Mechanopathology of biofilm-like Mycobacterium tuberculosis cords.

Mycobacterium tuberculosis (Mtb) cultured axenically without detergent forms biofilm-like cords, a clinical identifier of virulence. In lung-on-chip (LoC) and mouse models, cords in alveolar cells contribute to suppression of innate immune signaling via nuclear compression. Thereafter, extracellular cords cause contact-dependent phagocyte death but grow intercellularly between epithelial cells. The absence of these mechanopathological mechanisms explains the greater proportion of alveolar lesions with increased immune infiltration and dissemination defects in cording-deficient Mtb infections. Compression of Mtb lipid monolayers induces a phase transition that enables mechanical energy storage. Agent-based simulations demonstrate that the increased energy storage capacity is sufficient for the formation of cords that maintain structural integrity despite mechanical perturbation. Bacteria in cords remain translationally active despite antibiotic exposure and regrow rapidly upon cessation of treatment. This study provides a conceptual framework for the biophysics and function in tuberculosis infection and therapy of cord architectures independent of mechanisms ascribed to single bacteria.

Targeting mycobacterial membranes and membrane proteins: Progress and limitations.

Among the various bacterial infections, tuberculosis continues to hold center stage. Its causative agent, Mycobacterium tuberculosis, possesses robust defense mechanisms against most front-line antibiotic drugs and host responses due to their complex cell membranes with unique lipid molecules. It is now well-established that bacteria change their membrane composition to optimize their environment to survive and elude drug action. Thus targeting membrane or membrane components is a promising avenue for exploiting the chemical space focussed on developing novel membrane-centric anti-bacterial small molecules. These approaches are more effective, non-toxic, and can attenuate resistance phenotype. We present the relevance of targeting the mycobacterial membrane as a practical therapeutic approach. The review highlights the direct and indirect targeting of membrane structure and function. Direct membrane targeting agents cause perturbation in the membrane potential and can cause leakage of the cytoplasmic contents. In contrast, indirect membrane targeting agents disrupt the function of membrane-associated proteins involved in cell wall biosynthesis or energy production. We discuss the chronological chemical improvements in various scaffolds targeting specific membrane-associated protein targets, their clinical evaluation, and up-to-date account of their ''mechanisms of action, potency, selectivity'' and limitations. The sources of anti-TB drugs/inhibitors discussed in this work have emerged from target-based identification, cell-based phenotypic screening, drug repurposing, and natural products. We believe this review will inspire the exploration of uncharted chemical space for informing the development of new scaffolds that can inhibit novel mycobacterial membrane targets.

Cg1246, a new player in mycolic acid biosynthesis in Corynebacterium glutamicum.

Mycolic acids are key components of the complex cell envelope of Corynebacteriales. These fatty acids, conjugated to trehalose or to arabinogalactan form the backbone of the mycomembrane. While mycolic acids are essential to the survival of some species, such as Mycobacterium tuberculosis, their absence is not lethal for Corynebacterium glutamicum, which has been extensively used as a model to depict their biosynthesis. Mycolic acids are first synthesized on the cytoplasmic side of the inner membrane and transferred onto trehalose to give trehalose monomycolate (TMM). TMM is subsequently transported to the periplasm by dedicated transporters and used by mycoloyltransferase enzymes to synthesize all the other mycolate-containing compounds. Using a random transposition mutagenesis, we recently identified a new uncharacterized protein (Cg1246) involved in mycolic acid metabolism. Cg1246 belongs to the DUF402 protein family that contains some previously characterized nucleoside phosphatases. In this study, we performed a functional and structural characterization of Cg1246. We showed that absence of the protein led to a significant reduction in the pool of TMM in C. glutamicum, resulting in a decrease in all other mycolate-containing compounds. We found that, in vitro, Cg1246 has phosphatase activity on organic pyrophosphate substrates but is most likely not a nucleoside phosphatase. Using a computational approach, we identified important residues for phosphatase activity and constructed the corresponding variants in C. glutamicum. Surprisingly complementation with these non-functional proteins fully restored the defect in TMM of the Δcg1246 mutant strain, suggesting that in vivo, the phosphatase activity is not involved in mycolic acid biosynthesis.

The role of corynomycolic acids in Corynebacterium-host interaction.

Within the Actinobacteria, the genera Corynebacterium, Mycobacterium, Nocardia and Rhodococcus form the so-called CMNR group, also designated as mycolic acid-containing actinomycetes. Almost all members of this group are characterized by a mycolic acid layer, the mycomembrane, which covers the cell wall and is responsible for a high resistance of these bacteria against chemical and antibiotic stress. Furthermore, components of the mycomembrane are crucial for the interaction of bacteria with host cells. This review summarizes the current knowledge of mycolic acid synthesis and interaction with components of the immune system for the genus Corynebacterium with an emphasis on the pathogenic species Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis and Corynebacterium ulcerans as well as the biotechnology workhorse Corynebacterium glutamicum.

