The High Plasticity of Nonpathogenic Mycobacterium brumae Induces Rapid Changes in Its Lipid Profile during Pellicle Maturation: The Potential of This Bacterium as a Versatile Cell Factory for Lipid Compounds of Therapeutic Interest.

The immunomodulatory potential of mycobacteria to be used for therapeutic purposes varies by species and culture conditions and is closely related to mycobacterial lipid composition. Although the lipids present in the mycobacterial cell wall are relevant, lipids are mainly stored in intracellular lipid inclusions (ILIs), which have emerged as a crucial structure in understanding mycobacteria-host interaction. Little is known about ILI ultrastructure, production, and composition in nonpathogenic species. In this study, we compared the lipid profiles of the nonpathogenic immunomodulatory agent Mycobacterium brumae during pellicle maturation under different culture conditions with qualitative and quantitative approaches by using high-resolution imaging and biochemical and composition analyses to understand ILI dynamics. The results showed wax esters, mainly in early stages of development, and acylglycerols in mature ILI composition, revealing changes in dynamics, amount, and morphometry, depending on pellicle maturation and the culture media used. Low-glycerol cultures induced ILIs with lower molecular weights which were smaller in size in comparison with the ILIs produced in glycerol-enriched media. The data also indicate the simple metabolic plasticity of lipid synthesis in M. brumae, as well as its high versatility in generating different lipid profiles. These findings provide an interesting way to enhance the production of key lipid structures via the simple modulation of cell culture conditions.

γ Irradiated Mycobacteria Enhance Survival in Bladder Tumor Bearing Mice Although Less Efficaciously than Live Mycobacteria.

PURPOSE: γ Irradiated Mycobacterium bovis bacillus Calmette-Guérin has shown in vitro and ex vivo antitumor activity. However, to our knowledge the potential antitumor capacity has not been demonstrated in vivo. We studied the in vivo potential of γ irradiated bacillus Calmette-Guérin and γ irradiated M. brumae, a saprophytic mycobacterium that was recently described as an immunotherapeutic agent. MATERIALS AND METHODS: The antitumor capacity of γ irradiated M. brumae was first investigated by analyzing the in vitro inhibition of bladder tumor cell proliferation and the ex vivo cytotoxic effect of M. brumae activated peripheral blood cells. The effect of γ irradiated M. brumae or bacillus Calmette-Guérin intravesical treatment was then compared to treatment with live mycobacteria in the orthotopic murine model of bladder cancer. RESULTS: Nonviable M. brumae showed a capacity to inhibit in vitro bladder cancer cell lines similar to that of live mycobacteria. However, its capacity to induce cytokine production was decreased compared to that of live M. brumae. γ Irradiated M. brumae could activate immune cells to inhibit tumor cell growth, although to a lesser extent than live mycobacteria. Finally, intravesical treatment with γ irradiated M. brumae or bacillus Calmette-Guérin significantly increased survival with respect to that of nontreated tumor bearing mice. Both γ irradiated mycobacteria showed lower survival rates than those of live mycobacteria but the minor efficacy of γ irradiated vs live mycobacteria was only significant for bacillus Calmette-Guérin. CONCLUSIONS: Our results show that although γ irradiated mycobacteria is less efficacious than live mycobacteria, it induces an antitumor effect in vivo, avoiding the possibility of further mycobacterial infections.

Killed but metabolically active Mycobacterium bovis bacillus Calmette-Guérin retains the antitumor ability of live bacillus Calmette-Guérin.

