Biology of Mycobacterial Lipids

Biology of Mycobacterial Lipids covers various topics pertaining to the advancements and current research in the field of mycobacterial lipids, and on the significant progress in lipidomics, in recent times. The chapters present comprehensive, yet systematic cutting-edge research, comprising mycobacterial lipid terminologies, classifications, biosynthetic pathways, tools and techniques, and functional burgeoning. This unique reference book has contributions from pioneer researchers, experts, and eminent veterans from around the globe. It covers ground-breaking work that will bridge the gap between understanding biochemical patterns related to virulence, pathogenesis, and resistance and elucidating new targets for drug design, identifying biomarkers for predicting risk, early diagnosis, and therapeutic outcome.

• Covers major biochemical aspects of mycobacterial lipids, nomenclature, structure and classification, and metabolic pathways
• Focuses on recent trends and state-of-the-art technology, used in mycobacterial lipids study
• Brings together the functional aspects of mycobacterial lipidome, involvement of lipids in cellular network and signaling, its involvement in virulence and resistance, and host factor manipulations
• Highlights the discovery of lipid biomarkers, for diagnostic and therapeutic interventions, using mycobacterial lipidomic studies
• Presents lipids at the interface of various other biomolecules with integrative omics aspects

• Language: English
• Publisher: Academic Press
• Release date: Jun 3, 2022
• ISBN: 9780323919449

Book preview

Preface

Mycobacterial lipids "the unresolved miracle with the ray of hope"

Since antiquity mankind has fought a long way in pursuit to take charge over deadly Mycobacteria, still every year 1.5 million die with single bacterium Mycobacterium tuberculosis. Several breakthrough diagnosis and therapeutics researches have been made in last couple of centuries. Mycobacteria still remains the deadliest pathogen and the thorn in the flesh of researcher community. Nature has provided Mycobacteria a unique shield in the form of cell envelope and simultaneously handed over a target to mankind. Plethora of researches are going on around the globe on Mycobacterial lipids and tremendous amount of information have been fetched out which equipped the clinics with tools and ray of hope.

Mycobacterial diseases in general, tubercular or nontubercular, are clinically significant, causing a wide variety of infections in human as well as animals. Mycobacterium genus holds lipids that are distinct, complex, and unusually high and responsible for establishing Mycobacterium as a unique and challenging pathogen. The success of Mycobacteria relies on its thick waxy envelope which provides protective barrier enabling its efficient spread. This envelope also supports its survival within host macrophages acting as an efficient shield and providing resistance to antibiotic stress. Hence, in all circumstances, the researchers have to work to systematically analyze and understand the lipid domain of Mycobacterium in order to have an edge to win the battle against Mycobacterial pathogenesis and resistance.

The book entitled "Biology of mycobacterial lipids" dwell broadly on various topics pertaining to the advancements and current researches in the field of Mycobacterial lipids and significant progress in the area of lipidomics in recent times. It presents a comprehensive yet systematic cutting-edge researches comprising mycobacterial lipid terminologies, classification, biosynthetic pathways, state-of-the-art tools and techniques, functional burgeoning. This unique reference book have contributions from pioneer researchers, experts and eminent veterans around the globe covering ground breaking oeuvre that will bridge gap between understanding biochemical patterns related to virulence, pathogenesis, resistance, and elucidating new targets for drug design, identifying bio-markers for predicting risk, early diagnosis, and therapeutic outcome.

The book starts form paying tribute to Professor David E. Minnikin by Professor Gurdyal Besra who performed his PhD studies under his guidance and had long association with him. Prof. Minnikin was the pioneer worker in the field of Mycobacterial lipids and originator of a model for the essential role of lipids in the mycobacterial cell envelope in 1982. He was the very first person who supported the idea of this book and wanted to write a chapter on Evolution of the role of lipids in mycobacterial cell envelopes. However, unfortunately he left us recently, working for the science till his last breath and making a nonreplenishable void. We as editors pay deep homage to him, and he will be missed always and ever.

The initial section of the book provides the basic understanding of the Mycobacterial lipids specifically mycolic acids and lipid BioSynthetic Pathways. Role of Mycobacterial lipids in the host–pathogen interface has been discussed with elaboration on pathogenesis mechanisms and host immune response. Further lipids and Glycolipids as biomarkers of mycobacterial infections and multifaceted roles of Mycobacterium Cell Envelope Glycolipids during Host Cell Membrane Interactions have been discussed. Middle section of the book covers the mechanisms of host lipid access, import, and utilization in Mycobacterium. Also, the biological implications of NKT-cells stimulation by mycobacterial lipids and chemically synthetic mycobacterial mycolates on phospholipidome immunomodulation of murine macrophages are been covered. Additionally, the role of polyketide synthases and the role of serine enzymes in MTB lipid metabolism have been discussed. Last section covers the application of MTB lipid metabolic alterations in production of TB vaccine candidates. The overview of intrabacterial lipid inclusions and lipid toxin Mycolactone has also been included.

