Proteomic Analysis of the Outer Membrane subproteome of...
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Proteomic Analysis of the Outer Membrane subproteome of Stenotrophomonas maltophilia by two-dimensional gel electrophoresis and LC-MS/MS. Gerard Torrent, Elias Mongiardini, Mario Ferrer-Navarro, Raquel Planell, Pol Huedo, Paula Martínez, Isidre Gibert, Xavier Daura, Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès (Barcelona), Spain. Catalan Institution for Research and Advanced Studies, 08010 Barcelona. ABSTRACT METHODS Membrane protein extraction and sample preparation 2D-PAGE RESULTS AND DISCUSSION Cell disruption by French Press Centrifugacion to separate unbroken cells Ultracentrifugation at 100000 xg Resuspended in 10 mL sarcosine 2% 1h at RT. Ultracentrifugation at 100000 xg Resuspended pelled with lysis buffer Cell culture ATCC and M30 Protein identification 2D-PAGE 1D-LC-MS/MS CONCLUSIONS Acknowledgements: This project is supported by funding under Subprograma de proyectos de investigación fundamental from Ministerio de Ciencia e Innovación (Ref.: BFU2010-17199) and the Seventh Research Framework Programme of the European Union (ref. HEALTH-F3-2009-223101) We present a global view of subcellular outer membrane proteins in S. maltophilia Stenotrophomonas maltophilia is a gram-negative nosocomial pathogen. It has been associated with several clinical syndromes, primarily in relation to the opportunistic infection of immunocompromised patients. Although not an inherently virulent pathogen, its ability to colonize respiratory-tract epithelial cells and medical-device surfaces makes it a ready colonizer of hospital settings. Besides more specific resistance mechanisms, its capacity to form biofilms confers the bacterium additional protection in front of the immune system, antibiotics and disinfectants. Here we present the analysis of the purified OMPs of two different strains of S. maltophilia; a collection strain and a newly clinical isolate. Two-dimensional electrophoresis and LC-MS/MS have been the methods of choice to identify the components of this subproteome. The analysis of the OMPs and the comparison between strains with different infectivity capabilities shall provide a further understanding and identification of new targets for vaccine development and drug therapy. Outer membrane proteins (OMPs) of gram-negative bacteria play a key role in pathogenic bacteria during infection mechanisms and in resistance against antimicrobial agents. Proteins located on the outer membrane interact directly with the cell surface of the cell host. Some of these proteins act as adhesins while others act as receptors to facilitate the colonization as well as mediate the acquisition of nutrients or others molecules from this restricted niche. 1 1 1 1 1 1 1 1, 2 2 1 The virulence factors identified might be interesting targets for antimicrobial agents and they are interesting vaccine antigen candidate. We are constructing three knockouts of these virulence factors to elucidate its role in S. malthophilia 1D-LC-MS/MS Cytoplas mic 28% Cytoplas micMem brane 15% Extracellu lar 4% OuterMe mbrane 26% Periplasm ic 3% Unknown 24% MW marker ATCC strain M30 strain LC-MS/MS Cytoplasmic 12,5% Cytoplasmic Membrane 1% Extracellular 1,5% Outer Membane 64% Periplasmic; 1,5% Unknown; 18,7% KNOCKOUTS S. maltophilia Smlt 2834 Putative autotransporter protein Smlt 0880 Virulence-associated autotrasporter protein: Smlt 0821 Lps-assembly protein LptD (Organic solvent tolerance protein): ATCC M30 Autotransporters proteins of the type V secretion system of Gram-negative bacteria. Nonprotease autotransporters, Ag43, AIDA-1 and IcsA. Functions: host adhesion and biofilm formation. Autotransporter pathway Involved in outer membrane permeability It is well known that in vitro serial passage of microorganisms causes changes in physiology and virulence factors production. It is expected then that the comparison between clinical isolates and the collection strain shall allow us to identify proteins related with virulence. In order to perform these comparisons we have used the combination of two-dimensional electrophoresis (2DE) and mass spectrometry. In this case we haven´t differential proteins between two strains that can be related to pathogenicity of S. maltophilia. We were able to identify 37 outer membrane proteins between the two strains. Several proteins could be associated to the complex TonB related to iron transport. The most important omp is OmpA that its relates to many function like biofilm