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tibr.docx2. Introduction
2.1 Tuberculosis
Tuberculosis (TB) is an infectious disease caused by a bacterium called Mycobacterium tuberculosis(M. tuberculosis or MTB). It typically affects the lungs (pulmonary TB) but can affect other sites as well (extrapulmonary TB) (WHO, 2015). TB is still one of the most important infectious causes of morbidity and death in the world (Raviglioneet al., 2012). The disease is spread in the air when people who are sick with pulmonary TB expel bacteria, for example by coughing or sneezing (Gengenbacher and Kaufmann, 2012). A person needs to inhale only a few of these bacteria to become infected. Overall, a relatively small proportion of people infected with M. tuberculosis will develop TB disease. However, the probability of developing TB is much higher among people with compromised immune systems, such as people living with HIV, malnutrition or diabetes. TB is also more common among men than women, and affects mainly adults in the most economically productive age groups (WHO, 2015).
2.1.1History
TB is an ancient scourge that has claimed innumerable victims throughout the history of mankind. The disease has been studied and written about for several millennia. Over the centuries, various cultures of the world gave the illness different names: yaksma(India), phthisis (Greek), consumptione(Latin), and White Plague (British) each of which allude to the “cachexia-like” or “consuming” affect of the illness. Contrary to previous beliefs that TB passed from animals to humans, recent work suggests that the relationship between animals and human tuberculosis may have involved co-evolution rather than a direct transmission from animals to human beings (Donoghue et al., 2004 and Shet, 2012).
One can hypothesize that the genus Mycobacterium originated more than 150 million years ago. TB in Egypt can be documented more than 5000 years ago. Typical skeletal abnormalities of tuberculosis, including characteristic Pott's deformities, have been found in Egyptian mummies and are clearly depicted in early Egyptian art. A new era of TBtreatment had dawned. Treatment to cure became the goal sought for every afflicted person in the world (Daniel, 2006).
2.1.2Diagnostics
The most common method for diagnosing TB worldwide is sputum smear microscopy (developed more than 100 years ago), in which bacteria are observed in sputum samples examined under a microscope. Following recent breakthroughs in TB diagnostics, the use of rapid molecular tests to diagnose TB and drug-resistant TB is increasing (WHO, 2015). Automated liquid culture systems are now the gold standard for the diagnosis of TB; they are substantially faster and have a 10% greater yield than solid media(Lawn and Zumla, 2011).
2.1.3 Epidemiology
TB remains a major global health problem, responsible for ill health among millions of people each year. TB ranks as the second leading cause of death from an infectious disease worldwide, after the human immunodeficiency virus (HIV). The latest estimates included in World Health Organization (WHO) report in 2014 are that there were 9.0 million new TB cases in 2013, more than half (56%) were in the South-East Asia and Western Pacific Regions and 1.5 million TB deaths each year. About 60% of TB cases and deaths occur among men, but the burden of disease among women is also high (WHO, 2015).
These large numbers of TB cases and deaths notwithstanding, 21 years on from the 1993 WHO declaration of TB as a global public health emergency, major progress has been made. Globally, the TB mortality rate (deaths per 100 000 population per year) has fallen by 45% since 1990 and TB incidence rates (new cases per 100 000 population per year) are decreasing in most parts of the world. Between 2000 and 2013, an estimated 37 million lives were saved through effective diagnosis and treatment (WHO, 2015).
Figure 1:global situation and trends.The TB mortality rate has decreased 45% since 1990 (WHO, 2015).
2.2 Bacteriology
M. tuberculosis was first identified by the German scientist Robert Koch, who announced the discovery on March 24, 1882 (Tufarielloet al., 2003).M. tuberculosis is an obligate intracellular pathogen that can infect several animal species, although human beings are the principal hosts. It is an aerobic, acid-fast, non-motile, non-encapsulated, non-spore forming bacillus, but has the capacity to become dormant – a nonreplicating state characterized by low metabolic activity and phenotypic drug resistance. It grows most successfully in tissues with high oxygen content, such as the lungs. Compared with the cell walls of other bacteria, the lipid-rich cell wall is relatively impermeable to basic dyes unless combined with phenol. Thus, M. tuberculosis is neither gram positive nor gram negative but is instead described as acid-fast, since once stained it resists decolourisation with acidified organic solvents. M. tuberculosis is typically visualized by Ziehl–Neelsen (acid-fast) staining and appears as a rod-shaped red bacillus. At 37 °C and under optimal availability of oxygen and nutrients, a single M. tuberculosis organism has a generation time of 18–24 h and forms a white to light-yellow colony on agar within 3–4 weeks, which is extremely slow compared with other bacteria (Escherichia coli divides every 20 minutes). This slow replication rate and ability to persist in a latent state result in the need for long durations of both drug therapy of tuberculosis and for preventive therapy in people with M. tuberculosis infection (Lawn and Zumla, 2011 and Gengenbacher and Kaufmann, 2012).