PURPOSE: Mycobacterium bovis bacillus Calmette-Guérin is the most effective treatment for high risk noninvasive bladder cancer. Although bacillus Calmette-Guérin immunotherapy clearly decreases recurrence and progression rates, side effects are common and infection with the bacillus has been described. For these reasons it is necessary to find safer alternatives to the live bacillus. We explored the possibility of using killed but metabolically active bacillus Calmette-Guérin. MATERIALS AND METHODS: T24, J82 and RT4 bladder tumor cell lines were cultured with live and irradiation or heat treated bacillus Calmette-Guérin Connaught. We measured the inhibition of cell proliferation and the production of cytokines in cell culture supernatants. Peripheral mononuclear blood cells were also infected and the production of different cytokines in cell culture supernatants was analyzed. Peripheral blood mononuclear cell and cell culture supernatants activated by mycobacteria were then cultured with T24 cells to analyze whether they showed cytotoxic activity. RESULTS: Compared to the other bacillus Calmette-Guérin treatments, γ irradiated bacillus Calmette-Guérin showed activity similar to that of the live bacillus for inhibiting tumor growth and inducing cytokine production. Irradiated bacillus Calmette-Guérin showed metabolic activity and, thus, was considered killed but metabolically active. This is the treatment that most accurately preserved the mycobacterial structure. Killed but metabolically active bacillus Calmette-Guérin induced cytokine production by infected peripheral mononuclear blood cells. Mycobacteria activated peripheral blood mononuclear cell and cell supernatants showed cytotoxic activity against tumor cells, retaining the antitumor capacity of the live bacillus. CONCLUSIONS: Our results suggest that killed but metabolically active bacillus Calmette-Guérin could be considered a safer immunotherapy alternative to treatment with the live bacillus.

Connaught and Russian strains showed the highest direct antitumor effects of different Bacillus Calmette-Guérin substrains.

PURPOSE: Evolutionarily early and late bacillus Calmette-Guérin substrains are genetically distinct, showing different antigenic determinants. While it was suggested that this may influence the immunostimulatory effects of bacillus Calmette-Guérin as a vaccine in the context of tuberculosis, to our knowledge the impact of these genetic differences on the antitumor activity of bacillus Calmette-Guérin remains unknown. We compared the direct antitumor capacity and the ability to trigger cytokine production of 8 evolutionarily early and late BCG substrains in urothelial bladder cancer cell lines. MATERIALS AND METHODS: The T24, J82 and RT4 bladder tumor cell lines were cultured with different doses of 3 evolutionarily early bacillus Calmette-Guérin substrains (Japan, Moreau and Russian) and 5 evolutionarily late strains (Connaught, Danish, Glaxo, Phipps and Tice). The inhibition of cell proliferation at different time points and the production of interleukin-6 and 8 in cell culture supernatants were measured. RESULTS: For T24 and J82 cells Russian and Connaught induced the highest inhibition of cell proliferation and cytokine production, triggering values up to threefold higher than the other bacillus Calmette-Guérin strains. In contrast, Glaxo and Phipps (for T24 cells) and Glaxo and Tice (for J82 cells) were the least efficacious. For RT4 all bacillus Calmette-Guérin strains inhibited cell proliferation to a similar extent and induced low levels of only interleukin-8 except the Danish and Glaxo strains, which were less efficacious. CONCLUSIONS: Russian and Connaught, which are evolutionarily early and late substrains, respectively, are the most efficacious bacillus Calmette-Guérin strains for inhibiting cell proliferation and inducing cytokine production. Glaxo is the least efficacious strain.

Cyclopropanation of α-mycolic acids is not required for cording in Mycobacterium brumae and Mycobacterium fallax.

The capacity to form microscopic cords (cording) of Mycobacterium species has been related to their virulence. The compounds responsible for cording are unknown, but a recent study has shown that cording could be related to the fine structure of α-mycolic acids. This investigation attributes the need for a proximal cyclopropane in α-mycolic acids for cording in Mycobacterium tuberculosis and Mycobacterium bovis BCG and proposes cyclopropanases as good targets for new chemotherapeutic agents. As other Mycobacterium species in addition to M. tuberculosis and M. bovis form microscopic cords, it would be of major interest to know whether the relationship between proximal cyclopropanation of α-mycolic acids and cording could be extended to non-tuberculous mycobacteria. In this study, we have examined the correlation between the cording and cyclopropanation of α-mycolic acids in two species, Mycobacterium brumae and Mycobacterium fallax. Scanning electron microscopy images showed, for the first time to our knowledge, the fine structure of microscopic cords of M. brumae and M. fallax, confirming that these two species form true cords. Furthermore, NMR analysis performed on the same cording cultures corroborates the absence of cyclopropane rings in their α-mycolic acids. Therefore, we can conclude that the correlation between cording and cyclopropanation of α-mycolic acids cannot be extended to all mycobacteria. As M. brumae and M. fallax grow rapidly and have a simple pattern of mycolic acids (only α-unsaturated mycolic acids), we propose these two species as suitable models for the study of the role of mycolic acids in cording.