This book is a research-based reference book beneficial for biomedical scientists, researchers, and healthcare industries involved in various aspects of mycobacterial diseases. This book will also be useful for students of biomedicine, pathology, and pharmacy. The recent explosion in infectious diseases is accelerating the pace of research and development in all area of medical sciences. Additionally, the book will be a resource for enhanced cross-pollination in a multiapproach to future endeavors in the field of infectious diseases. It will provide a forum to foster academic exchange among researches across different domains of mycobacterial research and healthcare workers and beyond.

We are grateful to our esteemed contributors for their worthy and timely contributions without which this compilation would not become a ready reference for the researchers in this field. Patience and day-to-day support during the book preparation from Mr. Mica Ortega and Elsevier Publishing group is deeply acknowledged. We would like to thank Prof. Amit Chattopadhyay, a Global Leader in membrane and receptor biology and biophysics, for writing foreword for this book despite of his busy schedule. We are grateful and would like to thank Dr. G. Besra, a stalwart in the field of MTB cell wall assembly, who readily agreed and wrote deep heart throbbing words as tribute for Dr. Minnikin. We are also thankful to our respective institutions (Amity University Haryana and CNRS/Aix-Marseille University) for their overall support and providing the platform for display our academic rigor. Last but not the least, Dr. Fatima feel proud to dedicate this piece of work to her colleague and better half Dr. Saif Hameed who ignited the idea for this book and supported her constantly during the compilation.

Dr. Zeeshan Fatima

Dr. Stéphane Canaan

(Editors)

Chapter 1: An overview of mycolic acids

structure–function–classification, biosynthesis, and beyond

Shweta Singh, Damini Singh, Saif Hameed, and Zeeshan Fatima     Amity Institute of Biotechnology, Amity University Haryana, Gurugram, Haryana, India

Abstract

Mycobacterium tuberculosis (MTB) is the etiological agent of tuberculosis (TB), which causes morbidity and mortality throughout the world. However, the currently available drug regimens which are being used for the treatment of TB are associated with adverse side effects on patients, high medical costs, and emergence of multidrug resistance. Hence, novel antimycobacterial drug targets are urgently needed to address the issue. The cell wall of M ycobacterium comprises large amounts of unique lipids such as mycolic acids (MAs), phosphatidyl inositol mannosides, phthiocerol dimycocerate, lipoarabinomannan, trehalose monomycolate and dimycolate, phthiocerol dimycocerosate, cord factor, sulfolipids, and wax-D that are involved in its pathogenesis. Therefore, interest in targeting lipid metabolic pathways of MTB has emerged as a significant area of research in recent times. Hence, an in-depth understanding of the cell wall composition with special emphasis on MAs is pertinent to combat MTB drug resistance and virulence. This chapter covers the gist of Mycobacterial unique lipid constituent, i.e. MAs, briefly its structure, biological role, biosynthesis, and classical as well as advanced tools used for its study. Additionally, the association of Mycobacterial lipidomics with its genome and proteome has been discussed to understand the need of prospective research in the area.

Keywords

Lipid; Lipidomics; Mycolic acids; Mass spectrometry; Mycobacterium

Introduction

Tuberculosis (TB) is a highly contagious disease, caused by the bacterium Mycobacterium tuberculosis (MTB) which is second only to HIV in causing deaths during the 20th century [1]. It is a communicable disease that spreads from person to person through air by coughing, sneezing, or spitting. It mainly targets the pulmonary tissues but can spread to other body parts [2]. About one-third of the global population suffers from latent TB causing death of almost three million people worldwide [3]. Although TB is a curable disease, the tubercle bacterium has shown emergence of various multiple-drug resistant mechanisms against antibiotics leading to a more severe infection known as multidrug resistant tuberculosis.

Among the various factors associated with the virulence of the pathogen, the lipids composing the cell wall of the bacteria have drawn increasing interest due to the uniqueness in its composition consisting of mycolic acid (MA), glycolipids such as diacyltrehaloses, polyacyltrehalose, lipomannan (LM), lipoarabinomannan (LAM), mannose-capped-LAM, sulfolipids, and trehalose-6,6- dimycolate, all having implications in providing the pathogen an advantage in the host. Interestingly, the pathogen also alters its metabolism of fatty acids (FA) to survive the conditions existing inside the host that is reflected as an altered cell wall composition in terms of lipids [4]. Hence, an in-depth understanding of the cell wall composition particularly MAs is indispensable to understand and combat the MTB's drug resistance and virulence.

