between 1990 and 2013 , the global tuberculosis ( tb ) prevalence and mortality rates decreased by 41% and 45% , respectively ( 1 ) . despite the apparent progress in tb control , 9 million people developed tb and 1.5 million people succumbed to the disease in 2013 , reiterating that this disease is still a severe global public health challenge ( 1 ) . the world health organization s post-2015 global tb strategy aims to reduce the 2015 worldwide tb incidence and mortality rates by 90% and 95% , respectively , by the year 2035 ( 1 ) . the achievement of these goals is dependent on the identification of novel biomarkers and targets for which new innovations for tb diagnosis , treatment , and prevention can be elucidated . these goals may be realized by intensifying scientific research into the interaction of the tb etiologic agent , mycobacterium tuberculosis , with its human host . a vital precursor to host colonization and the deploying of bacterial virulence factors , such as toxins and effector proteins , is the adhesion of the pathogen to host cells . the initial binding to host cells occurs via weak and non - specific forces that are brought about by the physicochemical properties of the host surface . thereafter , specific and transient receptor interactions enable a stronger anchorage of the pathogen to the host surface . lastly , specific and high - affinity attachment is achieved by the interaction of bacterial adhesins with host receptors ( 2 ) . adhesins are monomeric or filamentous cell - surface molecules or structures that facilitate bacterial attachment to host surfaces . this process is a pivotal precursor to bacterial invasion and colonization of the host , by limiting the clearance of the pathogen through shear stress and by activating key virulence factors that are associated with infection ( 3 ) . furthermore , bacterial adhesion to host cells results in a manipulation of host cell signalling , which promotes the spread of the pathogen and allows for the microbe to evade the host immune response ( 3 ) . adhesins also bind to receptors of the extracellular matrix ( ecm ) , thereby activating signal transduction cascades in the host cell . these adhesins , termed microbial surface components recognizing adhesive matrix molecules ( mscramm ) , use receptors ( such as integrins ) to adhere to ecm molecules , including bone sialoprotein , collagens , elastin , fibronectin , glycosaminoglycans , laminin , thrombospodin , and vitronectin ( 4 ) . the ability of environmental bacteria to adhere to natural biopolymers ( e.g. , cellulose fibres ) can be beneficial to these organisms , such as by providing access to nutrient - rich sources . in addition , the adhesion of bacteria to synthetic biomaterials ( e.g. , medical devices ) , such as those biomaterials that have been exposed to body fluids and contain adhered serum proteins , results in severe clinical consequences ( 4 ) . adhesin - mediated adhesion to host surfaces also enables biofilm formation ( 5 ) , a multicellular lifestyle of bacteria that is associated with bacterial tolerance to antimicrobial agents , host defences , and other environmental challenges . due to their fundamental role in the host - pathogen interaction , bacteria have evolved a large variety of adherence - mediating components and strategies to enable their adhesion to the host . pili are polymeric hair - like surface structures that contain an adhesin at its distal tip . they are the most researched class of bacterial adhesins , particularly in gram - negative bacteria . gram - positive organisms were also subsequently found to elaborate pili , and their function in adhesion and pathogenesis is being deciphered ( 6 ) . these adhesins are capable of interacting with host molecules , including transmembrane proteins and ecm components ( 7 ) . it is well - established that during infection , m. tuberculosis interacts with and enters host cells , including macrophages , epithelial cells , and dendritic cells . this process is crucial to the organism s survival , replication , and dissemination ( 8) . however , the adhesins mediating these fundamental processes has been largely unexplored and underappreciated ( 9 ) . although several m. tuberculosis proteins have been documented to function in the attachment to host components ( table 1 ) , computational tools have predicted an abundance of additional uncharacterized m. tuberculosis adhesins ( table 2 ) . a list of the currently known mycobacterium tuberculosis adhesins a list of the putative mycobacterium tuberculosis