mycobacterium tuberculosis is an extraordinarily successful human pathogen with the ability to replicate within the normally hostile environment of host macrophages . after being phagocytosed by the macrophage ,
m. tuberculosis resides in a membrane - bound vacuole , the phagosome , which normally undergoes maturation into the phagolysosome that is essential for eliminating invading microbes and for antigen presentation.(1 ) however , m. tuberculosis is able to arrest phagosomal maturation by interfering with ca signaling and trafficking of the rab family of small gtpases , two important processes for organelle membrane fusion . when residing within a phagosome , the live bacilli secrets specific proteins such as tyrosine phosphatases to reduce the phagosomal level of phosphatidylinositol 3-phosphate or inhibit host proteins regulating vacuolar sorting , which all lead to impaired phagolysosomal fusion .
arrest of phagosomal maturation ( or block of phago - lysosome fusion ) is critical for m. tuberculosis persistence in host macrophages.(1 ) apart from secreted proteins , the exotic cell wall components of pathogenic mycobacteria are thought to be key modulators of host immune processes , but in most cases , their molecular effects on host cells are not well understood.(7 ) the best characterized are unique mycobacterial lipoglycans termed lipoarabinomannans ( lam ) , which are noncovalently associated with the bacterial plasma membrane and extend to the exterior of the cell wall .
notably , pathogenic m. tuberculosis produces mannose - capped lipoarabinomannan ( manlam ) structures,(10 ) whereas the fast - growing nonpathogenic species mycobacterium smegmatis synthesizes lam molecules capped with phosphatidyl - myo - inositol ( termed pilam)(11 ) ( figure 1 ) .
for instance , macrophages show significantly reduced phagosomelysosome fusion after engulfing manlam - coated microbeads.(12 ) manlam interacts with the mannose receptor and blocks the rise in cytosolic ca that would otherwise accompany mycobacterial entry into macrophages .
free manlam can insert into host cell membranes , leading to membrane reorganization and disruption of signaling pathways.(15 ) by contrast , pilam has none of these effects .
instead , it is an agonist of toll - like receptor 2 , which results in secretion of various cytokines and activation of apoptotic pathways , though the notion of its proinflammatory activity has been challenged recently.(19 ) motivated by reports suggesting different effects of manlam and pilam on macrophages , we sought to probe how the two mycobacterial lipoarabinans affect the molecular properties of host phagosomes .
previously , we developed a method to purify the membrane fraction from latex bead - containing ( lbc ) phagosomes in order to profile its proteome using mass spectrometry.(20 ) here we modified our method by combining tube - gel digestion(21 ) and itraq labeling(22 ) to study quantitative changes in the macrophage phagosomal proteome upon exposure of cells to manlam or pilam .
escherichia coli - derived lipopolysaccharide ( lps ) was used as a third stimulus in order to identify lam - specific effects . from a total list of 823 proteins found in the phagosomal membranes , we identified 47 proteins that were significantly up- or down - regulated ( > 1.25-fold , p < 0.05 ) by exposure of macrophages to manlam but not the other two lipoglycans .
motivated by this observation , we investigated the effects of mycobacterial lams on lc3 recruitment to the phagosome as well as autophagy activation in cultured macrophages .
manlam inhibited chemical - induced accumulation of autophagosomes , suggesting a previously unrecognized function of this virulence factor in undermining host defense responses .
the rat anti - lamp1 mab and mouse anti - syntaxin 6 mab were obtained from bd biosciences .
the goat anti - eea1 pab and goat anti - cathepsin d pab were obtained from santa cruz biotechnology .
the rabbit anti - lc3b pab and mouse anti - tubulin mab were obtained from sigma .
acrylamide / bisacrylamide solution ( 40% , 29:1 ) , ammonium persulfate ( aps ) , and tetramethylenediamine ( temed ) were obtained from bio - rad . latex beads ( 1.0 m microspheres , polysciences ) were washed twice in 0.05 m carbonatebicarbonate buffer ( ph 9.6 ) by centrifugation at 15  000 g for 5 min in 1.5-ml presiliconized low - retention microtubes ( fisher ) .
the beads in each tube were then resuspended in 900 l of carbonatebicarbonate buffer before a certain amount of a specific lipoglycan was added .
usually , 180 g of manlam or pilam , or 1.8 g of lps was used to coat a total of 4.55  10 latex beads .
