despite efforts to eradicate tuberculosis , it remains one of the most successful and deadly human diseases .
the tubercle bacilli resides within dendritic cells and macrophages , and in an immunocompetent host , the infection is walled off within a granuloma where it can remain dormant for years . while many factors contribute to its success , it is the thick , waxy cell wall of the bacillus that prevents dehydration , affords protection against varying levels of acidity and the detrimental affects of free radicals . in many respects ,
it is the unique architecture of the cell wall itself that makes mycobacterium tuberculosis ( mtb ) infection relatively difficult to treat with antibiotics .
since it is also a reservoir for many proteins and nonproteinaceous antigens , which are secreted into the extracellular milieu to stimulate and/or suppress the host immune response , its definition can be exploited for vaccine development . for decades
, the macromolecular features of the mycobacterial cell wall , including the mycolic acid and arabinogalactan core , have been studied in detail.(3 ) structurally , the cell wall of mtb is composed of a distinct inner core of mycolic acid - arabinogalactan - peptidoglycan ( magp ) . in addition to the covalently attached lipids and carbohydrates , it is well - known that the free lipids , lipoglycans , and phosphotidyl inositols that reside in the outer core of the cell wall play key roles in modulation of the host immune response.(4 ) specifically , the molecules , lipoarabinomannan ( lam ) , lipomannan ( lm ) , and phosphatidyl inositol mannoside ( pim ) are known to aid the process of host immune evasion.(2 ) in addition , virulence lipids such as trehalose dimycolate / monomycolate ( tdm / tmm ) , phthiocerol dimycocerosate ( pdim ) , and sulfolipids ( sl ) and the protein machinery , such as mmpls , required for their export are intercalated in the cell wall.(5 ) numerous cell wall associated proteins , including many lipoproteins and lipoglycoproteins , have also been described .
for example , the tlr2 agonists , lpqh ( 19 kda ) , psts1 ( 38 kda ) , and lprg ( rv14llc ) , are all found in the cell wall , where they function to regulate the action of macrophages and dendritic cells.(12 ) psts1 also plays a role in bacterial escape from the host macrophage through apoptosis.(13 ) many other lipoproteins of unknown function are identified in the secreted proteome of mtb culture filtrate .
the study of the secreted proteome of mtb was driven by the search for novel immunodominant antigens , drug targets , and biomarkers for disease . to build upon this work , a number of studies employed advances in proteomic technologies such as two - dimensional gel electrophoresis ( 2dge ) and liquid chromatography mass spectrometry ( lcms / ms ) to further mine additional subcellular compartments of mtb  cytosol , membrane , and cell wall .
historically , the proteins within the cell wall have been difficult to resolve and identify by traditional 2dge methods .
mawuenyega et al . , employing two - dimensional liquid chromatography coupled with mass spectrometry ( 2dlc / ms ) were the most successful at defining the cell wall proteome with the identification of 306 proteins.(24 ) in this study , we set out to comprehensively describe the mtb cell wall proteome in an effort to exploit additional proteins that may play a role in host - pathogen interactions and define new potential drug targets via discovery of unique biosynthetic or metabolic processes .
we used a combination of detergent extraction , 2dge , multidimensional liquid chromatography , and mass spectrometry to achieve our goal .
mtb strain h37rv was cultured in 2 l of glycerol alanine salts ( gas ) medium(28 ) in roller bottles for 14 d at 37 c with gentle agitation .
cells were harvested,(24 ) washed with phosphate - buffered saline ( pbs ) , ph 7.4 , and inactivated by -irradiation .
cells were disrupted in a french press , free lipids were removed , and the cell wall was obtained as previously described.(29 ) briefly , 1 g of lyophilized cell wall was subjected to two extractions of 2 h each followed by one 18 h extraction with chloroform / methanol ( 2:1 , v / v ) at a ratio of 30 ml / g of cell wall .
centrifugation at 27  000 g for 30 min was performed to collect cell wall material .
the 2:1 extracted cell wall was dried under n2 and further extracted twice for 2 h and one 18 h extraction with chloroform / methanol / water ( 10:10:3 , v / v / v ) each at 22 c .