What are lipids?

Michel-Eugène Chevreul, a French chemist of nineteenth century, is the father of lipid chemistry. He investigated the nature of fats on soap samples in 1811. Chevreul demonstrated that lard contained two types of triglycerides, one of which remained solid at room temperature and identified the nature of saponification reaction. The details of his findings are available in Chevreul's book "A Chemical Study of Oils and Fats of Animal Origin" 1823, translated by A.J. Dijkstra, edited G.R. List and J. Wisniak and published by Dijkstra-Tucker in 2009 (distributed by AOCS Press). Later on, in 1815, Henri Braconnot classified lipids (graisses) in two categories, suifs (solid greases or tallow) and huiles (fluid oils). In 1823, Michel-Eugène Chevreul developed detailed classification by including oils, greases, tallow, waxes, resins, balsams, and volatile oils (or essential oils). Chemists regarded fats as only simple lipids made of FA and glycerol (glycerides), till a century. Rosenbloom and Gies proposed the substitution of lipoid by lipin in 1912. Later on Bloor in 1920 introduced a new classification for lipoids: simple lipoids (greases and waxes), compound lipoids (phospholipoids and glycolipoids), and the derived lipoids (FA, alcohols, sterols) [5].

In 1947, T. P. Hilditch divided lipids into simple lipids with greases and waxes (true waxes, sterols, alcohols). Lipids have been described as discrete group of metabolites that have multiple biological functions which include energy storage, acting as a structural frame for cells and also intermediate in cell signaling pathways. They are insoluble in water as they are nonpolar, however, soluble in nonpolar solvents such as chloroform [6]. Lipids are mainly composed of hydrocarbons in their most reduced form, making them an excellent form of energy storage, as when metabolized the hydrocarbons oxidize to release large amounts of energy. Earlier, lipids were deemed only as energy storage sources and cell-skeletal frameworks, but in recent times, it has been realized that various classes of lipids are also involved in multiple disease progression due to which it became necessary to study them in a more comprehensive manner. Uniqueness of Mycobacterial cell envelope significantly attracted lipid biologist, and the pioneer studies of lipophilic nature of tubercle bacillus were coordinated by R.J. Anderson at Yale in 1930s. They have characterized the long-chain MAs for the first time. The essential structural details of MAs were determined in the laboratories of E. Lederer (Paris), J. Asselineau (Paris and Toulouse), and N. Polgar (Oxford) in 1950 and 1960s [7]. Lipids form the complex milieu in cell envelope of Mycobacterium and serve as potential drug target which has fascinated researchers till now. The myriad and biological importance of mycobacterial lipids gives rise to intensive studies to explore their structural and functional aspects contributing to virulence and pathogenesis [8].

Lipid profile of Mycobacterium

The cell wall morphology of Mycobacterium is well studied, and its components are substantially known. The macromolecular structure of mycobacterial cell wall shares similarities with both Gram-positive as well as Gram-negative bacteria. Generally, for staining of common bacterial species, a popular Gram staining technique is commonly used. However, due to the presence of unique lipids in its cell wall, Gram staining cannot be performed in Mycobacterium. Thus, a different method of staining called as Acid-fast staining or Ziehl-Neilsen staining procedure has been used for Mycobacterium cells. The cell wall comprises of lipid-rich outer layer of MA which lies on peptidoglycan layer and arabinogalactan attached to inner lipid membrane. However, thick waxy coat in Mycobacteria renders it resistant to Gram staining [9,10]. Though the cell wall is resistant to decolorization with acid alcohol but can be easily penetrated by phenol-based stains, giving mycobacteria another trait, viz. acid-fast Hence, this unique trait subsequently became the basis of staining for detection and diagnosis of TB [10].