the beads and lipoglycans were incubated for 1 h at 37 c on an eppendorf thermomixer ( shaking at 1400 rpm ) .
the beads were washed once and then incubated in 1 ml of pbs buffer containing 5% bsa ( sigma , endotoxin tested ) for 0.5 h at 37 c to block nonspecific binding sites .
the latex beads were washed with 0.5% bsa in pbs , resuspended in 1 ml of rpmi-1640 cell culture media , and stored at 4 c until use ( stable for 1 week ) .
the presence of mannose residues on latex beads after manlam coating was confirmed by immunofluorescence microscopy using fitc - conjugated con a. the other lipids were coated onto beads using the same protocol , which was presumed to be effective for all lipoglycans .
the murine macrophage cell line raw 264.7 was cultured as a monolayer in rpmi-1640 ( gibco , formulated with hepes and glutamine ) supplemented with 10% fetal bovine serum and 100 units / ml penicillin / streptomycin .
cells were first chilled with 4 c pbs for 5 min in order to synchronize phagocytosis .
four populations of cells were incubated ( 2 h , 37 c ) with latex beads coated with a specific bacterial lipoglycan or control beads without lipid coating at a multiplicity of infection ( moi ) of 50:1 to generate phagosomes .
after gentle cell lysis using a dounce homogenizer to reach 9095% breakage , the bead - containing phagosomes were isolated by ultracentrifugation on a sucrose gradient as described by desjardins and co - workers.(23 ) the latex bead - containing ( lbc ) phagosome fraction was collected from the top of the sucrose gradient ( 1020% interface ) and resuspended in pbs containing protease inhibitor cocktail ( calbiochem ) .
the lbc - phagosome fraction was washed by ultracentrifugation at 40  000 g for 20 min .
each lbc - phagosome pellet from a given treatment was resuspended in 1.5 ml of 0.2 m na2co3 ( ph 11.0 ) containing proteases inhibitors .
the lbc - phagosomes were disrupted by passaging the suspension 57 times through the needle of a 1-ml syringe ( 25 g ) .
the resulting sample was kept on ice for 30 min before the membrane fraction was pelleted by centrifugation for 45 min at 200  600 g at 4 c .
the pellet was washed with a low - salt buffer ( 10 mm triethylammonium bicarbonate ( teab ) , sigma ) .
a third centrifugation was performed at 100  000 g for 15 min to acquire the final membrane pellet .
the phagosomal pellet samples were stored at 80 c prior to protein extraction . a solution of 4% sds in 400 mm teab buffer ( 50 l )
the resuspended solution was agitated for 30 min at 10 c on a thermomixer to extract proteins from the membrane fraction .
after centrifugation at 14  000 g for 10 min , the supernatant was collected and diluted with additional ddh2o so that the final protein extract was in 1% sds and 100 mm teab buffer ( 4-fold dilution ) . for measuring protein concentration , we diluted the extracts by another 5-fold with 50 mm tris - hcl buffer before carrying out the bca assay ( pierce ) . for in - solution digestion , 4 g of protein derived from the phagosome membrane extract
was diluted either 5-fold or 20-fold in 100 mm teab , resulting in a final sds concentration of 0.2% or 0.01% , respectively .
each protein sample was treated with tcep ( 5 mm , 37 c for 30 min ) and iodoacetamide ( 15 mm , for 30 min in the dark ) to reduce and alkylate cysteine residues , then digested with trypsin overnight ( e / s = 1:20 w / w ) .
in addition to this single enzyme digestion , we tested the efficiency of dual enzyme digestion with both lys - c and trypsin .
a 5-fold diluted sample was first digested with lys - c ( promega ) for 4 h at 37 c ( e / s = 1:80 w / w ) before the trypsinization under the same condition as above .
all digests were acidified with 2% formic acid and desalted with a c18 ziptip ( millipore ) . to test the efficiency of in - tube digestion , 5 g of protein derived from the phagosome membrane extract
the protein sample was then mixed with 5 l of acrylamide / bisacrylamide ( 40% , 29:1 ) , 0.7 l of 1% aps , and 0.3 l of temed .
the gel impregnated with membrane proteins was cut into small pieces and washed three times with 50% acetonitrile ( acn ) in 100 mm teab buffer .
the gel pieces were then dehydrated with pure acn and dried using a speedvac . a total of 0.5 g of trypsin ( in 25 mm teab buffer , e / s = 1:10
w / w ) was absorbed by the dried gel , which was then incubated overnight at 37 c .
