Target For Anti-tuberculosis Drugs Research Articles

Identification and structural characterization of a histidinol phosphate phosphatase from Mycobacterium tuberculosis

The absence of a histidine biosynthesis pathway in humans, coupled with histidine essentiality for survival of the important human pathogen Mycobacterium tuberculosis (Mtb), underscores the importance of the bacterial enzymes of this pathway as major antituberculosis drug targets. However, the identity of the mycobacterial enzyme that functions as the histidinol phosphate phosphatase (HolPase) of this pathway remains to be established. Here, we demonstrate that the enzyme encoded by the Rv3137 gene, belonging to the inositol monophosphatase (IMPase) family, functions as the Mtb HolPase and specifically dephosphorylates histidinol phosphate. The crystal structure of Rv3137 in apo form enabled us to dissect its distinct structural features. Furthermore, the holo-complex structure revealed that a unique cocatalytic multizinc-assisted mode of substrate binding and catalysis is the hallmark of Mtb HolPase. Interestingly, the enzyme-substrate complex structure unveiled that although monomers possess individual catalytic sites they share a common product-exit channel at the dimer interface. Furthermore, target-based screening against HolPase identified several small-molecule inhibitors of this enzyme. Taken together, our study unravels the missing enzyme link in the Mtb histidine biosynthesis pathway, augments our current mechanistic understanding of histidine production in Mtb, and has helped identify potential inhibitors of this bacterial pathway.

Multisystem Analysis of Mycobacterium tuberculosis Reveals Kinase-Dependent Remodeling of the Pathogen-Environment Interface.

ABSTRACTTuberculosis is the leading killer among infectious diseases worldwide. Increasing multidrug resistance has prompted new approaches for tuberculosis drug development, including targeted inhibition of virulence determinants and of signaling cascades that control many downstream pathways. We used a multisystem approach to determine the effects of a potent small-molecule inhibitor of the essential Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB. We observed differential levels of phosphorylation of many proteins and extensive changes in levels of gene expression, protein abundance, cell wall lipids, and intracellular metabolites. The patterns of these changes indicate regulation by PknA and PknB of several pathways required for cell growth, including ATP synthesis, DNA synthesis, and translation. These data also highlight effects on pathways for remodeling of the mycobacterial cell envelope via control of peptidoglycan turnover, lipid content, a SigE-mediated envelope stress response, transmembrane transport systems, and protein secretion systems. Integrated analysis of phosphoproteins, transcripts, proteins, and lipids identified an unexpected pathway whereby threonine phosphorylation of the essential response regulator MtrA decreases its DNA binding activity. Inhibition of this phosphorylation is linked to decreased expression of genes for peptidoglycan turnover, and of genes for mycolyl transferases, with concomitant changes in mycolates and glycolipids in the cell envelope. These findings reveal novel roles for PknA and PknB in regulating multiple essential cell functions and confirm that these kinases are potentially valuable targets for new antituberculosis drugs. In addition, the data from these linked multisystems provide a valuable resource for future targeted investigations into the pathways regulated by these kinases in the M. tuberculosis cell.

Activity loss by H46A mutation in Mycobacterium tuberculosis isocitrate lyase is due to decrease in structural plasticity and collective motions of the active site

Mycobacterium tuberculosis isocitrate lyase (MtbICL) is a crucial enzyme of the glyoxylate cycle and is a validated anti-tuberculosis drug target. Structurally distant, non-active site mutation (H46A) in MtbICL has been found to cause loss of enzyme activity. The aim of the present work was to explore the structural alterations induced by H46A mutation that caused the loss of enzyme activity. The structural and dynamic consequences of H46A mutation were studied using multiple computational methods such as docking, molecular dynamics simulation and residue interaction network analysis (RIN). Principal component analysis and cross correlation analysis revealed the difference in conformational flexibility and collective modes of motions between the wild-type and mutant enzyme, particularly in the active site region. RIN analysis revealed that the active site geometry was disturbed in the mutant enzyme. Thus, the dynamic perturbation of the active site led to enzyme transition from its active form to inactive form upon mutation. The computational analyses elucidated the mutant-specific conformational alterations, differential dominant motions, and anomalous residue level interactions that contributed to the abrogated function of mutant MtbICL. An understanding of interactions of mutant enzymes may help in modifying the existing drugs and designing improved drugs for successful control of tuberculosis.