adhesins identified by computational analyses   putative adhesins identified from kumar et al ( 18 ) and the mycobacrv web server ( http://mycobacteriarv.igib.res.in ) a major challenge to the treatment of tb patients is the escalating rates of drug - resistant m. tuberculosis strains . a novel strategy for tb treatment and prevention involves targeting bacterial determinants of infection , rather than those of survival . this would incapacitate the pathogen and ensure a natural clearance of the bacilli , without imposing selective pressure and amplifying the number of drug - resistant forms of the bacterium . in addition , it would prevent the release of harmful products ( e.g. , endotoxins ) from lysed bacterial cells , which occurs with the use of bactericidal agents ( 2 ) . however , due to a plethora of m. tuberculosis adhesion molecules , anti - adhesion tb therapeutics should target a combination of the major adhesins , possibly as an adjunct to the conventional tb therapeutics , to be effective . for adhesion inhibition to be a viable intervention for tb treatment and prevention , further clarity on the entire repertoire of m. tuberculosis adhesins is needed . the purpose of this work is to review the major methods available for the identification of m. tuberculosis adhesins . these methods include adherence assays that utilize wild - type and gene knockout mutant strains , epitope masking and competitive inhibition analyses , ecm protein binding assays , microsphere adhesion assays , m. tuberculosis auto - aggregation assays , and in silico analyses . comparing the adhesion capacity of wild - type and gene knockout mutant strains is a widely used approach to identify genetic determinants of adhesion and infection . in this assay , wild - type and gene knockout mutant strains infect host cells ( macrophages or epithelial cells ) in microtitre plates . a multiplicity of infection of between 1:1 and 10:1 ( bacilli to host cells ) and an infection time of 1 - 4 hrs ( usually 1 hr ) is generally used . this is followed by washing to remove non - adherent bacilli and a lysis step ( usually with 0.1% triton x-100 ) to disrupt the host cells . thereafter , serial dilutions of the cell lysates are plated onto agar plates to enumerate viable adherent bacterial cells , including those that have invaded the host cells . since intracellular bacilli would have had to adhere prior to invading the host cells , the invaded bacteria also represent the total adhered population . however , one may distinguish between the adherent versus invaded fraction using the aminoglycoside protection assay ( 10 ) to quantify the invaded bacilli . this colony - forming unit ( cfu)-based adherence assay is advantageous in that it enables the cell association efficiency to be calculated from the total number of bacilli in the infection inocula . in addition , it is a simple and relatively low - cost method that requires no specialized equipment . this assay has been successfully used to identify the adhesin function of the pro - glu ( pe ) polymorphic gc - rich repetitive sequence ( pgrs ) protein encoded by rv1818c ( 11 ) , the heparin - binding hemagglutinin adhesin ( hbha ) ( 12 ) , and curli pili ( mtp ) ( 13 , 14 ) . in these studies , the deletion of the encoding genes ( rv1818c , rv0475 , and rv3312a ) resulted in a significant reduction in m. tuberculosis adhesion to macrophages and/or epithelial cells . this assay has also been used to measure the binding of m. tuberculosis to ecm proteins . be et al ( 15 ) incubated wild - type and pknd mutant m. tuberculosis strains with laminin for 1.5 hr . the matrices were then lysed and plated onto agar plates for cfu assessment . a two - fold reduction in the adhesion to laminin was observed for the mutant , indicating that protein kinase d is a laminin - binding adhesin . the deletion of essential ( and even some non - essential ) m. tuberculosis genes pose a technical challenge to the generation of gene knockout mutants . this prevents the utility of the previously described adherence assay to assess the possible adhesin function of the products that these genes encode . this strategy makes use of purified / recombinant proteins or antibodies against the purified / recombinant protein to block the binding of m. tuberculosis to the host components . hickey et al ( 16 ) used m. tuberculosis that was either untreated or treated with anti - cpn60.2 anti - bodies to infect macrophages . unattached bacteria were removed by washing and attached bacilli were identified by cfu