peptides were extracted from the gel with 50 l of 25 mm teab buffer , 100 l of 0.1% tfa , and 150 l of acn in 0.1% tfa , sequentially .
the solutions were combined and concentrated using a speedvac . for tube - gel digestion of phagosome membrane proteins before itraq labeling , 20 g of protein from each extract from cells treated with different lipid - coated beads was incorporated into gels using the same protocol , except that 10 mm methyl methanethiosulfonate ( mmts ) replaced iodoacetamide and the reagent amounts were increased 4-fold .
the residual protein solution on top of the gel was removed and subjected to a second solidification process .
each peptide extract was concentrated to roughly 20 l , and 1 m teab buffer was added to adjust the final buffer concentration to 400 mm .
four - plex itraq ( applied biosystems ) reagents were reacted with individual protein digests according to the manufacturer s protocol .
the phagosome membrane protein preparation and digestion procedures were performed twice to acquire two biological replicates . for comparison of the in - solution and tube - gel digestion efficiencies ,
0.5 g of each protein digest was analyzed by lcms using esi linear ion trap mass analyzer ( ltq , thermo , inc . ) in a data - dependent acquisition mode .
lc separation was performed on a house - made microcapillary column ( 0.100  100 mm , 3 m magic c18 packing material , michrom bioresources ) at a flow rate of 300 nl / min using buffers 0.1% formic acid ( fa ) in water ( a ) and 0.1% fa in acn ( b ) .
the gradient was 210% b for 5 min , 1040% b for 60 min , 4090% b for 10 min , and 90% b for 10 min .
the major parameters for ltq data acquisition were : scan range , 4002000 m / z ; precursor ion selection , six most abundant peaks per scan for ms / ms ; minimal ion signal , 500 ; and normalized collision energy , 35.0% .
the itraq - labeled peptide mixtures from tube - gel digestion of two experimental replicates were separated by two - dimensional liquid chromatography and analyzed by esi - q - tof mass spectrometry .
briefly , the peptide mixture was separated by off - line strong cation exchange ( scx ) chromatography using an ultimate hplc with a uv detector ( dionex - lc packings , sunnyvale , ca ) . labeled samples were resuspended in scx running buffer ( 5 mm kh2po4 , 25% acn , 0.1% fa ) and loaded onto a polylc polysulfethyl a column ( 2.1 mm  200 mm , the nest group , southborough , ma ) . peptides were eluted with increasing concentrations of 800 mm kcl , 5 mm kh2po4 , 25% acn , and 0.1% fa using a three step gradient : 010% for 10 min , 1025% for 30 min , and 25100% for 10 min .
fifteen fractions were collected at a flow rate of 300 l / min according peaks observed by uv absorption at 214 nm .
fractions were partially evaporated to remove acn on a speedvac and desalted using c18 macrospin columns ( the nest group , southborough , ma ) .
each fraction was injected onto a pepmap100 trapping column ( 0.3 mm 5 mm ) .
reversed - phase separation was performed on a lc packings pepmap c18 column ( 3 m , 0.075  150 mm ) at a flow rate of 300 nl / min using buffers 2% acn , 0.1% fa ( a ) and 80% acn , 0.1% fa ( b ) .
the gradient was 035% b for 100 min , 30100% b for 10 min , and 100% b for 10 min .
the samples from one experiment were injected into a qstar pulsar - i hybrid quadrupole tof ( applied biosystems , framingham , ma ) , while the samples from another replicate were analyzed by a qstar elite hybrid quadrupole tof ( applied biosystems , framingham , ma ) .
the major parameters for q - tof data acquisition were : ms scan range , 3501800 m / z ; ms / ms scan range , 702000 m / z ; precursor ion selection , three most abundant peaks per scan for ms / ms ; minimal ion counts , 30 ; and automated collision energy was applied ( fragment intensity multiplier = 2.0 ) .
lcms / ms data acquired using a ltq ion trap mass spectrometer ( thermo ) were searched against the mouse swiss - prot protein database using the sequest algorithm provided with bioworks 3.2 .
sequest results were filtered by xcorr ( + 1 > 1.8 ; + 2 > 2.5 ; + 3 > 3.5 ) , deltacn > 0.8 and the requirement of at least two different peptides from a protein .
parent and product ion mass errors were set at 1.2 and 0.8 da , respectively .