Physiological roles of sigma factor SigD in Corynebacterium glutamicum.

BACKGROUND: Sigma factors are one of the components of RNA polymerase holoenzymes, and an essential factor of transcription initiation in bacteria. Corynebacterium glutamicum possesses seven genes coding for sigma factors, most of which have been studied to some detail; however, the role of SigD in transcriptional regulation in C. glutamicum has been mostly unknown. RESULTS: In this work, pleiotropic effects of sigD overexpression at the level of phenotype, transcripts, proteins and metabolites were investigated. Overexpression of sigD decreased the growth rate of C. glutamicum cultures, and induced several physiological effects such as reduced culture foaming, turbid supernatant and cell aggregation. Upon overexpression of sigD, the level of Cmt1 (corynomycolyl transferase) in the supernatant was notably enhanced, and carbohydrate-containing compounds were excreted to the supernatant. The real-time PCR analysis revealed that sigD overexpression increased the expression of genes related to corynomycolic acid synthesis (fadD2, pks), genes encoding corynomycolyl transferases (cop1, cmt1, cmt2, cmt3), L, D-transpeptidase (lppS), a subunit of the major cell wall channel (porH), and the envelope lipid regulation factor (elrF). Furthermore, overexpression of sigD resulted in trehalose dicorynomycolate accumulation in the cell envelope. CONCLUSIONS: This study demonstrated that SigD regulates the synthesis of corynomycolate and related compounds, and expanded the knowledge of regulatory functions of sigma factors in C. glutamicum.

Mycoloyltransferases: A large and major family of enzymes shaping the cell envelope of Corynebacteriales.

Mycobacterium and Corynebacterium are important genera of the Corynebacteriales order, the members of which are characterized by an atypical diderm cell envelope. Indeed the cytoplasmic membrane of these bacteria is surrounded by a thick mycolic acid-arabinogalactan-peptidoglycan (mAGP) covalent polymer. The mycolic acid-containing part of this complex associates with other lipids (mainly trehalose monomycolate (TMM) and trehalose dimycolate (TDM)) to form an outer membrane. The metabolism of mycolates in the cell envelope is governed by esterases called mycoloyltransferases that catalyze the transfer of mycoloyl chains from TMM to another TMM molecule or to other acceptors such as the terminal arabinoses of arabinogalactan or specific polypeptides. In this review we present an overview of this family of Corynebacteriales enzymes, starting with their expression, localization, structure and activity to finally discuss their putative functions in the cell. In addition, we show that Corynebacteriales possess multiple mycoloyltransferases encoding genes in their genome. The reason for this multiplicity is not known, as their function in mycolates biogenesis appear to be only partially redundant. It is thus possible that, in some species living in specific environments, some mycoloyltransferases have evolved to gain some new functions. In any case, the few characterized mycoloyltransferases are very important for the bacterial physiology and are also involved in adaptation in the host where they constitute major secreted antigens. Although not discussed in this review, all these functions make them interesting targets for the discovery of new antibiotics and promising vaccines candidates. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Proteomic Analysis of the Mycobacterium tuberculosis Outer Membrane for Potential Implications in Uptake of Small Molecules.

Increased resistance to current antimycobacterial agents and a potential bias toward relatively hydrophobic chemical entities highlight an urgent need to understand how current anti-TB drugs enter the tubercle bacilli. While inner membrane proteins are well-studied, how small molecules cross the impenetrable outer membrane remains unknown. Here, we employed mass spectrometry-based proteomics to show that octyl-ß-d-glucopyranoside selectively extracts the outer membrane proteins of Mycobacterium tuberculosis. Differentially expressed proteins between nutrient-replete and nutrient-depleted conditions were enriched to identify proteins involved in nutrient uptake. We demonstrate cell surface localization of seven new proteins using immunofluorescence and show that overexpression of the proteins LpqY and ProX leads to hypersensitivity toward streptomycin, while overexpression of SubI, SpmT, and Rv2041 exhibited higher membrane permeability, assessed through an EtBr accumulation assay. Further, proton NMR metabolomics suggests the role of six outer membrane proteins in glycerol uptake. This study identifies several outer membrane proteins that are involved in the permeation of small hydrophilic molecules and are potential targets for enhancing the uptake and efficacy of anti-TB drugs.

CdbC: a disulfide bond isomerase involved in the refolding of mycoloyltransferases in Corynebacterium glutamicum cells exposed to oxidative conditions.

