Exploring the Cell Wall and Secretory Proteins of Mycobacterium leprae as Biomarkers.

The bacterial cell wall is composed of a wide variety of intricate proteins in addition to lipids, glycolipids, and polymers. Given the diversity of cell wall proteins among bacterial species, they are a feasible target for biomarker identification and characterization in clinical research and diagnosis of the disease. The slow growth rate of Mycobacterium leprae poses a major hurdle in the accurate diagnosis of leprosy before the onset of peripheral neuropathy. The use of biomarker- based diagnostic methods can help in preventing the spread and manifestation of leprosy. Despite many advances in research methods and techniques, there remains a knowledge gap regarding the cell wall proteomes of M. leprae that can be used as biomarkers. The cell wall and secretory proteins of M. leprae are the major focus of this review article. This article enfolds the characteristics and functions of M. leprae cell wall proteins and gives an insight into those cell wall proteins that are yet to be established as biomarkers. Tools and techniques used in cell wall extraction and biomarker identification can also be explored in this article.

TN strain proteome mediated therapeutic target mapping and multi-epitopic peptide-based vaccine development for Mycobacterium leprae.

Leprosy is a significant universal health problem that is remarkably still a concern in developing countries due to infection frequency. New therapeutic molecules and next-generation vaccines are urgently needed to accelerate the leprosy-free world. In this direction, the present study was performed using two routes: proteome-mediated therapeutic target identification and mapping as well as multi-epitopic peptide-based novel vaccine development using state of the art of computational biology for the TN strain of M. leprae. The TN strain was selected from 65 Mycobacterium strains, and TN strain proteome mediated 83 therapeutic protein targets were mapped and characterized according to subcellular localization. Also, drug molecules were mapped with respect to protein targets localization. The Druggability potential of proteins was also evaluated. For multi-epitope peptide-based vaccine development, the four common types of B and T cell epitopes were identified (SLFQSHNRK, VVGIGQHAA, MMHRSPRTR, LGVDQTQPV) and combined with the suitable peptide linker. The vaccine component had an acceptable protective antigenic score (0.9751). The molecular docking of vaccine components with TLR4/MD2 complex exhibited a low ACE value (-244.12) which signifies the proper binding between the two molecules. The estimated free Gibbs binding energy ensured accurate protein-protein interactions (-112.46 kcal/mol). The vaccine was evaluated through adaptive immunity stimulation as well as immune interactions. The molecular dynamic simulation was carried out by using CHARMM topology-based parameters to minimize the docked complex. Subsequently, the Normal Mode Analysis in the internal coordinates showed a low eigen-value (1.3982892e-05), which also signifies the stability of molecular docking. Finally, the vaccine components were adopted for reverse transcription and codon optimization in E. coli strain K12 for the pGEX-4T1 vector, which supports in silico cloning of the vaccine components against the pathogen. The study directs the experimental study for therapeutics molecules discovery and vaccine candidate development with higher reliability.

APRANK: Computational Prioritization of Antigenic Proteins and Peptides From Complete Pathogen Proteomes.

Availability of highly parallelized immunoassays has renewed interest in the discovery of serology biomarkers for infectious diseases. Protein and peptide microarrays now provide a rapid, high-throughput platform for immunological testing and validation of potential antigens and B-cell epitopes. However, there is still a need for tools to prioritize and select relevant probes when designing these arrays. In this work we describe a computational method called APRANK (Antigenic Protein and Peptide Ranker) which integrates multiple molecular features to prioritize potentially antigenic proteins and peptides in a given pathogen proteome. These features include subcellular localization, presence of repetitive motifs, natively disordered regions, secondary structure, transmembrane spans and predicted interaction with the immune system. We trained and tested this method with a number of bacteria and protozoa causing human diseases: Borrelia burgdorferi (Lyme disease), Brucella melitensis (Brucellosis), Coxiella burnetii (Q fever), Escherichia coli (Gastroenteritis), Francisella tularensis (Tularemia), Leishmania braziliensis (Leishmaniasis), Leptospira interrogans (Leptospirosis), Mycobacterium leprae (Leprae), Mycobacterium tuberculosis (Tuberculosis), Plasmodium falciparum (Malaria), Porphyromonas gingivalis (Periodontal disease), Staphylococcus aureus (Bacteremia), Streptococcus pyogenes (Group A Streptococcal infections), Toxoplasma gondii (Toxoplasmosis) and Trypanosoma cruzi (Chagas Disease). We have evaluated this integrative method using non-parametric ROC-curves and made an unbiased validation using Onchocerca volvulus as an independent data set. We found that APRANK is successful in predicting antigenicity for all pathogen species tested, facilitating the production of antigen-enriched protein subsets. We make APRANK available to facilitate the identification of novel diagnostic antigens in infectious diseases.