Cord factors from atypical mycobacteria (Mycobacterium alvei, Mycobacterium brumae) stimulate the secretion of some pro-inflammatory cytokines of relevance in tuberculosis.

The ability to induce several cytokines relevant to tuberculosis (TNF-α, IL-1ß, IL-6, IL-12p40 and IL-23) by cord factor (trehalose dimycolate) from Mycobacterium alvei CR-21(T) and Mycobacterium brumae CR-270(T) was studied in the cell lines RAW 264.7 and THP-1, and compared to the ability of cord factor from Mycobacterium tuberculosis H37Rv, where this glycolipid appears to be implicated in the pathogenesis of tuberculosis. Details of the fine structure of these molecules were obtained by NMR and MS. The mycoloyl residues were identified as α and (ω-1)-methoxy in M. alvei CR-21(T) and α in M. brumae CR-270(T); in both cases they were di-unsaturated instead of cyclopropanated as found in M. tuberculosis. In RAW 264.7 cells, cord factors from M. alvei CR-21(T), M. brumae CR-270(T) and M. tuberculosis differed in their ability to stimulate IL-6, the higher levels corresponding to the cord factor from M. tuberculosis. In THP-1 cells, a similar overall profile of cytokines was found for M. alvei CR-21(T) and M. brumae CR-270(T), with high proportions of IL-1ß and TNF-α, and different from M. tuberculosis, where IL-6 and IL-12p40 prevailed. The data obtained indicate that cord factors from the atypical mycobacteria M. alvei CR-21(T) and M. brumae CR-270(T) stimulated the secretion of several pro-inflammatory cytokines, although there were some differences with those of M. tuberculosis H37Rv. This finding seems to be due to their particular mycoloyl substituents and could be of interest when considering the potential adjuvanticity of these molecules.

Mycobacterial surface characters remodeled by growth conditions drive different tumor-infiltrating cells and systemic IFN-γ/IL-17 release in bladder cancer treatment.

The mechanism of action of intravesical Mycobacterium bovis BCG immunotherapy treatment for bladder cancer is not completely known, leading to misinterpretation of BCG-unresponsive patients, who have scarce further therapeutic options. BCG is grown under diverse culture conditions worldwide, which can impact the antitumor effect of BCG strains and could be a key parameter of treatment success. Here, BCG and the nonpathogenic Mycobacterium brumae were grown in four culture media currently used by research laboratories and BCG manufacturers: Sauton-A60, -G15 and -G60 and Middlebrook 7H10, and used as therapies in the orthotopic murine BC model. Our data reveal that each mycobacterium requires specific culture conditions to induce an effective antitumor response. since higher survival rates of tumor-bearing mice were achieved using M. brumae-A60 and BCG-G15 than the rest of the treatments. M. brumae-A60 was the most efficacious among all tested treatments in terms of mouse survival, cytotoxic activity of splenocytes against tumor cells, higher systemic production of IL-17 and IFN-É£, and bladder infiltration of selected immune cells such as ILCs and CD4TEM. BCG-G15 triggered an antitumor activity based on a massive infiltration of immune cells, mainly CD3+ (CD4+ and CD8+) T cells, together with high systemic IFN-É£ release. Finally, a reduced variety of lipids was strikingly observed in the outermost layer of M. brumae-A60 and BCG-G15 compared to the rest of the cultures, suggesting an influence on the antitumor immune response triggered. These findings contribute to understand how mycobacteria create an adequate niche to help the host subvert immunosuppressive tumor actions.

Demonstration of cord formation by rough Mycobacterium abscessus variants: implications for the clinical microbiology laboratory.

In low-income countries some infections caused by nontuberculous mycobacteria are misdiagnosed as multidrug-resistant tuberculosis. In most of these settings the observation of microscopic cords is the only technique used to identify Mycobacterium tuberculosis in the laboratory. In this article we definitively demonstrate that Mycobacterium abscessus, an emerging pulmonary pathogen, also forms microscopic cords.

Prevalence and concentration of non-tuberculous mycobacteria in cooling towers by means of quantitative PCR: a prospective study.