lcms / ms data acquired on a q - tof for itraq - based quantification were searched against a mouse international protein index ( ipi ) database ( mouse , version 3.27 , 56  000 entries ) using the paragon algorithm within proteinpilot version 2.0 software ( applied biosystems ) .
the major parameters in the software were explained by pierce et al.(24 ) protein identification was based on a confidence level > 95% and at least two different peptides assigned to the protein . a search performed against a concatenated database containing both forward and reversed sequences allowed estimation of the false discovery level ( below 1% ) .
the number of transmembrane helices in each protein was predicted using the tmhmm online program ( http://www.cbs.dtu.dk/services/tmhmm/ ) . for relative protein quantification ,
each protein ratio reported by protein pilot was associated with a p - value ( evaluating the statistical difference between the observed ratio and unity ) and ef ( error factor ) for each protein
id.(25 ) the ef term indicates that the actual ratio lies between ( reported ratio)/(ef ) and ( reported ratio )  ( ef ) at a 95% confidence .
the following criteria were required to consider a change in protein level significant : the protein i d had a p - value < 0.05 and a meaningful ef ( < 2 ) , at least two unique peptides were identified , and the fold difference was greater than 1.25 ( i.e. , the ratio was > 1.25 or < 0.80 ) as explained in results and discussion .
proteins from the lbc - phagosome membrane extracts were separated by sds - page ( 412% acrylamide for most experiments , 12% acrylamide gel for lc3 detection ) and analyzed by western blot using appropriate antibodies . for each sample , 3 g of protein was loaded .
raw264.7 cells stably expressing gfp - fused lc3 ( kindly provided by patrick fitzgerald , st .
jude s children s research hospital , memphis , tn ) were used to investigate lc3 trafficking after internalization of lipoglycan - coated latex beads .
the cells were cultured in dmem ( gibco , containing high - glucose , glutamine , and sodium pyruvate ) supplemented with nonessential amino acids ( gibco ) , 10% fetal bovine serum , and 100 units / ml penicillin / streptomycin .
cells were seeded onto 8-well , nunc lab - tek chambered coverglass microscopy slides ( fisher ) 20 h before images were acquired .
after incubation with lipoglycan - coated beads for 2 h , cells were gently washed with pbs twice to remove free beads .
the cells were then treated with lysotracker red dnd-99 ( molecular probes ) ( 100 nm ) for 15 min .
the cells were rinsed and incubated in fresh warm media and imaged using a zeiss 200 m epifluorescence deconvolution microscope .
all images were processed using the nearest neighbor deconvolution algorithm in the instrument software slidebook 4.2 ( intelligent imaging innovations ) . in a separate experiment
, the cells were incubated with the autophagy inducer chloroquine ( 50 m ) and a given bacterial lipoglycan at various concentrations . after 2 h
, the cells were washed with pbs and placed in warm media for fluorescence imaging .
previously , we reported a workflow for lbc phagosome isolation , membrane fractionation , and profiling of the phagosome membrane proteome by mass spectrometry.(20 ) here , we modified our platform to include the use of the itraq method for multiplexed protein quantitation . figure 2 illustrates the workflow of the entire experiment .
first , we coated latex beads with a lipoglycan from m. tuberculosis ( manlam ) , m. smegmatis ( pilam ) , or e. coli ( lps ) .
the efficiency of lipid coating was verified by specific staining of manlam - coated beads with fitc - conjugated concanavalin - a , a lectin that binds terminal mannose residues(27 ) ( supporting information si figure 1 ) . experimental design for quantitative analysis of the phagosomal membrane proteome under different lipoglycan treatments .
lbc - phagosomes were isolated using the method initially reported by desjardins and colleagues(23 ) and modified in our previous work.(20 ) to enrich membrane - bound components , we fractionated the phagosome based on our previous protocol with one modification : we performed an additional wash of the membrane pellet using a low - salt buffer to remove a greater proportion of highly abundant luminal proteins .
this adjustment allowed the identification of a greater number of membrane - associated proteins ( vide infra ) .
a consequence of enriching the samples for membrane - associated proteins is the requirement of high detergent concentrations ( > 4% sds ) for their solubilization , which poses a problem for in - solution trypsin digestion due to the loss of enzyme activity .
additionally , the in - gel digestion method , though compatible with high detergent concentrations , is not amenable to subsequent chemical labeling reactions as required by the itraq method .
