The Mycobacterial Cell Envelope

The cell envelope of mycobacteria, i.e., the compounds that surround the cytoplasm and protect the micro-organisms from their environment, is important for the bacterial physiology because inhibition of the production of some of its constituents, e.g., mycolic acids and arabinogalactan, kills the cells. It is also this structure that controls the transfer of materials into and out of the mycobacterium. The wall possesses a fundamental, covalently linked “cell-wall skeleton” (CWS) associated with a great variety of noncovalently linked substances, of which a majority are lipids and glycolipids. Isolated plasma membranes are typically obtained by breaking the cells by mechanical stress, e.g., sonication or shearing in the French pressure cell, followed by fractionation using differential centrifugation or density gradients. The wall of mycobacteria consists of a covalently linked CWS, and an abundant variety of wall-associated lipids and a few polypeptides. That the outermost layer of the mycobacterial cell envelope is partly polysaccharide in nature could be deduced from the staining of electron-transparent zone (ETZ) with peroxidase-conjugated concanavalin A and from old data showing that mycobacterial culture filtrates, notably the preparation called tuberculin, contain several polysaccharides. The polysaccharide/protein matrix in pathogens would also serve as a defense mechanism by facilitating survival in the host. The capsule and its constituents can also act by actively modifying the behavior of host cells in some way beneficial to the bacterium, as recently shown for the glucan from Mycobacterium tuberculosis.

This chapter concentrates on gram-positive bacteria, such as Bacillus subtilis and Staphylococcus aureus, that the author elucidated using new cryoTEM technology, which maintains the hydrated nature of the sample. For mycobacterial cell envelopes, more care is necessary because it is recognized that several of the wall constituents are soluble or deformable in the organic solvents that are used during dehydration. Ultrarapid freezing of cells is the central feature for cryoTEM. This freezing must occur very rapidly (less than milliseconds) so that vitrification of the sample occurs; amorphous ice, having the consistency of a glass, is formed and all macromolecular motion is stopped. In B. subtilis, new wall polymers enter the wall immediately above the plasma membrane where they accumulate and are (eventually) covalently bonded into the existing framework via transpeptidation of newly synthesized peptidoglycan strands to older strands. Above the periplasmic zone, there lies a darker zone of higher mass. This must be the cell wall constructed of peptidoglycan and secondary polymers. Careful densitometry of B. subtilis frozen hydrated walls revealed a graduation of high density to low density from the inside face of the cell wall to the outside. Mycobacteria possess an unusual cell wall consisting of an intermediate thickness of peptidoglycan and a set of unique additional components that, together, retain crystal violet within the cell when they undergo Gram staining.

Recent developments in genomics, bioinformatics, proteomics, and analytical techniques have resulted in a more detailed and thorough understanding of the structure of the mycobacterial cell wall core and its biosynthesis. Indeed, much of the current interest in its biosynthesis is driven by the need for novel, alternative drugs to counteract drug-resistant tuberculosis. The tetrapeptide side chains of peptidoglycan (PG) consist of L-alanyl-D-isoglutaminylmeso-diaminopimelyl-D-alanine (L-Ala-D-Glu-A2pm- D-Ala), with the Glu and A2pm being further amidated. Recent mass spectrometric analysis suggests that the arabinan chains released from the cell wall by an endogenous arabinase are also approximately 30 residues long and indicates that galactosamine (GalNH2) residues in Arabinogalactan (AG) isolated from the Mycobacterium tuberculosis CSU20 strain are found on the C2 position of some of the internal 3,5-branched Araf residues. Many of the insights to understanding the biosynthesis of the cell wall core are derived from earlier structural analysis. In general, polyprenyl diphosphate (Pol-P-P) molecules are synthesized through sequential condensation of isopentenyl diphosphate (IPP, derived from methylerythritol phosphate in mycobacteria) with allylic diphosphates, reactions that are catalyzed by prenyl diphosphate synthases. Synthesis of AG begins with the formation of the linker unit, and then there appears to be a concomitant extension of the galactan and arabinan moieties. The structural complexity of mycobacterial arabinofuran present in AG is likely to be reflected in a complex biosynthetic pathway, and arabinosyl transferases are now being rapidly identified in Mycobacterium spp.