counting and microscopy . the authors found that the m. tuberculosis strains with masked cpn60.2 displayed a significant decrease in the adhesion to macrophages compared with the unmasked m. tuberculosis . the authors also used macrophages that were either untreated or treated with recombinant cpn60.2 protein , prior to the addition of m. tuberculosis . the results indicated that m. tuberculosis displayed a decreased adhesion to cpn60.2-treated macrophages compared with untreated macrophages . a similar masking strategy was used by abou - zeid et al ( 17 ) to identify the bcg85 complex of mycobacterium bovis bcg , which is also present in m. tuberculosis as the antigen 85 complex , as a fibronectin - binding adhesin . in this report , the authors showed that m. bovis bcg that was pre - incubated with anti - bcg85 antibodies demonstrated a diminished capacity to adhere to fibronectin compared with unmasked m. bovis bcg . they also found that fibronectin that was pre - incubated with purified bcg85a and bcg85b proteins inhibited the adhesion of m. bovis bcg to fibronectin . in addition to their ability to bind to host cells , m. tuberculosis also adheres to ecm proteins , particularly collagens , fibronectin , and laminin . therefore , numerous studies have identified m. tuberculosis adhesins based on their ability to bind to ecm proteins . kumar et al ( 18 ) used an image - based enzyme - linked immunosorbent assay ( elisa ) to demonstrate the binding of the recombinant rv0309 , rv2599 , and rv3717 proteins to immobilized ecm proteins ( fibronectin , laminin , and/or collagen ) . detection of protein binding was achieved using horseradish peroxidase conjugated anti-6x histidine antibodies and ortho - phenylenediamine dihydro - chloride . alteri et al ( 19 ) used flow cytometry to measure the binding of wild - type and mtp - deficient m. tuberculosis strains to ecm proteins , by the detection of anti - ecm antibody complexes , using alexa fluor conjugate . the binding of mtp to laminin was confirmed using a sandwich elisa . in this experiment , the authors used varying concentrations of laminin to demonstrate that immobilized purified mtp protein bound to this ecm protein . similarly , using flow cytometry and elisa , pethe et al ( 20 ) showed that the mycobacterium smegmatis laminin - binding protein ( which is also expressed by m. tuberculosis ) binds to laminin . using elisa and reverse elisa , kinhikar et al ( 21 ) demonstrated that purified recombinant malate synthase bound to fibronectin and laminin . this was confirmed by fractioning the recombinant protein using sodium dodecyl sulphate polyacrylamide gel electrophoresis , treating the western blots with these ecm proteins , and detecting ecm protein binding using anti - ecm and secondary antibodies . these researchers also showed that recombinant malate synthase adhered to epithelial cells . this was done by detecting bound malate synthase using anti - malate synthase antibodies and flow cytometry . using a similar immunoblotting strategy to kinhikar et al ( 21 ) , espitia et al ( 22 ) and xolalpa et al ( 23 ) found that the fractioned recombinant rv1759c pe - pgrs protein and glutamine synthetase a1 bound to fibronectin , respectively . although microspheres have generally been used in m. tuberculosis phagocytosis assays , they also have application in the study of bacterial adhesion . esparza et al ( 24 ) used 1 m green fluorescent polystyrene microbeads coated with purified psts-1 abc phosphate transporter protein to interact with macrophages adhering to glass coverslips . non - adherent microbeads were removed by washing and adherent microbeads were identified by analyzing randomly selected cells by confocal microscopy . similarly , diaz - silvestre et al ( 25 ) used this technique to confirm the role of the 19-kda antigen ( lpqh glycolipoprotein ) as an adhesin . chitale et al ( 26 ) used 0.3 and 1.1 m latex beads coated with recombinant mycobacterium cell entry-1 ( mce1 ) protein to infect hela cells cultured on glass coverslips . the use of 1 m fluorescent latex beads has also been described , where fluorescence emission gives an indication of bead association ( 26 ) . apart from their function in bacteria - eukaryotic cell interactions , adhesins also mediate cell - to - cell contact between bacterial cells and are , thus , key contributors to bacterial aggregation and biofilm formation . menozzi et al ( 27 ) reported that incorporating purified hbha to m. tuberculosis cultures resulted in an aggregation of the bacilli , in a dose - dependent