Figure 2: High-power micrograph of acid-fast bacilli in the sputum of a patient with TB, shown by Ziehl-Neelsen staining(Lawn and Zumla, 2011, p.61).
2.2.1 The Mycobacterium tuberculosis complex (MTBC)
MTBC refers to group of species that cause TB in both humans and animals (M. tuberculosis, Mycobacterium canettii, Mycobacterium africanum, Mycobacterium microti, M. bovis, Mycobacterium caprae and Mycobacterium pinnipedii) that are genetically very similar. From those species, M. tuberculosis is the most well known member, infecting more than one-third of the world’s human population; it is also able to infect animals that have contact with humans. M. canettii and M. africanum, closely related to M. tuberculosis, can also cause human TB (lower pathogenicity) and are usually isolated from African patients or African ancestry. Mycobacterium bovis, which predominantly causes TB in cattle. Moreover, M bovis has 99.9% gene sequence identity with M. tuberculosis. M.bovis was responsible for about 6% of all human tuberculosis deaths in Europe before the introduction of milk pasteurisation; subsequent attenuation of a laboratory strain of M bovis led to the development of the BCG vaccine in 1921, is the only vaccine used in TB prevention during early childhood. However, the efficacy of BCG is variable across the world and is generally ineffective against adult pulmonary TB which is the most prevalent form of the disease. M. capraehas been isolated only from goats. Mycobacterium microti, a less frequently isolated pathogen that traditionally causes TB in voles. Finally, M. pinnipedii infects seals (Cousins et al., 2003,Sierra, 2006,Lawn and Zumla, 2011 and Forrelladet al., 2013).
2.2.2 Taxonomy
Taxonomically, the bacillus causing TB in humans belongs to the genus Mycobacterium that includes several other human pathogens, within the family of Mycobacteriaceae, in the order Actinomycetales (Rastogiet al., 2001 and Gengenbacher and Kaufmann, 2012).
The genus Mycobacterium is one of the oldest defined. The generic name Mycobacterium initially designated a group of organisms that grew as mould-like pellicles on liquid media (Rastogiet al., 2001).
2.3 Infection cycle
2.3.1 Infection of the alveolar macrophage
In lung infections, M. tuberculosis is typically inhaled into the body through the mouth or nose, passes through the airways and reaches the alveolar space in the lungs (Gupta et al., 2012).
Macrophages operate as prime defense cells against microbial intruders (Reljicet al., 2010). These microorganisms are ingested by phagocytosis, a process consisting of membrane invaginations finally culminating in phagosome formation. This organelle is a part of the intracellular trafficking and transport system and the site to which the entire arsenal of host defense is targeted. Microorganisms captured in the phagosome experience increasing acidification, reactive oxygen and nitrogen species (ROS and RNS), hydrolytic enzymes, and cationic antimicrobial peptides (CAMPs). Acidic pH inside the maturing phagosome activates enzymes that degrade bacterial lipids and proteins. Simultaneously microbial metabolism is suppressed by such conditions. ROS and RNS generated by the phagosomal enzymes NADPH phagocyte oxidase and inducible nitric oxide synthase (iNOS) damage captured microorganisms by modification of their DNA, lipids, thiols, tyrosine side chains, and active centers of metal-dependent proteins. Further damage of ingested pathogens is incurred by CAMPs via permeabilization of their cell membrane. The final steps of bacterial destruction and clearance require phagolysosome fusion. All of the described destruction pathways are influenced by the host's immune status. Macrophage activation via cytokines, notably, interferon-gamma (IFN-γ), for instance, allows these host cells to control their intracellular predators (Gengenbacher and Kaufmann, 2012). However, M. tuberculosis has developed mechanisms to subvert the killing ability of the macrophage and can replicate intracellularly unless the macrophage is robust and able to mount a successful microbicidal response (Welin and Lerm, 2012).
