Improve L-isoleucine production in Corynebacterium glutamicum WM001 by destructing the biosynthesis of trehalose dicorynomycolate.

Trehalose dicorynomycolates are structurally important constituents of the cell envelope in Corynebacterium glutamicum. The genes treS, treY, otsA, mytA and mytB are necessary for the biosynthesis of trehalose dicorynomycolates. In this study, the effect of biosynthesis of trehalose dicorynomycolates on L-isoleucine production in C. glutamicum has been investigated by deleting the genes treS, treY, otsA, mytA, and mytB in the L-isoleucine producing C. glutamicum WM001. L-isoleucine production was slightly improved in the mutants ΔtreY, ΔotsA, and ΔtreYA, and not improved in the single deletion mutant ΔtreS , but significantly improved in the triple deletion mutant ΔtreSYA. Deletion of mytA or mytB in ΔtreSYA could further improve L-isoleucine production. However, deletion of both mytA and mytB in ΔtreSYA significantly decreased L-isoleucine production. The final L-isoleucine producing C. glutamicum WL001 was constructed by deletion of treS, treY, otsA, and mytB, insertion of lrp, and replacement of the native promoter of ilvA with the L-isoleucine sensitive promoter PbrnFE7. WL001 grew worse than the control WM001, but produced 36.1% more L-isoleucine after 72 h shake flask cultivation than WM001.

Is acidemia at birth a risk factor for functional gastrointestinal disorders?

Functional gastrointestinal disorders (FGIDs) are common in early childhood. It has been demonstrated that neonatal acidemia at delivery can lead to significant neonatal morbidity. The primary aim of this study was to evaluate the relationship between acidemia at birth and the development of FGIDs, as regurgitation, colic, and constipation, in term infants. Term newborns born at the Foggia University Hospital, Italy during the year 2020 were included in the study. As per routine clinical practice, a cord blood gas analysis on a blood sample drawn from the umbilical artery (UA) of each infant immediately after birth was performed, and Apgar score was recorded. One year after birth, each infant's parents were interviewed through a phone call to investigate development of FGIDs, feeding practices, and morbidities. During the study period, 1574 term newborns met the inclusion criteria. The prevalence of infantile colic, regurgitation, and constipation was higher in infants with low UA pH (colic 51.5% vs. 25.4%, p < 0.001; regurgitation 30.6% vs. 15.2%, p < 0.001; constipation 24.6% vs. 16.0%, p = 0.015), with infants having moderate-severe acidemia facing the highest risk for all the examined FGIDs. In binary logistic regression analyses, UA pH and perinatal antibiotic exposure proved to be independently associated with the later diagnosis of each FGID. CONCLUSION: Newborns with acidemia at birth appear to face a higher risk of FGIDs in infancy. Avoiding low cord blood pH should continue to be the goal for obstetricians, while enhanced long-term surveillance for infants who experienced birth acidemia should be required. WHAT IS KNOWN: • Cord blood gas analysis is recommended in all high-risk deliveries, and in some centers, it is performed after all deliveries. • Neonatal acidemia at birth has been linked to adverse outcomes, mainly neurological. Recently, perinatal asphyxia has been reported to increase the risk of developing necrotizing enterocolitis in term infants. WHAT IS NEW: • An association between acidemia at birth and risk of developing FGIDs such as regurgitation and colic during the first year of life had never been described so far. • An increased surveillance of infants with low UA pH at birth may be beneficial and could allow for early detection of any of the reported FGIDs.

Mycobacterial membrane protein Large 3-like-family proteins in bacteria, protozoa, fungi, plants, and animals: A bioinformatics and structural investigation.