manner . ramsugit et al ( 28 ) showed that a mtp - deficient m. tuberculosis strain displayed a significant reduction in bacterial aggregation and pellicle formation in detergent - free sauton s media compared with the wild - type strain . both hbha and mtp were subsequently shown to also function as adhesins in the host - pathogen interaction ( 12 - 14 ) . the identification of adhesins that promote m. tuberculosis auto - aggregation could , therefore , enable the identification of potential adhesins that are associated with host interaction . kumar et al ( 18 ) screened all of the m. tuberculosis h37rv proteins through several bioinformatics tools and identified 20 novel putative adhesins ( table 2 ) . this was achieved using spaan adhesin predictor ( 29 ) at a threshold of pad > 0.65 , subcellular localization prediction algorithms [ loctree ( 30 ) , psortb ( 31 ) , and subloc ( 32 ) ] , and removing potential human homologues that were identified by blast . in addition , further filtering criteria for globular and low - molecular - weight proteins that were not previously reported in the literature were applied . three of the prospective adhesins identified in that study ( rv0309 , rv2599 , and rv3717 ) were subsequently experimentally confirmed to bind to ecm proteins ( 18 ) . the mycobacrv database of mycobacterial vaccine candidates provides information regarding possible adhesins of 22 human pathogenic mycobacterial species and their potential as vaccine candidates ( 33 ) . this database uses spaan ( 29 ) at pad>0.6 and psortb ( 34 ) at a very stringent cut - off to identify adhesin and adhesin - like proteins . using the mycobacrv web server ( http://mycobacteriarv.igib.res.in ) , 37 novel prospec - tive adhesins were identified in m. tuberculosis h37rv ( table 2 ) . comparing the adhesion capacity of wild - type and gene knockout mutant strains is a widely used approach to identify genetic determinants of adhesion and infection . in this assay , wild - type and gene knockout mutant strains infect host cells ( macrophages or epithelial cells ) in microtitre plates . a multiplicity of infection of between 1:1 and 10:1 ( bacilli to host cells ) and an infection time of 1 - 4 hrs ( usually 1 hr ) is generally used . this is followed by washing to remove non - adherent bacilli and a lysis step ( usually with 0.1% triton x-100 ) to disrupt the host cells . thereafter , serial dilutions of the cell lysates are plated onto agar plates to enumerate viable adherent bacterial cells , including those that have invaded the host cells . since intracellular bacilli would have had to adhere prior to invading the host cells , the invaded bacteria also represent the total adhered population . however , one may distinguish between the adherent versus invaded fraction using the aminoglycoside protection assay ( 10 ) to quantify the invaded bacilli . this colony - forming unit ( cfu)-based adherence assay is advantageous in that it enables the cell association efficiency to be calculated from the total number of bacilli in the infection inocula . in addition , it is a simple and relatively low - cost method that requires no specialized equipment . this assay has been successfully used to identify the adhesin function of the pro - glu ( pe ) polymorphic gc - rich repetitive sequence ( pgrs ) protein encoded by rv1818c ( 11 ) , the heparin - binding hemagglutinin adhesin ( hbha ) ( 12 ) , and curli pili ( mtp ) ( 13 , 14 ) . in these studies , the deletion of the encoding genes ( rv1818c , rv0475 , and rv3312a ) resulted in a significant reduction in m. tuberculosis adhesion to macrophages and/or epithelial cells . this assay has also been used to measure the binding of m. tuberculosis to ecm proteins . be et al ( 15 ) incubated wild - type and pknd mutant m. tuberculosis strains with laminin for 1.5 hr . the matrices were then lysed and plated onto agar plates for cfu assessment . a two - fold reduction in the adhesion to laminin was observed for the mutant , indicating that protein kinase d is a laminin - binding adhesin . the deletion of essential ( and even some non - essential ) m. tuberculosis genes pose a technical challenge to the generation of gene knockout mutants . this prevents the utility of the previously described adherence assay to assess the possible adhesin function of the products that these genes encode . this strategy makes use of purified / recombinant proteins or antibodies against the purified / recombinant protein to block the binding of m. tuberculosis to the host components . hickey