2.3.2 M. tuberculosis inhibit phagosomal maturation
The best characterized survival mechanism of M. tuberculosis inside the macrophage is the inhibition of phagosomal maturation (Welinet al., 2011).Based on the fact thatphagosomal maturation is inhibited by M. tuberculosis, it is widely accepted that effective maturation of the phagosome would be lethal to the bacillus(Welin and Lerm, 2012). Welin and Lerm (2012) showed that a low pH does not harm M. tuberculosis; rather, it is the phagosomal functions that depend on acidity that are important for M. tuberculosis control. Moreover, phagosomal maturation can lead to control of M. tuberculosis growth inside the human macrophage, but not to mycobacterial killing. In an acidified phagosome, M. tuberculosis relies on a membrane protein for the maintenance of intrabacterial pH and on the production of the catalase peroxidase KatG, which can deactivate reactive oxygen species (Welin and Lerm, 2012). Furthermore, the bacterium has developed resistance mechanisms against reactive nitrogen intermediates, involving the mycobacterial proteasome (Darwin et al., 2003). Thus, M. tuberculosis has evolved mechanisms to evade many of the damaging components of the mature phagosome, and it is not clear under which circumstances phagosomal maturation can control or actually kill M. tuberculosis inside human macrophages. The metabolic activity of M. tuberculosis is low in mature phagosomes, but high in immature phagosomes. However, the fact that the metabolic activity of M. tuberculosis is lower in mature phagosomes does not automatically mean that phagosomal maturation leads to death of the bacillus (Welin and Lerm, 2012). It has been reported that M. tuberculosis is able to disrupt its phagosome and to translocate into the host cell cytoplasm (Lee et al., 2008).
M tuberculosis has adopted a strategy to avoid destruction by lysosomal enzymes, that of interrupting the process of phagosome maturation. The studies showed that phagosomes containing M tuberculosis do not fuse with lysosomes. This failure of phagolysosomal fusion occurred only with the ingestion of viable bacteria; dead bacteria were effectively delivered to lysosomes (Tufarielloet al., 2003).
2.3.3 Interference with antigen presentation
Antigen presentation is a crucial part of activating killing mechanisms in both innate and adaptive immunity. There are three known routes of antigen presentation. One is seen when M. tuberculosis peptide antigens get processed and presented on MHC Class II molecules by (Antigen presenting cells) APCs to CD4+ T lymphocytes, which induce the killing of either infected cell and/or intracellular bacteria on their own by secreting IFN-γ or TNF-α. The second is seen when M. tuberculosis peptide antigens are processed and displayed on MHC Class I molecules to cytolytic CD8+ T cells, which then kill the infected cells and/or their intracellular bacteria by secreting toxic granules. The third is when M. tuberculosis lipid or glycolipid antigens (particularly mycolic acids and ManLAM) are recognised on CD1 molecules by CD8+ T cells or natural killer (NK) cells, which then kill the infected cell through the release of cytotoxic granules (Gengenbacher and Kaufmann, 2012 and Guptaet al., 2012).
2.3.4 The granuloma
Granulomas are the characteristic feature of human latent pulmonary TB, the hallmark of TB. They are formed when innate and adaptive immune responses are not able to rapidly eliminate the pathogen. As a result, immune cells and cytokines accumulate around infected cells to control the spread of infection. In TB, mature granulomas evolve when macrophagesare recruited in response to M. tuberculosis infection, collected around the infected cell(s), andactivated by cytokines such as IFN-γ. Human tuberculous granulomas are typically well-organised, with a central core of macrophages surrounded by T and B lymphocytes, dendritic cells (DCs), endothelial cells, fibroblasts and granulocytes(Gupta et al., 2012).
In humans, the granuloma shows high plasticity and three major types can be distinguished. These are (1) solid granulomas which contain M. tuberculosis; (2) necrotic granulomas typical for early stages of active TB; (3) caseous granulomas during end-stage or severe TB. These different stages are not distinct entities but form a continuum(Gengenbacher and Kaufmann, 2012).
2.3.5 Disease reactivation
Persons with latent TB infection do not feel sick and do not have any symptoms. They are infected with M. tuberculosis, but do not have TB disease. They are not infectious and cannot spread TB infection to others(CDC, 2015).
Reactivation disease is difficult to distinguish clinically from re-infection. It occurs when latent bacteria from old, scarred granulomatous lesions are reactivated into an active, virulent state. Reactivation disease is most frequently triggered when the host immune response weakens or is suppressed – probably the stark case is that of HIV+ individuals, who are low in CD4+ T counts and face 10% risk per year of reactivation disease (Guptaet al., 2012 and Ernst, 2012).