There is an increasing level of interest in non-tuberculous mycobacteria (NTM) due to the increasing reported rates of diseases caused by them. Although it is well known that NTM are widely distributed in the environment it is necessary to identify its reservoirs to prevent possible infections. In this study, we aimed to investigate the occurrence and levels of NTM in cooling towers to provide evidences for considering these settings as possible sources of respiratory infections. In the current study, we detected and quantified the presence of NTM by means of a rapid method in water samples taken from 53 cooling towers of an urban area (Barcelona, Spain). A genus-specific quantitative PCR (Q-PCR) assay with a quantification limit (QL) of 500 cells l(-1) was used. 56% (30) of samples were positive with a concentration range from 4.6 × 10(3) to 1.79 × 10(6) cells l(-1). In some cases (9/30), samples were positive but with levels below the QL. The colonization rate confirmed that cooling towers could be considered as a potential reservoir for NTM. This study also evaluated Q-PCR as a useful method to detect and quantify NTM in samples coming from environmental sources.

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography.

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Guérin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

BCG Substrains Change Their Outermost Surface as a Function of Growth Media.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) efficacy as an immunotherapy tool can be influenced by the genetic background or immune status of the treated population and by the BCG substrain used. BCG comprises several substrains with genetic differences that elicit diverse phenotypic characteristics. Moreover, modifications of phenotypic characteristics can be influenced by culture conditions. However, several culture media formulations are used worldwide to produce BCG. To elucidate the influence of growth conditions on BCG characteristics, five different substrains were grown on two culture media, and the lipidic profile and physico-chemical properties were evaluated. Our results show that each BCG substrain displays a variety of lipidic profiles on the outermost surface depending on the growth conditions. These modifications lead to a breadth of hydrophobicity patterns and a different ability to reduce neutral red dye within the same BCG substrain, suggesting the influence of BCG growth conditions on the interaction between BCG cells and host cells.

Dissemination of Mycobacterium tuberculosis is associated to a SIGLEC1 null variant that limits antigen exchange via trafficking extracellular vesicles.

The identification of individuals with null alleles enables studying how the loss of gene function affects infection. We previously described a non-functional variant in SIGLEC1, which encodes the myeloid-cell receptor Siglec-1/CD169 implicated in HIV-1 cell-to-cell transmission. Here we report a significant association between the SIGLEC1 null variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. Local spread of bacteria within the lung is apparent in Mtb-infected Siglec-1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. We find that Siglec-1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec-1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination.

Microscopic cords, a virulence-related characteristic of Mycobacterium tuberculosis, are also present in nonpathogenic mycobacteria.

The aggregation of mycobacterial cells in a definite order, forming microscopic structures that resemble cords, is known as cord formation, or cording, and is considered a virulence factor in the Mycobacterium tuberculosis complex and the species Mycobacterium marinum. In the 1950s, cording was related to a trehalose dimycolate lipid that, consequently, was named the cord factor. However, modern techniques of microbial genetics have revealed that cording can be affected by mutations in genes not directly involved in trehalose dimycolate biosynthesis. Therefore, questions such as "How does mycobacterial cord formation occur?" and "Which molecular factors play a role in cord formation?" remain unanswered. At present, one of the problems in cording studies is the correct interpretation of cording morphology. Using optical microscopy, it is sometimes difficult to distinguish between cording and clumping, which is a general property of mycobacteria due to their hydrophobic surfaces. In this work, we provide a new way to visualize cords in great detail using scanning electron microscopy, and we show the first scanning electron microscopy images of the ultrastructure of mycobacterial cords, making this technique the ideal tool for cording studies. This technique has enabled us to affirm that nonpathogenic mycobacteria also form microscopic cords. Finally, we demonstrate that a strong correlation exists between microscopic cords, rough colonial morphology, and increased persistence of mycobacteria inside macrophages.