we attempted to solve these problems using reduced amounts of sds ( 0.2% ) in solution as well as a more detergent - resistant protease , lys - c , prior to trypsin digestion , but neither of these modifications gave satisfactory results .
we turned our attention to the recent method of tube - gel digestion , developed by lu and zhu , which has proven effective for proteomic analysis of membrane proteins .
an advantage of this method is that detergent can be washed from the protein - impregnated gel prior to trypsinization .
furthermore , the resulting peptide products can be released from the gel into aqueous solution prior to chemical labeling .
we modified the tube - gel digestion method in three ways to better suit the analysis of phagosomal membrane extracts : ( 1 ) nh4hco3 was replaced with teab , a primary amine - free buffer compatible with itraq reagents ; ( 2 ) the residual protein solution that was excluded from the gel matrix was subjected to a second gel solidification process , so as to minimize sample loss ; and ( 3 ) we performed the cysteine reduction and alkylation steps prior to gel formation . using phagosomal membrane extracts as substrates , we compared the efficiency of this modified tube - gel digestion protocol to in - solution methods and analyzed the peptide products by lcms . the total number of proteins identified using different digestion protocols in three separate experiments are shown in supporting information table 1 .
the modified tube - gel protocol identified three times as many proteins as the best in - solution procedure ( using dual enzymes and 0.2% sds ) . a recent publication by han et al .
also highlights the robustness of the tube - gel method for processing membrane proteins prior to itraq labeling.(29 ) the digested peptides derived from untreated , manlam- , pilam- , or lps - treated samples were labeled with itraq114 , itraq115 , itraq116 , or itraq117 , respectively .
the pooled peptide mixture was subjected to 2d lcms / ms analysis both for protein identification and relative quantitation .
biological replicates were analyzed either on a qstar pulsar i or elite mass spectrometer , and the combined data sets produced a total of 823 nonredundant proteins ( supporting information table 2a ) .
notably , 540 of these proteins were not found in our earlier phagosome membrane proteomics study.(20 ) a summary of overlapping and unique protein ids from both data sets is present in supporting information table 3 .
we attribute the superior performance of the new method to ( 1 ) the additional low - salt wash , which depleted more soluble proteins and enriched membrane proteins accordingly ( supporting information figure 2b ) , and ( 2 ) the reduced sampling handling required of the tube - gel method , which minimized contamination with keratins .
the itraq isotopic labels were used to calculate relative protein levels from lipoglycan - treated or untreated samples . supporting information table
2b provides a list of 658 quantitative ratios ( lipoglycan treated : untreated ) measured in two experimental replicates . the criteria for selecting ratios that indicate significant changes in protein abundances are described in experimental procedures .
applying these filters allowed us to identify four protein subsets that were regulated by ( i ) manlam alone , ( ii ) manlam and pilam but not lps , ( iii ) manlam and lps but not pilam , or ( iv ) all three lipoglycans .
because manlam has been implicated in m. tuberculosis virulence , we focused our studies on the phagosomal proteins that were specifically regulated by this lipoglycan .
apart from the p - value filter , we applied a threshold of 1.25-fold difference because : ( 1 ) the average technical and biological variations for itraq - based quantitation have been reported to be 11% and 25% , respectively;(30 ) ( 2 ) precedents have established that in some cases changes in protein level as low as 1.2-fold can be biologically relevant ; ( 3 ) after applying these criteria , we found 42 proteins specifically regulated by manlam ( table 1 ) , with the remaining unchanged proteins covering 94% of all the proteins quantified in this study . this value is higher than the percent of unchanged proteins found in an analysis of biological replicates using itraq.(30 )
the protein ratios represent the relative abundance of a specific protein in the phagosome bearing latex beads coated with manlam , pilam , or lps vs uncoated beads .
macrophage cells were treated by latex beads coated with three different bacterial lipoglycans or uncoated beads , and proteins were extracted from phagosomal membranes for quantitative comparison using itraq isotopic labels .
the protein ratios indicate relative changes of protein levels in the phagosomes under a specific lipoglycan treatment in comparison with no treatment .
forty - two proteins were found to be significantly changed only by manlam treatment , which were summarized in this table .