Figure 3:Dynamic model of latent TB infection (LTBI) and active TB. In this model, LTBI is characterized by predominance of dormant bacilli and only very few active scouts capable of sensing the environment for growth attractiveness, which is low inside the solid granuloma. Some M. tuberculosiswake-up stochastically to maintain a small pool of scouts (left panel). Once the environment provides more favorable conditions, for example, in a caseating granuloma, scouts resuscitate dormant bacilli to become active probably by secretion of resuscitation-promoting factors (Rpfs) (right panel). A few organisms remain dormant and therefore phenotypically drug resistant, explaining the long treatment time required to cure active TB (Gengenbacher and Kaufmann, 2012, p. 524).
2.4 Treatment
Effective drug treatments were first developed in the 1940s. The most effective first-line anti-TB drug, rifampicin, became available in the 1960s. The currently recommended treatment for new cases of drug-susceptible TB is a six-month regimen of four first-line drugs: isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and pyrazinamide. Treatment success rates of 85% or more for new cases are regularly reported to WHO by its Member States (WHO, 2015).
Incomplete treatment or noncompliance of patients often leads to drug-resistant M. tuberculosis, which is conferred by genetic mutations (Gengenbacher and Kaufmann, 2012). Treatment for multidrug-resistant TB (MDR-TB), can be difficult, because loss of use of the 2 most potent anti-TB drugs (i.e., INH and RIF) means that only less effective “first-line” and “second-line” therapy, which is more toxic and less efficacious than INH and RIF, is available (Post et al., 2004). For most patients with MDR-TB, the current regimens recommended by WHO last 20 months, and treatment success rates are much lower (WHO, 2015).
The 20th WHO Expert Committee meeting on the selection and use of essential medicines has recommended the inclusion of five medicines (bedaquiline, delamanid, linezolid, rifapentine and terizidone) in the anti TB medicines section of the WHO Model List of Essential Medicines (EML). Rifapentine is indicated for the treatment of latent TB infection (LTBI). The other four medicines are used as part of treatment regimens for MDR-TB and extensively drug-resistant TB (XDR-TB) respectively, two conditions with high lethality and poor treatment outcomes. Bedaquiline and delamanid are two new drugs which have recently been granted conditional approval by stringent regulatory authorities for use in the treatment of MDR-TB. Linezolid and terizidone are old drugs and their off-label use for severe forms of drug-resistant TB has now also been approved by the EML (WHO, 2015).
2.4.1 Multidrug-resistant tuberculosis(MDR-TB)
Over the past two decades there has been the worldwide emergence of MDR-TB, then XDR-TB. MDR-TB is caused by M. tuberculosisthat is resistant to at least INH and RIF, and XDR-TB is caused by MDR-TB strains that have acquired additional resistance to any fluoroquinolone and one of three injectable aminoglycosides (capreomycin, kanamycin, and amikacin) (Gandhi et al., 2010, Lawn and Zumla, 2011 and Caminero and Daley, 2013). It is estimated that there were about 0·5 million incident cases of MDR-TB and 50 000 cases of XDR-TB in 2007 (Lawn and Zumla, 2011). Therefore, increasing a need to develop novel therapeutic strategies against TB infections(Jankuteet al., 2012).
In contrast to most bacteria, for M. tuberculosis, acquisition of drug resistance does not occur as a result of horizontal transfer of resistance-bearing genetic elements. Rather, acquisition of drug resistance by M. tuberculosis results from mutations (caused by nucleotide substitutions, insertions, or deletions) in specific resistance-determining regions of the genetic targets (or their promoters) or activating enzymes of anti-TB chemotherapeutic agents (Post et al., 2004).
With selection, drug-resistant organisms multiply to become the dominant strain; for example, isoniazid monotherapy selects for isoniazid-resistant mutants and allows them to multiply (Figure 4).Resistance to additional TB drugs can be added in a step-wise manner to create TB strains that are resistant to several drugs; for example, treatment of isoniazid-monoresistant TB with isoniazid and rifampicin selects for spontaneous rifampicin-resistant mutants. This process is referred to as acquired resistance (Figure 4), and is the rationale for the adage never add a single drug to a failing regimen (Gandhi et al., 2010).
Figure4:Acquisition of resistance.