Each Mycobacterium Requires a Specific Culture Medium Composition for Triggering an Optimized Immunomodulatory and Antitumoral Effect.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) remains the first treatment option for non-muscle-invasive bladder cancer (BC) patients. In research laboratories, M. bovis BCG is mainly grown in commercially available media supplemented with animal-derived agents that favor its growth, while biomass production for patient treatment is performed in Sauton medium which lacks animal-derived components. However, there is not a standardized formulation of Sauton medium, which could affect mycobacterial characteristics. Here, the impact of culture composition on the immunomodulatory and antitumor capacity of M. bovis BCG and Mycolicibacterium brumae, recently described as efficacious for BC treatment, has been addressed. Both mycobacteria grown in Middlebrook and different Sauton formulations, differing in the source of nitrogen and amount of carbon source, were studied. Our results indicate the relevance of culture medium composition on the antitumor effect triggered by mycobacteria, indicating that the most productive culture medium is not necessarily the formulation that provides the most favorable immunomodulatory profile and the highest capacity to inhibit BC cell growth. Strikingly, each mycobacterial species requires a specific culture medium composition to provide the best profile as an immunotherapeutic agent for BC treatment. Our results highlight the relevance of meticulousness in mycobacteria production, providing insight into the application of these bacteria in BC research.

Mycolicibacterium brumae Is a Safe and Non-Toxic Immunomodulatory Agent for Cancer Treatment.

Intravesical Mycobacterium bovis Bacillus Calmette-Guérin (BCG) immunotherapy remains the gold-standard treatment for non-muscle-invasive bladder cancer patients, even though half of the patients develop adverse events to this therapy. On exploring BCG-alternative therapies, Mycolicibacterium brumae, a nontuberculous mycobacterium, has shown outstanding anti-tumor and immunomodulatory capabilities. As no infections due to M. brumae in humans, animals, or plants have been described, the safety and/or toxicity of this mycobacterium have not been previously addressed. In the present study, an analysis was made of M. brumae- and BCG-intravenously-infected severe combined immunodeficient (SCID) mice, M. brumae-intravesically-treated BALB/c mice, and intrahemacoelic-infected-Galleria mellonella larvae. Organs from infected mice and the hemolymph from larvae were processed to count bacterial burden. Blood samples from mice were also taken, and a wide range of hematological and biochemical parameters were analyzed. Finally, histopathological alterations in mouse tissues were evaluated. Our results demonstrate the safety and non-toxic profile of M. brumae. Differences were observed in the biochemical, hematological and histopathological analysis between M. brumae and BCG-infected mice, as well as survival curves rates and colony forming units (CFU) counts in both animal models. M. brumae constitutes a safe therapeutic biological agent, overcoming the safety and toxicity disadvantages presented by BCG in both mice and G. mellonella animal models.

Surface spreading motility shown by a group of phylogenetically related, rapidly growing pigmented mycobacteria suggests that motility is a common property of mycobacterial species but is restricted to smooth colonies.

Motility in mycobacteria was described for the first time in 1999. It was reported that Mycobacterium smegmatis and Mycobacterium avium could spread on the surface of solid growth medium by a sliding mechanism and that the presence of cell wall glycopeptidolipids was essential for motility. We recently reported that Mycobacterium vaccae can also spread on growth medium surfaces; however, only smooth colonies presented this property. Smooth colonies of M. vaccae do not produce glycopeptidolipids but contain a saturated polyester that is absent in rough colonies. Here, we demonstrate that Mycobacterium chubuense, Mycobacterium gilvum, Mycobacterium obuense, and Mycobacterium parafortuitum, which are phylogenetically related to M. vaccae, are also motile. Such motility is restricted to smooth colonies, since natural rough mutants are nonmotile. Thin-layer chromatography analysis of the content of cell wall lipids confirmed the absence of glycopeptidolipids. However, compounds like the above-mentioned M. vaccae polyester were detected in all the strains but only in smooth colonies. Scanning electron microscopy showed great differences in the arrangement of the cells between smooth and rough colonies. The data obtained suggest that motility is a common property of environmental mycobacteria, and this capacity correlates with the smooth colonial morphotype. The species studied in this work do not contain glycopeptidolipids, so cell wall compounds or extracellular materials other than glycopeptidolipids are implicated in mycobacterial motility. Furthermore, both smooth motile and rough nonmotile variants formed biofilms on glass and polystyrene surfaces.

Molecule confirmation and structure characterization of pentatriacontatrienyl mycolate in Mycobacterium smegmatis.