Lipid transporters play an important role in most if not all organisms, ranging from bacteria to humans. For example, in Mycobacterium tuberculosis, the trehalose monomycolate transporter MmpL3 is involved in cell wall biosynthesis, while in humans, cholesterol transporters are involved in normal cell function as well as in disease. Here, using structural and bioinformatics information, we propose that there are proteins that also contain "MmpL3-like" (MMPL) transmembrane (TM) domains in many protozoa, including Trypanosoma cruzi, as well as in the bacterium Staphylococcus aureus, where the fatty acid transporter FarE has the same set of "active-site" residues as those found in the mycobacterial MmpL3s, and in T. cruzi. We also show that there are strong sequence and predicted structural similarities between the TM proton-translocation domain seen in the X-ray structures of mycobacterial MmpL3s and several human as well as fungal lipid transporters, leading to the proposal that there are similar proteins in apicomplexan parasites, and in plants. The animal, fungal, apicomplexan, and plant proteins have larger extra-membrane domains than are found in the bacterial MmpL3, but they have a similar TM domain architecture, with the introduction of a (catalytically essential) Phe > His residue change, and a Ser/Thr H-bond network, involved in H+ -transport. Overall, the results are of interest since they show that MMPL-family proteins are present in essentially all life forms: archaea, bacteria, protozoa, fungi, plants and animals and, where known, they are involved in "lipid" (glycolipid, phospholipid, sphingolipid, fatty acid, cholesterol, ergosterol) transport, powered by transmembrane molecular pumps having similar structures.

Structures of the mycobacterial membrane protein MmpL3 reveal its mechanism of lipid transport.

The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall.

[Pathogenesis of Secondary Pulmonary Tuberculosis and Role of Cord Factor in Secondary Infection].

The primary and secondary tuberculosis features two completely different pathogenesis.At present,the pathogenesis of primary tuberculosis has been clear,whereas that of secondary tuberculosis remains unclear.In order to decipher the mechanism of secondary infection of Mycobacterium tuberculosis and provide insights into vaccine research and drug development,this paper reviews the problems of the widely accepted mechanism of secondary infection,the new findings of the research on the mechanism,as well as the role of cord factors.

The liposome of trehalose dimycolate extracted from M. bovis BCG induces antitumor immunity via the activation of dendritic cells and CD8+ T cells.

Intravesical Bovis bacillus Calmette-Guérin (BCG) therapy is the most effective immunotherapy for bladder cancer, but it sometime causes serious side effects because of its inclusion of live bacteria. It is necessary to develop a more active but less toxic immunotherapeutic agent. Trehalose 6,6'-dimycolate (TDM), the most abundant hydrophobic glycolipid of the BCG cell wall, has been reported to show various immunostimulatory activities such as granulomagenesis and adjuvant activity. Here, we developed cationic liposomes incorporating TDM purified from Mycobacterium bovis BCG Connaught, and we investigated the antitumor effect of the cationic liposome TDM (Lip-TDM). Lip-TDM exerted an antitumor effect in bladder cancer, colon cancer, and melanoma-bearing mouse models that was comparable or even superior to that of BCG, with no body weight loss or granuloma formation. The antitumor effect of Lip-TDM disappeared in two types of mice: those with depletion of CD8+ T cells, and those with knockout of macrophage-inducible C-type lectin (Mincle) which recognize TDM. Lip-TDM treatment enhanced the maturation and migration of dendritic cells in the tumor microenvironment in a Mincle-dependent manner. Our results elucidate mechanisms that underlie Lip-TDM treatment and suggest that Lip-TDM has potential as a safe and effective treatment for various cancers.

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

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

Lactoferrin reduces mycobacterial M1-type inflammation induced with trehalose 6,6'-dimycolate and facilitates the entry of fluoroquinolone into granulomas.

Primary infection with Mycobacterium tuberculosis (Mtb) results in the formation of a densely packed granulomatous response that essentially limits the entry and efficacy of immune effector cells. Furthermore, the physical nature of the granuloma does not readily permit the entry of therapeutic agents to sites where organisms reside. The Mtb cell wall mycolic acid, trehalose 6,6'-dimycolate (TDM), is a physiologically relevant molecule for modelling macrophage-mediated events during the establishment of the tuberculosis-induced granuloma pathogenesis. At present, there are no treatments for tuberculosis that focus on modulating the host's immune responses. Previous studies showed that lactoferrin (LF), a natural iron-binding protein proven to modulate inflammation, can ameliorate the cohesiveness of granuloma. This led to a series of studies that further examined the effects of recombinant human LF (rHLF) on the histological progression of TDM-induced pathology. Treatment with rHLF demonstrated significant reduction in size and number of inflammatory foci following injections of TDM, together with reduced levels pulmonary pro-inflammatory cytokines TNF-α and IL-1ß. LF facilitated greater penetration of fluoroquinolone to the sites of pathology. Mice treated with TDM alone demonstrated exclusion of ofloxacin to regions of inflammatory response, whereas the animals treated with rHLF demonstrated increased penetration to inflammatory foci. Finally, recent findings support the hypothesis that this mycobacterial mycolic acid can specifically recruit M1-like polarized macrophages; rHLF treatment was shown to limit the level of this M1-like phenotypic recruitment, corresponding highly with decreased inflammatory response.

