against M. abscessus strains, their cytotoxicity towards human cell lines, and their hemolytic activity on human erythrocytes. The mechanism of action of the Ar-1 peptide was further investigated through membrane permeabilization assay, electron microscopy, lipid insertion assay via surface pressure measurement, and the induction of resistance assay.ResultsOur results demonstrated that Ar-1 was the safest peptide with no toxicity towards human cells and no hemolytic activity, and the most active against M. abscessus growth. Ar-1 acts by insertion into mycobacterial lipids, resulting in a rapid membranolytic effect that kills M. abscessus without induction of resistance.ConclusionOverall, the present study emphasized Ar-1 as a potential new alternative to conventional antibiotics in the treatment of CF-associated bacterial infection related to M. abscessus.

Decoding the role of mycobacterial lipid remodelling and membrane dynamics in antibiotic tolerance.

Current treatments for tuberculosis primarily target Mycobacterium tuberculosis (Mtb) infections, often neglecting the emerging issue of latent tuberculosis infection (LTBI) which are characterized by reduced susceptibility to antibiotics. The bacterium undergoes multiple adaptations during dormancy within host granulomas, leading to the development of antibiotic-tolerant strains. The mycobacterial membrane plays a crucial role in drug permeability, and this study aims to characterize membrane lipid deviations during dormancy through extensive lipidomic analysis of bacteria cultivated in distinct media and growth stages. The results revealed that specific lipids localize in different regions of the membrane envelope, allowing the bacterium to adapt to granuloma conditions. These lipid modifications were then correlated with the biophysical properties of the mycomembrane, which may affect interactions with antibiotics. Overall, our findings offer a deeper understanding of the bacterial adaptations during dormancy, highlighting the role of lipids in modulating membrane behaviour and drug permeability, ultimately providing the groundwork for the development of more effective treatments tailored to combat latent infections.

Biophysical Interaction Landscape of Mycobacterial Mycolic Acids and Phenolic Glycolipids with Host Macrophage Membranes.

Lipidic adjuvant formulations consisting of immunomodulatory mycobacterial cell wall lipids interact with host cells following administration. The impact of this cross-talk on the host membrane's structure and function is rarely given enough consideration but is imperative to rule out nonspecific perturbation underlying the adjuvant. In this work, we investigated changes in the plasma membranes of live mammalian cells after exposure to mycobacterial mycolic acid (MA) and phenolic glycolipids, two strong candidates for lipidic adjuvant therapy. We found that phenolic glycolipid 1 softened the plasma membrane, lowering membrane tension and stiffness, but MA did not significantly change the membrane characteristics. Further, phenolic glycolipid 1 had a fluidizing impact on the host plasma membrane, increasing the fluidity and the abundance of fluid-ordered-disordered coexisting lipid domains. Notably, lipid diffusion was not impacted. Overall, MA and, to a lesser extent, phenolic glycolipid 1, due to minor disruption of host cell membranes, may serve as appropriate lipids in adjuvant formulations.

A Loss of Function in LprG-Rv1410c Homologues Attenuates Growth during Biofilm Formation in Mycobacterium smegmatis.

MmpL (mycobacterial membrane protein large) proteins are integral membrane proteins that have been implicated in the biosynthesis and/or transport of mycobacterial cell wall lipids. Given the cellular location of these proteins, however, it is unclear how cell wall lipids are transported beyond the inner membrane. Moreover, given that mycobacteria grow at the poles, we also do not understand how new cell wall is added in a highly localized and presumably coordinated manner. Here, we examine the relationship between two lipid transport pathways associated with the proteins MmpL11 and LprG-Rv1410c. The lipoprotein LprG has been shown to interact with proteins involved in cell wall processes including MmpL11, which is required in biofilms for the surface localization of certain lipids. Here we report that deletion of mmpL11 (MSMEG_0241) or the lprG-rv1410c operon homologues MSMEG_3070-3069 in Mycobacterium smegmatis produced similar biofilm defects that were distinct from that of the previously reported mmpL11 transposon insertion mutant. Analysis of pellicle biofilms, bacterial growth, lipid profiles, and gene expression revealed that the biofilm phenotypes could not be directly explained by changes in the synthesis or localization of biofilm-related lipids or the expression of biofilm-related genes. Instead, the shared biofilm phenotype between ΔMSMEG_3070-3069 and ΔmmpL11 may be related to their modest growth defect, while the origins of the distinct mmpL11::Tn biofilm defect remain unclear. Our findings suggest that the mechanisms that drive pellicle biofilm formation in M. smegmatis are not connected to crosstalk between the LprG-Rv1410c and MmpL11 pathways and that any functional interaction between these proteins does not relate directly to their lipid transport function.