the fully delipidated cell wall was dried under n2 and resuspended in pbs , ph 7.4 .
cell wall protein was quantified by bicinchoninic acid ( bca ) assay ( thermo pierce ) .
cell wall protein ( cwp ) was solubilized by one of three methods using ( i ) 6 m guanidine hcl ( guhcl ) , ( ii ) 2% sodium dodecyl sulfate ( sds ) , or ( iii ) 4% tritionx-114 ( tx-114 ) . for method ( i ) , 75 mg of cwp was incubated at 22 c for 4 h with agitation .
solubilized proteins were exchanged into 0.01 m nh4hco3 , and the protein amount was determined by bca assay .
the average protein recovery following guhcl extraction of cwp was 40% . for method ( ii )
, sds soluble proteins were generated by extraction of 100 mg of cwp with 2% sds in pbs ( w / v ) as described previously.(29 ) briefly , the sample was bath sonicated for 3 h at 90 c .
the sample was centrifuged at 27  000 g at 22 c for 30 min , collected , and centrifuged again .
the fully cleared supernatant was then subjected to paired - ion extraction for removal of sds.(30 ) proteins were concentrated by centrifugation as above and pellets washed with acetone at 20 c for 4 h. the final pellet was resuspended in 0.01 m nh4hco3 and protein amount was determined by bca .
the average protein recovery following sds extraction of cwp was 60% . in method ( iii ) , a stock solution of 32% tx-114 in pbs was added to 300 mg of cwp to a final concentration of 4% detergent .
primary extraction occurred at 4 c for 16 h. the extract was allowed to biphase at 37 c for 30 min and was fully separated by centrifugation at 27  000 g for 30 min .
the tx-114 detergent phases were pooled , and proteins collected by cold acetone precipitation.(31 ) final tx-114 proteins were resuspended as above and protein amount was determined by bca assay .
samples of 200 g of each cwp preparation were solubilized for 914 h in rehydration buffer ( 7 m urea/2 m thiourea , 1% amidosulfobetaine-14 ( asb-14 ) , 1% 3-[(3-cholamidopropyl ) dimethylammonio]-1-propanesulfonate ( chaps ) , 1 mm dithiothreitol ( dtt ) , 0.25% np-40 , 0.625% zoom carrier ampholytes 310 , and 1.9%
cwp was applied on zoom precast immobilized ph 47 linear gradient strips ( 7.0 cm ; invitrogen ) according to manufacturer s instructions .
focusing was achieved using a stepwise voltage gradient of 200 , 450 , 700 , and 1000 v , for 10 min each followed by focusing at 2000 v for 2 h. sds - page of the isoelectric focusing ( ief ) strips was performed using zoom 412% bis - tris sds - page gradient gels ( invitrogen ) .
images were captured using a gel - doc system ( bio - rad , hercules , ca ) , and spot detection was performed using delta 2d software ( greifswald , germany ) .
a total of 5.0 mg of tx-114 cwp was digested with modified trypsin ( roche diagnostics ) at a ratio of 1:20 ( e / s ) , in 0.1 m urea and 20 mm methylamine .
the digest was desalted using sep - pak light c18 cartridge ( waters , inc . ) and concentrated under vacuum .
the resultant peptides were separated by strong cation - exchange ( scx ) chromatography using a polysulfylethyl a column ( 460 m  200 mm , 300  ; poly lc , inc . )
the digest was applied in buffer a ( 5 mm k2po4 , 20% acetonitrile ( acn ) , ph 3.0 ) using a gradient of 080% buffer b ( a with 0.5 m kcl ) over 75 min with a flow rate of 1 ml / min .
the elution of peptides was monitored at 214 nm , and fractions ( 3 ml each ) were pooled based on uv absorbance .
each fraction ( 6 total ) was concentrated under vacuum and resuspended into reverse phase buffer a ( 0.1% tfa in h2o ) .
