Phenolic Glycolipid Research Articles

The loss of the PDIM/PGL virulence lipids causes differential secretion of ESX-1 substrates in Mycobacterium marinum.

The mycobacterial cell envelope is a major virulence determinant in pathogenic mycobacteria. Specific outer lipids play roles in pathogenesis, modulating the immune system and promoting the secretion of virulence factors. ESX-1 (ESAT-6 system-1) is a conserved protein secretion system required for mycobacterial pathogenesis. Previous studies revealed that mycobacterial strains lacking the outer lipid PDIM have impaired ESX-1 function during laboratory growth and infection. The mechanisms underlying changes in ESX-1 function are unknown. We used a proteo-genetic approach to measure phthiocerol dimycocerosate (PDIM)- and phenolic glycolipid (PGL)-dependent protein secretion in M. marinum, a non-tubercular mycobacterial pathogen that causes tuberculosis-like disease in ectothermic animals. Importantly, M. marinum is a well-established model for mycobacterial pathogenesis. Our findings showed that M. marinum strains without PDIM and PGL showed specific, significant reductions in protein secretion compared to the WT and complemented strains. We recently established a hierarchy for the secretion of ESX-1 substrates in four (I-IV) groups. Loss of PDIM differentially impacted secretion of Group III and IV ESX-1 substrates, which are likely the effectors of pathogenesis. Our data suggest that the altered secretion of specific ESX-1 substrates is responsible for the observed ESX-1-related effects in PDIM-deficient strains.IMPORTANCEMycobacterium tuberculosis, the cause of human tuberculosis, killed an estimated 1.3 million people in 2022. Non-tubercular mycobacterial species cause acute and chronic human infections. Understanding how these bacteria cause disease is critical. Lipids in the cell envelope are essential for mycobacteria to interact with the host and promote disease. Strains lacking outer lipids are attenuated for infection, but the reasons are unclear. Our research aims to identify a mechanism for attenuation of mycobacterial strains without the PDIM and PGL outer lipids in M. marinum. These findings will enhance our understanding of the importance of lipids in pathogenesis and how these lipids contribute to other established virulence mechanisms.

Bioorthogonal Metabolic Labeling of the Virulence Factor Phenolic Glycolipid in Mycobacteria.

Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid that varies among clinical Mycobacterium tuberculosis strains. PGL is also found in Mycobacterium marinum, where it promotes infection of zebrafish through effects on the innate immune system. Given the important role this lipid plays in the host-pathogen relationship, tools for profiling its abundance, spatial distribution, and dynamics are needed. Here, we report a strategy for imaging PGL in live mycobacteria using bioorthogonal metabolic labeling. We functionalized the PGL precursor p-hydroxybenzoic acid (pHB) with an azide group (3-azido pHB). When fed to mycobacteria, 3-azido pHB was incorporated into the cell surface, which could then be visualized via the bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido pHB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing M. marinum and M. tuberculosis strains. Finally, we applied this metabolic labeling strategy to study the dynamics of PGL within the mycobacterial membrane. This new tool enables visualization of PGL that may facilitate studies of mycobacterial pathogenesis.

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.

The spectrum of central nervous system manifestations in leprosy: a systematic review of published case reports and case series.

This systematic review aimed to investigate central nervous system (CNS) involvement in leprosy by analysing multiple cohort studies, individual cases and case series. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed. PubMed, Scopus and Embase databases were searched up to 8 July 2023, using a predefined search strategy. Inclusion criteria included patients diagnosed with leprosy with evidence of CNS involvement. The quality of the included cases was evaluated using the Joanna Briggs Institute checklist. A total of 34 records were identified, including 18 cohort studies and 16 reports describing 27 isolated cases. Autopsies revealed macroscopic changes in the spinal cord, neurofibrillary tangles and senile plaques. Mycobacterium leprae was detected in neurons of the medulla oblongata and spinal cord using PCR and phenolic glycolipid 1 staining. Cerebrospinal fluid (CSF) analysis showed inflammatory changes, increased gamma globulins and detection of Mycobacterium leprae antigens and antibodies. In 21 patients (78%), spinal cord/brachial plexus abnormities were detected. In the majority, MRI revealed T2/fluid-attenuated inversion recovery (FLAIR) hyperintensity in the cervical cord. In patients with brainstem involvement, T2/FLAIR hyperintensity was noted in the cerebellar peduncles, facial nerve nuclei and/or other cranial nerve nuclei. Brain parenchymal involvement was noted in three patients. This systematic review provides evidence of CNS involvement in leprosy, based on autopsy findings, neuroimaging, CSF analysis and neurophysiological studies.