Mycobacterial DNA-binding protein 1 is critical for long term survival of Mycobacterium smegmatis and simultaneously coordinates cellular functions.

Bacteria can proliferate perpetually without ageing, but they also face conditions where they must persist. Mycobacteria can survive for a long period. This state appears during mycobacterial diseases such as tuberculosis and leprosy, which are chronic and develop after long-term persistent infections. However, the fundamental mechanisms of the long-term living of mycobacteria are unknown. Every Mycobacterium species expresses Mycobacterial DNA-binding protein 1 (MDP1), a histone-like nucleoid associated protein. Mycobacterium smegmatis is a saprophytic fast grower and used as a model of mycobacterial persistence, since it shares the characteristics of the long-term survival observed in pathogenic mycobacteria. Here we show that MDP1-deficient M. smegmatis dies more rapidly than the parental strain after entering stationary phase. Proteomic analyses revealed 21 upregulated proteins with more than 3-fold in MDP1-deficient strain, including DnaA, a replication initiator, NDH, a NADH dehydrogenase that catalyzes downhill electron transfer, Fas1, a critical fatty acid synthase, and antioxidants such as AhpC and KatG. Biochemical analyses showed elevated levels of DNA and ATP syntheses, a decreased NADH/NAD+ ratio, and a loss of resistance to oxidative stress in the MDP1-knockout strain. This study suggests the importance of MDP1-dependent simultaneous control of the cellular functions in the long-term survival of mycobacteria.

The Intergenic Recombinant HLA-B∗46:01 Has a Distinctive Peptidome that Includes KIR2DL3 Ligands.

HLA-B∗46:01 was formed by an intergenic mini-conversion, between HLA-B∗15:01 and HLA-C∗01:02, in Southeast Asia during the last 50,000 years, and it has since become the most common HLA-B allele in the region. A functional effect of the mini-conversion was introduction of the C1 epitope into HLA-B∗46:01, making it an exceptional HLA-B allotype that is recognized by the C1-specific natural killer (NK) cell receptor KIR2DL3. High-resolution mass spectrometry showed that HLA-B∗46:01 has a low-diversity peptidome that is distinct from those of its parents. A minority (21%) of HLA-B∗46:01 peptides, with common C-terminal characteristics, form ligands for KIR2DL3. The HLA-B∗46:01 peptidome is predicted to be enriched for peptide antigens derived from Mycobacterium leprae. Overall, the results indicate that the distinctive peptidome and functions of HLA-B∗46:01 provide carriers with resistance to leprosy, which drove its rapid rise in frequency in Southeast Asia.

Excavating the surface-associated and secretory proteome of Mycobacterium leprae for identifying vaccines and diagnostic markers relevant immunodominant epitopes.

For centuries, Mycobacterium leprae, etiological agent of leprosy, has been afflicting mankind regardless of extensive use of live-attenuated vaccines and antibiotics. Surface-associated and secretory proteins (SASPs) are attractive targets against bacteria. We have integrated biological knowledge with computational approaches and present a proteome-wide identification of SASPs. We also performed computational assignment of immunodominant epitopes as coordinates of prospective antigenic candidates in most important class of SASPs, the outer membrane proteins (OMPs). Exploiting the known protein sequence and structural characteristics shared by the SASPs from bacteria, 17 lipoproteins, 11 secretory and 19 novel OMPs (including 4 essential proteins) were identified in M. leprae As OMPs represent the most exposed antigens on the cell surface, their immunoinformatics analysis showed that the identified 19 OMPs harbor T-cell MHC class I epitopes and class II epitopes against HLA-DR alleles (54), while 15 OMPs present potential T-cell class II epitopes against HLA-DQ alleles (6) and 7 OMPs possess T-cell class II epitopes against HLA-DP alleles (5) of humans. Additionally, 11 M. leprae OMPs were found to have B-cell epitopes and these may be considered as prime candidates for the development of new immunotherapeutics against M. leprae.