Pathogenesis of Secondary Pulmonary Tuberculosis and Role of Cord Factor in Secondary Infection / 中国医学科学院学报

The primary and secondary tuberculosis features two completely different pathogenesis.At present,the pathogenesis of primary tuberculosis has been clear,whereas that of secondary tuberculosis remains unclear.In order to decipher the mechanism of secondary infection of

Mycobacterial Cord Factor Reprograms the Macrophage Response to IFN-γ towards Enhanced Inflammation yet Impaired Antigen Presentation and Expression of GBP1.

Mycobacteria survive in macrophages despite triggering pattern recognition receptors and T cell-derived IFN-γ production. Mycobacterial cord factor trehalose-6,6-dimycolate (TDM) binds the C-type lectin receptor MINCLE and induces inflammatory gene expression. However, the impact of TDM on IFN-γ-induced macrophage activation is not known. In this study, we have investigated the cross-regulation of the mouse macrophage transcriptome by IFN-γ and by TDM or its synthetic analogue trehalose-6,6-dibehenate (TDB). As expected, IFN-γ induced genes involved in Ag presentation and antimicrobial defense. Transcriptional programs induced by TDM and TDB were highly similar but clearly distinct from the response to IFN-γ. The glycolipids enhanced expression of a subset of IFN-γ-induced genes associated with inflammation. In contrast, TDM/TDB exerted delayed inhibition of IFN-γ-induced genes, including pattern recognition receptors, MHC class II genes, and IFN-γ-induced GTPases, with antimicrobial function. TDM downregulated MHC class II cell surface expression and impaired T cell activation by peptide-pulsed macrophages. Inhibition of the IFN-γ-induced GTPase GBP1 occurred at the level of transcription by a partially MINCLE-dependent mechanism that may target IRF1 activity. Although activation of STAT1 was unaltered, deletion of Socs1 relieved inhibition of GBP1 expression by TDM. Nonnuclear Socs1 was sufficient for inhibition, suggesting a noncanonical, cytoplasmic mechanism. Taken together, unbiased analysis of transcriptional reprogramming revealed a significant degree of negative regulation of IFN-γ-induced Ag presentation and antimicrobial gene expression by the mycobacterial cord factor that may contribute to mycobacterial persistence.

Unveiling the biodiversity of lipid species in Corynebacteria- characterization of the uncommon lipid families in C. glutamicum and pathogen C. striatum by mass spectrometry.

Uncommon lipids in biotechnologically important Corynebacterium glutamicum and pathogen Corynebacterium striatum in genus Corynebacterium are isolated and identified by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high resolution mass measurement. We redefined several lipid structures that were previously mis-assigned or not defined, including cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), (α-d-mannopyranosyl)-(1 â†’ 4)-(α-D-glucuronyl diacyglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), acyl trehalose monomycolate (acyl-TMM). We also report the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in which many isomeric structures are present. The LIT MSn approaches afford identification of the functional group, the fatty acid substituents and their regiospecificity in the molecules, revealing the biodiversities of the lipid species in two Corynebacterium strains that have played very different and important roles in human nutrition and health.

MmpL3 inhibitors as antituberculosis drugs.

The mycobacterial membrane protein Large 3 (MmpL3) is an inner membrane protein that transports trehalose-monomycolates, precursors for trehalose-dimycolates and mycolic acids that make up essential components of the mycobacterial outer membrane. Inhibition of MmpL3 weakens the mycobacterial cell wall and ultimately results in cell death in both in vitro and in vivo infection models. This highlights the therapeutic potential of MmpL3 as a drug target. High-throughput whole-cell screening along with whole genome sequencing of resistant mutants has identified numerous classes of compounds that can be classified as MmpL3 inhibitors. In this review, we provide insights into the current development of various MmpL3 inhibitors and discuss the potential challenges in this area.

A TLR4-TRIF-dependent signaling pathway is required for protective natural tumor-reactive IgM production by B1 cells.