This chapter focuses on the major metabolic steps and essential enzymatic players in the mycolic acid biosynthetic pathway, providing a historical perspective and highlighting the key advances of the last few years in this dynamic area. Information relative to the mycolic acid structure has been brought through the application of early and modern chemical techniques, in particular thin-layer chromatography (TLC), gas chromatography (GC), high-pressure liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The structures of mycolic acids of genera other than mycobacteria were found to be relatively simple in terms of chemical functions, being composed only of homologous series with various numbers of double bonds, up to 7 for some Gordona species. The pathway for synthesis of mycolic acids could be virtually divided into three major steps: (1) the synthesis and elongation of fatty acids to give precursors of both the α-branch and the very long meromycolic chain; (2) the elongation and introduction of functional modifications on the meromycolic chain; and (3) the condensation of two long-chain fatty acids, followed by a reduction to yield the mycolic acid specific motif. More recently, the involvement of the AccD4 carboxyltransferase in mycolic acid synthesis and its essentiality for mycobacterial survival have been demonstrated. Fatty acid synthase (FAS)-II has been shown to elongate medium-chain-length C12 to C16 fatty acids to yield C18-C30 acyl-ACPs in vitro, which are most likely the precursors of the very long-chain meromycolic acids.

This chapter has two purposes. The first is to review the chemistry, biosynthesis, biologic activity, and pathogenic function for trehalose dimycolate (TDM). The second purpose of the chapter is to review the history, genetic determinants, and biochemical basis of the cording morphology. The historical controversy about the relationship between cording and TDM has been partially clarified by recent studies, which are used as the basis for the attempts to summarize the importance of TDM and cording in mycobacterial pathogenesis. Cording must be distinguished from clumping, which is a general property of mycobacteria owing to their hydrophobic surface. Characterization of umaA2 null mutants of BCG and Mycobacterium tuberculosis revealed a strain that lacked the proximal cyclopropane ring of the alpha mycolic acid. Genetic analyses of the cording morphology in M. tuberculosis have identified multiple genes that are necessary for the expression of the cording morphology. Chemical characterization of the active compound revealed, a glycolipid whose common name became “cord factor” based on its derivation from Bloch’s petroleum ether extracts. A biochemical screen for the molecular basis for mycolic acid transfer from glucose monomycolate (GMM) to free trehalose identified antigen 85 A-C. Each of the proteins transferred a mycolic acid from glucose monomycolate to free trehalose or GMM to produce GMM or TDM respectively. TDM can be isolated from all mycobacteria that synthesize mycolic acids. Cording remains a reliable predictor of attenuation and in many cases is due to loss of specific cell envelope lipids.

Mycobacterium tuberculosis bacilli are phagocytozed mostly by alveolar macrophages following entry into the lung. M. tuberculosis interactions with phagocytes are central to both host protective immunity and tuberculosis pathogenesis. The M. tuberculosis envelope lipids include phosphatidyl-myo-inositol mannosides (PIM) and their multiglycosylated counterparts, lipomannans (LM) and mannosylated lipoarabinomannans (ManLAM). These molecules are involved in the modulation of the host immune responses. PIM are found in the plasma membrane among other phospholipids and also in the capsule, where they seem to be randomly distributed from the cell surface to its innermost layers. The biosynthetic pathway of polar and apolar PIM, although incomplete, is by far the best documented aspect of the biosynthesis of PI-based lipoglycans. Defective or deficient PIM/LM/LAM synthesis is associated with lethality or growth defects, and this raises the issue of the contribution of these complex molecules to the physiology of Mycobacterium sp. The ability of soluble lipoglycans to bind C-type lectins and TLR2 is of particular interest because mycobacterial compounds, including lipoglycans and PIM, are delivered from infected macrophages, through exosomes or apoptotic vesicles, to noninfected bystander dendritic cells (DCs). Toll-like receptors (TLRs) play a crucial role in innate immunity by the recognition of molecular patterns associated with mycobacteria. ManLAM binding to the C-type lectins, MR and DC-SIGN elicits cell signaling pathways. PIM and LM stimulate non-conventional αβT cells restricted by the CD1 proteins and innate immunity through TLR2 binding.