individual scx pools were subjected to further separation by reversed - phase hplc ( rp - hplc ) using a monomeric c18 column ( 4.6 mm  150 mm , vydac ) .
peptides were eluted using a gradient of 050% reverse - phase buffer b ( 90% acn in a ) over 40 min at a flow rate of 1 ml / min .
a total of 139 rp - hplc peptide fractions were collected and concentrated under vacuum .
coomassie blue stained spots were excised from 2-de gels and subjected to in - gel digestion with modified trypsin ( roche diagnostics ) . digests and 2dlc peptide fractions were resolved by liquid chromatographymass spectrometry ( lcms ) using either an lcq ( 2dge ) or ltq ( 2dlc ) as described previously .
tandem mass spectra were extracted , charge state deconvoluted , and deisotoped by bioworks version 3.2 ( thermo finnigan , san jose , ca ) .
all ms / ms samples were analyzed using sequest ( thermo finnigan ; version 27 , rev .
12 ) sequest was set up to search the tb genome database ( version 2.0 , genbank accession no .
al123456 , 3912 entries ) assuming the digestion enzyme trypsin , a fragment ion mass tolerance of 1.0 da , a parent ion tolerance of 2.5 da , and 3 allowable missed cleavages .
oxidation of methionine and acrylamide adduct of cysteine ( 2dge spots ) were specified in sequest as variable modifications .
sequest identifications required at least deltacn scores of greater than 0.3 and xcorr scores of greater than 1.5 , 2.2 , 2.5 , and 2.5 for singly , doubly , triply , and quadruply charged peptides ( supplementary table s1 ) .
scaffold ( version scaffold-01_05_21 , proteome software , inc . , portland , or ) was used to validate ms / ms based peptide and protein identifications .
peptide identifications were accepted if they could be established at greater than 90% probability as specified by the peptide prophet algorithm.(35 ) protein identifications were accepted if they could be established at greater than 90% probability as assigned by the protein prophet algorithm(35 ) and contained at least two unique peptides .
the protein probability false discovery rate for the cwp identifications was 3.5%.(36 ) proteins that contained similar peptides and could not be differentiated based on ms / ms analysis alone were grouped to satisfy the principles of parsimony ( supplementary table s1 ) .
fasta files for each annotated mtb protein were collected and organized into small groups of 50 .
each group was then submitted to the signalp server ( http://www.cbs.dtu.dk/services/signalp/ ) using the gram - positive option .
the output was saved in html format and a perl script written to combine the results from all html files into one file and then converted to tab delimited format .
this generated a complete list of results that were sorted in a spreadsheet to create the subsets of neural network positive , hidden markov model positive , and the union of the nn and hmm sets .
these lists were input into a java application along with the list of proteins isolated from the cell wall fractions .
this application simply compared the predicted list to those isolated and identified the proteins found in the cell wall that were also predicted in each subset as having a signal peptide .
to predict lipoproteins , the mtb peptide fasta file from above was broken down into 1000 sequences per file .
the output files were combined in a spreadsheet , and from the sorted data , a list was extracted with every signal peptidase ii cleavage site generating a predicted lipoprotein list .
this list was input into a java application that would compare the cell wall proteins , identifying the predicted lipoproteins from each cell wall fraction .
protein preparations from the three cwp fractions resolved by 2dge or sds - page were transferred to pvdf membranes and incubated with mouse monoclonal antibodies ( -glcb [ rv1837c ] , -psts1 [ rv0934 ; it-23 ] , -lprg [ rv1411c ] , -ald [ rv2780 ] , -lpqh [ rv3763 ; it-19 ] ; all available through the nih , niaid contract , tuberculosis vaccine testing and research materials ) and polyclonal antibody against fructose bisphosphate aldolase ( -fba [ rv0363c ] , kindly provided by dr .
mary jackson , colorado state university ) in tbs and 0.25% tween 80 ( tbst ) .
after incubation , membranes were rinsed with tbst and developed using amersham ecl advance western blotting detection kit ( ge healthcare life science , piscataway , nj ) as per manufacturers instructions .
protein immunoblots were visualized with the typhoon 9400 multiplex fluorescent imager ( ge healthcare life science , piscataway , nj ) .
to maximize the resolution and protein identification of cell wall proteins , the delipidated cell wall of mtb was subjected to extraction based on solubility within three reagents  an anionic ( sds ) , a chaotrophic ( guhcl ) , and a nonionic ( tx-114 ) detergent .