Identification of new antibacterial targets in RNA polymerase of Mycobacterium tuberculosis by detecting positive selection sites

Bacterial RNA polymerase (RNAP) is an effective target for antibacterial treatment. In order to search new potential targets in RNAP of Mycobacterium, we detected adaptive selections of RNAP related genes in 13 strains of Mycobacterium by phylogenetic analysis. We first collected sequences of 17 genes including rpoA, rpoB, rpoC, rpoZ, and sigma factor A-M. Then maximum likelihood trees were constructed, followed by positive selection detection. We found that sigG shows positive selection along the clade (M. tuberculosis, M. bovis), suggesting its important evolutionary role and its potential to be a new antibacterial target. Moreover, the regions near 933Cys and 935His on the rpoB subunit of M. tuberculosis showed significant positive selection, which could also be a new attractive target for anti-tuberculosis drugs.

Alterations in conformational topology and interaction dynamics caused by L418A mutation leads to activity loss of Mycobacterium tuberculosis isocitrate lyase

Mycobacterium tuberculosis isocitrate lyase (MtbICL) is a key enzyme of the glyoxylate cycle that catalyzes the cleavage of isocitrate to succinate and glyoxylate and is a potential antituberculosis drug target. The aim of this research was to explore the structural alterations induced by L418A point mutation that caused the loss of enzyme activity. In-depth structural analyses were carried out for understanding the influence of L418A mutation using techniques, viz. molecular dynamics, principal component analysis, time-dependent secondary structure, residue interaction network and molecular docking. Since L418A mutation site is structurally far from the active site, it cannot influence the binding of the substrate directly. Our results showed that collective motions, residual mobility, and flexibility of the enzyme increased upon mutation. The mutated residue changed the global conformational dynamics of the system along with the residue-residue interaction network, leading to a loss of the enzyme activity. The docking results suggest that L418A mutation influenced the binding interactions of the substrate with several residues in the active site of MtbICL. This study provides information on the structural dynamics of MtbICL and highlights the importance of residue level interactions in the protein. Thus, our results may provide significant guidance to the scientific community engaged in designing potent inhibitors targeting MtbICL.

Structural and Dynamics Perspectives on the Binding of Substrate and Inhibitors in Mycobacterium tuberculosis DHFR.

Dihydrofolate reductase (DHFR), an essential enzyme in the folate pathway, is a potential target for new anti-tuberculosis drugs. Fifteen crystal structures of Mycobacterium tuberculosis DHFR complexed with NADPH and various inhibitors are available in the RCSB Protein Data Bank, but none of them is a substrate binding structure. Therefore, we performed molecular dynamics simulations on ternary complexes of M. tuberculosis DHFR:NADPH with a substrate (dihydrofolate) and each of three competitive inhibitors in 2,4-diaminopyrimidine series (P1, P157, and P169), in order to gain insight into the inhibition-mechanism of DHFR in the folate pathway. The binding energy and thermodynamics values of each system were calculated by the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method. The dynamics of the enzyme and the motion of each amino acid residue at the active site were examined. The key factors that promote the binding of P157 and P169 on M. tuberculosis DHFR (mtbDHFR) reveal opportunities for using these compounds as novel anti-tuberculosis drugs.

Inhibition of M. tuberculosis β-ketoacyl CoA reductase FabG4 (Rv0242c) by triazole linked polyphenol-aminobenzene hybrids: comparison with the corresponding gallate counterparts.

Herein we report six novel triazole linked polyphenol-aminobenzene hybrids (3-8) as inhibitors of Mycobacterium tuberculosis FabG4 (Rv0242c), a less explored β-ketoacyl CoA reductase that has immense potential to be the future anti-tuberculosis drug target due to its possible involvement in drug resistance and latent infection. Novel triazole linked polyphenol-aminobenzene hybrids have been synthesized, characterized and evaluated for their inhibitory activity against FabG4. All of them inhibit FabG4 at low micromolar concentrations. In silico docking study has been carried out to explain the experimental findings. A comparative study of these new inhibitors with previously reported gallate counterparts leads to structure-activity relations (SAR) of substituent linked to N-1 of triazole ring.

