Investigating Bacterial Volatilome for the Classification and Identification of Mycobacterial Species by HS-SPME-GC-MS and Machine Learning.

Species of Mycobacteriaceae cause disease in animals and humans, including tuberculosis and leprosy. Individuals infected with organisms in the Mycobacterium tuberculosis complex (MTBC) or non-tuberculous mycobacteria (NTM) may present identical symptoms, however the treatment for each can be different. Although the NTM infection is considered less vital due to the chronicity of the disease and the infrequency of occurrence in healthy populations, diagnosis and differentiation among Mycobacterium species currently require culture isolation, which can take several weeks. The use of volatile organic compounds (VOCs) is a promising approach for species identification and in recent years has shown promise for use in the rapid analysis of both in vitro cultures as well as ex vivo diagnosis using breath or sputum. The aim of this contribution is to analyze VOCs in the culture headspace of seven different species of mycobacteria and to define the volatilome profiles that are discriminant for each species. For the pre-concentration of VOCs, solid-phase micro-extraction (SPME) was employed and samples were subsequently analyzed using gas chromatography-quadrupole mass spectrometry (GC-qMS). A machine learning approach was applied for the selection of the 13 discriminatory features, which might represent clinically translatable bacterial biomarkers.

Bi-PSSM: Position specific scoring matrix based intelligent computational model for identification of mycobacterial membrane proteins.

Mycobacterium is a pathogenic bacterium, which is a causative agent of tuberculosis (TB) and leprosy. These diseases are very crucial and become the cause of death of millions of people every year in the world. So, the characterize structure of membrane proteins of the protozoan play a vital role in the field of drug discovery because, without any knowledge about this Mycobacterium's membrane protein and their types, the scientists are unable to treat this pathogenic protozoan. So, an accurate and competitive computational model is needed to characterize this uncharacterized structure of mycobacterium. Series of attempts were carried out in this connection. Split amino acid compositions, Unbiased-Dipeptide peptide compositions (Unb-DPC), Over-represented tri-peptide compositions, compositions & translation were the few recent encoding techniques followed by different researchers in their publications. Although considerable results have been achieved by these models, still there is a gap which is filled in this study. In this study, an evolutionary feature extraction technique position specific scoring matrix (PSSM) is applied in order to extract evolutionary information from protein sequences. Consequently, 99.6% accuracy was achieved by the learning algorithms. The experimental results demonstrated that the proposed computational model will lead to develop a powerful tool for anti-mycobacterium drugs as well as play a promising rule in proteomic and bioinformatics.

Mycobacterial tlyA gene product is localized to the cell-wall without signal sequence.

The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms.

Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies.

Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over-express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.

[Mycolic acids--biological role and potential application in Mycobacterium detection and differentiation]. / Kwasy mikolowe--rola biologiczna i potencjalne zastosowanie w wykrywaniu oraz róznicowaniu rodzaju Mycobacterium.

Mycolic acids are one of the basic structural elements of the cell wall of bacteria from Corynebacterineae suborder. These compounds are long-chain α-hydroxy ß-alkyl fatty acids with two hydrocarbon chains: longer meromycolic and shorter α-chain meromycolic α-chain. The genus Mycobacterium is characterized by the presence of mycolic acids in length from 60 to 90 carbon atoms having a fully saturated α-chain with a defined length of 22, 24 or 26 carbon atoms. Current research indicates that not only the presence of mycolic acids in the cell wall of mycobacteria is essential for the virulence of mycobacteria. It is proved that the relationship between different types of mycolic acids, their length and the degree of cyclopropanation may vary depending on the stage of infection and mycobacterial culture conditions. At the same time it has been shown that some mycolic acid types are crucial for biofilm formation, antimycobacterial drug resistance or interactions with the immune system. Recent studies also indicate that analysis of mycolic acid profiles could be an alternative to conventional methods of diagnosis of diseases such as tuberculosis, leprosy or mycobacteriosis.

Mycolic acids: structures, biosynthesis, and beyond.

Mycolic acids are major and specific lipid components of the mycobacterial cell envelope and are essential for the survival of members of the genus Mycobacterium that contains the causative agents of both tuberculosis and leprosy. In the alarming context of the emergence of multidrug-resistant, extremely drug-resistant, and totally drug-resistant tuberculosis, understanding the biosynthesis of these critical determinants of the mycobacterial physiology is an important goal to achieve, because it may open an avenue for the development of novel antimycobacterial agents. This review focuses on the chemistry, structures, and known inhibitors of mycolic acids and describes progress in deciphering the mycolic acid biosynthetic pathway. The functional and key biological roles of these molecules are also discussed, providing a historical perspective in this dynamic area.