Contrasted evolutionary constraints on secreted and non-secreted proteomes of selected Actinobacteria.

BACKGROUND: Actinobacteria have adapted to contrasted ecological niches such as the soil, and among others to plants or animals as pathogens or symbionts. Mycobacterium genus contains mostly pathogens that cause a variety of mammalian diseases, among which the well-known leprosy and tuberculosis, it also has saprophytic relatives. Streptomyces genus is mostly a soil microbe known for its secondary metabolites, it contains also plant pathogens, animal pathogens and symbionts. Frankia, a nitrogen-fixing actinobacterium establishes a root symbiosis with dicotyledonous pionneer plants. Pathogens and symbionts live inside eukaryotic cells and tissues and interact with their cellular environment through secreted proteins and effectors transported through transmembrane systems; nevertheless they also need to avoid triggering host defense reactions. A comparative genome analysis of the secretomes of symbionts and pathogens allows a thorough investigation of selective pressures shaping their evolution. In the present study, the rates of silent mutations to non-silent mutations in secretory proteins were assessed in different strains of Frankia, Streptomyces and Mycobacterium, of which several genomes have recently become publicly available. RESULTS: It was found that secreted proteins as a whole have a stronger purifying evolutionary rate (non-synonymous to synonymous substitutions or Ka/Ks ratio) than the non-secretory proteins in most of the studied genomes. This difference becomes statistically significant in cases involving obligate symbionts and pathogens. Amongst the Frankia, secretomes of symbiotic strains were found to have undergone evolutionary trends different from those of the mainly saprophytic strains. Even within the secretory proteins, the signal peptide part has a higher Ka/Ks ratio than the mature part. Two contrasting trends were noticed amongst the Frankia genomes regarding the relation between selection strength (i.e. Ka/Ks ratio) and the codon adaptation index (CAI), a predictor of the expression rate, in all the genes belonging to the core genome as well as the core secretory protein genes. The genomes of pathogenic Mycobacterium and Streptomyces also had reduced secretomes relative to saprophytes, as well as in general significant pairwise Ka/Ks ratios in their secretomes. CONCLUSION: In marginally free-living facultative symbionts or pathogenic organisms under consideration, secretory protein genes as a whole evolve at a faster rate than the rest and this process may be an adaptive life-strategy to counter the host selection pressure. The higher evolutionary rate of signal peptide part compared to mature protein provides an indication that signal peptide parts may be under relaxed purifying selection, indicative of the signal peptides not being secreted into host cells. Codon usage analysis suggests that in actinobacterial strains under host selection pressure such as symbiotic Frankia, ACN, FD and the pathogenic Mycobacterium, codon usage bias was negatively correlated to the selective pressure exerted on the secretory protein genes.

Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

Proteomic changes in response to potassium starvation in the extremophilic yeast Debaryomyces hansenii.

In this work, we performed for the first time a proteomic approach to the processes induced by long-term potassium starvation in the halotolerant yeast Debaryomyces hansenii. The proteomic profile under this ionic stress conditions shows that important changes in gene expression take place as an adaptive response. We found a significant protein expression repression as well as metabolic changes such as the inhibition of the upper part of the glycolysis, the amino acid synthesis, and the Krebs cycle. On the other hand, genes related to stress responses, protein degradation, and sterols synthesis were upregulated in response to potassium deprivation. The findings in this study provide important information about how this particular yeast copes with ionic stress at molecular levels, which might further enrich the global understanding of salt tolerance processes in eukaryal systems and moreover highlighting the importance of the 'omics' approaches as a complement to the classical physiological studies.

Advances in Proteomics of Mycobacterium leprae.