et al ( 16 ) used m. tuberculosis that was either untreated or treated with anti - cpn60.2 anti - bodies to infect macrophages . unattached bacteria were removed by washing and attached bacilli were identified by cfu counting and microscopy . the authors found that the m. tuberculosis strains with masked cpn60.2 displayed a significant decrease in the adhesion to macrophages compared with the unmasked m. tuberculosis . the authors also used macrophages that were either untreated or treated with recombinant cpn60.2 protein , prior to the addition of m. tuberculosis . the results indicated that m. tuberculosis displayed a decreased adhesion to cpn60.2-treated macrophages compared with untreated macrophages . a similar masking strategy was used by abou - zeid et al ( 17 ) to identify the bcg85 complex of mycobacterium bovis bcg , which is also present in m. tuberculosis as the antigen 85 complex , as a fibronectin - binding adhesin . in this report , the authors showed that m. bovis bcg that was pre - incubated with anti - bcg85 antibodies demonstrated a diminished capacity to adhere to fibronectin compared with unmasked m. bovis bcg . they also found that fibronectin that was pre - incubated with purified bcg85a and bcg85b proteins inhibited the adhesion of m. bovis bcg to fibronectin . in addition to their ability to bind to host cells , m. tuberculosis also adheres to ecm proteins , particularly collagens , fibronectin , and laminin . therefore , numerous studies have identified m. tuberculosis adhesins based on their ability to bind to ecm proteins . kumar et al ( 18 ) used an image - based enzyme - linked immunosorbent assay ( elisa ) to demonstrate the binding of the recombinant rv0309 , rv2599 , and rv3717 proteins to immobilized ecm proteins ( fibronectin , laminin , and/or collagen ) . detection of protein binding was achieved using horseradish peroxidase conjugated anti-6x histidine antibodies and ortho - phenylenediamine dihydro - chloride . alteri et al ( 19 ) used flow cytometry to measure the binding of wild - type and mtp - deficient m. tuberculosis strains to ecm proteins , by the detection of anti - ecm antibody complexes , using alexa fluor conjugate . the binding of mtp to laminin was confirmed using a sandwich elisa . in this experiment , the authors used varying concentrations of laminin to demonstrate that immobilized purified mtp protein bound to this ecm protein . similarly , using flow cytometry and elisa , pethe et al ( 20 ) showed that the mycobacterium smegmatis laminin - binding protein ( which is also expressed by m. tuberculosis ) binds to laminin . using elisa and reverse elisa , kinhikar et al ( 21 ) demonstrated that purified recombinant malate synthase bound to fibronectin and laminin . this was confirmed by fractioning the recombinant protein using sodium dodecyl sulphate polyacrylamide gel electrophoresis , treating the western blots with these ecm proteins , and detecting ecm protein binding using anti - ecm and secondary antibodies . this was done by detecting bound malate synthase using anti - malate synthase antibodies and flow cytometry . using a similar immunoblotting strategy to kinhikar et al ( 21 ) , espitia et al ( 22 ) and xolalpa et al ( 23 ) found that the fractioned recombinant rv1759c pe - pgrs protein and glutamine synthetase a1 bound to fibronectin , respectively . although microspheres have generally been used in m. tuberculosis phagocytosis assays , they also have application in the study of bacterial adhesion . esparza et al ( 24 ) used 1 m green fluorescent polystyrene microbeads coated with purified psts-1 abc phosphate transporter protein to interact with macrophages adhering to glass coverslips . non - adherent microbeads were removed by washing and adherent microbeads were identified by analyzing randomly selected cells by confocal microscopy . similarly , diaz - silvestre et al ( 25 ) used this technique to confirm the role of the 19-kda antigen ( lpqh glycolipoprotein ) as an adhesin . chitale et al ( 26 ) used 0.3 and 1.1 m latex beads coated with recombinant mycobacterium cell entry-1 ( mce1 ) protein to infect hela cells cultured on glass coverslips . the use of 1 m fluorescent latex beads has also been described , where fluorescence emission gives an indication of bead association ( 26 ) . apart from their function in bacteria - eukaryotic cell interactions , adhesins also mediate cell - to - cell contact between bacterial cells and are , thus , key contributors to bacterial aggregation and biofilm formation . menozzi et al ( 27 ) reported