A “golden age” of tuberculosis (TB) chemotherapy was heralded by the discovery of streptomycin in 1944. The chemotherapeutic regimen consists of an initial 2-month phase of treatment with isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB), followed by a continuation phase of treatment lasting four months with INH and RIF. Important considerations for new agents include enhancement of penetration of infection sites, such as lung cavities, and long biological half-lives; achieving either might represent a significant advance toward shortening therapy and lead to simpler treatment regimens with improved patient compliance. The products of the emb locus of Mycobacterium avium were identified as the targets for EMB using a strategy of target overexpression. The locus contains three genes, embR, embA, and embB; the former encodes a putative regulator of embA and embB and is expendable for the resistant phenotype, which is copy number dependent. Pharmacoproteomic studies with M. tuberculosis H37Rv revealed that similar protein profiles were catalogued after both EMB and SQ109 treatments. A spontaneous mutant of Mycobacterium smegmatis designated mc2651 is resistant to INH, but retains wild-type KatG activity. Analyses of treated sensitive bacteria using electron microscopy revealed dysfunction in cell wall biosynthesis and incomplete septation. The increased abundance of CmaA2, involved in mycolic acid biosynthesis under anaerobic conditions suggests a level of metabolic activity related to mycolic acid biosynthesis under conditions usually associated with a transition to dormancy that may be linked, resulting in modulation of mycolic acid chain length during a dormant or persistent anaerobic state.

Erp protein has been detected in culture supernatants or cell wall preparations but not in cell extracts. Erp has been detected in protein samples of several mycobacterial species by Western blot. The Erp family is an expanding group of proteins originally identified and characterized from mycobacteria causing tuberculosis (TB) and leprosy. The availability of Mycobacterium tuberculosis genome sequence and its comparison with other mycobacterial genomes has accelerated the study of pathogenesis as never before and has raised a number of questions concerning the roles and functions of a large group of putative unknown proteins, in which Erp is included. Analysis of the Cterminal variable regions of the PE proteins identified the presence of 41 to 43 amino acid-long tandem repeats in two PE proteins, namely Rv0978 and Rv0980. The PE and PPE family of proteins have been shown to be differentially expressed in M. tuberculosis during in vitro growth and, at the same time, exhibit gene expression diversity in different clinical isolates. The possible function of the PE/PPE protein family also became apparent. It was hypothesized that these antigens play a role in evading the host immune response, thereby preventing the establishment of an effective cellular response that is required to contain the disease. Erp, PE, PPE, and PE_PGRS proteins have a highly conserved N-terminal domain that, though different for Erp, PE, and PPE, appears to serve the purpose of exporting these proteins to the surface.

Mycobacteria devote a large part of the coding capacity of its genome to fatty acid biosynthesis. Porins of Neisseria meningitidis induce actin nucleation in the host cell, suggesting a role in cell actin reorganization, which might influence the invasive ability of the bacteria. The growth and nutritional requirements of mycobacteria have been intensely studied since the discovery of Mycobacterium tuberculosis. Nutrient uptake mechanisms obviously depend on the permeability barriers imposed by the cell envelope. Mycobacteria have evolved a complex cell wall, comprising a peptidoglycan-arabinogalactan polymer with covalently bound mycolic acids of considerable size (up to 90 carbon atoms), a variety of extractable lipids, and pore-forming proteins. The unique mycolic acid bilayer is an extremely efficient permeability barrier protecting the cell from toxic compounds and is generally thought to be the major determinant of the intrinsic resistance of mycobacteria to most common antibiotics, chemotherapeutic agents and chemical disinfectants. The susceptibility of both Mycobacterium bovis BCG and M. tuberculosis to zwitterionic β-lactam antibiotics was substantially enhanced by MspA, decreasing the minimal inhibitory concentration up to 16-fold. The accelerated growth of M. bovis BCG on expression of MspA identified slow nutrient uptake as one of the determinants of slow growth in mycobacteria.