Metastatic cancer involving spread to the peritoneal cavity is referred to as peritoneal carcinomatosis and has a very poor prognosis. Our previous studies demonstrated a toll-like receptor 4 (TLR4) and C-type lectin receptor (CLR; Mincle/MCL) agonist pairing of monophosphoryl lipid A (MPL) and trehalose-6,6'-dicorynomycolate (TDCM) effectively inhibits peritoneal tumor growth and ascites development through a mechanism dependent upon B1a cell-produced natural IgM, complement, and phagocytes. In the current study, we investigated the requirement for TLR4 and Fc receptor common γ chain (FcRγ), required for Mincle/MCL signaling, in the MPL/TDCM-elicited response. MPL/TDCM significantly increased macrophages and Ly6Chi monocytes in the peritoneal cavity of both TLR4-/- and FcRγ-/- mice, suggesting redundancy in the signals required for monocyte/macrophage recruitment. However, B1 cell activation, antibody secreting cell differentiation, and tumor-reactive IgM production were defective in TLR4-/-, but not FcRγ-/- mice. TRIF was required for production of IgM reactive against tumor- and mucin-related antigens, but not phosphorylcholine, whereas TLR4 was required for production of both types of reactivities. Consistent with this, B1 cells lacking TLR4 or TRIF did not proliferate or differentiate into tumor-reactive IgM-producing cells in vitro and did not reconstitute MPL/TDCM-dependent protection against peritoneal carcinomatosis in CD19-/- mice. Our results indicate a TLR4/TRIF-dependent pathway is required by B1 cells for MPL/TDCM-elicited production of protective tumor-reactive natural IgM. The dependency on TRIF signaling for tumor-reactive, but not phosphorylcholine-reactive, IgM production reveals unexpected heterogeneity in TLR4-dependent regulation of natural IgM production, thereby highlighting important differences to consider when designing vaccines or therapies targeting these specificities.

Protein kinases PknA and PknB independently and coordinately regulate essential Mycobacterium tuberculosis physiologies and antimicrobial susceptibility.

The Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB are essential for growth and have been proposed as possible drug targets. We used a titratable conditional depletion system to investigate the functions of these kinases. Depletion of PknA or PknB or both kinases resulted in growth arrest, shortening of cells, and time-dependent loss of acid-fast staining with a concomitant decrease in mycolate synthesis and accumulation of trehalose monomycolate. Depletion of PknA and/or PknB resulted in markedly increased susceptibility to ß-lactam antibiotics, and to the key tuberculosis drug rifampin. Phosphoproteomic analysis showed extensive changes in protein phosphorylation in response to PknA depletion and comparatively fewer changes with PknB depletion. These results identify candidate substrates of each kinase and suggest specific and coordinate roles for PknA and PknB in regulating multiple essential physiologies. These findings support these kinases as targets for new antituberculosis drugs and provide a valuable resource for targeted investigation of mechanisms by which protein phosphorylation regulates pathways required for growth and virulence in M. tuberculosis.

Immune Recognition of Pathogen-Derived Glycolipids Through Mincle.

Mincle (macrophage inducible C-type lectin, Clec4e, Clecsf9) was originally identified as a member of the C-type lectin receptor family in 1999. Then, the function of Mincle to control antifungal immunity by binding to Candida albicans was reported in 2008. Around the same time, it was reported that Mincle recognized damaged cells and induced sterile inflammation by coupling with the ITAM-adaptor molecule FcRγ. In the following year, a breakthrough discovery reported that Mincle was an essential receptor for mycobacterial cord factor (trehalose-6,6'-dimycolate, TDM). Mincle gained increasing attention immediately after this critical finding. Although our understanding of the recognition of Mycobacteria has been advanced significantly, it was also revealed that Mincle interacts with pathogens other than Mycobacteria. In addition, endogenous ligands of Mincle were identified recently. Therefore, Mincle is now considered a danger receptor both for self and non-self ligands, so-called damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). This chapter will give an overview of the accumulated knowledge of the multi-task danger receptor Mincle from its discovery to the latest findings.

Type I IFN, Ly6C+ cells, and Phagocytes Support Suppression of Peritoneal Carcinomatosis Elicited by a TLR and CLR Agonist Combination.