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.

Field-friendly anti-PGL-I serosurvey in children to monitor Mycobacterium leprae transmission in Bihar, India.

It has been amply described that levels of IgM antibodies against Mycobacterium leprae (M. leprae) phenolic glycolipid I (PGL-I) correlate strongly with the bacterial load in an infected individual. These findings have generated the concept of using seropositivity for antibodies against M. leprae PGL-I as an indicator of the proportion of the population that has been infected. Although anti-PGL-I IgM levels provide information on whether an individual has ever been infected, their presence cannot discriminate between recent and past infections. Since infection in (young) children by definition indicates recent transmission, we piloted the feasibility of assessment of anti-PGL-I IgM seroprevalence among children in a leprosy endemic area in India as a proxy for recent M. leprae transmission. A serosurvey for anti-PGL-I IgM antibodies among children in highly leprosy endemic villages in Bihar, India, was performed, applying the quantitative anti-PGL-I UCP-LFA cassette combined with low-invasive, small-volume fingerstick blood (FSB). Local staff obtained FSB of 1,857 children (age 3-11 years) living in 12 leprosy endemic villages in Bihar; of these, 215 children (11.58%) were seropositive for anti-PGL-I IgM. The anti-PGL-I seroprevalence level of 11.58% among children corresponds with the seroprevalence levels described in studies in other leprosy endemic areas over the past decades where no prophylactic interventions have taken place. The anti-PGL-I UCP-LFA was found to be a low-complexity tool that could be practically combined with serosurveys and was well-accepted by both healthcare staff and the population. On route to leprosy elimination, quantitative anti-PGL-I serology in young children holds promise as a strategy to monitor recent M. leprae transmission in an area.

Levels of TGF- Serum Positively Correlated with Levels of IgM Anti PGL-1 In Household Contacts of Multibacillary Leprosy Patients

Background Leprosy is a chronic progressive infectious disease. Phenolic glycolipid-1 (PGL-1) is an antigen of Mycobacterium leprae, which can trigger the host’s antibody response. Transforming growth factorβ (TGF-β) plays an immunosuppressive role when the host is exposed to PGL-1 antigen or other M. leprae antigens. This study aims to determine the correlation of TGF-β levels with IgM anti-PGL-1 levels in patients with multibacillary leprosy household contacts. Methods Observational analytical study with a cross-sectional approach. The study subjects consisted of 48 household contact subjects and 24 non-household contact subjects aged 15-65 years old who were selected by consecutive sampling based on inclusion and exclusion criteria. 3 mL of venous blood samples were taken and then examined for IgM anti-PGL-1 and TGF-β with the ELISA kit. Data analysis was carried out using SPSS version 23, and a p-value <0.05 was significant. Results The mean level of IgM anti-PGL-1 in the household contact group was 685.46 ± 290.79 u/mL, while in the non-household contact group was 345.50 ± 206.58 u/mL. The mean TGF-β level in household contact groups was 256.69 ± 127.41 pg/mL, while in the non-household contact group was 144.85 ± 36.73 pg/mL (p<0.001). This study found a moderate positive relationship (r=0.450, p<0.001) between levels of TGF-β and IgM anti-PGL-1 household contacts and non-household contacts group. Conclusions The mean level of IgM anti-PGL-1 and TGF-β in household contacts is higher than in non-household contacts, with a significant difference. There is a moderate positive significant relationship between levels of TGF-β and IgM anti-PGL-1 household contacts and non-household contacts group.