I=isoniazid. R=rifampicin. P=pyrazinamide. MDR=multidrug resistant. TB=tuberculosis. (Gandhi et al., 2010, p.1834).
2.5 The Cell wall
The mycobacterial bacillus is encompassed by a remarkably elaborate cell wall structure (Alderwicket al., 2015).The compositional complexity of the mycobacterial cell envelope differs significantly from both Gram-negative and Gram-positive bacteria. Its cell wall has an unusually high content of lipids that act as a low permeability barrier and; hence, contribute to resistance to common antibiotics and chemotherapeutic agents (Jankuteet al., 2012). The cell wall is composed of two segments, upper and lower. Beyond the membrane is peptidoglycan (PG) in covalent attachment to arabinogalactan (AG), which in turn is attached to the mycolic acids with their long meromycolate and short α-chains. This is termed the cell wall core—the mycolyl arabinogalactan–peptidoglycan (mAGP) complex. The upper segment is composed of free lipids, some with longer fatty acids complementing the shorter α-chains, and some with shorter fatty acids complementing the longer chains. Interspersed somehow are the cell-wall proteins, the phosphatidylinositol mannosides (PIMs), the phthiocerol-containing lipids, lipomannan (LM), and lipoarabinomannan (LAM). When cell walls are disrupted, for instance extracted with various solvents, the free lipids, proteins, LAM, and PIMs are solubilized, and the mAGPcomplex remains as the insoluble residue. In simplistic terms, it can be considered that these lipids, proteins, and lipoglycans are the signaling, effector molecules in the disease process(Brennan, 2003), whereas the insoluble core is essential for the virulence and persistence of M. tuberculosis in hosts, and therefore the enzymes responsible for its biosynthesis are possible antibiotic targets(Jankuteet al., 2012 and Chen et al., 2013).
The lipid content of the cell envelope of mycobacteria may represent up to 40% of the cell dry mass, compared to less than 5% in other Gram-positive bacteria and only 10% in Gram-negative bacteria. The permeability of cell walls of mycobacteria was found to be 10–100-fold lower than that of the notably impermeable bacillus P. aeruginosa (Daffé, 2015).
Figure 5: Cell envelope of M. tuberculosis. The cell wall core consists of peptidoglycan connected to arabinogalactan and covalently attached hydrophobic mycolic acids, which are intercalated with glycolipids and a diverse repertoire of complex lipids. LAM is linked to the plasma membrane via the lipid portion of the phosphatidylinositol anchor and serves as a modulin with immunoregulatory effects (Jankuteet al., 2012, p.130).
2.6 Peptidoglycan(PG)
2.6.1 Structure of PG
PG (or murein) is a complex macromolecular structure located on the outside of the cytoplasmic membrane of almost all eubacteria, and it is a major component of the cell wall of almost all eubacteria(van Heijenoort, 2001 and Barreteauet al., 2008). Its main function is to preserve cell integrity by withstanding the internal osmotic pressure. It is also responsible for the maintenance of a defined cell shape and is intimately involved in the cell division process(van Heijenoort, 2001).
Mycobacterial PG forms the backbone of the mAGP complex (Chatterjee, 1997) and is composed of alternating N-acetylglucosamine (GlcNAc) and modified N-acetylmuramic acid (MurNAc) residues, linked in a β (1→4) configuration(Alderwicket al., 2015). The carboxyl group of each N-acetylmuramic acid residue is substituted by a peptide subunit (van Heijenoort, 2001).
Unlike E. coli PG, the muramic acid residues in both M. tuberculosis and Mycobacterium smegmatis contain a mixture of N-acetyl and N-glycolyl derivatives, whereby the N-acetyl function has been oxidized to aN-glycolyl function to form MurNGly(Mahapatraet al., 2000 and Alderwicket al., 2015).
Although the precise function of the N-glycolyl modifications is yet to be elucidated, it has been hypothesized that these additional glycolyl-containing residues might have the potential for additional hydrogen bonding interactions, strengthening the mesh-like structure of the PG layer, as well as possibly protecting the organism from degradation via lysozyme. An additional deviation from E. coli PG is the use of the muramic acid residues as attachment sites for the galactan domain of the arabinogalactan(Alderwicket al., 2015).