This chapter highlights both the well established and the yet poorly understood aspects of siderophoremediated iron acquisition in pathogenic and nonpathogenic mycobacteria, with a particular emphasis in the siderophore system of Mycobacterium tuberculosis. The M. tuberculosis siderophore system is believed to play a crucial role in the procurement of a suitable iron supply to support bacterial multiplication in vivo and to be a key factor in the ability of this human pathogen to produce successful infections. The mycobacteria examined for iron-acquisition systems appear to rely on siderophores with high affinity for the ferric ion as the primary mechanism for iron acquisition. Transcription of genes of the exochelin (EXC) and mycobactin/carboxymycobactin (MBT/CMBT) systems is derepressed when the bacterium experiences iron limitations, thus leading to siderophore biosynthesis and siderophore-mediated iron uptake. Several Mycobacterium species produce two structurally related families of high-affinity Fe3+-binding siderophores, the MBTs and the CMBTs. Mutational analysis has conclusively linked the mbt gene cluster to production of both MBTs and CMBTs. Mutational analysis has conclusively linked the mbt gene cluster to production of both MBTs and CMBTs. Several Mycobacterium species that are normally found as environmental saprophytes release EXCs, the nonribosomally synthesized pentapeptidebased or hexapeptide-based siderophores, into the extracellular environment. More recently, analogues of salicyl-AMS have also been demonstrated to block MBT/CMBT biosynthesis and M. tuberculosis multiplication in iron-limiting conditions.

The ATP-binding cassette (ABC) transporter systems are a conserved superfamily of multisubunit permeases that are found in all living organisms. Bacterial ABC transporters can be either importers or exporters. The ABC proteins form the largest paralogous family of proteins in Escherichia coli. It was initially predicted as a daunorubicin exporter on the basis of its similarity to the drrAB locus from Streptomyces peucetius. Transcriptome analysis of Mycobacterium tuberculosis expression in macrophages showed that 454 genes were induced and 147 genes were repressed in activated or resting macrophages at 24 hours compared with broth culture. In several bacteria, peptide transport can be important for nutrition of the cell, signaling processes such as regulation of gene expression, sporulation, chemotaxis, competence, and virulence. Phosphate is another essential anion that is also transported by one or several multisubunits ABC permeases in mycobacteria. The mkl gene has been shown to be required for macrophage survival and also has been identified twice as being essential for survival of M. tuberculosis in mice. The phosphate transport system (Pst) from M. tuberculosis is a tightly regulated high-affinity system encoded by three putative pst operons, suggesting that the bacteria are involved in subtle biochemical adaptations of M. tuberculosis for their survival under varying conditions during the infectious cycle. Active multidrug efflux pumps and the mycobacterial cell wall permeability barrier are the mechanisms that are thought to be potentially involved in the natural drug resistance of mycobacteria.

The diverse array of Mycobacterium tuberculosis lipids, many of which are critical for virulence and mediate interactions with host cells, exist primarily on the outermost surface of the bacterium. Fourteen mmpL genes have been identified by analysis of the two completed M. tuberculosis genomic sequences. These genes encode for large, multitransmembrane containing proteins and were thus given the moniker MmpL (for mycobacterial membrane protein, large). Initial models of MmpL function were based on the RND transporters from gram-negative bacteria. MmpL7 was the first MmpL family member to be studied and was identified as required for virulence and bacterial growth in vivo in a mouse model of M. tuberculosis infection. Mutants in mmpL8 were also isolated in a signature- tagged mutagenesis screen, and MmpL8 is indispensable for growth and virulence in mice. First, mmpL4 transcription is induced, albeit modestly, during infection of activated macrophages. The eukaryotic larger resistance, nodulation, and division (RND-family member, Niemann-Pick C1 (NPC1) utilizes the proton motive force to translocate cholesterol across the cell membrane and it is widely thought that RND family members use proton antiport or symport as a mechanism of facilitated transport of their substrates. The Lol system functions to export lipoproteins across the periplasm of Escherichia coli to the inner leaflet of the outer membrane. LolCDE bind specifically to lipoproteins destined for the outer membrane and export them across the cell membrane, where they bind the periplasmic shuttle LolA.

The search for important antigens among secreted proteins from Mycobacterium tuberculosis present in culture filtrates led to the identification of ESAT-6 (early secretory antigenic target of 6 kDa), a small molecule that migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 6 kDa. When administered as a subunit vaccine in mice, the mixture of culture filtrate proteins induced protection at the same level as the live vaccine Mycobacterium bovis BCG (BCG). This chapter talks about ESAT-6 and related proteins in the past few years since their discovery. The original ESAT-6 protein was purified from M. tuberculosis culture filtrates by ammonium sulfate precipitation, hydrophobic interaction chromatography, followed by anion exchange chromatography. CFP-10 is another important T-cell antigen present in culture filtrates and identified as a member of the ESX protein family. The active secretion of ESAT-6 and CFP-10 and the surface features of the CFP-10-ESAT-6 complex suggested a function based on specific binding to host proteins rather than a pore-forming role of the complex to mediate cell lysis activity. The ESAT-6 family proteins are also found to be important in the development of vaccines against tuberculosis. The infectious cycle of M. tuberculosis ranges from lifelong asymptomatic parasitism to the rapid development of tuberculous cavities in the lungs of patients that have become susceptible to develop the disease.