Effect of Host Cholesterol on the Membrane Dynamics of Outer Membrane Lipids of Mycobacteria.

The ability of Mycobacterium tuberculosis to remain dormant after primary infection represents the prime cause of new TB cases throughout the world. Hence, diagnosis and treatment of individuals hosting dormant mycobacterium is one of the crucial strategies to be adopted for the prevention of Tuberculosis. Among many strategies unleashed by the latent bacterium, one of them is scavenging host cholesterol for carbon source. Cholesterol modifies lipid membranes over many scales and here, its effect on mycobacterial membrane biophysics and the subsequent effect on partitioning of antibiotics into cholesterol- enriched mycobacterial membranes was investigated. Our research showed that cholesterol alters the phase state behavior of mycobacterial outer membrane lipids by enhancing the overall membrane order at the headgroup and acyl chain region and is integrated into both ordered and disordered domains/phases, with a preference for the latter. Exogenous cholesterol further alters the drug partitioning behavior of structurally different drugs, pointing to a larger clinical potential of using more hydrophobic medications to target dormant bacteria.

Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells

Mycobacterium tuberculosis (Mtb) infection elicits both protein and lipid antigen-specific T cell responses. However, the incorporation of lipid antigens into subunit vaccine strategies and formulations has been underexplored, and the characteristics of vaccine-induced Mtb lipid-specific memory T cells have remained elusive. Mycolic acid (MA), a major lipid component of the Mtb cell wall, is presented by human CD1b molecules to unconventional T cell subsets. These MA-specific CD1b-restricted T cells have been detected in the blood and disease sites of Mtb-infected individuals, suggesting that MA is a promising lipid antigen for incorporation into multicomponent subunit vaccines. In this study, we utilized the enhanced stability of bicontinuous nanospheres (BCN) to efficiently encapsulate MA for in vivo delivery to MA-specific T cells, both alone and in combination with an immunodominant Mtb protein antigen (Ag85B). Pulmonary administration of MA-loaded BCN (MA-BCN) elicited MA-specific T cell responses in humanized CD1 transgenic mice. Simultaneous delivery of MA and Ag85B within BCN activated both MA- and Ag85B-specific T cells. Notably, pulmonary vaccination with MA-Ag85B-BCN resulted in the persistence of MA, but not Ag85B, within alveolar macrophages in the lung. Vaccination of MA-BCN through intravenous or subcutaneous route, or with attenuated Mtb likewise reproduced MA persistence. Moreover, MA-specific T cells in MA-BCN-vaccinated mice differentiated into a T follicular helper-like phenotype. Overall, the BCN platform allows for the dual encapsulation and in vivo activation of lipid and protein antigen-specific T cells and leads to persistent lipid depots that could offer long-lasting immune responses.

Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells.