Measurements and Accuracy of IgM and IgG Anti Phenolic Glycolipid-1 Levels in Blood Serum for Early Detection Mycobacterium leprae by using Enzyme-Linked Immunosorbent Assay (ELISA): A Reality of a Laboratory

Indonesia was the third most recent case of leprosy globally in 2020 with 11,173 people, after India and Brazil. Most of the leprosy manifestations are asymptomatic. This is possibly as subclinical leprosy which individuals without leprosy symptoms but have leprosy specific antibodies high levels, so it has the potential to become a transmission and disability. Therefore, an ELISA test need for early detection in preventing leprosy transmission. This study aims to measure IgM and IgG antibody levels in leprosy patients and assess the accuracy of the measurement results. This research is a cross-sectional study. Five patients' blood samples have analyzed for IgM and IgG anti-PGL-1 antibody levels by ELISA. Accuracy interpretation of this measurement based on the %CV. Antibody levels were classified based on the cut-off <605 u/ml as IgM seronegative or <630 u/ml as IgG seronegative, 605–1000 u/ml as low seropositive IgM or 630-1000 u/ml as low seropositive IgG, and >1000 u/ml as high seropositive IgM and IgG. Among five patients examined, 40% had high seropositive leprosy with anti-PGL-1 IgM and IgG antibody titers>1000 u/ml, and 60% of patients had seronegative leprosy. Accuracy in this ELISA test shows high accuracy with %CV <10% in the conversion of OD to antibody titer levels. IgM and IgG Anti PGL-1 antibody titers by ELISA as one of the parameters in identifying patients at higher risk of leprosy. A significant portion of patients with high seropositive leprosy with high accuracy.

Mycobacterium leprae's Infective Capacity Is Associated with Activation of Genes Involved in PGL-I Biosynthesis in a Schwann Cells Infection Model.

Peripheral nerves and Schwann cells (SCs) are privileged and protected sites for initial colonization, survival, and spread of leprosy bacillus. Mycobacterium leprae strains that survive multidrug therapy show a metabolic inactivation that subsequently induces the recurrence of typical clinical manifestations of leprosy. Furthermore, the role of the cell wall phenolic glycolipid I (PGL-I) in the M. leprae internalization in SCs and the pathogenicity of M. leprae have been extensively known. This study assessed the infectivity in SCs of recurrent and non-recurrent M. leprae and their possible correlation with the genes involved in the PGL-I biosynthesis. The initial infectivity of non-recurrent strains in SCs was greater (27%) than a recurrent strain (6.5%). In addition, as the trials progressed, the infectivity of the recurrent and non-recurrent strains increased 2.5- and 2.0-fold, respectively; however, the maximum infectivity was displayed by non-recurrent strains at 12 days post-infection. On the other hand, qRT-PCR experiments showed that the transcription of key genes involved in PGL-I biosynthesis in non-recurrent strains was higher and faster (Day 3) than observed in the recurrent strain (Day 7). Thus, the results indicate that the capacity of PGL-I production is diminished in the recurrent strain, possibly affecting the infective capacity of these strains previously subjected to multidrug therapy. The present work opens the need to address more extensive and in-depth studies of the analysis of markers in the clinical isolates that indicate a possible future recurrence.

Positive Correlation of Plasma Interleukin-6 Levels with Immunoglobulin M (IgM) Anti Phenolic Glycolipid-1 (PGL-1) Levels in Household Contacts of Multibacillary Leprosy Patients