Sigma factors are interchangeable subunits of bacterial RNA polymerase that are required for promoter selectivity and transcription initiation. Sigma factors regulate processes involved in cell surface biology in many bacterial species. The characterization of stress response systems used by bacteria to respond to surface stress is extremely important because it can contribute to a better understanding of both bacterial sensing and signaling through different cell compartments, and cell envelope physiology and biogenesis. Cell integrity stress, like other forms of stress, may lead to increased expression of chaperone/heat shock genes, and the activity of these heat shock proteins (Hsps) is likely to be essential for maintaining cell envelope homeostasis. Mpt53 is a secreted DsbE-like protein containing a thioredoxin-active site. This protein was recently characterized and hypothesized to be a functional homologue of DsbA, able to catalyze the formation of disulfide bonds in unfolded secreted proteins. Even though at the current state of knowledge it is difficult to assign many of the sigma factors specific functions, one can hypothesize that some of them, such as σF, σD and σE, regulate genes responsible for the maintenance of surface homeostasis following damage that can occur during stationary phase or following exposure to surface damaging host effector molecules. Others such as σL and σM appear to be involved in the regulation of the composition of the mycobacterial surface in response to still unknown stimuli and can play an important role in modulating Mycobacterium tuberculosis interactions with the host.

The cell envelope of mycobacterium consists of a dense network of unusual lipids and sugars. This impermeable barrier imparts resistance against hostile environments and to the commonly used antimicrobial agents. Long-chain α-alkyl β-hydroxy mycolic acids are the core lipid constituents of mycobacterial cell envelope. These are esterified to the arabinogalactan to form the mycolic arabinogalactan (MAG), which link to the peptidoglycan through a phosphodiester bond. In addition, mycolic acids also exist as free glycolipids in the form of trehalose monomycolate (TMM) and trehalose dimycolate (TDM). Other mycobacterial cell envelope lipids include sulfolipids (SL), polyacyl trehalose (PAT), mannosyl-β-1-phosphomycoketide (MPM), and diacyl trehalose (DAT), all of which require polyketide enzymatic machinery for their biosynthesis. Some polyketide derived lipids include two structurally related members, phenolphthiocerol glycolipid (PGL) and phthiocerol dimycoserosates (PDIMs), mycobacterial siderophores that are involved in iron sequestration and glycopeptidolipids (GPLs) isolated from opportunistic environmental mycobacteria. In 1998, the Mycobacterium tuberculosis genome sequencing project revealed a large number of proteins homologous to polyketide synthases (PKSs) which typically produce secondary metabolites in Streptomyces and fungi. Polyketide synthases function as multifunctional enzymatic assembly lines. NRPS-PKS server provides novel clues about putative polyketide products of new modular PKS clusters based on comparison with sequence and structural features of PKS domains in PKSDB. Sulfolipids consist of phthioceronic acid and hydroxyphthioceronic acids acylated along with fatty acids to a sulfated trehalose core to form sulfolipids.

This chapter focuses on recent developments in one’s understanding of how different components of the cell envelope from virulent mycobacteria, in particular Mycobacterium tuberculosis, interact with each stage of innate and adaptive immune responses. Human dendritic cells express an additional C-type lectin, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), which is not present on macrophages but serves as a major receptor for M. tuberculosis on dendritic cells (DCs). Cell wall components of mycobacteria bind to these pathogen recognition receptors on DCs and macrophages, and initiate the host response to infection. The biological relevance of TLR activation by cell wall components has been examined by mycobacterial infection of gene-deficient mice. Inactivation of protein kinase G (PknG) by gene disruption or chemical inhibition resulted in delivery of pathogenic mycobacteria to lysosomes and mycobacterial killing, and conversely, expression of PknG in nonpathogenic prevented phagosomal maturation. The induction of adaptive immunity to mycobacteria and activation of infected macrophages by IFN-γ can overcome the maturation arrest of mycobacteria containing phagosomes. CD1 proteins, which are antigen presenting molecules encoded by genes located outside of the major histocompatibility complex (MHC), recognize nonpeptide lipid or glycolipid structures, including components of mycobacteria. The containment of mycobacterial infection requires the formation of granulomas (or tubercles), which are nodular aggregations of lymphocytes, macrophages, and epithelioid cells. In human M. tuberculosis infection, the propensity of trehalose dimycolate (TDM) and other cell wall components to promote granuloma formation in association with a chronic T-cell response leads to caseating granulomas, which may erode into airways.