Although Mycobacterium leprae was the first bacterial pathogen identified causing human disease, it remains one of the few that is non-cultivable. Understanding the biology of M. leprae is one of the primary challenges in current leprosy research. Genomics has been extremely valuable, nonetheless, functional proteins are ultimately responsible for controlling most aspects of cellular functions, which in turn could facilitate parasitizing the host. Furthermore, bacterial proteins provide targets for most of the vaccines and immunodiagnostic tools. Better understanding of the proteomics of M. leprae could also help in developing new drugs against M. leprae. During the past nearly 15 years, there have been several developments towards the identification of M. leprae proteins employing contemporary proteomics tools. In this review, we discuss the knowledge gained on the biology and pathogenesis of M. leprae from current proteomic studies.

alpha1-acid glycoprotein as a putative biomarker for monitoring the development of the type II reactional stage of leprosy.

Leprosy, a spectral disease manifested on the basis of host immune responses, is complicated by its reactional stages, namely type I reversal reaction (RR) and type II erythema nodosum leprosum (ENL). These reactional stages are characterized by uncontrolled and aberrant immune responses. Biomarkers for reactional stages would aid in early diagnosis, efficient treatment, prevention of neurological complications and prediction of predisposition to reactional stages. In this study, comparative analysis of the serum proteome of leprosy patients by two-dimensional electrophoresis (2DE) followed by mass spectrometry showed differential expression of acute-phase protein alpha (1)-acid glycoprotein (AGP; also known as orosomucoid). AGP levels in untreated ENL cases were significantly higher than in lepromatous leprosy (LL; a non-reactional disease stage) (P=0.0126), RR (P=0.0176) and healthy controls (P=0.0030). These data were confirmed using ELISA. The levels of AGP decreased to normal levels after treatment with multidrug therapy and thalidomide (P =0.0167). In a follow-up study, AGP levels, which were high in the untreated ENL stage, decreased significantly at 5 days ( P=0.0084) and 21 days (P=0.0027) post-treatment. A stage-dependent increase in AGP in an LL patient who progressed into the ENL stage was also shown. Glycosylation analysis by 2DE showed differential expression of acidic glycoforms of AGP in untreated ENL cases. Changes in AGP concentration and differential expression of isoforms correlated with the inflammatory condition in ENL and also with the treatment regimen. Thus, initial validation of AGP as an ENL-specific biomarker and treatment indicator was shown in this study.

Saccharomyces cerevisiae CCMI 885 secretes peptides that inhibit the growth of some non-Saccharomyces wine-related strains.

The nature of the toxic compounds produced by Saccharomyces cerevisiae CCMI 885 that induce the early death of Hanseniaspora guilliermondii during mixed fermentations, as well as their ability to inhibit the growth of other non-Saccharomyces wine-related strains, was investigated. The killing effect of mixed supernatants towards H. guilliermondii was inactivated by protease treatments, thus revealing the proteinaceous nature of the toxic compounds. Analysis of the protein pattern of mixed supernatants on Tricine SDS-PAGE showed that this S. cerevisiae strain secretes peptides (<10 kDa), which were detected only when death of H. guilliermondii was already established. Death-inducing supernatants were ultrafiltrated by 10 and 2 kDa membranes, respectively, and the inhibitory effect of those permeates were tested in H. guilliermondii cultures. Results indicated that the (2-10) kDa protein fraction of those supernatants seemed to contain antimicrobial peptides active against H. guilliermondii. Thus, the (2-10) kDa protein fraction was concentrated and its inhibitory effect tested against strains of Kluyveromyces marxianus, Kluyveromyces thermotolerans, Torulaspora delbrueckii and H. guilliermondii. Under the growth conditions used for these tests, the (2-10) kDa protein fraction of S. cerevisiae CCMI 885 supernatants exhibited a fungistatic effect against all the strains and a fungicidal effect against K. marxianus.

Validating divergent ORF annotation of the Mycobacterium leprae genome through a full translation data set and peptide identification by tandem mass spectrometry.