the ratio with an asterisk is the mean of two replicate measurements . of the 42 proteins found to be regulated by manlam ,
lysosome - associated membrane protein 1 ( lamp1 ) , the late endosome membrane marker cd63 , and the late endosome - specific small gtpase rab7 were all in this group , a finding that agrees with previous microscopy studies showing diminished recruitment of these proteins to phagosomes containing manlam - coated beads(13 ) or live m. tuberculosis.(3 ) in addition , lysosomal enzymes such as the cysteine protease cathepsin d ( catd ) , and subunits of lysosomal vacuolar atpase ( v - atpase ) were down - regulated by 3040% ( table 1 ) .
loss of v - atpase , a proton transporter , could explain the reduced lysotracker staining of phagosomes containing manlam - coated beads reported earlier.(15 ) interestingly , we noticed that the phagosomes containing manlam - coated beads partially share the features of those harboring live m. tuberculosis .
russell group has reported earlier that mycobacterium phagosomes exclude v - atpase to restrict vacuole acidification(33 ) as well as reduce acquisition of the mature form of catd.(34 ) in western blot analysis , we also observed reduced production of the mature catd in the manlam - containg phagosome ( figure 3a ) , though to a less extent than that observed in the live mycobacterium phagosome.(34 ) in summary , our proteomic data are consistent with classical cell - based or biochemical assays and suggest that manlam contributes to phagosome maturation arrest .
( a ) immunoblot of phagosomal membrane extracts from cells treated with different lipoglycans using antibodies specific for endosomal markers and a lysosomal protease .
each ratio represents the changes of a particular protein in the presence of manlam- , pilam- , or lps - coated beads relative to the uncoated beads .
( c ) immunoblot of syntaxin 6 in phagosome membrane extracts under different lipoglycan treatments .
note : this protein was not identified in the proteomic analysis . among the down - regulated species , we were particularly interested in a protein of unknown function containing a zinc finger fyve domain ( ipi00554920.5 ) .
this domain is known to bind phosphatidylinositol 3-phosphate ( pi3p),(35 ) a regulatory phospholipid that mediates membrane trafficking , endosomal protein sorting , and multisubunit enzyme assembly via multiple effectors .
importantly , pi3p plays an essential role in phagosomal acquisition of lysosomal constituents , and it is excluded from phagosomes containing live mycobacteria.(4 ) early endosome antigen 1 ( eea1 ) , a protein that is known to be required for phagolysosome biogenesis and that also contains a fyve domain , was also down - regulated by manlam . by analogy , the uncharacterized protein may be a new pi3p - binding effector involved in phagosome maturation and is possibly modulated by the m. tuberculosis lipoglycan .
eighteen proteins increased in abundance when cells were treated with manlam - coated beads ( table 1 ) .
the small gtpase rab14 is critical for stimulating vesicle fusion specifically between phagosomes and early endosomes rather than late endosomes.(40 ) overexpression of this regulatory protein is known to prevent phagosomes containing dead mycobacteria from maturing into bactericidal phagolysosomes.(40 ) manlam - induced upregulation of rab14 in phagosomes may promote phagosome - early endosome fusion thereby stalling the maturation process .
another protein in this group , vacuolar protein sorting - associated protein 41 ( vam2p homologue ) , is also known to concentrate in early endosomes.(41 ) the protein interacts with multiple snare family members , which are important vesicle fusion regulators.(41 ) the observation that manlam up - regulates at least two known early endosomal proteins suggests that its host phagosome resembles early endosomes , a proposal that been previously put forth for m. tuberculosis - containing phagosomes .
we performed immunoblotting to validate quantitative changes of certain phagosomal proteins in response to manlam treatment .
lamp1 , catd , and eea1 were down - regulated in the presence of manlam - coated beads compared to samples treated pilam - coated , lps - coated or uncoated beads ( figure 3a ) .
the observed down - regulation of eea1 concurs with deretic and co - workers report that eea1 is depleted from phagosomes containing live m. tuberculosis.(44 ) by contrast , transferrin receptor ( tfr ) , an early endosome marker , was found to be up - regulated in phagosomes containing manlam - coated beads .
this coincides with a previous report by clemens and horwitz , showing that only the phagosomes harboring live mycobacterium acquire transferrin added exogenously.(43 ) these trends revealed by immunoblotting matched those observed by quantitative mass spectrometry ( figure 3b ) .
the reduced levels of eea1 in phagosomes containing manlam - coated bead may have broad consequences for vesicle fusion and membrane trafficking .
eea1 is recruited to organelles via its association with pi3p on organelle membranes(45 ) and also interacts with syntaxin 6 , a snare protein that participates in the vesicular traffic between the tgn and the endocytic system including phagosomes .