Background/aim: Leprosy is a chronic infectious disease that can be cured but causes high morbidity because it causes disability. Leprosy is caused by the bacterium Mycobacterium leprae, an obligate intracellular virulence factor in the form of phenolic glycolipid-1 (PGL-1), a capsule component in M. leprae. This study aims for known Increased IL-6 correlation with IgM anti PGL-1 level in household contacts of multibacillary leprosy patient. Methods: Analytical observational study with a cross-sectional approach. The sample consisted of 42 household contact persons and 22 non-contact samples aged 18-65 years who were selected through consecutive sampling according to the inclusion and exclusion criteria. Samples of 3 millilitres of venous blood were taken, which would then be examined for PGL-1 and IL-6 with the ELISA kit. Data analysis with SPSS version 23 and p-value <0.05 was significant Results: The average anti-PGL-1 IgM level in the household contact group was 636.76±288.45 u/ml, while in the non-contact group, it was 211.00±237.43 u/ml. The average IL-6 level in the household contact group was 20.28 ± 12.54 pg/ml, while in the non-contact group, it was 8.29 ± 2.88 pg/ml. There was a significant difference in the levels of IL-6 and anti-PGL-1 IgM in the household contact and non-contact groups (p<0.001). This study found a strong positive relationship (r=0.620, p<0.001) between IL-6 levels and anti-PGL-1 IgM levels. Conclusion: IgM anti-PGL-1 and IL-6 levels in household contacts were higher than in non-contacts, with a significant difference. There is a significant positive relationship between IL-6 and anti-PGL-1 IgM levels. Keywords: leprosy, contacts, IgM anti-PGL-1, IL-6

Issue Information

Front cover Axonopathy is considered a hallmark of leprosy neuropathy, a chronic infectious disease caused by Mycobacterium leprae infection in Schwann cells. Leprosy neuropathy is associated with irreversible motor and sensory loss seen in infected patients. In the present work, immortalized human Schwann cell line ST8814 were infected with M.leprae. The neurotoxicity of infected Schwann cell-conditioned medium (SCCM) was then evaluated in a human neuroblastoma cell lineage and mouse neurons. ST8814 Schwann cells exposed to M. leprae affected neuronal viability by deviating glial 14C-labeled lactate, important fuel of neuronal central metabolism, to de novo lipid synthesis. We observed that transgenic M. bovis (BCG) cells expressing the M. leprae phenolic glycolipid-1 (PGL-1), a specific cell wall antigen proposed to mediate bacterial-Schwann cell interaction - (BCG-PGL-1) - was able to induce a phenotype similar to M. leprae, unlike the wild type BCG strain. This Schwann cell neurotoxic phenotype, induced by M. leprae PGL-1, occurs through the protein kinase B (Akt) pathway. The pharmacological inhibition of Akt by triciribine significantly reduced free fatty acid content in the SCCM from M. leprae- and BCG-PGL-1-infected Schwann cells and, hence, prevented neuronal death. Overall, these findings provide novel evidence that both M. leprae and PGL-1 induce a toxic Schwann cell phenotype, by modifying the host lipid metabolism, resulting in profound implications for neuronal loss as a basis of leprosy neuropathy. Image content Sensory neurons dissociated from mouse dorsal root ganglion, visualized by immunofluorescence using anti-ß-III tubulin evidencing neurotoxicity of M. leprae-infected Schwann cells conditioned medium over arborization. Read the full article ‘Phenolic glycolipid-1 of Mycobacterium leprae is involved in human Schwann cell line ST8814 neurotoxic phenotype’ by K. do Carmo de Vasconcelos Girardi, B. S. Mietto, K. dos Anjos Lima, G. C. Atella, D. S. da Silva, A. M. R. Pereira, P. S. Rosa, F. A. Lara. 2023, vol.164 (2), pp. 158–171) on doi:10.1111/jnc.15722

Immunological hyporesponsiveness in tuberculosis: The role of mycobacterial glycolipids.

Glycolipids constitute a major part of the cell envelope of Mycobacterium tuberculosis (Mtb). They are potent immunomodulatory molecules recognized by several immune receptors like pattern recognition receptors such as TLR2, DC-SIGN and Dectin-2 on antigen-presenting cells and by T cell receptors on T lymphocytes. The Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic relatives, phosphatidylinositol mannosides (PIMs) and lipomannan (LM), as well as other Mtb glycolipids, such as phenolic glycolipids and sulfoglycolipids have the ability to modulate the immune response, stimulating or inhibiting a pro-inflammatory response. We explore here the downmodulating effect of Mtb glycolipids. A great proportion of the studies used in vitro approaches although in vivo infection with Mtb might also lead to a dampening of myeloid cell and T cell responses to Mtb glycolipids. This dampened response has been explored ex vivo with immune cells from peripheral blood from Mtb-infected individuals and in mouse models of infection. In addition to the dampening of the immune response caused by Mtb glycolipids, we discuss the hyporesponse to Mtb glycolipids caused by prolonged Mtb infection and/or exposure to Mtb antigens. Hyporesponse to LAM has been observed in myeloid cells from individuals with active and latent tuberculosis (TB). For some myeloid subsets, this effect is stronger in latent versus active TB. Since the immune response in individuals with latent TB represents a more protective profile compared to the one in patients with active TB, this suggests that downmodulation of myeloid cell functions by Mtb glycolipids may be beneficial for the host and protect against active TB disease. The mechanisms of this downmodulation, including tolerance through epigenetic modifications, are only partly explored.