Mycobacterium tuberculosis (Mtb) infection elicits both protein and lipid antigen-specific T cell responses. However, the incorporation of lipid antigens into subunit vaccine strategies and formulations has been underexplored, and the characteristics of vaccine-induced Mtb lipid-specific memory T cells have remained elusive. Mycolic acid (MA), a major lipid component of the Mtb cell wall, is presented by human CD1b molecules to unconventional T cell subsets. These MA-specific CD1b-restricted T cells have been detected in the blood and disease sites of Mtb-infected individuals, suggesting that MA is a promising lipid antigen for incorporation into multicomponent subunit vaccines. In this study, we utilized the enhanced stability of bicontinuous nanospheres (BCN) to efficiently encapsulate MA for in vivo delivery to MA-specific T cells, both alone and in combination with an immunodominant Mtb protein antigen (Ag85B). Pulmonary administration of MA-loaded BCN (MA-BCN) elicited MA-specific T cell responses in humanized CD1 transgenic mice. Simultaneous delivery of MA and Ag85B within BCN activated both MA- and Ag85B-specific T cells. Notably, pulmonary vaccination with MA-Ag85B-BCN resulted in the persistence of MA, but not Ag85B, within alveolar macrophages in the lung. Vaccination of MA-BCN through intravenous or subcutaneous route, or with attenuated Mtb likewise reproduced MA persistence. Moreover, MA-specific T cells in MA-BCN-vaccinated mice differentiated into a T follicular helper-like phenotype. Overall, the BCN platform allows for the dual encapsulation and in vivo activation of lipid and protein antigen-specific T cells and leads to persistent lipid depots that could offer long-lasting immune responses.

Telacebec Interferes with Virulence Lipid Biosynthesis Protein Expression and Sensitizes to Other Antibiotics.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a public health issue, particularly due to multi-drug-resistant Mtb. The bacillus is wrapped in a waxy envelope containing lipids acting as essential virulence factors, accounting for the natural antibiotic resistance of mycobacteria. Telacebec (previously known as Q203) is a promising new anti-TB agent inhibiting the cytochrome bc1 complex of a mycobacterial electron transport chain (ETC). Here, we show that the telacebec-challenged M. bovis BCG exhibited a reduced expression of proteins involved in the synthesis of phthiocerol dimycocerosates (PDIMs)/phenolic glycolipids (PGLs), lipid virulence factors associated with cell envelope impermeability. Consistently, telacebec, at concentrations lower than its MIC, downregulated the transcription of a PDIM/PGL-synthesizing operon, suggesting a metabolic vulnerability triggered by the drug. The drug was able to synergize on BCG with rifampicin or vancomycin, the latter being a drug exerting a marginal effect on PDIM-bearing bacilli. Telacebec at a concentration higher than its MIC had no detectable effect on cell wall PDIMs, as shown by TLC analysis, a finding potentially explained by the retaining of previously synthesized PDIMs due to the inhibition of growth. The study extends the potential of telacebec, demonstrating an effect on mycobacterial virulence lipids, allowing for the development of new anti-TB strategies.

A conserved membrane protein negatively regulates Mce1 complexes in mycobacteria

Tuberculosis continues to pose a serious threat to global health. Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that relies on various mechanisms to survive and persist within the host. Among their many virulence factors, mycobacteria encode Mce systems. Some of these systems are implicated in lipid uptake, but the molecular basis for Mce function(s) is poorly understood. To gain insights into the composition and architecture of Mce systems, we characterized the putative Mce1 complex involved in fatty acid transport. We show that the Mce1 system in Mycobacterium smegmatis comprises a canonical ATP-binding cassette transporter associated with distinct heterohexameric assemblies of substrate-binding proteins. Furthermore, we establish that the conserved membrane protein Mce1N negatively regulates Mce1 function via a unique mechanism involving blocking transporter assembly. Our work offers a molecular understanding of Mce complexes, sheds light on mycobacterial lipid metabolism and its regulation, and informs future anti-mycobacterial strategies.

Mycobacterial phage TM4 requires a eukaryotic-like Ser/Thr protein kinase to silence and escape anti-phage immunity

In eukaryotic cells, serine/threonine protein kinases (StpKs) play important roles in limiting viral infections. StpKs are commonly activated upon infections, inhibiting the expression of genes central for viral replication. Here, we report that a eukaryotic-like StpK7 encoded by MSMEG_1200 in M.smegmatis is required for mycobacteriophage TM4 to escape bacterial defense. stpK7 is located within a gene island, MSMEG_1191-MSMEG_1200, containing multiple anti-phage genes resembling the BREX (bacteriophage exclusion) phage-resistance system. StpK7 negatively regulates the expression of this gene island. Following phage TM4 infection, StpK7 is induced, directly phosphorylating the transcriptional regulator MSMEG_1198 and inhibiting its positive regulatory activity, thus reducing the expression of multiple downstream genes in the BREX-like gene island. Further analysis showed that genes within this anti-phage island critically regulate mycobacterial lipid hemostasis and phage adsorption. Collectively, this work characterizes a regulatory network driven by StpK7, which is utilized by phage TM4 to escape from the host defense against mycobacteria.