that incorporating purified hbha to m. tuberculosis cultures resulted in an aggregation of the bacilli , in a dose - dependent manner . ramsugit et al ( 28 ) showed that a mtp - deficient m. tuberculosis strain displayed a significant reduction in bacterial aggregation and pellicle formation in detergent - free sauton s media compared with the wild - type strain . both hbha and mtp were subsequently shown to also function as adhesins in the host - pathogen interaction ( 12 - 14 ) . the identification of adhesins that promote m. tuberculosis auto - aggregation could , therefore , enable the identification of potential adhesins that are associated with host interaction . kumar et al ( 18 ) screened all of the m. tuberculosis h37rv proteins through several bioinformatics tools and identified 20 novel putative adhesins ( table 2 ) . this was achieved using spaan adhesin predictor ( 29 ) at a threshold of pad > 0.65 , subcellular localization prediction algorithms [ loctree ( 30 ) , psortb ( 31 ) , and subloc ( 32 ) ] , and removing potential human homologues that were identified by blast . in addition , further filtering criteria for globular and low - molecular - weight proteins that were not previously reported in the literature were applied . three of the prospective adhesins identified in that study ( rv0309 , rv2599 , and rv3717 ) were subsequently experimentally confirmed to bind to ecm proteins ( 18 ) . the mycobacrv database of mycobacterial vaccine candidates provides information regarding possible adhesins of 22 human pathogenic mycobacterial species and their potential as vaccine candidates ( 33 ) . this database uses spaan ( 29 ) at pad>0.6 and psortb ( 34 ) at a very stringent cut - off to identify adhesin and adhesin - like proteins . using the mycobacrv web server ( http://mycobacteriarv.igib.res.in ) , 37 novel prospec - tive adhesins were identified in m. tuberculosis h37rv ( table 2 ) . tb remains a major challenge . despite global efforts to improve our understanding of m. tuberculosis pathogenesis , insufficient knowledge has been gained on the m. tuberculosis adherence mechanisms , the first ( and arguably the most critical ) step in the pathogenesis of this bacterium . several m. tuberculosis adhesins have been identified ; however , computational analyses have predicted a large number of additional adhesin - encoding genes in this pathogen s genome . several of these genes encode proteins belonging to the pe - pgrs and pro - pro - glu ( ppe ) protein families . it , therefore , seems likely that key information regarding this organism s adherence mechanisms could be obtained by the study of the adhesin capability of proteins belonging to these families . with several methods available for the study of m. tuberculosis adhesion , as highlighted in this review , identifying novel m. tuberculosis adhesins is possible and should be explored further . advances in gene disruption technologies open up the possibility of screening mutant libraries to identify m. tuberculosis genes involved in the pathogen s adherence . adapting the adhesion methodologies to enable high - throughput screening of libraries of potential anti - adhesion therapeutics is needed and may have value in the control of this ravaging disease .
anti - adhesion therapy represents a potentially promising avenue for the treatment and prevention of tuberculosis in a post - antibiotic era . 
 adhesins are surface - exposed microbial structures or molecules that enable pathogenic organisms to adhere to host surfaces , a fundamental step towards host infection . 
 although several mycobacterium tuberculosis adhesins have been identified , it is predicted that numerous additional adherence - mediating components contribute to the virulence and success of this pathogen . 
 significant further research to discern and characterize novel m. tuberculosis adhesins is , therefore , required to gain a holistic account of m. tuberculosis adhesion to the host . 
 this would enable the identification of potential drug and vaccine targets for attenuating m. tuberculosis adherence and infectivity . 
 several methods have been successfully applied to the study and identification of m. tuberculosis adhesins . in this manuscript , we review these methods , which include adherence assays that utilize wild - type and gene knockout mutant strains , epitope masking and competitive inhibition analyses , extracellular matrix protein binding assays , microsphere adhesion assays , m. tuberculosis auto - aggregation assays , and in silico analyses .