Phenolic glycolipids (PGLs) consist of a conserved lipid core and a variable carbohydrate moiety; the lipid core is composed of a family of long-chain β-diols, phenolphthiocerol, and related molecules. The structure of the backbone of PGLs and the related DIMs was established more than 30 years ago, but their synthesis remained elusive until the last decade. Early work by Gastambide-Odier and colleagues to decipher the phthiocerol and phenolphthiocerol biosynthesis pathway were based on metabolic labeling either with [1- or 3-14C] propionate or with [14C] methionine, followed by chemical degradation and tracing the radioactivity. DIM and PGL share a common lipid core synthesized by the same enzymatic machinery, but unlike PGL, DIM has no saccharide domain. The gene drrC encodes an integral membrane protein predicted to be part of an ABC transporter involving two other subunits encoded by genes drrA and drrB mapping upstream from drrC. Unlike DIM, which have been found in all clinical isolates of Mycobacterium tuberculosis tested, PGL-tb are produced by only a small proportion of strains. The availability of the M. tuberculosis genome sequence and the concomitant development of powerful genetic tools for mycobacteria were enormous leaps forward for studying the biology of M. tuberculosis, including the biosynthesis and role of PGL-tb and related compounds.

Sulfated metabolites are abundant in higher eukaryotes, where they play roles in cell-to-cell communication. This chapter highlights recent advances in the understanding of sulfolipid-1 (SL-1) biosynthesis and discusses the potential biological significance of SL-1 and other sulfated metabolites in the context of both historical observations and modern experiments. Sequencing of the Mycobacterium tuberculosis genome in 1998 provided the tools necessary to draw links from gene to protein and metabolite. A signaturetag mutagenesis (STM) screen identified mmpL7 as a gene essential for M. tuberculosis growth in a mouse model of infection. Sulfate ester functionality distinguishes SL-1 from other well-characterized mycobacterial lipids. Recently, Gap, a small integral membrane protein from Mycobacterium smegmatis was shown to be required for transport of glycopeptidolipids to the cell surface. In vivo analysis of the Δpks2 mutants left the SL-1 community questioning the importance of the molecule that had previously garnered so much attention. Attenuation of the ΔmmpL8 mutant in the persistent stage of M. tuberculosis infection contrasts with the reported phenotype of the Δpks2 mutants. Although M. tuberculosis is the most extensively studied species of the mycobacterial genus, several other mycobacteria have medical importance owing to their synergism with human immunodeficiency virus. One such species is the opportunistic environmental mycobacterium Mycobacterium avium, which preferentially infects individuals with compromised immunity. This pathogen also encodes nine putative sulfotransferases, the largest number of sulfotransferases of any sequenced mycobacterial species. As one's knowledge of human immunity grows, better cellular and in vivo models for M. tuberculosis virulence will be created.

One of the most recently characterized mycobacterial cell wall proteins is the heparin-binding hemagglutinin (HBHA). Initially found on the surface of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG), HBHA is also present on the surface of other mycobacteria, including other pathogenic and nonpathogenic species. The direct role of HBHA in bacterial adherence to epithelial cells was confirmed by the use of isogenic M. tuberculosis mutant strains. When the mutation is complemented by the natural hbhA gene under the control of its own promoter, binding to the A549 cells is restored to wild-type levels. These observations confirm that HBHA is one of the key adhesins for cells other than professional phagocytes, suggesting that interactions of M. tuberculosis with cells other than macrophages may play an important role in the pathogenesis of tuberculosis. Purified HBHA also binds to concanavalin A, which was initially proposed to indicate that HBHA may be a glycoprotein. The fact that HBHA is surface exposed despite the lack of a cleavable N-terminal signal peptide suggests that its cellular localization is Sec independent. The localization of HBHA at the mycobacterial membrane may be mediated by the putative hydrophobic N-proximal transmembrane domain. Cytotoxicity appeared to be independent of host factors and was thus proposed to be driven by bacterial virulence factors.