2.7 Biosynthesis of PG
2.7.1 Biosynthesis of UDP- N-acetylglucosamine
The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasmof all bacteria (Barreteauet al., 2008), and the activated sugar nucleotide UDP-GIcNAc is usually considered to be the “start point” of PG anabolism. UDP-GIcNAcis synthesized from D-fructose-6-phosphate in four steps and requires three enzymes. GlmS is an aminotransferase that converts D-fructose-6-phosphate to D-glucosamine-6-phosphate, and although a clear ortholog is present in M. tyberculosis, there has been no investigation of this gene or protein to date (Durand et al., 2008 and Alderwicket al., 2015). GlmM is a mutase enzyme that converts D-glucosamine-6-phosphate to D-glucosamine-1-phosphate and has been shown to be an essential gene in M. smegmatis. Indeed, the conversion of D-glucosamine-6-phosphate to D-glucosamine-1-phosphate is unique to prokaryotes and is considered a potential drug target(Li et al., 2012 and Alderwicket al., 2015). GlmUis a bifunctional enzyme that carries out both acetylation and uridylation reactions, ultimately forming UDP-GIcNAc(Alderwicket al., 2015). Activity of GlmU is required for growth of M. smegmatis as the bacteria did not grow in the absence of active GlmU enzyme(Zhang et al., 2008).
2.7.2 Biosynthesis of UDP- N-acetylmuramic acid
UDP-MurNAc is formed from UDP-GIcNAc in a two-steps process involving the catalytic activities of two enzymes, MurA and MurB. MurA is a UDP-GIcNAcenolpyruvyl transferase that catalyzes the transfer of enolpyruvate from phosphoenoylpyruvate to the 3-position of the N-acetylglucosamine moiety of UDP-GIcNAc, liberating inorganic phosphate in the process. MurB is a reductase that uses NADPH as an electron donor to convert the enolpyruvate moiety to D-lactoyl, thus delivering UDP-MurNAc as a final product(Alderwicket al., 2015).
In mycobacteria, MurX(ususally termed MraY in other prokaryotes) is an integral membrane protein that transfers the phosphor-MurNAc-pentapeptide to a decaprenol phosphate lipid. The result is the displacement of the UDP nucleotide moiety with a decaprenyl lipid carrier, thus forming lipid I from Park's nucleotide. MurG is a GT-B glycosyltransferase that uses UDP-GIcNAc as a substrate to form a β (1→4) glycosidic bond between GIcNAc and either MurNAc or MurNGIyc sugar of lipid I. MurG marks the final “step” in the cytoplasmic pathway of PG biosynthesis, and its product is a cell wall intermediate termed lipid II (Alderwicket al., 2015). Therefore, MurG it is a target for the development of new antibacterial agents (Trunkfieldet al., 2010).
2.7.3 The latter stages of peptidoglycan assembly
Lipid II is transferred by an as-yet-unknown mechanism through the hydrophobic environment of the cytoplasmic membrane to externally located sites where polymerization of the disaccharide-peptide monomer involves two major types of membrane-bound activities: glycosyltransferases (GTs), which catalyze the formation of the linear glycan chains, and transpeptidases, which catalyze the formation of the peptide cross-bridges and the binding of nascent peptidoglycan to the preexisting cell wall (van Heijenoort, 2007).
Several biochemical and molecular genetic investigations in other non-mycobacterial prokaryotes have identified both MurJand FtsW proteins as being candidate proteins that translocate lipid II across the bacterial cytoplasmic membrane (Ruiz, 2008, Mohammadiet al., 2011 and Alderwicket al., 2015).
FtsW belongs to the shape, elongation, division, and sporulation (SEDS) family of proteins, all of which are integral membrane proteins involved in translocation of molecules across the cytoplasmic membrane. Interestingly, RodA is also a member of the SEDS family, and a recent study has shown that this protein also contributes lipid II flippase activity (Alderwicket al., 2015).
The final stages of peptidoglycan synthesis are carried out by penicillin-binding proteins (PBPs), a group of serine acyl transferases involved in cell wall expansion, cell shape maintenance, septum formation and cell division (Dasguptaet al., 2006).
2.8 Arabinogalactan(AG)
2.8.1 StructureofAG
AG is a key structural component of the mAGP complex that constitutes approximately 35% of the mycobacterial cell wall. This heteropolysaccharide plays an important role in covalently anchoring the mycolic acid layer to the inner PG layer, and is unique in that all of its sugars(arabinose and galactose) are present in the furanose ring form (Jankuteet al., 2012).In addition, AG is a highly branched structure is covalently attached to peptidoglycan (PG) via a phosphodiester bond to approximately 10 to 12% of the muramic acid residues (Jankuteet al., 2014). Moreover, unlike most bacterial polysaccharides, AG lacks repeating units and is instead composed of a few distinct structural motifs (Jankuteet al., 2012).