Synthesis of a poly-hydroxypyrolidine-based inhibitor of Mycobacterium tuberculosis GlgE.

Long treatment times, poor drug compliance, and natural selection during treatment of Mycobacterium tuberculosis (Mtb) have given rise to extensively drug-resistant tuberculosis (XDR-TB). As a result, there is a need to identify new antituberculosis drug targets. Mtb GlgE is a maltosyl transferase involved in α-glucan biosynthesis. Mutation of GlgE in Mtb increases the concentration of maltose-1-phosphate (M1P), one substrate for GlgE, causing rapid cell death. We have designed 2,5-dideoxy-3-O-α-d-glucopyranosyl-2,5-imino-d-mannitol (9) to act as an inhibitor of GlgE. Compound 9 was synthesized using a convergent synthesis by coupling thioglycosyl donor 14 and 5-azido-3-O-benzyl-5-deoxy-1,2-O-isopropylidene-β-d-fructopyranose (23) to form disaccharide 24. A reduction and intramolecular reductive amination transformed the intermediate disaccharide 24 to the desired pyrolidine 9. Compound 9 inhibited both Mtb GlgE and a variant of Streptomyces coelicolor (Sco) GlgEI with Ki = 237 ± 27 μM and Ki = 102 ± 7.52 μM, respectively. The results confirm that a Sco GlgE-V279S variant can be used as a model for Mtb GlgE. In conclusion, we designed a lead transition state inhibitor of GlgE, which will be instrumental in further elucidation of the enzymatic mechanism of Mtb GlgE.

Screening essential genes of Mycobacterium tuberculosis with the pathway enrichment method

The number of effective drugs for the prevention and control of tuberculosis is very limited. Therefore, high-throughput screening for Mycobacterium tuberculosis drug targets is critical. In addition, determining the essential gene cluster is important for both understanding a survival mechanism and finding novel molecular targets for anti-tuberculosis drugs. In this study, we applied the pathway enrichment method to perform high throughput screening of genes encoding key molecules for potential drug targets for M. tuberculosis. Our results indicated 122 genes that existed in more than three pathways, while four existed in 11 pathways. We predicted 55 genes that are potentially essential genes. Four of them, namely, Rv0363c, Rv0408, Rv0409 and Rv0794c, had the highest probability to be essential genes, and thus further experimental validation is warranted.

Synthesis of a C-phosphonate mimic of maltose-1-phosphate and inhibition studies on Mycobacterium tuberculosis GlgE

The emergence of extensively drug-resistant tuberculosis (XDR-TB) necessitates the need to identify new anti-tuberculosis drug targets as well as to better understand essential biosynthetic pathways. GlgE is a Mycobacterium tuberculosis (Mtb) encoded maltosyltransferase involved in α-glucan biosynthesis. Deletion of GlgE in Mtb results in the accumulation of M1P within cells leading to rapid death of the organism. To inhibit GlgE a maltose-C-phosphonate (MCP) 13 was designed to act as an isosteric non-hydrolysable mimic of M1P. MCP 13, the only known inhibitor of Mtb GlgE, was successfully synthesized using a Wittig olefination as a key step in transforming maltose to the desired product. MCP 13 inhibited Mtb GlgE with an IC50=230±24μM determined using a coupled enzyme assay which measures orthophosphate release. The requirement of M1P for the assay necessitated the development of an expedited synthetic route to M1P from an intermediate used in the MCP 13 synthesis. In conclusion, we designed a substrate analogue of M1P that is the first to exhibit Mtb GlgE inhibition.