Phenolic glycolipid-1 of Mycobacterium leprae is involved in human Schwann cell line ST8814 neurotoxic phenotype.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae infection in Schwann cells. Axonopathy is considered a hallmark of leprosy neuropathy and is associated with the irreversible motor and sensory loss seen in infected patients. Although M. leprae is recognized to provoke Schwann cell dedifferentiation, the mechanisms involved in the contribution of this phenomenon to neural damage remain unclear. In the present work, we used live M. leprae to infect the immortalized human Schwann cell line ST8814. The neurotoxicity of infected Schwann cell-conditioned medium (SCCM) was then evaluated in a human neuroblastoma cell lineage and mouse neurons. ST8814 Schwann cells exposed to M. leprae affected neuronal viability by deviating glial 14 C-labeled lactate, important fuel of neuronal central metabolism, to de novo lipid synthesis. The phenolic glycolipid-1 (PGL-1) is a specific M. leprae cell wall antigen proposed to mediate bacterial-Schwann cell interaction. Therefore, we assessed the role of the PGL-1 on Schwann cell phenotype by using transgenic M. bovis (BCG)-expressing the M. leprae PGL-1. We observed that BCG-PGL-1 was able to induce a phenotype similar to M. leprae, unlike the wild-type BCG strain. We next demonstrated that this Schwann cell neurotoxic phenotype, induced by M. leprae PGL-1, occurs through the protein kinase B (Akt) pathway. Interestingly, the pharmacological inhibition of Akt by triciribine significantly reduced free fatty acid content in the SCCM from M. leprae- and BCG-PGL-1-infected Schwann cells and, hence, preventing neuronal death. Overall, these findings provide novel evidence that both M. leprae and PGL-1, induce a toxic Schwann cell phenotype, by modifying the host lipid metabolism, resulting in profound implications for neuronal loss. We consider this metabolic rewiring a new molecular mechanism to be the basis of leprosy neuropathy.

ELISA Test Based on the Phenolic Glycolipid-I (PGL-I) of Mycobacterium leprae: A Reality of a Laboratory from a Non-Endemic Country

Background: Leprosy is a neglected tropical disease caused by Mycobacterium leprae, leading to disabilities if untreated. The ELISA based on phenolic glycolipid I (PGL-I), or its synthetic version ND-O-BSA, is almost universally positive in multibacillary leprosy and thus extensively used in endemic countries. Household contacts with a positive antibody titer have ~6-fold higher probability to develop the disease than those with a negative titer. Thus, the aim of the study was to evaluate the performance of this ELISA in the setting of a non-endemic country. Methods: We calculate the cut-off using optimized O.D. thresholds, generated by receiver operating characteristics (ROC) curve analysis, testing 39 well-characterized sera obtained from lepromatous leprosy patients with strongly positive ND-O-BSAELISA titer and 39 sera from healthy non-endemic patients never exposed to M. leprae or M. tuberculosis. Indeed, we tested a second set of sera from suspected or confirmed leprosy or household contacts (SLALT group, n=50), and patients with tuberculosis (control group, n=40). Results: We detected 56.4% of SLALT and 22.5% of tuberculosis as positive, consistent with the literature. Conclusion: The ELISA based on ND-O-BSA may thus be considered a good option to be used in a non-endemic area as a screening tool in at risk population usually coming to our center.

Laboratory perspectives for Leprosy: Diagnostic, prognostic and predictive tools.

