Membrane and membrane‐associated proteins in Triton X‐114 extracts of <b><i>Mycobacterium bovis</i></b> BCG identified using a combination of gel‐based and gel‐free fractionation strategies

Målen, Hiwa; Berven, Frode S.; Søfteland, Tina; Arntzen, Magnus Ø.; D’Santos, Clive S.; Souza, Gustavo; Wiker, Harald G.

doi:10.1002/pmic.200700528

Cited by 50 publications

(56 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Triton X-114 phase-separation was used to isolate lipophilic proteins following the method of Bordier [13,14]. In brief, 3-4 week old bacilli were lysed by bead beating and centrifuged, initially at 2300 g to remove unbroken cells and cell-wall debris.…”

Section: Membrane Protein Fraction (Mpf)mentioning

confidence: 99%

Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway

Souza¹,

Leversen²,

Målen³

et al. 2011

Journal of Proteomics

Self Cite

174

135

View full text Add to dashboard Cite

Correct protein compartmentalization is a key step for molecular function and cell viability, and this is especially true for membrane and externalized proteins of bacteria. Recent proteomic reports of Bacillus subtilis have shown that many proteins with Sec-like signal peptides and absence of a transmembrane helix domain are still observed in membrane-enriched fractions, but further evidence about signal peptide cleavage or soluble protein contamination is still needed. Here we report a proteomic screening of identified peptides in culture filtrate, membrane fraction and whole cell lysate of Mycobacterium tuberculosis. We were able to detect peptide sequencing evidence that shows that the predicted signal peptide was kept uncleaved for several types of proteins such as mammalian cell entry (Mce) proteins and PE or PE-PGRS proteins. Label-free quantitation of all proteins identified in each fraction showed that the majority of these proteins with uncleaved signal peptides are, indeed, enriched in the Triton X-114 lipid phase. Some of these proteins are likely to be located in the inner membrane while others may be outer membrane proteins.

show abstract

Section: Membrane Protein Fraction (Mpf)mentioning

confidence: 99%

Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway

Souza¹,

Leversen²,

Målen³

et al. 2011

Journal of Proteomics

Self Cite

174

135

View full text Add to dashboard Cite

show abstract

“…Using this approach in independent studies, 349 and 739 proteins out of the possible 4,015 proteins encoded by the genome were identified in M. tuberculosis membrane fractions prepared by differential centrifugation 18, 19 . With BCG a similar effort involving Triton X-114 fractions, which enriches for lipophilic proteins including hydrophobic membrane proteins, identified 351 proteins and 1,766 M. tuberculosis proteins were identified in Triton X-114 fractions 20 . Triton X-114 can be considered an alternative to differential centrifugation for enriching membrane and membrane-associated proteins 20 .…”

Section: Introductionmentioning

confidence: 99%

Comparison of the Membrane Proteome of Virulent Mycobacterium tuberculosis and the Attenuated Mycobacterium bovis BCG Vaccine Strain by Label-Free Quantitative Proteomics

et al. 2013

View full text Add to dashboard Cite

The Mycobacterium tuberculosis (MTB) membrane is rich in antigens that are potential targets for diagnostics and the development of new vaccines. To better understand the mechanisms underlying MTB virulence and identify new targets for therapeutic intervention we investigated the differential composition of membrane proteomes between virulent M. tuberculosis H37Rv (MTB) and the Mycobacterium bovis BCG vaccine strain. To compare the membrane proteomes, we used LC-MS/MS analysis in combination with label-free quantitative (LFQ) proteomics, utilizing the area-under-curve (AUC) of the extracted ion chromatograms (XIC) of peptides obtained from m/z and retention time alignment of MS1 features. With this approach, we obtained relative abundance ratios for 2,203 identified membrane-associated proteins in high confidence. Of these proteins, 294 showed statistically significant differences of at least 2 fold, in relative abundance between MTB and BCG membrane fractions. Our comparative analysis detected several proteins associated with known genomic regions of difference between MTB and BCG as being absent, which validated the accuracy of our approach. In further support of our label-free quantitative data, we verified select protein differences by immunoblotting. To our knowledge we have generated the first comprehensive and high coverage profile of comparative membrane proteome changes between virulent MTB and its attenuated relative BCG, which helps elucidate the proteomic basis of the intrinsic virulence of the MTB pathogen.

show abstract

“…M. bovis BCG is an attenuated strain of M. bovis lacking the RD-1 region, which contains important virulence determinants (39). However, it still shares many features with M. tuberculosis and M. bovis, including the expression of lipoproteins (40). Similar to infection with M. tuberculosis, RP105…”

Section: Rp105 Mediates Cytokine Production By Macrophages In Responsmentioning

confidence: 99%

RP105 Engages Phosphatidylinositol 3-Kinase p110δ To Facilitate the Trafficking and Secretion of Cytokines in Macrophages during Mycobacterial Infection

Micaroni

Puyskens

et al. 2015

The Journal of Immunology

View full text Add to dashboard Cite

Cytokines are key regulators of adequate immune responses to infection with Mycobacterium tuberculosis. We demonstrate that the p110δ catalytic subunit of PI3K acts as a downstream effector of the TLR family member RP105 (CD180) in promoting mycobacteria-induced cytokine production by macrophages. Our data show that the significantly reduced release of TNF and IL-6 by RP105−/− macrophages during mycobacterial infection was not accompanied by diminished mRNA or protein expression. Mycobacteria induced comparable activation of NF-κB and p38 MAPK signaling in wild-type (WT) and RP105−/− macrophages. In contrast, mycobacteria-induced phosphorylation of Akt was abrogated in RP105−/− macrophages. The p110δ-specific inhibitor, Cal-101, and small interfering RNA–mediated knockdown of p110δ diminished mycobacteria-induced TNF secretion by WT but not RP105−/− macrophages. Such interference with p110δ activity led to reduced surface-expressed TNF in WT but not RP105−/− macrophages, while leaving TNF mRNA and protein expression unaffected. Activity of Bruton’s tyrosine kinase was required for RP105-mediated activation of Akt phosphorylation and TNF release by mycobacteria-infected macrophages. These data unveil a novel innate immune signaling axis that orchestrates key cytokine responses of macrophages and provide molecular insight into the functions of RP105 as an innate immune receptor for mycobacteria.

show abstract

Membrane and membrane‐associated proteins in Triton X‐114 extracts of Mycobacterium bovis BCG identified using a combination of gel‐based and gel‐free fractionation strategies

Cited by 50 publications

References 37 publications

Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway

Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway

Comparison of the Membrane Proteome of Virulent Mycobacterium tuberculosis and the Attenuated Mycobacterium bovis BCG Vaccine Strain by Label-Free Quantitative Proteomics

RP105 Engages Phosphatidylinositol 3-Kinase p110δ To Facilitate the Trafficking and Secretion of Cytokines in Macrophages during Mycobacterial Infection

Contact Info

Product

Resources

About