Haemoglobins of Mycobacteria: structural features and biological functions.

The genus Mycobacterium is comprised of Gram-positive bacteria occupying a wide range of natural habitats and includes species that range from severe intracellular pathogens to economically useful and harmless microbes. The recent upsurge in the availability of microbial genome data has shown that genes encoding haemoglobin-like proteins are ubiquitous among Mycobacteria and that multiple haemoglobins (Hbs) of different classes may be present in pathogenic and non-pathogenic species. The occurrence of truncated haemoglobins (trHbs) and flavohaemoglobins (flavoHbs) showing distinct haem active site structures and ligand-binding properties suggests that these Hbs may be playing diverse functions in the cellular metabolism of Mycobacteria. TrHbs and flavoHbs from some of the severe human pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae have been studied recently and their roles in effective detoxification of reactive nitrogen and oxygen species, electron cycling, modulation of redox state of the cell and facilitation of aerobic respiration have been proposed. This multiplicity in the function of Hbs may aid these pathogens to cope with various environmental stresses and survive during their intracellular regime. This chapter provides recent updates on genomic, structural and functional aspects of Mycobacterial Hbs to address their role in Mycobacteria.

Rhomboid homologs in mycobacteria: insights from phylogeny and genomic analysis.

BACKGROUND: Rhomboids are ubiquitous proteins with diverse functions in all life kingdoms, and are emerging as important factors in the biology of some pathogenic apicomplexa and Providencia stuartii. Although prokaryotic genomes contain one rhomboid, actinobacteria can have two or more copies whose sequences have not been analyzed for the presence putative rhomboid catalytic signatures. We report detailed phylogenetic and genomic analyses devoted to prokaryotic rhomboids of an important genus, Mycobacterium. RESULTS: Many mycobacterial genomes contained two phylogenetically distinct active rhomboids orthologous to Rv0110 (rhomboid protease 1) and Rv1337 (rhomboid protease 2) of Mycobacterium tuberculosis H37Rv, which were acquired independently. There was a genome-wide conservation and organization of the orthologs of Rv1337 arranged in proximity with glutamate racemase (mur1), while the orthologs of Rv0110 appeared evolutionary unstable and were lost in Mycobacterium leprae and the Mycobacterium avium complex. The orthologs of Rv0110 clustered with eukaryotic rhomboids and contained eukaryotic motifs, suggesting a possible common lineage. A novel nonsense mutation at the Trp73 codon split the rhomboid of Mycobacterium avium subsp. Paratuberculosis into two hypothetical proteins (MAP2425c and MAP2426c) that are identical to MAV_1554 of Mycobacterium avium. Mycobacterial rhomboids contain putative rhomboid catalytic signatures, with the protease active site stabilized by Phenylalanine. The topology and transmembrane helices of the Rv0110 orthologs were similar to those of eukaryotic secretase rhomboids, while those of Rv1337 orthologs were unique. Transcription assays indicated that both mycobacterial rhomboids are possibly expressed. CONCLUSIONS: Mycobacterial rhomboids are active rhomboid proteases with different evolutionary history. The Rv0110 (rhomboid protease 1) orthologs represent prokaryotic rhomboids whose progenitor may be the ancestors of eukaryotic rhomboids. The Rv1337 (rhomboid protease 2) orthologs appear more stable and are conserved nearly in all mycobacteria, possibly alluding to their importance in mycobacteria. MAP2425c and MAP2426c provide the first evidence for a split homologous rhomboid, contrasting whole orthologs of genetically related species. Although valuable insights to the roles of rhomboids are provided, the data herein only lays a foundation for future investigations for the roles of rhomboids in mycobacteria.

Structural studies of cord factors from Mycobacterium simiae related to the capacity for tumour necrosis factor alpha (α-TNF) induction.