inhibition of eea1 s function causes reduced accumulation of syntaxin 6 in phagosomes.(12 ) likewise , we considered the possibility that syntaxin 6 is depleted from phagosomes bearing manlam - coated beads .
indeed , western blot analysis showed that syntaxin 6 was down - regulated in the presence of manlam - coated beads ( figure 3c ) while four other syntaxins ( stx3 , 7 , 8 , 12 ) and two syntaxin - binding proteins ( stxbp2 , 5 ) identified by our proteomic analysis did not exhibit reduced levels upon manlam treatment ( supporting information table 2b ) .
loss of syntaxin 6 from phagosomal membranes may cause a global reduction in the delivery of lysosomal components , including v - atpase and catd , from the tgn to phagosomes . taken together , the proteomic and biochemical data are consistent with the proposal that manlam undermines trafficking from the tgn to the phagosome , which is reported to depend on the production of pi3p by the kinase vps34.(48 ) interestingly , we also observed lamp1 down - regulated by manlam treatment , yet this protein is thought to be delivered to phagosomes in a pi3p - independent manner .
therefore , manlam may affect multiple trafficking pathways that contribute to maturation of the phagosome and its proteome .
a connection was recently revealed between the pathways of phagosome maturation and autophagy activation.(20 ) autophagy serves as a means for the removal of intracellular bacteria and viruses , apart from its primary function of maintaining cytoplasmic homeostasis.m .
tuberculosis survival in infected macrophages is suppressed by artificial induction of the autophagy response.(53 ) we previously observed that the autophagosomal marker lc3 is enriched on lbc phagosomal membranes upon autophagy activation.(20 ) lc3 is not only a widely used marker but also an essential component of the autophagy machinery.(54 ) upon the induction of autophagy , the 18-kda cytosolic precursor lc3-i is cleaved at its c - terminus and conjugated to phosphatidylethanolamine , generating a 16-kda form termed lc3-ii.(55 ) lipid - modified lc3-ii integrates into the membranes of autophagosomes and undergoes either recycling or degradation when autophagosomes fuse with lysosomes.(56 ) given the importance of the autophagy pathway in the macrophage response to m. tuberculosis and the role of lc3 in this process , we sought to determine whether manlam affects the cellular distribution of lc3 .
our proteomic data suggested that lc3 levels were reduced by about 30% in phagosomes containing manlam - coated beads ( figure 4a ) , an observation that was confirmed by western blot analysis of phagosomal membrane extracts prepared in the same manner ( figure 4b ) .
by contrast , lc3 was up - regulated in phagosomes containing lps - coated beads and essentially unchanged in phagosomes containing pilam - coated beads ( figure 4 ) .
( a ) proteomic quantitation ; ( b ) and immunoblot of phagosomal membrane extracts using an anti - lc3 antibody . to determine the effects of manlam treatment on subcellular localization of lc3 , we monitored a gfp - lc3 fusion protein in raw264.7 murine macrophages upon treatment with various lipid - coated and uncoated beads ( figure 5a ) .
the average number of microbeads bearing bright fluorescence of gfp per cell provided a metric of the relative amount of lc3 in the phagosomes ( figure 5b ) .
basal levels of gfp - lc3 were observed in phagosomes containing uncoated beads , reflecting the endogenous low autophagy activity in resting macrophages .
additionally , these phagosomes stained with lysotracker , indicating successful fusion with lysosomes to become acidic vacuoles .
phagosomes bearing pilam - coated beads showed similar levels of gfp - lc3 fluorescence and lysotracker staining as in the presence of uncoated beads .
in contrast , phagosomes containing manlam - coated beads exhibited weaker gfp - lc3 fluorescence than observed with uncoated beads , as well as weaker lysotracker staining .
lps - coated beads affected lc3 translocation to phagosomes in an opposite manner compared to manlam - coated beads . in response to lps - coated beads , gfp - lc3 levels were increased in phagosomes , consistent with previous observations that lps activates the autophagy pathway.(57 ) manlam reduces gfp - lc3 fluorescence in lbc phagosomes .
( a ) translocation of gfp - lc3 to lbc phagosomes under different lipoglycan treatment .
raw cells stably expressing gfp - lc3 were allowed to internalize latex beads ( 3 m ) coated with a lipoglycan for 2 h. control cells were treated with lipid - free beads .