Further, Mycobacterial cell envelope comprises of higher percentage of lipids which contributes to 40% of the weight of cell envelope [11,12]. The cell envelope of Mycobacterial strains differs in structural composition [7]. Pathogenic MTB contains outer capsule layer surrounding the cell wall. Membrane outside the cell wall is known as mycomembrane. It is divided into outer and inner leaflets. The outer leaflet is noncovalently attached to lipids and glycolipids. Inner leaflets have arabinogalactan which extends its penta-saccharide termini by esterified unique long-chain FA called MAs forming mycomembrane, followed by a peptidoglycan layer underneath [13]. It also contains a periplasmic space between the plasma membrane and the peptidoglycan, consisting of intrinsic and extrinsic proteins [14]. The capsular layer is made up of matrix of glucan with noncovalently linked glycophospholipids, for example, phosphatidyl-myo-inositol mannosides (PIMs), and the LM and LAM derivatives, and other extractable lipids, such as diacyl-trehalose (DAT), polyacyl-trehalose, phthiocerol dimycocerosate, and sulfoglycolipid and the core is composed of mycolyl-arabinogalactan-peptidoglycan complex. Polysaccharide capsule and proteins constitute the outermost layer with porins traversing the hydrophobic outer membrane. Mycobacterial species are known to produce a wide variety of different classes of FA having high molecular weight (60–90 carbons) [15,16]. MTB lipids are amphipathic hydrocarbons and exist in low to high molecular mass ranges. Their diverse molecular nature has been extensively narrowed down by mass spectrometric analysis.

What are MAs?

MAs are unique lipids which constitute up to 60% of the cell wall's contents and 50% of its dry weight. It mainly consists of long-chain α-alkyl β-hydroxy FA which contributes to the extremely low permeability of the mycobacterial cell wall [17]. The estimated length of FAs is 60–90 atoms, which are consisted of fully saturated α-chains of length 22, 24 or 26 carbon atoms. MAs have three distinctive classes named as α-, methoxy-, and keto-MAs (Table 1.1). Among them, the most abundant form is α-MA (>70%), whereas the minor components are methoxy- and keto-MA (10%–15%) [18]. The α-MA is a cis, cis-dicyclopropyl FA whereas, both keto- and methoxy-MAs are either cis- or trans-cyclopropane rings. Variations in the length are present in alkyl groups terminally and number of groups of methylene between carboxyl group and cyclopropane rings decides the structural variations in MA depending on the source [19]. In addition, MAs can also exist in oxidized form [16].

Table 1.1

MA is also present in the outer cell envelope lipids containing trehalose dimycolate (TDM), trehalose monomycolate (TMM), glucose monomycolate (GMM) and as free MA. Physiologically, the varying amounts and composition of MAs affect the growth rate, permeability, virulence, and colony morphology of MTB [16,20,21]. New insights into MA revealed its different types, length, and degree of cyclopropanation variations which could potentially occur in varying infection stages and culture conditions [22]. The flexibility and fluidity attributes of the cell envelope in MTB are known to be contributed majorly by MA molecules, which in turn are arranged in the form of a bilayer associated with cell wall lipids and serve as low fluid permeability barrier for the bacterium.

Structure of MAs

Anderson isolated MAs more than 100 years ago. In 1950s, Asselineau and Lederer described the first structures of MAs as α-alkyl-β-hydroxy FA of long chains in which two were branched and three were hydroxylated. Depending on the mycobacterial species, MA exhibits structural diversity in their functional groups and carbon chain length. Mycobacterial cells produce MAs as free acids and esterified to the arabinogalactan layer of the cell envelope as well as mycolate esters of trehalose, that is, TMM and TDM, respectively, and glycerol monomycolate. GMM, a mycolate ester is interestingly produced by the Mycobacteria during infection by acquisition of host glucose as substrate and hence acts as a local indicator of the mycobacterial invasion in the host [23].