2dge of all cwp fractions demonstrated the majority of proteins resolving within a range of mw 2575 kda ( supplementary figure 1 ) . on average ,
210 spots were resolved for guhcl and sds cwp fractions and 170 from tx-114 . from the 2dge analysis ,
290 proteins were identified with 122 , 131 , and 37 proteins identified in the guhcl , sds , and tx-114 fractions , respectively .
for all fractions , over half were unique to each subset ( figure 1a ) .
analysis of the data demonstrated 80% of proteins had known functions ( figure 2 ) .
in contrast , the tx-114 extracted fraction was less amenable to gel separation most likely due to its increased hydrophobic content .
a multidimensional chromatography approach was employed to better resolve proteins in this fraction , in order to exploit any unique or uncharacterized protein families within the tx-114 subset .
while 2dge was insufficient in identifying a significant number of proteins in the tx-114 detergent extract ( figure 1a ) , scx chromatography combined with reverse phase chromatography of a digest of this cwp preparation allowed the resolution of 364 proteins , including an additional 294 proteins not found in any of the 2dge preparations ( figure 1b ) .
( a ) identification of cell wall proteins by two - dimensional gel electrophoresis and two - dimensional liquid chromatography . detergent extraction and separation of cell wall
protein subsets via 2dge demonstrated the majority of proteins to be resolved using sds and guhcl .
( b ) cumulatively , separation of cell wall proteins by 2dge resulted in the identification of 234 proteins . separating the tx-114 protein subset by multidimensional chromatography ( 2dlcms / ms ) resolved an additional 294 proteins .
proteins identified for each extraction method ( guhcl , sds , tx114 ) were sorted by functional category as a percentage of total proteins per sample .
representation of all protein groups was identified with the exception of groups 4 and 8 ( data not shown ) .
seventy percent of the proteins identified in the cell wall are associated with lipid metabolism ( group 1 ) , cell wall processes ( group 3 ) , and intermediary metabolism ( group 7 ) .
black , sds ; white , guhcl ; gray , tx114 ; hash , total .
validation of a few selected proteins , by western blot analysis of samples resolved by 2dge ( supplementary figure 1 ) and sds - page ( supplementary figure 2 ) , corroborated the identification of these proteins by mass spectrometry .
all identified proteins were grouped by functional category as defined by institute pasteur , which demonstrated mtb protein families present in each extract .
proteins in categories 3 ( cell wall and cell wall processes ) and 7 ( intermediary metabolism ) were consistently overrepresented among the cwp preparations ( figure 2 ) . combining the data sets of both gel and non - gel based protein identifications led to the detection of 528 proteins ( supplementary table s2 ) .
these were further classified into functional groups as defined by the sanger institute ( figure 3ac ) .
one hundred and five of the 528 cell wall proteins identified were not reported in previous mtb proteome publications , including a comprehensive proteomic database http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi,(19 ) and http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi(42 ) ( table 1 ) .
the distributions are among the major functional groups and the subgroups within functional groups i and ii .
the percentage for each subgroup indicates the percentage of the total number of identified proteins in its major functional group .
i.x is defined by proteins involved in central intermediary metabolism , amino acid biosynthesis , polyamine synthesis , biosynthesis of cofactors , prosthetic groups , and carriers , lipid biosynthesis , polyketide and non - ribosomal peptide synthesis , and broad regulatory functions .
cell wall protein fractions : a , 2% sds ; b , 6 m guhcl ; c , 4% tx-114 ; d , 2dlc tx-114 .
a majority of proteins were classified in either category i , small molecule metabolism ( 35% ) , or category ii , macromolecule synthesis and degradation ( 25% ) ( figure 3 ) .