The structure of cord factor was studied in several strains of Mycobacterium simiae, including 'habana' TMC 5135, considered as highly immunogenic in experimental tuberculosis and leprosy. The mycolic acids liberated from cord factor were identified in all cases as α'-, α- and keto-mycolates. According to the general NMR and MS data, α'-mycolates were mono-unsaturated and contained from 64 to 68 carbon atoms, whereas α-mycolates mainly presented two 2,3-disubstituted cyclopropane rings and a chain length of 80-91 carbon atoms; keto-mycolates mostly contained one cyclopropane ring and 85-91 carbon atoms. Taking into account the (1)H-NMR results, strains varied in the ratio of the different mycolates, and the high levels of keto-mycolates found in the cord factors of TMC 5135 and ATCC 25275(T) stood out. Notably, MS revealed that the odd carbon number series of α-mycolates (C87-C89) predominated in the cord factor of TMC 5135, in contrast to the remaining studied strains, in which the even (C84-C86) and odd carbon number series appeared more equal. The fine structural differences detected among the cord factors studied did not seem to be relevant to the general capacity of these molecules to induce the secretion of tumour necrosis factor alpha, as the cord factors from several strains of M. simiae (TMC 5135, IPK-342 and ATCC 25275(T)) induced similar amounts of this cytokine in RAW 264.7 cells.

Structural insights into antibody recognition of mycobacterial polysaccharides.

Mycobacteria are major human pathogens responsible for such serious and widespread diseases as tuberculosis and leprosy. Among the evolutionary adaptations essential for pathogenicity in mycobacteria is a complex carbohydrate-rich cell-wall structure that contains as a major immunomodulatory molecule the polysaccharide lipoarabinomannan (LAM). We report here crystal structures of three fragments from the non-reducing termini of LAM in complex with a murine antibody Fab fragment (CS-35Fab). These structures reveal for the first time the three-dimensional structures of key components of LAM and the molecular basis of LAM recognition at between 1.8- and 2.0-A resolution. The antigen-binding site of CS-35Fab forms three binding pockets that show a high degree of complementarity to the reducing end, the branch point and one of the non-reducing ends of the Y-shaped hexasaccharide moiety found at most of the non-reducing termini of LAM. Structures of CS-35Fab bound to two additional tetrasaccharides confirm the general mode of binding seen in the hexasaccharide and indicate how different parts of LAM are recognized. Altogether, these structures provide a rational basis for understanding the overall architecture of LAM and identify the key elements of an epitope that may be exploited for the development of novel and more effective anti-mycobacterial vaccines. Moreover, this study represents the first high-resolution X-ray crystallographic investigation of oligofuranoside-protein recognition.

Efficient chemical synthesis of a dodecasaccharidyl lipomannan component of mycobacterial lipoarabinomannan.

Lipomannan (LM) is one of the domains of lipoarabinomannan (LAM) glycolipids, the latter being one of several cell surface organic molecules that fortify mycobacterial species against external attack. Some members of mycobacterial families are pathogenic, most notably Mycobacterium tuberculosis and Mycobacterium leprae, while others are nonpathogenic, and used in the clinic, such as Mycobacterium smegmatis. Additional biological significance arises from the fact that LM has been implicated in several health disorders outside of those associated with mycobacterial pathogens, notably for treatment of bladder cancer. LM is comprised of a heavily lipidated phosphoinositide dimannoside headgroup, from which a mannan array, of varied complexity, extends. The latter consists of a 1,6-alpha-linked backbone flanked at position O2, not necessarily regularly, with alpha-linked mannosides. This paper gives an example of lipomannan synthesis in which all of the sugar components, whether functioning as donors or acceptors, are obtained from n-pentenyl orthoesters, themselves in turn prepared in three easy steps from D-mannose. Assembly of the mannan array is facilitated by the exquisite regioselectivity occasioned by the use of ytterbium triflate/N-iodosuccinimide as the trigger for reaction of n-pentenyl orthoesters.

Breaking down the wall: fractionation of mycobacteria.

Mycobacterium spp. possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan-arabinogalactan complex which in turn is esterified by mycolic acids that form with other non-bound lipids an asymmetric permeability barrier and an outer layer, also called a capsule in the case of pathogenic species. In order to investigate the functional roles of the cell envelope components, especially those of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae, it is necessary to fractionate the envelope by breaking the unusual wall that covers these bacteria. To this aim we first compared the efficiency of high pressure (cell disrupter/French press) with those of pathogen-compatible breakage methods such as sonication, bead beater and lysozyme treatment using the non-pathogenic Mycobacterium smegmatis. When the distribution of various specific markers of the cell envelope compartments, which include mycolic acids, arabinose, NADH oxidase activity, cell wall and cytosolic proteins, were determined sonication combined with lysozyme treatment was found to be the best option. The protocol of subcellular fractionation was then validated for pathogenic species by applying the method to Mycobacterium bovis BCG cells, an attenuated strain of the M. tuberculosis complex.