This chapter summarizes studies on the role of mycobacterial kinases and phosphatases (i) in the growth and pathogenesis of mycobacterium and (ii) in the cell wall metabolism of the pathogen. Earlier, the two-component systems (TCSs) involving a histidine kinase (HK) and a response regulator (RR) were considered to play a key role in phosphotransfer mechanism for signal transduction in bacteria, whereas serine/threonine protein kinases (STPKs) and their associated phosphatases were more relevant to signal transduction pathways in eukaryotes. However, with the inflow of bacterial genome sequences, it is now known that these eukaryotic-like protein kinases and phosphatases are present in prokaryotes also and play an important role in bacterial metabolism and pathogenesis. Transposon-insertion mutagenesis experiments carried out to identify the genes required for optimal in vitro mycobacterial growth resulted in the identification of only three out of the 11 mycobacterial kinases, namely PknA, PknB, and PknG. In view of the unique cell wall structure of mycobacteria and the presence of a large repertoire of polyketides and complex lipids, kinases and phosphatases are bound to play an important role in the regulation of the cell wall metabolism of this pathogen. Future work will expose the mechanistic details and proteins used by this pathogen to downregulate the host signaling pathways. Structural analysis of complexes of these signaling proteins may provide the key to designing molecules for selective disruption of signal transduction.

This chapter describes the major advances in the understanding of the biology and biosynthesis of glycopeptidolipids (GPLs). Strains from the Mycobacterium fortuitum complex contain surface species-specific lipids, allowing their precise identification. In M. fortuitum bv. Peregrinum, two major GPLs were characterized by a combination of chemical analyses. The M. avium-M. intracellulare-M. scrofulaceum complex (MAC) is among the most common nontuberculous mycobacteria recovered from clinical specimens and is also a prevalent pathogen in AIDS patients. An immunogenic GPL, named GPL X-I, was isolated from M. xenopi, a nontuberculous mycobacterium responsible for pulmonary and disseminated infectious diseases mainly occurring in immunocompromised patients. M. avium subsp. paratuberculosis is closely related to M. avium subsp. avium and is responsible for cattle infections. The isolation of GPL-nonproducing mutants after a transposon mutagenesis of M. smegmatis was greatly facilitated thanks to the characteristic morphotypes of these mutants. The lipopeptide core can be modified by glycosylation, O methylation, and O acetylation, and each of the genes responsible for these modifications has been characterized. Freeze-fracture electron microscopy has been used to study the structure of the envelope of M. avium cells growing inside mouse liver macrophages and has revealed an onion-like structure. A study investigated the consequence of drug treatment with a regimen of clarithromycin and ethambutol on the chemical alterations of GPLs in M. avium. Small metabolites such as sulfolipids, phenolglycolipids, or glycopeptidolipids use the same building blocks that are Pks, FadD, FadE, MmpL, and Gtf and that have evolved substrate specificity.

Mycolactones are a family of lipophilic small molecules and virulence factors produced as secondary metabolites by Mycobacterium ulcerans and some highly related aquatic mycobacteria. This chapter describes what is known about mycolactones and their unique role in the pathogenesis of Buruli ulcer, explains their unusual biosynthetic locus, and highlights the key questions that remain to be answered. A recent report describes an abundant extracellular matrix produced by M. ulcerans that harbors vesicles that are rich in mycolactones. Mycolactones are related to antibacterial macrolides such as erythromycin and immunosuppressants such as FK506 because they all share a polyketide-derived macrolactone core. The apoptotic ability of mycolactone F is significantly less than that of the other mycolactones; a feature that may be associated with its shorter acyl side chain. All of these mycolactone-producing mycobacteria, including M. ulcerans, form a distinct lineage within a genetically more diverse assemblage of Mycobacterium marinum that have clearly evolved from a common M. marinum-like progenitor. All mycolactone-producing mycobacteria found so far have been discovered through their ability to cause disease in vertebrates, but they may be acting as “indicator” species and represent only a proportion of the mycolactones that exist in nature.