The diagnosis of leprosy poses several challenges. The bacillary load, serology, and tissue response are determined by the host immune status, which make individual tests unsuitable across the spectrum. The sensitivity of tests for identifying paucibacillary cases remains limited, on the other hand, many tests lack specificity in differentiating contacts from diseased cases. Nonetheless, a plethora of laboratory tests have been added to the armamentarium of the clinicians dealing with leprosy. In the current review, we critically analyze the tests available for diagnosis, prognostication, and prediction of treatment response in leprosy. We discuss in brief the conventional tests available and detail the newer serologic and molecular tests added over the past few years with an attempt to suggest the pros and cons of each, and the tests best fit for each clinical scenario. Slit skin smears and skin or nerve biopsies are primarily performed to exclude clinical mimics, confirm a diagnosis, and immunologically subtype the case. Antibody titres of phenolic glycolipid-1 and its synthetic variants can be measured in serum and saliva and provide noninvasive means to detect leprosy with good specificity. Conventional, quantitative, real-time, and other variants of PCR can detect M. leprae DNA and have been used to effect in blood, tissue, and urine samples. T helper I and II cytokine signatures can be used to differentiate the subtypes of leprosy. Newer machine learning algorithms use combinations of these tests to predict the development of leprosy in contacts. Tests to detect treatment response, antimicrobial drug resistance, and predict the onset of reactions in leprosy can be used to advantage. We compare the characteristics of these tests and suggest an algorithm for leprosy diagnosis optimally utilizing them in various clinical settings.

On the molecular mechanism of nonspecific antimicrobial action of protonated diallylammonium polymers on mycobacterial cells

The protonated diallylammonium polymers (PDAA) are known to possess a high antimicrobial activity relative to a broad scope of pathogens, unlike their quaternary analog poly(N, N-diallyl-N, N-dimethylammonium chloride) (PDADMAC). Moreover, PDAA exhibits nonspecific antimicrobial activity relative to Mycobacterium tuberculosis, in contrast to known quaternary polymeric and low-molecular-weight biocides. The present paper is devoted to studying, using several physicochemical methods, the different facets of the interaction of secondary poly (diallylammonium trifluoroacetate) (PDAATFA) with Mycobacterium smegmatis – the nearest fast-growing relative of M. tuberculosis in comparison with the action of PDADMAC. We studied the interaction between polymer and phenolic glycolipids molecules (PGL) (i.e., molecules of the outer leaflet of both M. smegmatis and M. tuberculosis cell wall) by FTIR. We used phenyl-β-D-glucuronide as a model compound whose structure is close to that of the glycosylated phenolic part of the PGL molecule. It was assessed the polymer effect on transmembrane potential (TM) and permeability barrier of M. smegmatis. It was studied the biocidal activity of the polymers relative to M. smegmatis. The cytotoxic effect of PDAATFA on M. smegmatis cells was analyzed by TEM. We have proved the direct interaction of polymers with the outer membrane molecules for the first time. FTIR and the rest data show a formation of the non-covalent intermolecular hydrogen-bonded complex between protonated PDAATFA polymers and phenolic glycolipids that leads to the destruction of the mycobacterial outer membrane and, as a result, cell death. We believe that because of the structural similarity of cell wall chemistry and organization, this conclusion is proper for model organism M. smegmatis and pathogenic M. tuberculosis. Contrary to expected, the action of more hydrophobic PDADMAC does not lead to a destruction of the outer membrane but to a gradual suppression of the TM potential and cell death due to inhibition of general bioenergetic processes over the extended treatment time.

Sub-Lineage Specific Phenolic Glycolipid Patterns in the Mycobacterium tuberculosis Complex Lineage 1.