[Molecular pathogenesis of mycobacterial diseases].

Mycobacterial diseases, including tuberculosis, leprosy, and disease due to nontuberculous mycobacteria, are the major cause of death from infectious diseases around the world. About one-third of the world population is latently infected with Mycobacterium tuberculosis. Over 8 million new cases and nearly 2 million deaths occur each year. Tuberculosis presents a significant health threat to the world. The pathogenicity of mycobacteria is related to their ability to escape killing by ingested macrophages, latent infection, and induce delayed type hypersensitivity. This has been attributed to several components of the mycobacterial cell wall, such as surface glycolipids, lipoarabinomannan, complement activation factor, heat-shock protein, and mycobacterial DNA binding protein. From the aspect of my research interests, I have focused on mycobacterial glycolipids and mycobacterial DNA binding protein in this article. Surface molecules of mycobacteria exert pleiotropic activities in both the microbe and host, and thus participate in the pathogenesis of mycobacterial diseases. The better understanding of mycobacterial pathogenicity may open the new avenue for the development of therapeutic and prophylactic interventions.

Iron chelation properties of an extracellular siderophore exochelin MN.

The coordination chemistry of an extracellular siderophore produced by Mycobacterium neoaurum, exochelin MN (ExoMN), is reported along with its pK(a) values, Fe(III) and Fe(II) chelation constants, and aqueous solution speciation as determined by spectrophotometric and potentiometric titration techniques. Exochelin MN is of particular interest as it can efficiently transport iron into pathogenic M. leprae, which is responsible for leprosy, in addition to its own parent cells. The Fe(III) coordination properties of ExoMN are important with respect to understanding the Fe(III) acquisition and uptake mechanism in pathogenic M. leprae, as the siderophores from this organism are very difficult to isolate. Exochelin MN has two hydroxamic acid groups and an unusual threo-beta-hydroxy-l-histidine available for Fe(III) chelation. The presence of threo-beta-hydroxy-l-histidine gives rise to a unique mode of Fe(III) coordination. The pK(a) values for the two hydroxamic acid moieties, the histidine imidazole ring and the alkylammonium groups on ExoMN, correspond well with the literature values for these moieties. Proton-dependent Fe(III)- and Fe(II)-ExoMN equilibrium constants were determined using a model involving sequential protonation of the Fe(III)- and Fe(II)-ExoMN complexes. These data were used to develop a model whereby deprotonation reactions on the surface of the complex in the second coordination shell result in first coordination shell isomerization. The overall formation constants were calculated: log beta(110) = 39.12 for Fe(III)-ExoMN and 16.7 for Fe(II)-ExoMN. The calculated pFe value of 31.1 is one of the highest among all siderophores and their synthetic analogues and indicates that ExoMN is thermodynamically capable of removing Fe(III) from transferrin. The E(1/2) for the Fe(III)ExoMN/Fe(II)ExoMN(-) couple was determined to be -595 mV from quasi-reversible cyclic voltammograms at pH = 10.8, and the pH-dependent E(1/2) profile was used to determine the Fe(II)-ExoMN protonation constants.