Comparative Genomic and Proteomic Anatomy of Mycobacterium Ubiquitous Esx Family Proteins: Implications in Pathogenicity and Virulence

Secreted proteins are among the most important molecules involved in host-pathogen interaction of Mycobacterium tuberculosis, the etiological agent of human tuberculosis (TB). M. tuberculosis encodes five types of VII secretion systems (ESX-1 to ESX-5) responsible for the exportation of many proteins. This system mediated substrates including members of the Esx family implicated in tuberculosis pathogenesis and survival within host cells. However, the distribution and evolution of this family remain elusive. To explore the evolution and distribution of Esx family proteins, we analyzed all available Mycobacteria genomes. Interestingly, amino mutations among M. tuberculosis esx family proteins may relate to their functions. We further analyzed the differences between pathogenic Mycobacteria, the attenuated Mycobacteria and non-pathogenic Mycobacteria. The stability, the globular domains and the phosphorylation of serine/threonine residues of M. tuberculosis esx proteins with their homologies among other Mycoabcteria were analyzed. Our comparative genomic and proteomic analysis found that the change of stability, gain or loss of globular domains and phosphorylation of serine/threonine might be responsible for the difference between the pathogenesis and virulence of the esx proteins and its homolog widespread among Mycobacteria and related species, which may provide clues for novel anti-tuberculosis drug targets.

Discrimination of large maltooligosaccharides from isobaric dextran and pullulan using ion mobility mass spectrometry

RATIONALEIon mobility mass spectrometry (IMMS) has previously been shown to resolve small isobaric oligosaccharides, but larger alpha-oligoglucans are also abundant in biology and are of industrial importance. If conformational differences between such isomers are retained in the gas phase, IMMS could be used to address questions in biology and industry.METHODSNegative mode electrospray ionization (ESI) travelling-wave IMMS was used to resolve large isobaric α-glucan ions on the basis of their different gas-phase conformations. α,ω-Dicarboxy-terminated polystyrene was used to calibrate the instrument allowing the collision cross-sections (CCSs) of ions to be determined.RESULTSα-1,4-Linked maltooligosaccharides with a degree of polymerisation of up to 35 could be discriminated from α-1,6-linked dextran and α-1,4/1,6-linked pullulan using IMMS. Fragmentation spectra of ions separated by IMMS could also distinguish isomers. Two conformational isomers of maltohexaose were resolvable by IMMS, likely reflecting extended and V6 helical conformations. IMMS was also able to identify a product within a mixture of maltooligosaccharides treated with the potential anti-tuberculosis drug target Mycobacterium tuberculosis GlgB branching enzyme.CONCLUSIONSBiological samples of complex isobaric oligosaccharides can be analysed using IMMS in the negative mode providing facile analyses and high sensitivity without the need for either derivatisation or chromatographic separation. © 2013 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.

Targeting Type VII/ESX Secretion Systems for Development of Novel Antimycobacterial Drugs

The emergence of multi- and extensively-drug resistant strains of Mycobacterium tuberculosis makes the development of novel anti-tubercular compounds and the identification of alternative mycobacterial drugable targets urgent priorities. Recently, type VII secretion systems (T7SS) have been discovered in mycobacteria. The genome of M. tuberculosis encodes 5 of such systems (ESX-1 to -5), three of which have been characterized and shown to be essential for viability (ESX-3, ESX-5) or virulence (ESX-1, ESX-5). Because of their crucial role in host-pathogen interactions as well as their involvement in basic biological processes of tubercle bacilli, T7SS/ESX represent promising targets for novel anti-tuberculosis drugs. Here, we review the current knowledge of the T7SS/ESX and their impact on M. tuberculosis physiology and virulence. Finally, we discuss the possible approaches to develop T7SS/ESX inhibitors.

Biochemical characterization and ligand-binding properties of trehalose-6-phosphate phosphatase from <italic>Mycobacterium tuberculosis</italic>

Trehalose-6-phosphate phosphatase (TPP) is an essential enzyme for growth of mycobacteria, which has been identified to be a potential anti-tuberculosis drug target. However, the biochemical and ligand-binding properties and the 3D structure of TPP remain unclear so far. In the present study, we expressed the recombinant TPP protein from Mycobacterium tuberculosis (otsB2/Rv3372). Results from the far-ultraviolet circular dichroism experiments indicated that the secondary structure of TPP was rich in α-helix with a lower structural stability (Cm = 2.099 ± 0.134 M). Ligand-binding assay by isothermal titration calorimetry demonstrated that the recombinant TPP protein could bind with trehalose-6-P in the presence of Mg(2+) (Kd = 39.52 ± 1.78 μM) with a molar ratio of 1 : 1. In addition, the 3D structure of TPP was modeled by I-TASSER, indicating that the TPP protein was composed of a hydrolase domain, a cap domain, and an N-terminal domain. Flexible docking was further conducted by using the Simulations/Dock module of the Molecular Operating Environment software. The binding pocket of TPP for both trehalose-6-P and Mg(2+) was determined, which was located on the interface between the hydrolase domain and the cap domain. Asp149, Gly186, Arg187, Arg291, and Glu295 were identified to be the key residues for TPP binding with trehalose-6-P. This work may lay the basis for further structural and functional studies of TPP and TPP-related novel drug development.