( b ) quantitation of gfp - lc3 colocalization with phagosomes containing beads coated with different lipoglycans .
a total of 200250 cells were sampled in each experiment to find out the average number of lbc phagosomes bearing gfp fluorescence in each cell .
next , we investigated the effects of the bacterial lipids on lc3 distribution in the presence of chloroquine , an anti - inflammatory drug that causes accumulation of autophagosomes by preventing their fusion with lysosomes.(58 ) the drug has been previously employed to facilitate measurements of autophagic flux in vivo.(58 ) raw264.7 cells stably expressing gfp - lc3 were treated by chloroquine in the absence or presence of a given lipoglycan suspended in medium .
trypan blue staining was performed to confirm that the chemical and lipid treatments did not affect cell viability ( supporting information figure 3 ) .
cells treated with chloroquine alone showed punctate fluorescence derived from gfp - lc3-ii ( figure 6a ) , an indicator of elevated autophagy activity .
such punctate fluorescence was barely observed in untreated cells due to lysosomal degradation of autophagosomal lc3 in the absence of chloroquine.(58 ) cells treated with pilam and chloroquine demonstrated a distribution of gfp - lc3-ii that was similar to that observed with chloroquine treatment alone .
however , gfp - lc3ii fluorescence was significantly more diffuse in cells that were treated with manlam ( figure 6a ) .
quantitation of the average number of gfp - lc3-ii puncta ( > 1 m ) per cell in the presence of the various bacterial lipids is shown in figure 6b .
similar data were obtained from cells treated with three different doses of manlam , suggesting that the minimal effective concentration could be even lower than the 2 g / ml used in our assay .
collectively , these data indicate that manlam , but not pilam or lps , suppresses the accumulation of autophagosomes induced by chloroquine .
thus , manlam appears to interfere with the endogenous formation of autophagic vacuoles , an early stage of autophagy activation .
( a ) macrophages expressing gfp - lc3 were incubated with chloroquine ( 50 m ) in the presence or absence of either manlam or pilam for 2 h. arrows , representative lc3 punctate stains .
( b ) quantitation of lc3 punctate structures ( > 1 m ) in cells incubated with chloroquine in the absence or presence of a specific lipoglycan .
using a new platform for quantitative comparison of the membrane proteomes of macrophage phagosomes , we identified 823 proteins of which 42 were significantly regulated by manlam from m. tuberculosis but not pilam derived from nonpathogenic mycobacteria or e. coli lps .
several manlam - regulated proteins are known to be involved in vesicle trafficking pathways and phagosome maturation .
others have unknown function ( e.g. , fyve domain - containing protein ) , but their regulation by manlam suggests a role in membrane trafficking events important for endosomal fusion and interaction with phagosomes .
we also found that manlam suppresses the accumulation of lc3-ii in both lbc phagosomes and autophagosomes , whereas pilam has no such effects .
given the established importance of phagocytosis and autophagy in the macrophage response to m. tuberculosis infection , it is possible that manlam s functions include interference in these critical processes of innate immunity .
proposed vps34 , the kinase responsible for pi3p production , as a possible target,(44 ) and indeed phagosomes containing m. tuberculosis show retarded and reduced acquisition of this critical traffic - regulating lipid.(45 ) delineating the downstream effectors of manlam is likely to reveal new players in phagosome maturation , autophagy activation , and other pathways .
as well , understanding the mechanisms by which manlam undermines the macrophage response may reveal new therapeutic avenues .
 the mycobacterial cell wall component lipoarabinomannan ( lam ) has been described as one of the key virulence factors of mycobacterium tuberculosis . 
 modification of the terminal arabinan residues of this lipoglycan with mannose caps in m. tuberculosis or with phosphoinositol caps in mycobacterium smegmatis results in distinct host immune responses . given that m. tuberculosis typically persists in the phagosomal vacuole after being phagocytosed by macrophages 
 , we performed a proteomic analysis of that organelle after treatment of macrophages with lams purified from the two mycobacterial species . 
 the quantitative changes in phagosomal proteins suggested a distinct role for mannose - capped lam in modulating protein trafficking pathways that contribute to the arrest of phagosome maturation . 
 enlightened by our proteomic data , we performed further experiments to show that only the lam from m. tuberculosis inhibits accumulation of autophagic vacuoles in the macrophage , suggesting a new function for this virulence - associated lipid . 
Introduction
Experimental Procedures
Results and Discussion
Conclusions