In Corynebacterium glutamicum cells, cdbC, which encodes a protein containing the CysXXCys motif, is regulated by the global redox-responsive regulator OsnR. In this study, we assessed the role of the periplasmic protein CdbC in disulfide bond formation and its involvement in mycomembrane biosynthesis. Purified CdbC efficiently refolded scrambled RNaseA, exhibiting prominent disulfide bond isomerase activity. The transcription of cdbC was decreased in cells grown in the presence of the reductant dithiothreitol (DTT). Moreover, unlike wild-type and cdbC-deleted cells, cdbC-overexpressing (P180-cdbC) cells grown in the presence of DTT exhibited retarded growth, abnormal cell morphology, increased cell surface hydrophobicity and altered mycolic acid composition. P180-cdbC cells cultured in a reducing environment accumulated trehalose monocorynomycolate, indicating mycomembrane deformation. Similarly, a two-hybrid analysis demonstrated the interaction of CdbC with the mycoloyltransferases MytA and MytB. Collectively, our findings suggest that CdbC, a periplasmic disulfide bond isomerase, refolds misfolded MytA and MytB and thereby assists in mycomembrane biosynthesis in cells exposed to oxidative conditions.

Targeting intracellular nontuberculous mycobacteria and M. tuberculosis with a bactericidal enzymatic cocktail.

To address intracellular mycobacterial infections, we developed a cocktail of four enzymes that catalytically attack three layers of the mycobacterial envelope. This cocktail is delivered to macrophages, through a targeted liposome presented here as ENTX_001. Endolytix Cocktail 1 (EC1) leverages mycobacteriophage lysin enzymes LysA and LysB, while also including α-amylase and isoamylase for degradation of the mycobacterial envelope from outside of the cell. The LysA family of proteins from mycobacteriophages has been shown to cleave the peptidoglycan layer, whereas LysB is an esterase that hydrolyzes the linkage between arabinogalactan and mycolic acids of the mycomembrane. The challenge of gaining access to the substrates of LysA and LysB provided exogenously was addressed by adding amylase enzymes that degrade the extracellular capsule shown to be present in Mycobacterium tuberculosis. This enzybiotic approach avoids antimicrobial resistance, specific receptor-mediated binding, and intracellular DNA surveillance pathways that limit many bacteriophage applications. We show this cocktail of enzymes is bactericidal in vitro against both rapid- and slow-growing nontuberculous mycobacteria (NTM) as well as M. tuberculosis strains. The EC1 cocktail shows superior killing activity when compared to previously characterized LysB alone. EC1 is also powerfully synergistic with standard-of-care antibiotics. In addition to in vitro killing of NTM, ENTX_001 demonstrates the rescue of infected macrophages from necrotic death by Mycobacteroides abscessus and Mycobacterium avium. Here, we demonstrate shredding of mycobacterial cells by EC1 into cellular debris as a mechanism of bactericide.IMPORTANCEThe world needs entirely new forms of antibiotics as resistance to chemical antibiotics is a critical problem facing society. We addressed this need by developing a targeted enzyme therapy for a broad range of species and strains within mycobacteria and highly related genera including nontuberculous mycobacteria such as Mycobacteroides abscessus, Mycobacterium avium, Mycobacterium intracellulare, as well as Mycobacterium tuberculosis. One advantage of this approach is the ability to drive our lytic enzymes through encapsulation into macrophage-targeted liposomes resulting in attack of mycobacteria in the cells that harbor them where they hide from the adaptive immune system and grow. Furthermore, this approach shreds mycobacteria independent of cell physiology as the drug targets the mycobacterial envelope while sidestepping the host range limitations observed with phage therapy and resistance to chemical antibiotics.

Role of the Mycobacterium tuberculosis ESX-4 Secretion System in Heme Iron Utilization and Pore Formation by PPE Proteins.