“Ancestral” Mycobacterium tuberculosis complex (MTBC) strains of Lineage 1 (L1, East African Indian) are a prominent tuberculosis (TB) cause in countries around the Indian Ocean. However, the pathobiology of L1 strains is insufficiently characterized. Here, we used whole genome sequencing (WGS) of 312 L1 strains from 43 countries to perform a characterization of the global L1 population structure and correlate this to the analysis of the synthesis of phenolic glycolipids (PGL) – known MTBC polyketide-derived virulence factors. Our results reveal the presence of eight major L1 sub-lineages, whose members have specific mutation signatures in PGL biosynthesis genes, e.g., pks15/1 or glycosyltransferases Rv2962c and/or Rv2958c. Sub-lineage specific PGL production was studied by NMR-based lipid profiling and strains with a completely abolished phenolphthiocerol dimycoserosate biosynthesis showed in average a more prominent growth in human macrophages. In conclusion, our results show a diverse population structure of L1 strains that is associated with the presence of specific PGL types. This includes the occurrence of mycoside B in one sub-lineage, representing the first description of a PGL in an M. tuberculosis lineage other than L2. Such differences may be important for the evolution of L1 strains, e.g., allowing adaption to different human populations.

Design of a specific peptide against phenolic glycolipid-1 from Mycobacterium leprae and its implications in leprosy bacilli entry.

BACKGROUND Mycobacterium leprae, the causative agent of Hansen’s disease, causes neural damage through the specific interaction between the external phenolic glycolipid-1 (PGL-1) and laminin subunit alpha-2 (LAMA2) from Schwann cells.OBJECTIVE To design a LAMA2-based peptide that targets PGL-1 from M. leprae.METHODS We retrieved the protein sequence of human LAMA2 and designed a specific peptide using the Antimicrobial Peptide Database and physicochemical parameters for antimycobacterial peptide-lipid interactions. We used the AlphaFold2 server to predict its three-dimensional structure, AUTODOCK-VINA for docking, and GROMACS programs for molecular dynamics simulations.FINDINGS We analysed 52 candidate peptides from LAMA2, and subsequent screening resulted in a single 60-mer peptide. The mapped peptide comprises four β-sheets and a random coiled region. This peptide exhibits a 45% hydrophobic ratio, in which one-third covers the same surface. Molecular dynamics simulations show that our predicted peptide is stable in aqueous solution and remains stable upon interaction with PGL-1 binding. In addition, we found that PGL-1 has a preference for one of the two faces of the predicted peptide, which could act as the preferential binding site of PGL-1.MAIN CONCLUSIONS Our LAMA2-based peptide targeting PGL-1 might have the potential to specifically block this key molecule, suggesting that the preferential region of the peptide is involved in the initial contact during the attachment of leprosy bacilli to Schwann cells.

Developments in the Synthesis of Mycobacterial Phenolic Glycolipids.

The highly lipophilic outer barrier of mycobacteria, such asM. tuberculosisandM. leprae, is key to their virulence and intrinsic antibiotic resistance. Various components of this mycomembrane interact with the host immune system but many of these interactions remain ill-understood. This review covers several chemical syntheses of one of these components, mycobacterial phenolic glycolipids (PGLs), and outlines the interaction of these PGLs with the human immune system, as established using these well-defined pure compounds.

Structure-function analysis of MmpL7-mediated lipid transport in mycobacteria

Mycobacterial membrane protein Large (MmpL7) is a Resistance-Nodulation-Division (RND) family transporter required for the export of the virulence lipid, phthiocerol dimycocerosate (PDIM), in Mycobacterium tuberculosis. Using a null mutant of the related, vaccine strain Mycobacterium bovis BCG, we show that MmpL7 is also involved in the transport of the structurally related phenolic glycolipid (PGL), which is also produced by the hypervirulent M. tuberculosis strain HN878, but absent in M. tuberculosis H37Rv. Furthermore, we generated an in silico model of M. tuberculosis MmpL7 that revealed MmpL7 as a functional outlier within the MmpL-family, missing a canonical proton-relay signature sequence, suggesting that it employs a yet-unidentified mechanism for energy coupling for transport. In addition, our analysis demonstrates that the periplasmic porter domain 2 insert (PD2-insert), which doesn't share any recognisable homology, is highly alpha-helical in nature, suggesting an organisation similar to that seen in the hopanoid PD3/4 domains. Using the M. bovis BCG mmpL7 mutant for functional complementation with mutated alleles of mmpL7, we were able to identify residues present in the transmembrane domains TM4 and TM10, and the PD2 domain insert that play a crucial role in PDIM transport, and in certain cases, biosynthesis of PDIM.