The galactan component of AG is present in the form of approximately 30 alternating β(1→5) and β(1→6) galactofuranosyl (Galf) residues combined in a linear fashion(Figure 5). Three arabinan chains, each containing approximately 30 arabinofuranosyl (Araf) residues, are linked to this linear galactan at the C-5 of some of the β(1→6) Galf residues. The arabinan domain, in contrast to the linear galactan, is highly branched (Jankuteet al., 2012).
At the nonreducing end of AG, the highly branched arabinan domain is covalently attached to the mycolic acids of the outer membrane, thus completing the whole mAGP complex (Figure 5)(Jankuteet al., 2012). The molecular structure of AG can be segmented into three separate constituents: the liker unit (LU), galactan, and arabinan (Alderwicket al., 2015).
2.9 Biosynthesis of AG
2.9.1 Linker unit biosynthesis
The whole AG component of the mAGP complex is synthesized on a decaprenyl phosphate (C50-P) lipid carrier, followed by an attachment to both PG and mycolates. The biosynthesis of the linker unit begins with the transfer of GlcNAc-1-P from UDP-GlcNAc to the lipid carrier by a GlcNAc transferase WecA (Rv1302). M. tuberculosis WecA shows significant homology to other WecA proteins from Gram-negative bacteria, such as Escherichia coli, where it catalyzes the first step in the biosynthesis of lipopolysaccharide O-antigen. Recent knockout mutant studies together with scanning electron microscopy and transmission electron microscopy (TEM) have shown that wecA of M. smegmatis (MSMEG_4947) is essential for mycobacterial growth . The rhamnosyltransferaseWbbL encoded by Rv3265c then attaches the rhamnosyl residue (Rha) to the C50-P-P-GlcNAc yielding C50-P-P-GlcNAc-Rha, thus forming the linker unit of AG(Birch et al., 2007, Jinet al., 2010 and Jankuteet al., 2012).
2.9.2 Galactan biosynthesis
The assembly of the linker unit serves as an acceptor for the sequential addition of up to a total of 30 Galf residues combined in a linear fashion. Two bifunctionalgalactofuranosyltransferases involved in cell wall biosynthesis have been identified to date, both of which utilize UDP-Galf as its high-energy substrate. GlfT1 (Rv3782) specifically recognizes the linker unit, C50-P-P-GlcNAc-Rha, and catalyzes the transfer of the first two Galf residues to the C-4 position of Rha. The remaining Galf residues are added by the second transferase GlfT2 (Rv3808c). It was demonstrated that GlfT2 acts both as a UDP-Galf:β-D-(1→5) galactofuranosyltransferase and a UDP-Galf:β-D-(1→6) galactofuranosyltransferase, and is responsible for the bulk of galactan biosynthesis by sequentially polymerizing the galactan polysaccharide in alternating β(1→5) and β(1→6) linkages(Kremer et al., 2001, Birch et al., 2007 and Jankuteet al., 2012).
2.9.3 Arabinan biosynthesis
Studies in vitro suggest that Araf residues are transferred directly onto a previously generated intermediate, C50-P-P-GlcNAc-Rha-Galf 30, utilizing the only known mycobacterial Araf donor, decaprenylmonophosphoryl-D-arabinose (DPA). The biosynthesis of this sugar donor has recently been investigated in detail, leading to a better understanding of AG formation in mycobacteria. DPA is synthesized from 5-phosphoribosyl-1-pyrophosphate (pRpp) to decaprenylphosphoryl-5-phosphoribose (DPPR), a reaction catalyzed by UbiA (Rv3806c). The disruption of CorynebacteriumglutamicumubiA (NCgl2781) resulted in a complete loss of cell wall arabinan, indicating that DPA is the only Araf sugar donor in AG biosynthesis (Jankuteet al., 2012).
EMB is a bacteriostatic agent extensively used as a front-line drug for the treatment of TB since 1966 (Jankuteet al., 2012). EMB specifically inhibits the synthesis of the arabinan of AG and of lipoarabinomannan, apparently through its action on a family of arabinosyltransferases(Takayama and Kilburn, 1989 and Mikusováet al., 1996). Moreover, EMBinhibits of mycolic acid transfer into the cell wall and the simultaneous accumulation of trehalose-monomycolate (TMM), trehalose-dimycolate (TDM), and free mycolic acids (Alderwicket al., 2015).
