Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73 % of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-γ production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses.
ArandM.,
FriedbergT.,
OeschF.
1992; Colorimetric quantitation of trace amounts of sodium lauryl sulfate in the presence of nucleic acids and proteins. Anal Biochem 207:73–75[CrossRef]
AsselineauC.,
AsselineauJ.,
LaneelleG.,
LaneelleM. A.
2002; The biosynthesis of mycolic acids by mycobacteria: current and alternative hypotheses. Prog Lipid Res 41:501–523[CrossRef]
BroquetA. H.,
ThomasG.,
MasliahJ.,
TrugnanG.,
BacheletM.
2003; Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release. J Biol Chem 278:21601–21606[CrossRef]
CaccamoN.,
MilanoS.,
Di SanoC.,
CignaD.,
IvanyiJ.,
KrenskyA. M.,
DieliF.,
SalernoA.
2002; Identification of epitopes of Mycobacterium tuberculosis 16-kDa protein recognized by human leukocyte antigen-A*0201 CD8+ T lymphocytes. J Infect Dis 186:991–998[CrossRef]
ColeS. T.,
BroschR.,
ParkhillJ.39 other authors1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544[CrossRef]
DriessenA. J.,
MantingE. H., van der DoesC. 2001; The structural basis of protein targeting and translocation in bacteria. Nat Struct Biol 8:492–498[CrossRef]
FujikiY.,
HubbardA. L.,
FowlerS.,
LazarowP. B.
1982; Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol 93:97–102[CrossRef]
FutaiM.,
NoumiT.,
MaedaM.
1989; ATP synthase (H+-ATPase): results by combined biochemical and molecular biological approaches. Annu Rev Biochem 58:111–136[CrossRef]
GalevaN.,
AltermannM.
2002; Comparison of one-dimensional and two-dimensional gel electrophoresis as a separation tool for proteomic analysis of rat liver microsomes: cytochromes P450 and other membrane proteins. Proteomics 2:713–722[CrossRef]
GorgA.,
ObermaierC.,
BoguthG.,
HarderA.,
ScheibeB.,
WildgruberR.,
WeissW.
2000; The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:1037–1053[CrossRef]
GuS.,
ChenJ.,
DobosK. M.,
BradburyE. M.,
BelisleJ. T.,
ChenX.
2003; Comprehensive proteomic profiling of the membrane constituents of a Mycobacterium tuberculosis strain. Mol Cell Proteomics 2:1284–1296[CrossRef]
IborraS.,
SotoM.,
CarrionJ.,
NietoA.,
FernandezE.,
AlonsoC.,
RequenaJ. M.
2003; The Leishmania infantum acidic ribosomal protein P0 administered as a DNA vaccine confers protective immunity to Leishmania major infection in BALB/c mice. Infect Immun 71:6562–6572[CrossRef]
LalvaniA.,
NagvenkarP.,
UdwadiaZ.7 other authors2001; Enumeration of T cells specific for RD1-encoded antigens suggests a high prevalence of latent Mycobacterium tuberculosis infection in healthy urban Indians. J Infect Dis 183:469–477[CrossRef]
LopezM.,
SlyL. M.,
LuuY.,
YoungD.,
CooperH.,
ReinerN. E.
2003; The 19-kDa Mycobacterium tuberculosis protein induces macrophage apoptosis through Toll-like receptor-2. J Immunol 170:2409–2416[CrossRef]
MarkwellM. A.,
HaasS. M.,
BieberL. L.,
TolbertN. E.
1978; A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210[CrossRef]
MatsuokaM.,
NomaguchiH.,
YukitakeH.,
OharaN.,
MatsumotoS.,
MiseK.,
YamadaT.
1997; Inhibition of multiplication of Mycobacterium leprae in mouse foot pads by immunization with ribosomal fraction and culture filtrate from Mycobacterium bovis BCG. Vaccine 15:1214–1217[CrossRef]
McIntoshT. J.,
VidalA.,
SimonS. A.
2003; Sorting of lipids and transmembrane peptides between detergent-soluble bilayers and detergent-resistant rafts. Biophys J 85:1656–1666[CrossRef]
MehrotraJ.,
BishtD.,
TiwariV. D.,
SinhaS.
1995; Serological distinction of integral plasma membrane proteins as a class of mycobacterial antigens and their relevance for human T cell activation. Clin Exp Immunol 102:626–634
MehrotraJ.,
MittalA.,
DhindsaM. S.,
SinhaS.
1997; Fractionation of mycobacterial integral membrane proteins by continuous elution SDS-PAGE reveals the immunodominance of low molecular weight subunits for human T cells. Clin Exp Immunol 109:446–450[CrossRef]
MehrotraJ.,
MittalA.,
RastogiA. K.,
JaiswalA. K.,
BhandariN. K.,
SinhaS.
1999; Antigenic definition of plasma membrane proteins of Bacillus Calmette-Guérin: predominant activation of human T cells by low-molecular-mass integral proteins. Scand J Immunol 50:411–419[CrossRef]
PessolaniM. C.,
SmithD. R.,
RivoireB.,
McCormickJ.,
HeftaS. A.,
ColeS. T.,
BrennanP. J.
1994; Purification, characterization, gene sequence, and significance of a bacterioferritin from Mycobacterium leprae
. J Exp Med 180:319–327[CrossRef]
SchmidtF.,
DonahoeS.,
HagensK.,
MattowJ.,
SchaibleU. E.,
KaufmannS. H.,
AebersoldR.,
JungblutP. R.
2004; Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology. Mol Cell Proteomics 3:24–42
ShinB. K.,
WangH.,
YimA. M.9 other authors2003; Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function. J Biol Chem 278:7607–7616[CrossRef]
TekaiaF.,
GordonS. V.,
GarnierT.,
BroschR.,
BarrellB. G.,
ColeS. T.
1999; Analysis of the proteome of Mycobacterium tuberculosis in silico
. Tuber Lung Dis 79:329–342[CrossRef]
WesselD.,
FluggeU. I.
1984; A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143[CrossRef]
YuanY.,
CraneD. D.,
SimpsonR. M.,
ZhuY.,
HickeyM. J.,
ShermanD. R., BarryC. E.III1998; The 16-kDa alpha-crystallin (Acr) protein of Mycobacterium tuberculosis is required for growth in macrophages. Proc Natl Acad Sci U S A 95:9578–9583[CrossRef]