4′-Phosphopantetheinyl Transferase PptT, a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo

The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, contains lipids with unusual structures. These lipids play a key role in both virulence and resistance to the various hostile environments encountered by the bacteria during infection. They are synthesized by complex enzymatic systems, including type-I polyketide synthases and type-I and -II fatty acid synthases, which require a post-translational modification to become active. This modification consists of the covalent attachment of the 4′-phosphopantetheine moiety of Coenzyme A catalyzed by phosphopantetheinyl transferases (PPTases). PptT, one of the two PPTases produced by mycobacteria, is involved in post-translational modification of various type-I polyketide synthases required for the formation of both mycolic acids and lipid virulence factors in mycobacteria. Here we identify PptT as a new target for anti-tuberculosis drugs; we address all the critical issues of target validation to demonstrate that PptT can be used to search for new drugs. We confirm that PptT is essential for the growth of M. bovis BCG in vitro and show that it is required for persistence of M. bovis BCG in both infected macrophages and immunodeficient mice. We generated a conditional expression mutant of M. tuberculosis, in which the expression of the pptT gene is tightly regulated by tetracycline derivatives. We used this construct to demonstrate that PptT is required for the replication and survival of the tubercle bacillus during the acute and chronic phases of infection in mice. Finally, we developed a robust and miniaturized assay based on scintillation proximity assay technology to search for inhibitors of PPTases, and especially of PptT, by high-throughput screening. Our various findings indicate that PptT meets the key criteria for being a therapeutic target for the treatment of mycobacterial infections.

Development of a Colorimetric Assay and Kinetic Analysis for Mycobacterium tuberculosis D-glucose-1-phosphate Thymidylyltransferase

dTDP-L-rhamnose as a sugar donor provides L-rhamnosyl residue in the synthesis of disaccharide linker (D-N-acetylglucosamine-L-rhamnose), the key structure of the Mycobacterium tuberculosis cell wall. Four enzymes are involved in the formation of dTDP-L-rhamnose and D-glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the first step of D-glucose-1-phosphate and dTTP to dTDP-D-glucose and PPi. The previous studies on RmlA essentiality proved RmlA as a potential target for antituberculosis drugs. However, there has not been a suitable assay for RmlA to screen inhibitors currently. In this study, the authors reported a microtiter plate–based colorimetric assay for RmlA enzyme activity. Using this assay, the kinetic properties of M. tuberculosis RmlA including initial velocity, optimal temperature, optimal pH, the effect of Mg2+, and kinetic parameters were determined. The establishment of the accurate and rapid colorimetric assay and kinetic analysis of M. tuberculosis RmlA will facilitate high-throughput screening of RmlA inhibitors.

Insights into the Distribution and Functions of the Eukaryotic GPI-like Anchored Genes Among Mycobacterium from a Comparative Genomic Perspective

Glycosylphosphatidylinositol (GPI)-anchored proteins range from small peptides to larger antigens and fulfill a variety of cellular functions in eukaryotes. We speculated there should be such molecules in intracellular pathogens such as Mycobacterium due to their complex interplay with the host. However, no prior publications have touched this topic. To explore the existence and distribution of GPI-like molecules among Mycobacterium, we exhaustively analyzed all publicly available Mycobacterium genomes and found that the GPI-like signal sequences are prevalent among Mycobacterium, and a significant dichotomy between nonpathogenic Mycobacterium (exemplified by Mycobacterium smegmatis) and pathogenic Mycobacterium (exemplified by Mycobacterium tuberculosis), through genome-wide GPI-SOM analysis. Some well-documented anti-tuberculosis drug targets are predicted to have GPI-like anchored signals, such as KasA and atpE. Interestingly, Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins predicted to have GPI-anchoring sequence are unique to pathogenic Mycobacterium. These results can be further explored for better control measures against tuberculosis.