MA is cleavable at high temperature into meromycolic chain and FA. MA extract was reduced under pressure of 250–300°C resulted into generation of hexacosanoic acid and unidentified long chain of formula C88H172O4 which presented its first structural information. Afterwards, intensive advancements in chromatography techniques in late 1950s and 1960s helped us to investigate and determine the structure of MA as long chain of α-alkyl β-hydroxy FA. The pyrolytic cleavage of MAs resulted into two products: a long-chain meroMA chain and a short alkyl chain called as α branch. There are two centers in α and β positions that are relative to carboxylic group found to be R configuration for all MA despite the other functional groups [24]. It has been reported that specific MA profile is associated with each mycobacterial species. MA of mycobacteria shows large diversity of chemical functions and chain lengths which defines the different classes of MA and also leads to complex thin-layer chromatography (TLC) patterns (Table 1.1) [25]. Apolar MAs referred to as α-MAs contain 74-80 carbon atoms and two double bonds in general (cis or trans configuration) or having cis-cyclopropyl groups which are located in meromycolic chain. A small fraction of α-MAs may contain more unsaturation, as observed in some strains of the MTB complex, with three unsaturation and longer chains (more than six to eight carbons) [26]. Till date, most of the Mycobacterial MAs have been found to contain supplementary oxygen functions in distal part of meromycolic chain that defines the wax-ester, epoxy, hydroxy, methoxy, and keto types of MA. Notably, in MTB, the oxygenated MAs such as keto-, methoxy- and hydroxyl- MAs contains 84–88 carbon atoms and therefore have four to six carbons longer chain length. MAs are major and specific long-chain FA that represent essential components of the MTB cell envelope. They play a crucial role in the cell wall architecture and permeability; hence, the natural resistance of mycobacteria to most antibiotics and represent key factors in mycobacterial virulence. Biosynthesis of MA precursors requires two types of FA synthases (FASs), the eukaryotic-like multifunctional enzyme FAS I and the acyl carrier protein (ACP)–dependent FAS II systems, which consists of a series of discrete mono-functional proteins, each catalyzing one reaction in the pathway. Unlike FAS II synthases of other bacteria, the mycobacterial FAS II is incapable of de novo FA synthesis from acetyl-coenzyme A, but instead elongates medium-chain-length FA previously synthesized by FAS I, leading to meroMAs. In addition, MA subspecies with defined biological properties can be distinguished according to the chemical modifications decorating the meromycolate. Nearly all the genetic components involved in both elongation and functionalization of the meroMA have been identified and are generally clustered in distinct transcriptional units. A large body of information has been generated on the enzymology of the MA biosynthetic pathway and on their genetic and biochemical/structural characterization as targets of several antitubercular drugs. The following sections are a comprehensive overview of MA structure, function, and biosynthesis. Special emphasis is given to recent work addressing the regulation of MA biosynthesis, adding new insights to our understanding of how pathogenic mycobacteria adapt their cell wall composition in response to environmental changes.

Classification of Mycobacterial lipids

Advancements in the field of mass spectrometry have enabled a much greater understanding as well as a detailed characterization of the Mycobacterium lipids. Sartain et al. [26] and Layre et al. [27] independently classified MTB lipids in three independent databases based on LIPID MAPS organizational tree (http://www.lipidmaps.org) and used the MTB lipid at the subclass level through LC-MS-based approaches [25,27,28]. Sartain et al. [26] created a database called MTB LipidDB representing 2512 lipid entities including searchable database files consisting of 14,489 mass entries whereas Layre et al. [27] developed two databases, viz. MycoMap (https://www.brighamandwomens.org/assets/BWH/research/pdfs/mycomap-database083111.pdf) and MycoMass (https://www.brighamandwomens.org/assets/BWH/research/pdfs/mycomass-database083111.pdf), representing more than 5000 molecular species of MTB lipids with high mass accuracy and precision representing 32,438 entries [28]. These lipids are classified into eight classes, namely, FA, glycerophospholipids, glycerolipids, saccharolipids, sphingolipids, polyketides (PK), prenol lipids (PR), and sterol lipids [29] based on LIPID MAPS. However, Sartain et al. (MTB LipidDB) considered only six out of eight categories from LIPID MAPS. Thus, MTB LipidDB consists of 15 main lipid classes, 46 lipid subclasses in addition to 16 level four lipid classes. In instances where MTB lipids did not fit into the LIPID MAPs classification system, 30 novel lipid subclasses (e.g., diacyltrehaloses) and 16 level 4 classes, for example, α-MAs were created. These novel subclasses and level 4 classes were developed for MTB LipidDB for its organization purposes only and have not been accepted by LIPID MAPS as such. The major hierarchical difference between LIPID MAPS and MTB LipidDB exists at the species level of classification [25]. MTB LipidDB uses lipid groups, which possess a unique chemical formula corresponding to a unique exact mass of instead of lipid species, which are used in case of LIPID MAPS. The lipid groups are characterized based on the head group composition. Unlike lipid species, an individual lipid group was not distinguished by stereochemistry, unsaturated bond position, or the length and position of individual fatty acyl substituents.

Sabareesh and Singh [30] developed a standalone software MS-LAMP through integration of MTB LipidDB and LIPID MAPS for the analysis of MTB lipids—"Mycobacterium tuberculosis Lipidome MS-LAMP specifically [31]. Additionally, one was generated for the general lipids and named as General Lipidome MS-LAMP." MTB lipidome contains 2518 lipids (Tables 1.2A and B), whereas General Lipidome consists of 37,572 lipids. Out of 37,572 lipids present in the General Lipidome, the masses of only 37,056 lipids are known, while the molecular masses of remaining 516 lipids are still