formation and adhesion. We also found different outer membrane proteins that could be related to lipoproteins family. The LC-MS/MS method identified several citoplasmatic and unknown proteins and the omps identified were 26 % and 64% to ATCC and M30 strains. Functional classification (PSORT) revealed that a large proportion of the OMPs (60%) were involved in cell process and a 17% in virulence factors that can be related in the pathogenesis of S. maltophilia. Several virulence factors are related to TonB (iron transport and other substances system) and It has been shown to play a role in efflux of multidrug resistence in P. aeroginosa. Other virulence factors are proteins related to biogenesis and permeability of the outer membrane (OstA) and two autotransporter proteins or Type V secretory system that are associated with pathogenic functions. 40 Omps were identified with LC/MS approach and 37 additional ones in 2-DE. Functional classificatnio of the 73 identified S. maltophilia OMPs distribuied in four functional groups. Virulence factors:- Adhesions; Toxins; and Hemolytic molecule. Information storage and processing:- Translational, ribosomal and biogenesis; Transcription; DNA replication and repair. Cellular process:- cell division and chromosome partitioning; Cell envelope biogenesis; Cell motility; Posttranslational modification; Inorganic ion transport. Metabolism:- Energy production and conversion; Amino acid transport and metabolism; Nucleotide transport and metabolism; Carbohydrate transport and metabolism; Lipid metabolism. 60,3 5,5 16,4 17,8 0,0 25,0 50,0 75,0 cell proces information and storage metabolisme Virulence factor Protein profiles of subcellular fractions resolved on SDS-PAGE. The lines were slice and digested followed by LC-MS/MS. Distribution of proteins identified in subcellular fraction via LC-MS/MS Saha, S. and Raghava, G.P.S. (2006) Genomics Proteomics & Bioinformatics. 4(1):42-7 The two separation and analysis methods (2D-PAGE and LD-MS/MS) are complementary to obtain the profile of Omp of S. maltophilia. OmpA is expressed to very high levels and its characteristic multifunctionality confers it and interesting protein to be studied. ATCC M30 Smlt 0955: OmpA Ton B complex OmpA 10 3 IEF pI SDS-PAGE SDS-PAGE 10 3 IEF pI
Transcript of Proteomic Analysis of the Outer Membrane subproteome of...
Proteomic Analysis of the Outer Membrane subproteome of Stenotrophomonas maltophilia by two-dimensional gel
electrophoresis and LC-MS/MS.Gerard Torrent, Elias Mongiardini, Mario Ferrer-Navarro, Raquel Planell, Pol Huedo, Paula Martínez, Isidre Gibert, Xavier Daura,Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès (Barcelona), Spain.
Catalan Institution for Research and Advanced Studies, 08010 Barcelona.
ABSTRACT
METHODS
Membrane protein extraction and sample preparation
2D-PAGE
RESULTS AND DISCUSSION
Cell disruption byFrench Press
Centrifugacion toseparate unbroken
cells
Ultracentrifugationat 100000 xg
Resuspended in 10 mLsarcosine 2%
1h at RT.
Ultracentrifugationat 100000 xg
Resuspended pelledwith lysis buffer
Cell cultureATCC and M30
Protein identification 2D-PAGE 1D-LC-MS/MS
CONCLUSIONS
Acknowledgements: This project is supported by funding under Subprograma de proyectos de investigación fundamental from Ministerio de Ciencia e Innovación (Ref.: BFU2010-17199) and the Seventh Research Framework Programme of the European Union (ref. HEALTH-F3-2009-223101)
We present a global view of subcellular outer membrane proteins in S. maltophilia
Stenotrophomonas maltophilia is a gram-negative nosocomial pathogen. It has been associated with several clinical syndromes, primarily in relation to the opportunistic infection of immunocompromised patients. Although not an inherently virulent pathogen, its ability to colonize respiratory-tract epithelial cells and medical-device surfaces makes it a ready colonizer of hospital settings. Besides more specific resistance mechanisms, its capacity to form biofilms confers the bacterium additional protection in front of the immune system, antibiotics and disinfectants.
Here we present the analysis of the purified OMPs of two different strains of S. maltophilia; a collection strain and a newly clinical isolate. Two-dimensional electrophoresis and LC-MS/MS have been the methods of choice to identify the components of this subproteome. The analysis of the OMPs and the comparison between strains with different infectivity capabilities shall provide a further understanding and identification of new targets for vaccine development and drug therapy.