subclasses of category i showed 19% classified in small molecule metabolism - other ( i.x ) , which included the classes : central intermediary metabolism and amino acid biosynthesis ( figure 3 ; upper right ) ; and subclasses of category ii demonstrated an even distribution of proteins between synthesis / degradation of macromolecules ( ii.a , ii.b , 12.5% ) and cell envelope proteins ( ii.c , 12.1% ) ( figure 3 ; lower right ) .
the secreted proteins of mtb have traditionally been characterized as important antigens and immune - modulators . prior to being exported , many of these proteins are resident within the cell wall where their function remains largely unknown . to find the putative secreted proteins identified within the cell wall proteome , all identified proteins were subject to interrogation against neural network ( nn ) and hidden markov model ( hmm ) algorithms ( signalp , http://www.cbs.dtu.dk/services/signalp/ ) which revealed 18% , 19% ,
27% putative secreted proteins in guhcl , sds , tx-114 cwp samples , respectively , and 13% in the 2dlc resolved tx-114 cwp fraction .
cumulatively , of the 528 proteins identified in this study , 87 proteins were predicted to contain secretion signals and included the identification of 23 proteins uniquely found in this study ( table 2 ) .
a majority ( 60% ) of the cwp associated with secretion are indeed within the category of the cell wall and its processes ( figure 4 ) .
further , many of these cwp are also membrane associated , either by description or functional annotation . of
the 23 secreted cwp unique to this study , 11 have been described to be involved with small molecule and peptide binding .
additionally , 23 of the 87 secreted proteins are classified as hypothetical or unknown ( categories v and vi of sanger institute ) illustrating that to a large extent , the functions of these exported proteins are poorly understood .
eighty - seven cell wall proteins were predicted to contain secretion signals and putative lipoprotein motifs .
sixty percent of these proteins are classified within the cell wall and cell wall processes functional category .
next , we interrogated the cwp to identify those with putative lipoprotein motifs . here , a total of 16 proteins were predicted by lipop ( lipop , http://www.cbs.dtu.dk/services/lipop/)(43 ) to contain a signal peptidase ii cleavage motif , and an additional 8 proteins were identified when compared to the 99 putative lipoproteins ( 2.5% of the genome ) that reportedly exist within the mtb proteome.(7 ) a majority of the lipoproteins contain no additional functional classification ; however , 4 proteins ( rv0928 , rv0934 , rv2864c , rv3666c ) are involved with substrate binding and transport within the periplasm .
one of two superoxide dismutases in mtb , sodc ( rv0432 ) , was also identified within the lipoprotein - enriched tx-114 cwp fraction .
this protein has recently been defined as a highly glycosylated putative lipoprotein and is a defined b - cell antigen .
other putative lipoproteins identified in this study contain o - glycosylation motifs as predicted by netoglyc and were found within the mtb glycoproteome ( table 2).(46 ) the function of these dual - modified lipo - glycoproteins , as well as the nature of their modification remain largely undefined .
in this study , we focused on the gel - based two - dimensional separation of the cell wall and complemented that separation by liquid chromatography of digested tx-114 cwp . identified protein families included known antigens , fbp a , b , and c ( rv 3804c , rv1886c , and rv0129c ) , cfp10 ( rv3874 ) and esat-6 like proteins esxj and esxl ( rv1038c , rv1198 ) along with antigens lpqh ( rv3763 ) , lprg ( rv1411c ) , and lpra ( rv1270c ) .
in concordance with previous work , a number of ribosomal proteins are reported here , which must indicate a high level of protein synthesis occurring at the cytosolcell wall interface , most likely to facilitate the entry of proteins into the cellular envelope . for over a decade , the advancement of techniques in 2dge , mass spectrometry , and increased access to bioinformatic tools greatly enhanced proteomic studies of mtb . largely descriptive , these studies were undertaken to identify novel virulence factors and drug targets .
subsequent studies set out to understand functional relationships between proteins and discover antigens responsible for adaptive t - cell responses .
