mycobacterium avium complex ( mac ) contains clinically important nontuberculous mycobacteria and is the second largest medical complex in the mycobacterium genus after the mycobacterium tuberculosis ( mtb ) complex . mac affects patients with chronic obstructive pulmonary disease and cystic fibrosis , as well as immunosuppressed individuals with hiv / aids . mac is currently composed of 12 species which are very close phylogenetically related , but they are diverse regarding to their host preference , course of disease , virulence and immune response , such as m.  avium , m. intracellulare and m.  colombiense . m.  colombiense ( mcol ) infections were initially described in hiv patients in bogot , colombia . in this initial study , mcol was confirmed to be the causative agent of pulmonary disease and bacteraemia in this group of people who died of hiv / mcol coinfection . after this study mcol has been isolated in other scenarios such as lymphadenopathy , subcutaneous infections and disseminated disease in hiv - negative patients in countries like spain , france , china , canada and russia . in this study we attempted to find the mcol virulence factors from the genome sequence to decipher its possible skills as a mac opportunistic pathogen . the mcol cect 3035 genome was sequenced using pacbio technology ( institute for genomes sciences , university of maryland , college park , md , usa ) , then automatically annotated using the gendb 2.4 platform . to study the mcol pathogenome , we used different bioinformatics tools to search for prophages sequences ( phast tool ) , genomic islands ( islandviewer   and gipsy / pips   tools ) , protein families ( pathogenfinder ) and mycobacterial virulence factors ( vfdatabase ( vfdb ) ) . the islands detected with islandviewer were classified using the national center for biotechnology information functional annotation and the conserved domain database , and for the gipsy / pips analysis we used the m.  indicus pranii genome as the closest related nonpathogenic species . to search for the mcol singletons paratuberculosis map4 ( refseq nc_021200 ) , m.  intracellulare atcc 13950 ( refseq nc_016946 ) , m.  intracellulare mott-36y ( refseq nc_017904 ) , m.  intracellulare mott-02 ( refseq nc_016947 ) , m.  intracellulare mott-64 ( refseq nc_016948 ) , m.  indicus pranii mtcc 9506 ( refseq nc_018612 ) , m.  yongonense 051390 ( refseq nc_021715 ) , mtb h37rv ( refseq nc_000962 ) and mcol cect 3035 with the edgar tool . then we detected the secretory singletons using pred - tat   and pred - lipo   tools . for the detection of transmembrane singletons we used tmhmm server 2.0 , and for the detection of the cell wall singletons we used cw - pred . using the tools phast , islandviewer and gipsy / pips , we did not detect prophage sequences in the mcol genome . in addition , we did not find structures compatible with crispr / cas systems by applying the crispr finder tool . however , we detected a total of 45 genomic islands ( nine of these are classified as pathogenicity island ) , which contain a total of 320 predicted genes . interestingly , 92.8% of these genes code for hypothetical proteins with unknown functions ( fig .  1 ) . in addition , the mcol pathogenicity islands contain genes that code for the drra - c transporter , which is involved in the transportation of phthiocerol dimycocerosates ( pdims ) and also contains the rmt2 and mmpl4 genes , which are involved in the synthesis and transmembrane transportation of glycopeptidolipids ( gpls ) . using the edgar tool for comparative genomics analysis of a selected set of mycobacterial genomes we detected a total of 452 singletons , which represent 8.4% of the mcol genome . according to a classification of these singletons into functional clusters of orthologous groups ( cogs ) categories 87 genes are involved in cellular processing and signaling , 75 are involved in information storage and processes , 163 are involved in metabolism of mcol and 122 are grouped as poorly characterized ( fig .  2 ) . the majority of singletons ( 85% ) contain typical sec signal peptides for recognition of the preprotein by the general secretory pathway , and 22% contain varying numbers of transmembrane helical segments for a putative integration into the cytoplasmic membrane . we also detected one singleton with an lpxtg motif that is an indication of cell wall localization of the respective protein . among these mcol singletons encoding gene sufs2 is inducible in response to oxidative stress and is important for the survival of agrobacterium tumefaciens 