Outer membrane proteins (OMPs) of gram-negative bacteria play a key role in pathogenic bacteria during infection mechanisms and in resistance against antimicrobial agents. Proteins located on the outer membrane interact directly with the cell surface of the cell host. Some of these proteins act as adhesins while others act as receptors to facilitate the colonization as well as mediate the acquisition of nutrients or others molecules from this restricted niche.
1 1 1 1 1 1 1 1, 2
2
1
The virulence factors identified might be interesting targets for antimicrobial agents and they are interesting vaccine antigen candidate.
We are constructing three knockouts of these virulence factors to elucidate its role in S. malthophilia
1D-LC-MS/MS
Cytoplasmic28%
CytoplasmicMem
brane
15%
Extracellular4%
OuterMembrane
26%
Periplasmic
3%
Unknown24%
MWmarker
ATCC strain
M30 strain
LC-MS/MSCytoplasmic
12,5%Cytoplasmic Membrane
1%
Extracellular1,5%
Outer Membane
64%
Periplasmic; 1,5%
Unknown; 18,7%
KNOCKOUTS S. maltophilia
Smlt 2834 Putative autotransporter protein
Smlt 0880 Virulence-associated autotrasporter protein:
Smlt 0821 Lps-assembly protein LptD (Organic solvent tolerance protein):
ATCC M30
Autotransporters proteins of the type V secretion system of Gram-negative bacteria.Nonprotease autotransporters, Ag43, AIDA-1 and IcsA. Functions: host adhesion and biofilm formation.
Autotransporter pathway
Involved in outer membrane permeability
It is well known that in vitro serial passage of microorganisms causes changes in physiology and virulence factors production. It is expected then that the comparison between clinical isolates and the collection strain shall allow us to identify proteins related with virulence. In order to perform these comparisons we have used the combination of two-dimensional electrophoresis (2DE) and mass spectrometry. In this case we haven´t differential proteins between two strains that can be related to pathogenicity of S. maltophilia.
We were able to identify 37 outer membrane proteins between the two strains. Several proteins could be associated to the complex TonB related to iron transport. The most important omp is OmpA that its relates to many function like biofilm formation and adhesion. We also found different outer membrane proteins that could be related to lipoproteins family.
The LC-MS/MS method identified several citoplasmatic and unknown proteins and the omps identified were 26 % and 64% to ATCC and M30strains. Functional classification (PSORT) revealed that a large proportion of the OMPs (60%) were involved in cell process and a 17% in virulence factors that can be related in the pathogenesis of S. maltophilia. Several virulence factors are related to TonB (iron transport and other substances system) and It has been shown to play a role in efflux of multidrug resistence in P. aeroginosa. Other virulence factors are proteins related to biogenesis and permeability of the outer membrane (OstA) and two autotransporter proteins or Type V secretory system that are associated with pathogenic functions.
40 Omps were identified with LC/MS approach and 37 additional ones in 2-DE.
Functional classificatnio of the 73 identified S. maltophilia OMPs distribuied in four functional groups.
Virulence factors:- Adhesions; Toxins; and Hemolytic molecule.
Information storage and processing:- Translational, ribosomal and biogenesis; Transcription; DNA replication and repair.
Cellular process:- cell division and chromosome partitioning; Cell envelope biogenesis; Cell motility; Posttranslational modification; Inorganic ion transport.
Metabolism:- Energy production and conversion; Amino acid transport and metabolism; Nucleotide transport and metabolism; Carbohydrate transport and metabolism; Lipid metabolism.
60,3
5,5
16,4 17,8
0,0
25,0
50,0
75,0
cell proces information and storage
metabolisme Virulence factor
Protein profiles of subcellularfractions resolved on SDS-PAGE.The lines were slice and digested followed by LC-MS/MS.
Distribution of proteins identified in subcellular fraction via LC-MS/MS
Saha, S. and Raghava, G.P.S. (2006) Genomics Proteomics & Bioinformatics. 4(1):42-7
The two separation and analysis methods (2D-PAGE and LD-MS/MS) are complementary to obtain the profile of Omp of S. maltophilia.
OmpA is expressed to very high levels and its characteristic multifunctionality confers it and interesting proteinto be studied.
ATCC
M30
Smlt 0955: OmpA
Ton B
complex
OmpA
103
IEF
pI
SD
S-P
AG
E
SD
S-P
AG
E
103
IEF
pI