Mycobacterium leprae has undergone extensive degenerative evolution, with a large number of pseudogenes. It is also the organism with the greatest divergence between gene annotations from independent institutes. Therefore, M. leprae is a good model to verify the currently predicted coding sequence regions between different annotations, to identify new ones and to investigate the expression of pseudogenes. We submitted a total extract of the bacteria isolated from Armadillo to Gel-LC-MS/MS using a linear quadrupole ion trap-Orbitrap mass spectrometer. Spectra were analyzed using the Leproma (1614 genes and 1133 pseudogenes) and TIGR (5446 genes) databases and a database containing the full genome translation. We identified a total of 1046 proteins, including five proteins encoded by previously predicted pseudogenes, which upon closer inspection appeared to be proper genes. Only 11 of the additional annotations by TIGR were verified. We also identified six tryptic peptides from five proteins from regions not considered to be coding sequences, in addition to peptides from two unannotated gene candidates that overlap with other genes. Our data show that the Leproma annotation of M. leprae is quite accurate, and there were no peptide observations corresponding to true pseudogenes, except for a new gene candidate, overlapping with an essential enolase on the complementary strand.

Heparin-binding hemagglutinin (HBHA) of Mycobacterium leprae is expressed during infection and enhances bacterial adherence to epithelial cells.

A heparin-binding hemagglutinin (HBHA) expressed on the surface of Mycobacterium tuberculosis is an antigenic protein that has been implicated in bacterial adherence to epithelial cells and systemic dissemination. In this study, the potential role of the Mycobacterium leprae HBHA (ML-HBHA) homologue in leprosy was investigated. Initially, the in vivo expression of HBHA and its association with the M. leprae cell envelope was confirmed by immunoblotting and proteomic analysis. Mycobacterium leprae recombinant HBHA (rML-HBHA) bound to a heparin-Sepharose column, and its capacity to act as an adhesin was demonstrated in experiments showing that the exogenous addition of the protein to latex beads or to M. leprae cells promotes a dramatic increase in association with epithelial cells. Finally, serum anti-HBHA immunoglobulin G levels were investigated in individuals infected with M. leprae. Altogether, our data indicate that HBHA is recognized during the course of bacterial infection in humans and may play a role in leprosy pathogenesis.

Deciphering the proteomic profile of Mycobacterium leprae cell envelope.

The complete sequence of the Mycobacterium leprae genome, an obligate intracellular pathogen, shows a dramatic reduction of functional genes, with a coding capacity of less than 50%. Despite this massive gene decay, the leprosy bacillus has managed to preserve a minimal gene set, most of it shared with Mycobacterium tuberculosis, allowing its survival in the host with ensuing pathological manifestations. Thus, the identification of proteins that are actually expressed in vivo by M. leprae is of high significance in understanding obligate, intracellular mycobacterial pathogenesis. In this study, a high-throughput proteomic approach was undertaken resulting in the identification of 218 new M. leprae proteins. Of these, 60 were in the soluble/cytosol fraction, 98 in the membrane and 104 in the cell wall. Although several proteins were identified in more than one subcellular fraction, the majority were unique to one. As expected, a high percentage of these included enzymes responsible for lipid biosynthesis and degradation, biosynthesis of the major components of the mycobacterial cell envelope, proteins involved in transportation across lipid barriers, and lipoproteins and transmembrane proteins with unknown functions. The data presented in this study contribute to our understanding of the in vivo composition and physiology of the mycobacterial cell envelope, a compartment known to play a major role in bacterial pathogenesis.

Proteomic changes in Debaryomyces hansenii upon exposure to NaCl stress.

The proteome of the highly NaCl-tolerant yeast Debaryomyces hansenii was investigated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), and 47 protein spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) followed by mass spectrometry (MS). The influence of NaCl on the D. hansenii proteome was investigated during the first 3 h of NaCl exposure. The rate of protein synthesis was strongly decreased by exposure to 8% and 12% (w/v) NaCl, as the average incorporation rates of l-[(35)S]methionine within the first 30 min after addition of NaCl were only 7% and 4% of the rate in medium without NaCl. In addition, the number of protein spots detected on 2D gels prepared from cells exposed to 8% and 12% (w/v) NaCl exceeded less than 28% of the number of protein spots detected on 2D gels prepared from cells without added NaCl. Several proteins were identified as being either induced or repressed upon NaCl exposure. The induced proteins were enzymes involved in glycerol synthesis/dissimilation and the upper part of glycolysis, whereas the repressed proteins were enzymes involved in the lower part of glycolysis, the route to the Krebs cycle, and the synthesis of amino acids. Furthermore, one heat shock protein (Ssa1p) was induced, whereas others (Ssb2p and Hsp60p) were repressed.