 . the presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases and is an important virulence factor . additionally , cryptococcus gattii knockout mutants for genes encoding the urease accessory proteins ure4 and ure6 showed reduced multiplication within macrophages . the vfdb analysis allowed us to detect a total of 204 mcol genes that are homologous with previously described mycobacterial virulence factors . the largest groups of genes comprise deduced virulence factors assigned to the class 's cell surface components and secretion system . analysis of protein families with the pathogenfinder 1.1 tool allowed us to predict 32 virulence factors in the mcol genome sequence . of these genes 65.6% ( n  =  21 ) encoded hypothetical proteins and 5.5% ( n  =  2 ) encoded potentially secreted proteins with characteristic sec signal peptides . among these ten were also found in the vfdb , and 23 are new potential virulence factors . among these new potential mcol virulence factors we found  several genes homologous to the m.  avium subsp . k10 genome : map_0847 , map_1945c , map_0092 , map_3697c , map_1909 , map_3223c , map_0908c , map_2811c and map_2636 . according to basic local alignment search tool ( blast ) searches , these genes ( except map_1909 ) are likely not to be present in the mtb h37rv genome . interestingly , the map_1909 gene encodes the lppm lipoprotein , which in mtb is an important virulence factor involved in the manipulation of the phagosomal maturation in macrophages . hominissuis 104 genome : mav_4325 ( codes for abc transporter , atp - binding protein ) , mav_1611 ( codes for transcriptional regulator , tetr family protein ) , mav_4258 ( codes for the rsbw protein ) and mav_2193 ( codes for acyl carrier protein ) , mav_2835 ( codes for enoyl - coa hydratase / isomerase family protein ) , as well as the mav_3699 , mav_0045 , mav_1178 , mav_1177 , mav_4234 , mav_0139 , and mav_3009 genes ( code for hypothetical proteins ) . according to blast search , all these genes ( except mav_4258 ) are likely to not be present in the mtb h37rv genome . in addition , the rsbw protein in mtb is involved in the response to heat , cold , oxidative , starvation and anaerobic stresses . finally , we found a homologue to the mmar_4989 gene from the m.  marinum m genome . this gene encodes the yrbe4a conserved membrane protein , which has an unknown role in virulence but is predicted to be involved in lipid catabolism in mtb . experimental studies are necessary to decipher the role of these new potential virulence factors in opportunistic pathogens such as mcol . one of the hostile environments to which mycobacteria have to adapt during infection in the host is the macrophage . therefore , among all of the probable mcol virulence factors these have special importance because they are involved or are possibly implicated in macrophage modulation mechanisms ( fig .  4 ) . trehalose is an important compound for several mycobacterial cell wall glycolipids , including sulfolipid-1 ( sl-1 ) , trehalose monomycolate and trehalose dimycolate ( tdm ) . on the other hand , mcol has the gene that encodes the whib3 protein , which is an important virulence factor that is involved in several processes , including up - regulation of the synthesis of sl-1 and tdm , probably through the sensing of the reactive oxygen species ( ros ) and nitric oxide . this complex constitutes a group of extracellular mycolyl transferases ( ag85a , ag85b and ag85c ) , which play important roles in the biosynthesis of major components of the mycobacterial cell envelope , such as tdm and mycolylarabinogalactan . mcol has the genes that code for the sec export systems , seca1 and seca2 . the housekeeping seca1 protein of mtb is involved in the secretion of the virulence factors lprg and lpqh . in addition , the seca2 export system secretes important virulence factors such as superoxide dismutase ( soda ) involved in the survival inside macrophages   and catalase  peroxidase protein ( katg ) involved in isoniazid resistance . mcol has the gene encode protein kinase g ( pkng ) , which prevents phagosome  lysosome fusion by blocking lysosomal delivery . this process blocks the intracellular degradation of mycobacteria in lysosomes and mediates intracellular survival of mycobacteria within macrophages . mcol has genes that code for proteins that in mtb are associated with protection against ros ; these are ndk , nuog , sod and katg . in mtb ndk damages the phagocyte nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase complex in macrophages . nadph oxidase is responsible for the production of ros , which are radical molecules that are crucial for the control of mycobacterial infections . in addition ndk also prevents phagosome  lysosome fusion through a mechanism that involves the inactivation of macrophages rab proteins . it has been suggested that the ndk protein could be exported through esx-1 , which is lacking in mac members , including mcol . therefore , experimental research is necessary to investigate ndk secretion in mcol . in mtb nuog is an important virulence factor that can neutralize ros produced by nadph oxidase and with this inhibits macrophage apoptosis . in addition , soda and katg proteins are secreted by the seca2 export system ; they protect against ros by forming h2o2 , which is further detoxified by katg . two - component systems ( tcss ) are major regulatory systems for bacterial adaptation to environmental changes . the prra / b , mpra / b , phop / r and dosr / s tcss are the most studied and are involved in important virulence mechanisms in mtb . the prra / b system has been implicated in macrophage phagocytosis and is transiently required for the early stages of macrophage infection . the mpra / b is a stress - responsive tcs that directly regulates expression of several sigma factors , and it also regulates the espa operon in mtb and modulates esx-1 function . in mtb phop / r controls 30 genes directly , whereas regulatory cascades are responsible for signal amplification and downstream effects through proteins like espr , which controls esx1 function . phop also controls the noncoding rna ( ncrna ) mcr7 , which controls the secretion of the twin arginine translocation ( tat ) substrates , such as antigen ag85 complex proteins ( ag85a and ag85c ) . in mtb the dosr / s tcs regulates the expression of around 48 genes under hypoxic and nitric oxide stress and is required for full virulence and pathogenicity in different animal models . mcol has genes that code for proteins that are involved in the transportation and regulation of iron , such as irta , irtb , viub , esx-3 and ider . to obtain iron mtb synthesizes a cell - associated siderophore named mycobactin and a secreted one , carboxymycobactin , which sequesters iron and delivers it to the bacterium via specialized fe siderophore transporters . in mtb fe - carboxymycobactin can be secreted through irta transporters and is translocated by the irtb - viub abc transporter . the secretion and translocation of mycobactin is not entirely understood , but in m.  smegmatis the esx-3 secretion system plays an important role for the mycobactin - mediated iron uptake . sulfolipid-1 ( sl-1 ) is a cell wall glycolipid and important virulence factor that restricts mtb growth in macrophages . sl-1 has been also proposed to alter superoxide ( o ) production   and blocks phagosome several enzymatic steps are necessary for the sl-1 biosynthesis ; mmpl8 and sap proteins then transport sl1 across the membrane . mcol possesses the genes involved in the sl-1 biosynthesis and transport , except the pks2 . therefore , it is highly probable that mcol does not produce sl-1 . additionally , the production of this glycolipid is suggested to be restricted to pathogenic mycobacteria , especially to members of the mtb complex . mtb produces phospholipase c ( plc ) , which is an important virulence factor that induces the inhibition of cyclooxygenase-2 expression ( cox-2 ) , and this blocks prostaglandin e2 ( pge2 ) synthesis by macrophages . ( pge2 is an essential factor involved in the activation of the membrane repair mechanism , and therefore its inhibition is associated with necrosis of macrophages . ) analysis of the mcol genome suggests that the genes involved in the phospholipase c production ( plca , plcb , plcc and plcd ) are missing . therefore , it is a feasible that this species does not utilize this virulence mechanism during macrophage infection . members of mtb complex also produce p - hydroxybenzoic acid derivatives ( p - hbads ) , which are precursors of pgl biosynthesis . according to vfdb , several genes participating in pdim / pgl / p - hbad synthesis and transportation are lacking in mcol . the esx secretion system is a specialized secretion system for the transport of extracellular proteins across the mycobacterial cell wall . in mycobacteria there are five esx systems ( esx-1 , esx-2 , esx-3 , esx-4 , esx-5 ) , and all of them are present in the mtb genome . the esx-1 secretion system exports important mtb virulence factors such as esat-6 and cfp-10 , and it also induces apoptosis in macrophages and generates a semiporous phagosome , . mcol possesses all of the esx systems except esx-1 , and mcol also lacks the esat-6 and cfp-10 genes . the heat shock protein hspx is under the control of the dosr / s tcs during hypoxic conditions , and is suggested to be involved in the long - term survival of mtb inside macrophages . however , the mmps and mmpl proteins assemble into a complex that contribute to gpls biosynthesis and export . mcol possesses a gpl locus ; it is highly possible that this species is able to produce gpl . however , experimental studies are necessary to establish the mcol gpl serovar structure . in conclusion , mcol possesses many of the mtb virulence mechanisms that have been involved in apoptosis , necrosis and inhibition of phagosome  lysosome fusion . however , mcol has deletions in the genes involved in the p - hba / pdim / pgl , plc , sl-1 and hspx production , and loss of the esx-1 . the inability to produce these compounds may be related to its lower virulence in mice infected with mcol compared to mice infected with mtb . thus , the opportunistic nature of mcol could be associated with its inability to produce significant virulence mechanisms used by mtb during its adaptation to the host . in addition , the singletons and the virulence - associated genes conserved in the mcol genome could be used to design molecular diagnostic tools .
mycobacterium avium complex ( mac ) contains clinically important nontuberculous mycobacteria worldwide and is the second largest medical complex in the mycobacterium genus after the mycobacterium tuberculosis complex . 
 mac comprises several species that are closely phylogenetically related but diverse regarding their host preference , course of disease , virulence and immune response . in this study we provided immunologic and virulence - related insights into the m.  colombiense genome as a model of an opportunistic pathogen in the mac . by using bioinformatic tools we found that m.  colombiense has deletions in the genes involved in p - hba / pdim / pgl , plc , sl-1 and hspx production , and loss of the esx-1 locus . 
 this information not only sheds light on our understanding the virulence mechanisms used by opportunistic mac pathogens but also has great potential for the designing of species - specific diagnostic tools .