Iron chelation properties of an extracellular siderophore exochelin MN

The coordination chemistry of an extracellular siderophore produced by Mycobacterium neoaurum, exochelin MN (ExoMN), is reported along with its pK(a) values, Fe(III) and Fe(II) chelation constants, and aqueous solution speciation as determined by spectrophotometric and potentiometric titration techniques. Exochelin MN is of particular interest as it can efficiently transport iron into pathogenic M. leprae, which is responsible for leprosy, in addition to its own parent cells. The Fe(III) coordination properties of ExoMN are important with respect to understanding the Fe(III) acquisition and uptake mechanism in pathogenic M. leprae, as the siderophores from this organism are very difficult to isolate. Exochelin MN has two hydroxamic acid groups and an unusual threo-beta-hydroxy-l-histidine available for Fe(III) chelation. The presence of threo-beta-hydroxy-l-histidine gives rise to a unique mode of Fe(III) coordination. The pK(a) values for the two hydroxamic acid moieties, the histidine imidazole ring and the alkylammonium groups on ExoMN, correspond well with the literature values for these moieties. Proton-dependent Fe(III)- and Fe(II)-ExoMN equilibrium constants were determined using a model involving sequential protonation of the Fe(III)- and Fe(II)-ExoMN complexes. These data were used to develop a model whereby deprotonation reactions on the surface of the complex in the second coordination shell result in first coordination shell isomerization. The overall formation constants were calculated: log beta(110) = 39.12 for Fe(III)-ExoMN and 16.7 for Fe(II)-ExoMN. The calculated pFe value of 31.1 is one of the highest among all siderophores and their synthetic analogues and indicates that ExoMN is thermodynamically capable of removing Fe(III) from transferrin. The E(1/2) for the Fe(III)ExoMN/Fe(II)ExoMN(-) couple was determined to be -595 mV from quasi-reversible cyclic voltammograms at pH = 10.8, and the pH-dependent E(1/2) profile was used to determine the Fe(II)-ExoMN protonation constants.

CD1 proteins: targets of T cell recognition in innate and adaptive immunity.

The CD1 family consists of antigen presenting molecules encoded by genes located outside of the major histocompatibility complex. CD1 proteins are conserved among mammalian species and are expressed on the surface of cells involved in antigen presentation. The CD1 system has been shown to be involved in activation of cell-mediated responses, and T cells specific for either CD1 molecules or antigens presented by CD1 have been isolated. Structural and biochemical analyses demonstrate that antigens presented by CD1 are nonpeptide lipid or glycolipid structures, including examples found in the cell walls of pathogenic mycobacteria. The hydrophobic part of these antigens most likely binds in the CD1 ligand-binding groove, whereas the polar headgroup of these antigens appears to make direct contact with the T cell receptor and determines specific recognition. Presentation of antigens by CD1 molecules requires uptake and intracellular processing by antigen presenting cells and can be achieved for both exogenous and endogenous antigens. T cells recognizing CD1 restricted antigens have a broad range of functional activities that suggest that the CD1 system is involved in both innate and adaptive immune responses against microbial infections.

Complete DNA sequence analysis for 16S ribosomal RNA gene of the leproma-derived, cultivable and nerve-invading mycobacterium HI-75.

The complete 1493 nucleotide sequence of the 16SrRNA gene of the leproma-derived and cultivable mycobacterium HI-75 strain was analyzed to elucidate the taxonomic characteristics by direct sequencing of the polymerase chain reaction (PCR) products. The results revealed that the sequence of mycobacterium HI-75 was mostly similar to that of Mycobacterium scrofulaceum with 5 bases differences in the sequenced 1493 bases (0.35%) of the 16SrRNA gene. M. leprae differed from the strain with 47 bases (3.3%). Sasaki and Hamit reported the nerve-invasive activity of the inoculated mycobacterium HI-75 in nude mice or the 131I-treated immunocompromised Swiss mice. The results indicate that mycobacterium HI-75 could be a mutant of M. scrofulaceum possessing the ability to invade the peripheral nerve in addition to developing leproma-like lesions.

Molecular characterization of the thioredoxin system of Mycobacterium smegmatis.

Thiol-disulphide exchanges are involved in many important biological processes; they are normally regulated by the glutaredoxin and thioredoxin systems. The thioredoxin system (TX) is composed of thioredoxin (TrxA) and thioredoxin reductase (TrxB) and requires NADPH as a cofactor. The thioredoxin genes trxA and trxB of Mycobacterium smegmatis mc2(6) were cloned and sequenced. The complete nucleotide sequences revealed that the TX genes of M. smegmatis were clustered, similar to the organization of trxA and trxB of S. clavuligerus, M. tuberculosis and M. leprae. Alignment with the M. tuberculosis and M. leprae protein sequences showed that the deduced amino acid sequences for M. smegmatis trxA and trxB have a very high degree of similarity. Sequence alignments and phylogenetic analysis of known TrxAs and TrxBs clearly identify the two gene products of M. smegmatis as members of the TX family grouped with other mycobacteria.

Characterization of a haemolysin from Mycobacterium tuberculosis with homology to a virulence factor of Serpulina hyodysenteriae.

Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysin/cytotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenteriae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tuberculosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium avium and Mycobacterium bovis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His6-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bovis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in vitro.