Structure of Streptomyces Maltosyltransferase GlgE, a Homologue of a Genetically Validated Anti-tuberculosis Target

GlgE is a recently identified (1→4)-α-d-glucan:phosphate α-d-maltosyltransferase involved in α-glucan biosynthesis in bacteria and is a genetically validated anti-tuberculosis drug target. It is a member of the GH13_3 CAZy subfamily for which no structures were previously known. We have solved the structure of GlgE isoform I from Streptomyces coelicolor and shown that this enzyme has the same catalytic and very similar kinetic properties to GlgE from Mycobacterium tuberculosis. The S. coelicolor enzyme forms a homodimer with each subunit comprising five domains, including a core catalytic α-amylase-type domain A with a (β/α)(8) fold. This domain is elaborated with domain B and two inserts that are specifically configured to define a well conserved donor pocket capable of binding maltose. Domain A, together with domain N from the neighboring subunit, forms a hydrophobic patch that is close to the maltose-binding site and capable of binding cyclodextrins. Cyclodextrins competitively inhibit the binding of maltooligosaccharides to the S. coelicolor enzyme, showing that the hydrophobic patch overlaps with the acceptor binding site. This patch is incompletely conserved in the M. tuberculosis enzyme such that cyclodextrins do not inhibit this enzyme, despite acceptor length specificity being conserved. The crystal structure reveals two further domains, C and S, the latter being a helix bundle not previously reported in GH13 members. The structure provides a framework for understanding how GlgE functions and will help guide the development of inhibitors with therapeutic potential.

Novel inhibitors of Mycobacterium tuberculosis dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) identified by virtual screening.

The complex and highly impermeable cell wall of Mycobacterium tuberculosis (Mtb) is largely responsible for the ability of the mycobacterium to resist the action of chemical therapeutics. An L-rhamnosyl residue, which occupies an important anchoring position in the Mtb cell wall, is an attractive target for novel anti-tuberculosis drugs. In this work, we report a virtual screening (VS) study targeting Mtb dTDP-deoxy-L-lyxo-4-hexulose reductase (RmlD), the last enzyme in the L-rhamnosyl synthesis pathway. Through two rounds of VS, we have identified four RmlD inhibitors with half inhibitory concentrations of 0.9-25 μM, and whole-cell minimum inhibitory concentrations of 20-200 μg/ml. Compared with our previous high throughput screening targeting another enzyme involved in L-rhamnosyl synthesis, virtual screening produced higher hit rates, supporting the use of computational methods in future anti-tuberculosis drug discovery efforts.

Unexpected and widespread connections between bacterial glycogen and trehalose metabolism

Glycogen, a large α-glucan, is a ubiquitous energy storage molecule among bacteria, and its biosynthesis by the classical GlgC-GlgA pathway and its degradation have long been well understood - or so we thought. A second pathway of α-glucan synthesis, the four-step GlgE pathway, was recently discovered in mycobacteria. It requires trehalose as a precursor, and has been genetically validated as a novel anti-tuberculosis drug target. The ability to convert glycogen into trehalose was already known, so the GlgE pathway provides a complementary way of cycling these two metabolites. As well as containing cytosolic storage glycogen, mycobacteria possess an outer capsule containing a glycogen-like α-glucan that is implicated in immune system evasion, so the GlgE pathway might be linked to capsular α-glucan biosynthesis. Another pathway (the Rv3032 pathway) for α-glucan biosynthesis in mycobacteria generates a methylglucose lipopolysaccharide thought to be associated with fatty acid metabolism. A comparative genomic analysis was carried out to evaluate the occurrence and role of the classical pathway, the new GlgE pathway and the Rv3032 pathway across bacteria occupying very different ecological niches. The GlgE pathway is represented in 14 % of sequenced genomes from diverse bacteria (about half as common as the classical pathway), while the Rv3032 pathway is restricted with few exceptions to mycobacteria, and the GlgB branching enzyme, usually presumed to be associated with the classical pathway, correlates more strongly with the new GlgE pathway. The microbiological implications of recent discoveries in the light of the comparative genomic analysis are discussed.