the quantitative techniques of mass spectrometric profiling , isotope coded affinity tag ( icat ) and isobaric tagging for relative and absolute quantification ( itraq ) , have afforded the accurate monitoring of up- and down - regulation of proteins on a global scale .
previous work mining subcellular fractions of mtb , focused on the cytosol and culture filtrate fractions , resulted in thorough characterization and the creation of 2d gel databases .
efforts have been made more recently to resolve the insoluble cell wall fraction . as mentioned previously , mawuenyega et al .
was able to identify 300 proteins within the cell wall using 2dlc as a separation method .
their study demonstrated functional relationships among key protein families within the fatty - acid synthesis pathway . using these studies as experimental platforms
, we were able to focus on the mtb cell wall and were successful in reporting over 100 additional proteins that had not been identified previously .
the elucidation of the cell wall proteome can facilitate subsequent studies of a more targeted nature where specific proteins can be monitored in response to various environmental , nutritional , or drug pressures .
one of the most interesting yet poorly understood protein families residing within the cell wall of mtb are the triacylated lipoproteins . as stated above , sutcliffe et al . predicted 2.5% of the genome to encode for lipoproteins .
defining these proteins beyond their antigenic and immune modulatory potential remains quite elusive with only a few studies predicting functional roles for mtb lipoproteins .
in this study , a vast majority of proteins found were involved in small molecule and macromolecule metabolism .
this builds upon the evidence that clearly defines two mtb cw lipoproteins , lppx and lprg , as aiding and/or facilitating the transport of lipids(57 ) and small molecules,(58 ) respectively .
the presence of these lipoproteins and perhaps other secreted , cw resident proteins provides a conduit between various nutrient transportation pathways that are required for cellular survival and growth while allowing the mycobacterial cell wall to maintain its rigid hydrophobic integrity . while bacterial cell walls have historically been thought of as structural scaffolding with little active interplay between the extracellular milieu and inner cytosolic environment , this perception of a static bacterial structure is fading and we are beginning to understand the true complexity and dynamic nature of the biological processes being facilitated within the cell wall .
for instance , these proteins may contribute to bacterial cell wall remodeling events in response to environmental stressors such as nutrient depletion , antibiotic pressures , the host immune response , and tissue remodeling events throughout the course of infection .
for mtb , the highly complex framework of the magpouter lipid architecture can now be definitively complemented with a highly diverse protein population
. this new perspective will provide further insight into cell wall remodeling processes during mycobacterial infection as well as give a more comprehensive list of potential drug targets and diagnostic candidates .
several classes of proteins were identified , including putative secreted proteins , lipoproteins , and known t and b cell antigens .
many proteins have unknown function ; however , their presence within the cell wall may be of biological relevance .
 the cell envelope of mycobacterium tuberculosis ( mtb ) is complex and diverse ; composed of proteins intermingled in a matrix of peptidoglycan , mycolic acids , lipids , and carbohydrates . 
 proteomic studies of the mtb cell wall have been limited ; nonetheless , the characterization of resident and secreted proteins associated with the cell wall are critical to understanding bacterial survival and immune modulation in the host . 
 in this study , the cell wall proteome was defined in order to better understand its unique biosynthetic and secretion processes . 
 mtb cell wall was subjected to extraction with organic solvents to remove noncovalently bound lipids and lipoglycans and remaining proteins were solubilized with either sds , guanidine - hcl , or tx-114 . 
 these extracts were analyzed by two - dimensional gel electrophoresis and mass - spectrometry and resulted in the identification of 234 total proteins . 
 the lipoproteome of mtb , enriched in the tx-114 extract , was further resolved by multidimensional chromatography and mass spectrometry to identify an additional 294 proteins . 
 a query of the 528 total protein identifications against neural network or hidden markov model algorithms predicted secretion signals in 87 proteins . 
 classification of these 528 proteins also demonstrated that 35% are involved in small molecule metabolism and 25% are involved in macromolecule synthesis and degradation building upon evidence that the mtb cell wall is actively engaged in mycobacterial survival and remodeling . 
Introduction
Methods
Results
Discussion
Conclusion