Fusion Landscape of Mycobacterial Envelope-Derived Lipid Vesicles with Intact Bacteria Dictates High Intracellular Drug Retention.

Membrane vesicles are critical regulators of pathogenic diseases. In tubercular infections, the use of mycobacteria derived vesicles as delivery vehicles to overcome drug resistance and complex treatment regimens has never been attempted. Here, we first address how these vesicles interact with their target cells, especially via membrane fusion. Membrane fusion between alike mycobacterial outer and inner membrane layer-derived lipid vesicles is shown to be driven by the structural, geometrical, and biophysical attributes of constituent lipids. The increased fusion of outer-membrane-derived vesicles with intact bacteria ensures enhanced intracellular drug levels and is presented as a "natural" antitubercular drug delivery vehicle.

Latent Tuberculosis Patients Have an Increased Frequency of IFN-γ-Producing CD5+ B Cells, Which Respond Efficiently to Mycobacterial Proteins.

Tuberculosis (TB) remains a public health problem worldwide and is one of the deadliest infectious diseases, only after the current COVID-19 pandemic. Despite significant advances in the TB field, there needs to be more immune response comprehension; for instance, the role played by humoral immunity is still controversial. This study aimed to identify the frequency and function of B1 and immature/transitional B cells in patients with active and latent TB (ATB and LTB, respectively). Here we show that LTB patients have an increased frequency of CD5+ B cells and decreased CD10+ B cells. Furthermore, LTB patients stimulated with mycobacteria's antigens increase the frequency of IFN-γ-producing B cells, whereas cells from ATB do not respond. Moreover, under the mycobacterial protein stimulus, LTB promotes a pro-inflammatory environment characterized by a high level of IFN-γ but also can produce IL-10. Regarding the ATB group, they cannot produce IFN-γ, and mycobacterial lipids and proteins stimulate only the IL-10 production. Finally, our data showed that in ATB, but not in LTB, B cell subsets correlate with clinical and laboratory parameters, suggesting that these CD5+ and CD10+ B cell subpopulations have the potential to be biomarkers to differentiate between LTB and ATB. In conclusion, LTB has increased CD5+ B cells, and these cells can maintain a rich microenvironment of IFN-γ, IL-10, and IL-4. In contrast, ATB only maintains an anti-inflammatory environment when stimulated with mycobacterial proteins or lipids.

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.

Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions.

For decades, investigators have studied the interaction of Mycobacterium tuberculosis (Mtb) with macrophages, which serve as a major cellular niche for the bacilli. Because Mtb are prone to aggregation, investigators rely on varied methods to disaggregate the bacteria for these studies. Here, we examined the impact of routinely used preparation methods on bacterial cell envelope integrity, macrophage inflammatory responses, and intracellular Mtb survival. We found that both gentle sonication and filtering damaged the mycobacterial cell envelope and markedly impacted the outcome of infections in mouse bone marrow-derived macrophages. Unexpectedly, sonicated bacilli were hyperinflammatory, eliciting dramatically higher TLR2-dependent gene expression and elevated secretion of IL-1β and TNF-α. Despite evoking enhanced inflammatory responses, sonicated bacilli replicated normally in macrophages. In contrast, Mtb that had been passed through a filter induced little inflammatory response, and they were attenuated in macrophages. Previous work suggests that the mycobacterial cell envelope lipid, phthiocerol dimycocerosate (PDIM), dampens macrophage inflammatory responses to Mtb. However, we found that the impact of PDIM depended on the method used to prepare Mtb. In conclusion, widely used methodologies to disaggregate Mtb may introduce experimental artifacts in Mtb-host interaction studies, including alteration of host inflammatory signaling, intracellular bacterial survival, and interpretation of bacterial mutants.