Metastatic cancer involving spread to the peritoneal cavity is referred to as peritoneal carcinomatosis and has a very poor prognosis. Our previous study demonstrated a Toll-like receptor and C-type lectin receptor agonist pairing of monophosphoryl lipid A (MPL) and trehalose-6,6'-dicorynomycolate (TDCM) effectively inhibits tumor growth and ascites development following TA3-Ha and EL4 challenge through a mechanism dependent on B-1a cell-produced natural IgM and complement. In this study, we investigated additional players in the MPL/TDCM-elicited response. MPL/TDCM treatment rapidly increased type I IFN levels in the peritoneal cavity along with myeloid cell numbers, including macrophages and Ly6Chi monocytes. Type I IFN receptor (IFNAR1-/-) mice produced tumor-reactive IgM following MPL/TDCM treatment, but failed to recruit Ly6C+ monocytes and were not afforded protection during tumor challenges. Clodronate liposome depletion of phagocytic cells, as well as targeted depletion of Ly6C+ cells, also ablated MPL/TDCM-induced protection. Cytotoxic mediators known to be produced by these cells were required for effects. TNFα was required for effective TA3-Ha killing and nitric oxide was required for EL4 killing. Collectively, these data reveal a model whereby MPL/TDCM-elicited antitumor effects strongly depend on innate cell responses, with B-1a cell-produced tumor-reactive IgM and complement pairing with myeloid cell-produced cytotoxic mediators to effectively eradicate tumors in the peritoneal cavity.

Mycobacterial Trehalose 6,6'-Dimycolate-Induced M1-Type Inflammation.

Murine models of Mycobacterium tuberculosis (Mtb) infection demonstrate progression of M1-like (proinflammatory) and M2-like (anti-inflammatory) macrophage morphology following primary granuloma formation. The Mtb cell wall cording factor, trehalose 6,6'-dimycolate (TDM), is a physiologically relevant and useful molecule for modeling early macrophage-mediated events during establishment of the tuberculosis-induced granuloma pathogenesis. Here, it is shown that TDM is a major driver of the early M1-like macrophage response as seen during initiation of the granulomas of primary pathology. Proinflammatory cytokines tumor necrosis factor-α, IL-1ß, IL-6, and IL-12p40 are produced in lung tissue after administration of TDM to mice. Furthermore, CD11b+CD45+ macrophages with a high surface expression of the M1-like markers CD38 and CD86 were found present in regions of pathology in lungs of mice at 7 days post-TDM introduction. Conversely, only low phenotypic marker expression of M2-like markers CD206 and EGR-2 were present on macrophages. These findings suggest that TDM plays a role in establishment of the M1-like shift in the microenvironment during primary tuberculosis.

The crystal structure of the mycobacterial trehalose monomycolate transport factor A, TtfA, reveals an atypical fold.

Trehalose monomycolate (TMM) represents an essential element of the mycobacterial envelope. While synthesized in the cytoplasm, TMM is transported across the inner membrane by MmpL3 but, little is known regarding the MmpL3 partners involved in this process. Recently, the TMM transport factor A (TtfA) was found to form a complex with MmpL3 and to participate in TMM transport, although its biological role remains to be established. Herein, we report the crystal structure of the Mycobacterium smegmatis TtfA core domain. The phylogenetic distribution of TtfA homologues in non-mycolate containing bacteria suggests that TtfA may exert additional functions.

A piperidinol-containing molecule is active against Mycobacterium tuberculosis by inhibiting the mycolic acid flippase activity of MmpL3.

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc26230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3, encoding an inner membrane-associated mycolic acid flippase in M. tuberculosis Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.

Identification of New MmpL3 Inhibitors by Untargeted and Targeted Mutant Screens Defines MmpL3 Domains with Differential Resistance.

The Mycobacterium tuberculosis mycolate flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole-genome sequencing of isolated resistant mutants. Here, we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel M. tuberculosis growth inhibitors for differential growth inhibition of wild-type M. tuberculosis compared to the growth of a pool of 24 unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular M. tuberculosisM. tuberculosis cells treated with these compounds were shown to accumulate trehalose monomycolates, have reduced levels of trehalose dimycolate, and displace an MmpL3-specific probe, supporting MmpL3 as the target. The inhibitors were mycobacterium specific, with several also showing activity against the nontuberculous mycobacterial species M. abscessus Cluster analysis of cross-resistance profiles generated by dose-response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance-conferring substitutions to the MmpL3 crystal structure revealed clade-specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small-molecule inhibitors.