Emergence of species-specific transporters during evolution of the hemiascomycete phylum.

We have traced the evolution patterns of 2480 transmembrane transporters from five complete genome sequences spanning the entire Hemiascomycete phylum: Saccharomyces cerevisiae, Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica. The use of nonambiguous functional and phylogenetic criteria derived from the TCDB classification system has allowed the identification within the Hemiascomycete phylum of 97 small phylogenetic transporter subfamilies comprising a total of 355 transporters submitted to four distinct evolution patterns named "ubiquitous," "species specific," "phylum gains and losses," or "homoplasic." This analysis identifies the transporters that contribute to the emergence of species during the evolution of the Hemiascomycete phylum and may aid in establishing novel phylogenetic criteria for species classification.

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses.

Continued proteomic analysis of Mycobacterium leprae subcellular fractions.

Recently the sequence of the Mycobacterium leprae chromosome, the only known obligate intracellular mycobacterium, was completed. It has a dramatic reduction in functional genes, with a coding capacity of only 49.5%, the lowest one so far observed among bacterial genomes. The leprosy bacillus seems to preserve a minimal set of genes that allows its survival in the host. The identification of genes that are actually expressed by the bacterium is of high significance in the context of mycobacterial pathogenesis. In this current study, a proteomic approach was undertaken to identify the proteins present in the soluble/cytosol and membrane subcellular fractions obtained from armadillo derived M. leprae. Proteins from each fraction were separated by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry. A total of 147 protein spots were identified from 2-DE patterns and shown to comprise products of 44 different genes, twenty eight of them corresponding to new proteins. Additionally, two highly basic proteins (with pI >10.0) were isolated by heparin affinity chromatography and identified by N-terminal sequencing. This study constitutes the first application of proteomics to a host-derived Mycobacterium.

Studying genomes through the aeons: protein families, pseudogenes and proteome evolution.

Protein families can be used to understand many aspects of genomes, both their "live" and their "dead" parts (i.e. genes and pseudogenes). Surveys of genomes have revealed that, in every organism, there are always a few large families and many small ones, with the overall distribution following a power-law. This commonality is equally true for both genes and pseudogenes, and exists despite the fact that the specific families that are enlarged differ greatly between organisms. Furthermore, because of family structure there is great redundancy in proteomes, a fact linked to the large number of dispensable genes for each organism and the small size of the minimal, indispensable sub-proteome. Pseudogenes in prokaryotes represent families that are in the process of being dispensed with. In particular, the genome sequences of certain pathogenic bacteria (Mycobacterium leprae, Yersinia pestis and Rickettsia prowazekii) show how an organism can undergo reductive evolution on a large scale (i.e. the dying out of families) as a result of niche change. There appears to be less pressure to delete pseudogenes in eukaryotes. These can be divided into two varieties, duplicated and processed, where the latter involves reverse transcription from an mRNA intermediate. We discuss these collectively in yeast, worm, fly, and human. The fly has few pseudogenes apparently because of its high rate of genomic DNA deletion. In the other three organisms, the distribution of pseudogenes on the chromosome and amongst different families is highly non-uniform. Pseudogenes tend not to occur in the middle of chromosome arms, and tend to be associated with lineage-specific (as opposed to highly conserved) families that have environmental-response functions. This may be because, rather than being dead, they may form a reservoir of diverse "extra parts" that can be resurrected to help an organism adapt to its surroundings. In yeast, there may be a novel mechanism involving the [PSI+] prion that potentially enables this resurrection. In worm, the pseudogenes tend to arise out of families (e.g. chemoreceptors) that are greatly expanded in it compared to the fly. The human genome stands out in having many processed pseudogenes. These have a character very different from those of the duplicated variety, to a large extent just representing random insertions. Thus, their occurrence tends to be roughly in proportion to the amount of mRNA for a particular protein and to reflect the extent of the intergenic sequences. Further information about pseudogenes is available at http://genecensus.org/pseudogene