Mycobacterium tuberculosis (Mtb) is transmitted through aerosols and primarily colonizes within the lung. The World Health Organization estimates that Mtb kills ~1.4 million people every year. A key aspect that makes Mtb such a successful pathogen is its ability to overcome iron limitation mounted by the host immune response. In our previous studies, we have shown that Mtb can utilize iron from heme, the largest source of iron in the human host, and that it uses two redundant heme utilization pathways. In this study, we show that the ESX-4 type VII secretion system (T7SS) is necessary for extracellular heme uptake into the Mtb cell through both heme utilization pathways. ESX-4 influences the secretion of the culture filtrate proteins Rv0125 and Rv1085c, which are also necessary for efficient heme utilization. We also discovered that deletion of the alternative sigma factor SigM significantly reduced Mtb heme utilization through both pathways and predict that SigM is a global positive regulator of core heme utilization genes of both pathways. Finally, we present the first direct evidence that some mycobacterial PPE (proline-proline-glutamate motif) proteins of the PPE protein family are pore-forming membrane proteins. Altogether, we identified core components of both Mtb Heme utilization pathways that were previously unknown and identified a novel channel-forming membrane protein of Mtb. IMPORTANCE M. tuberculosis (Mtb) is completely dependent on iron acquisition in the host to cause disease. The largest source of iron for Mtb in the human host is heme. Here, we show that the ancestral ESX-4 type VII secretion system is required for the efficient utilization of heme as a source of iron, which is an essential nutrient. This is another biological function identified for ESX-4 in Mtb, whose contribution to Mtb physiology is poorly understood. A most exciting finding is that some mycobacterial PPE (proline-proline-glutamate motif) proteins that have been implicated in the nutrient acquisition are membrane proteins that can form channels in a lipid bilayer. These observations have far-reaching implications because they support an emerging theme that PPE proteins can function as channel proteins in the outer mycomembrane for nutrient acquisition. Mtb has evolved a heme uptake system that is drastically different from all other known bacterial heme acquisition systems.

A Bifunctional Chemical Reporter for in Situ Analysis of Cell Envelope Glycan Recycling in Mycobacteria.

In mycobacteria, the glucose-based disaccharide trehalose cycles between the cytoplasm, where it is a stress protectant and carbon source, and the cell envelope, where it is released as a byproduct of outer mycomembrane glycan biosynthesis and turnover. Trehalose recycling via the LpqY-SugABC transporter promotes virulence, antibiotic recalcitrance, and efficient adaptation to nutrient deprivation. The source(s) of trehalose and the regulation of recycling under these and other stressors are unclear. A key technical gap in addressing these questions has been the inability to trace trehalose recycling in situ, directly from its site of liberation from the cell envelope. Here we describe a bifunctional chemical reporter that simultaneously marks mycomembrane biosynthesis and subsequent trehalose recycling with alkyne and azide groups. Using this probe, we discovered that the recycling efficiency for trehalose increases upon carbon starvation, concomitant with an increase in LpqY-SugABC expression. The ability of the bifunctional reporter to probe multiple, linked steps provides a more nuanced understanding of mycobacterial cell envelope metabolism and its plasticity under stress.

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.

Metabolic Labeling of Live Mycobacteria with Trehalose-Based Probes.

The mycobacterial cell envelope includes a unique outer membrane, also known as the mycomembrane, which is the major defense barrier that confers intrinsic drug tolerance to Mycobacterium tuberculosis (Mtb) and related bacteria. The mycomembrane is typified by long-chain mycolic acids that are esterified to various acceptors, including: (1) trehalose, forming trehalose mono- and di-mycolate; (2) arabinogalactan, forming arabinogalactan-linked mycolates; and (3) in some species, protein serine residues, forming O-mycoloylated proteins. Synthetic trehalose and trehalose monomycolate analogs have been shown to specifically and metabolically incorporate into mycomembrane components, facilitating their analysis in native contexts and opening new avenues for the specific detection and therapeutic targeting of mycobacterial pathogens in complex settings. This chapter highlights trehalose-based probes that have been developed to date, briefly discusses their applications, and describes protocols for their use in mycobacteria research.

Study of the conformational behaviour of trehalose mycolates by FT-IR spectroscopy.

Mycolic acids are fundamental cell wall components, found in the outer membrane barrier (mycomembrane) of Mycobacterium related genera, that have shown antigenic, murine innate immunity inducting and inflammatory activity triggering action. The mycolic acid derivatives, such as the lipid extractable trehalose monomycolates (TMM) and dimycolates (TDM), have been extensively investigated by several biochemical and biological methods and, more recently, we have performed the first neutron scattering measurements on these molecules in order to characterize their dynamical behavior as well as their rigidity properties. In the present paper, we show the first systematic FT-IR study on TMM, TDM and glucose monomycolate (GMM). It includes the analysis of individual lipids but also mixtures of TMM/TDM (ratio of 1:1) or TMM/GMM (ratio of 1:2). The present work is aimed to the first characterization of the vibrational behavior of mycolates and their mixtures enabling us to elucidate the molecular mechanisms responsible for the capability of mycolic acids to affect the flexibility and permeability properties of the mycomembrane. As a whole, the present FT-IR findings provide information that have relevant biological implications, allowing to demonstrate that the membrane fluidity is not only linked to the chain length, but also to the specific conformational behavior adopted by mycolates, which in the mixtures is strongly affected by their mutual interactions. In addition, the capability of trehalose to drive the mycolate conformational behavior and then the chain order and packing is emphasized; due to the TDM relevant evidences shown by our data, this trehalose effect could be related to the TDM toxicity and inflammation action.

Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria.

Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.