3. PIMs, LM, and LAM
Other cell wall-associated lipids, such as PIMs,LM and LAM, are also found in the cell wall(Mishra et al., 2011). They are the most abundant noncovalently linked glycophospholipids in the mycobacterial cell wall. These phospholipids have a phosphatidyl-myo-inositol (PI) backbone and are essential for the viability of mycobacteria in the host. PIMs are the key precursors of LAM, the end product of the biosynthetic pathway, which plays a role in recognition of M. tuberculosis by macrophages and also accounts for its cross-protective immunity. Modifications of the PI anchor follow the order: PI → PIM → LM → LAM as deduced from pulse chase labeling experiments, which were further supported by mutational studies (Jankuteet al., 2012).
In addition to their physiological function, these complex glycoconjugates play a key role in modulating the host response during infection. PIMs, LM and LAM all display several immunomodulatory properties by interaction with different receptors of the immune system(Mishra et al., 2011 and Jankuteet al., 2012). LAM is a major immunomodulatory found in the cell envelope of M. tuberculosis and one of the virulence factor associated with M. tuberculosis(Vergneet al., 2014).
LAM are lipoglycans ubiquitously found in the envelope of mycobacteria. They are composed of a carbohydrate backbone made of a D-mannan core and a D-arabinan domain, a mannosyl-phosphatidylinositol (MPI) anchor at the reducing end of the mannan core and capping motifs. The arabinan domain is capped by either mannosyl (ManLAM) or phosphoinositide residues (PILAM). ManLAMhave been found in the slow-growing mycobacteria, such as M. tuberculosis, whereas PILAM have been identified in fast-growing and avirulent species, such as Mycobacterium smegmatis (Quesniauxet al., 2004 and Jankuteet al., 2012).ManLAM immunosuppressive activities rely on the presence of the mannose caps (Vergneet al., 2014).
In M. tuberculosis, ManLAM and its precursors initiate an immunosuppressive response including inhibition of pro-inflammatory cytokines production, inhibition of phagosome maturation and inhibition of macrophage apoptosis. Therefore, enable the persistence of mycobacteria in phagosomes(Nigouet al., 2002, Mishra et al., 2011, Jankuteet al., 2012 and Vergneet al., 2014).
The LM- and LAM-induced response to inhibit macrophage activation affects the signaling cascades initiated by Toll-like receptors and T-cell receptors. For example, LM activates TLR2 receptors on the antigen presenting cells via the adaptor protein myeloid differentiation factor 88 (MyD88), which shifts the immune response from a protective Th1 adaptive immune response to the nonprotective Th2 response. Since the Th2 response confers humoral immunity, which is ineffective against the intracellular pathogens, this shift favors the survival of M. tuberculosis in the macrophage (Jankuteet al., 2012).
3.1 Mycolic Acid
Mycolic acids are vital components of the waxy cell wall of M. tuberculosis. Synthesized by the concerted and sequential action of three enzymatic units, fatty acid synthase I (FAS-I), fatty acid synthase II (FAS-II), and a polyketide synthase (Pks13), these very long-chain (C30-C90) α-alkyl, β-hydroxy fatty acids are essential for viability and virulence (Varela et al., 2012).
Three distinct structural classes of mycolic acids are found in M. tuberculosis, and they are α-, methoxy- and keto-mycolic acids, as shown in (Figure 6). The α-mycolic acid is the most abundant form (>70%), whereas methoxy- and keto-mycolic acids are the minor components (10 to 15%) (Takayamaet al., 2005).
Figure 6:Chemical structures of mycolic acids from M. tuberculosis(Takayamaet al., 2005, p.82).
3.2 Cord Factor (Trehalose-6,6-dimycolate) (TDM)
TDM, the mycobacterial cord factor, is the most abundant cell wall lipid of virulent mycobacteria, is sufficient to cause granuloma formation, and has long been known to be a major virulence factor of M. tuberculosis. Moreover, is toxic to mammalian cells(Rajni, Rao and Meena, 2011 and Lang, 2013).
TDM has long chain lipids as structural component of the hydrophobic cell wall which is found to be crucial for the survival of mycobacteria within phagosomes of host.TDM inhibits the process of phagosome-lysosome fusion and is thus a key compound for the survival of the bacillus inside the host´s phagosomes.