Mycobacterium smegmatis is often used to study the different components of mycobacterial cell wall. Mycolic acids are important components of mycobacterial cell wall that have been associated with virulence. Recently, a novel lipid containing mycolic acids has been described in M. smegmatis. However, some uncertainties regarding the structure of this molecule named mycolate ester wax have been reported. The objective of this work was to perform an in depth structural study of this molecule for its precise characterization. Using 1H and 13C NMR spectroscopy, the molecular structure of mycolate ester wax found in M. smegmatis has been elucidated. The characterization was complemented with MS analyses. This molecule is formed by a carbon chain with three methyl substituted olefinic units and a mycolate structure with trans double bonds and cis cyclopropane rings. The present molecular study will facilitate the detection and identification of pentatriacontatrienyl mycolate in future studies by the performance of a simple 1D 1H NMR experiment.

Intravesical Mycobacterium brumae triggers both local and systemic immunotherapeutic responses against bladder cancer in mice.

The standard treatment for high-risk non-muscle invasive bladder cancer (BC) is the intravesical administration of live Mycobacterium bovis BCG. Previous studies suggest improving this therapy by implementing non-pathogenic mycobacteria, such as Mycobacterium brumae, and/or different vehicles for mycobacteria delivery, such as an olive oil (OO)-in-water emulsion. While it has been established that BCG treatment activates the immune system, the immune effects of altering the mycobacterium and/or the preparation remain unknown. In an orthotopic murine BC model, local immune responses were assessed by measuring immune cells into the bladder and macromolecules in the urine by flow cytometry and multiplexing, respectively. Systemic immune responses were analyzed by quantifying sera anti-mycobacteria antibody levels and recall responses of ex vivo splenocytes cultured with mycobacteria antigens. In both BCG- and M. brumae-treated mice, T, NK, and NKT cell infiltration in the bladder was significantly increased. Notably, T cell infiltration was enhanced in OO-in-water emulsified mycobacteria-treated mice, and urine IL-6 and KC concentrations were elevated. Furthermore, mycobacteria treatment augmented IgG antibody production and splenocyte proliferation, especially in mice receiving OO-in-water emulsified mycobacteria. Our data demonstrate that intravesical mycobacterial treatment triggers local and systemic immune responses, which are most significant when OO-in-water emulsified mycobacteria are used.

Trehalose Polyphleates, External Cell Wall Lipids in Mycobacterium abscessus, Are Associated with the Formation of Clumps with Cording Morphology, Which Have Been Associated with Virulence.

Mycobacterium abscessus is a reemerging pathogen that causes pulmonary diseases similar to tuberculosis, which is caused by Mycobacterium tuberculosis. When grown in agar medium, M. abscessus strains generate rough (R) or smooth colonies (S). R morphotypes are more virulent than S morphotypes. In searching for the virulence factors responsible for this difference, R morphotypes have been found to form large aggregates (clumps) that, after being phagocytozed, result in macrophage death. Furthermore, the aggregates released to the extracellular space by damaged macrophages grow, forming unphagocytosable structures that resemble cords. In contrast, bacilli of the S morphotype, which do not form aggregates, do not damage macrophages after phagocytosis and do not form cords. Cording has also been related to the virulence of M. tuberculosis. In this species, the presence of mycolic acids and surface-exposed cell wall lipids has been correlated with the formation of cords. The objective of this work was to study the roles of the surface-exposed cell wall lipids and mycolic acids in the formation of cords in M. abscessus. A comparative study of the pattern and structure of mycolic acids was performed on R (cording) and S (non-cording) morphotypes derived from the same parent strains, and no differences were observed between morphotypes. Furthermore, cords formed by R morphotypes were disrupted with petroleum ether (PE), and the extracted lipids were analyzed by thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry. Substantial amounts of trehalose polyphleates (TPP) were recovered as major lipids from PE extracts, and images obtained by transmission electron microscopy suggested that these lipids are localized to the external surfaces of cords and R bacilli. The structure of M. abscessus TPP was revealed to be similar to those previously described in Mycobacterium smegmatis. Although the exact role of TPP is unknown, our results demonstrated that TPP are not toxic by themselves and have a function in the formation of clumps and cords in M. abscessus, thus playing an important role in the pathogenesis of this species.