24 research outputs found
Global Acetylomics of Campylobacter jejuni Shows Lysine Acetylation Regulates CadF Adhesin Processing and Human Fibronectin Binding
Lysine acetylation (KAc) is a reversible post-translational
modification
(PTM) that can alter protein structure and function; however, specific
roles for KAc are largely undefined in bacteria. Acetyl-lysine immunoprecipitation
and LC–MS/MS identified 5567 acetylated lysines on 1026 proteins
from the gastrointestinal pathogen Campylobacter jejuni (∼63% of the predicted proteome). KAc was identified on proteins
from all subcellular locations, including the outer membrane (OM)
and extracellular proteins. Label-based LC–MS/MS identified
proteins and KAc sites during growth in 0.1% sodium deoxycholate (DOC,
a component of gut bile salts). 3410 acetylated peptides were quantified,
and 784 (from 409 proteins) were differentially abundant in DOC growth.
Changes in KAc involved multiple pathways, suggesting a dynamic role
for this PTM in bile resistance. As observed elsewhere, we show KAc
is primarily nonenzymatically mediated via acetyl-phosphate; however,
the deacetylase CobB also contributes to a global elevation of this
modification in DOC. We observed several multiply acetylated OM proteins
and altered DOC abundance of acetylated peptides in the fibronectin
(Fn)-binding adhesin CadF. We show KAc reduces CadF Fn binding and
prevalence of lower mass variants. This study provides the first system-wide
lysine acetylome of C. jejuni and contributes
to our understanding of KAc as an emerging PTM in bacteria
Global Acetylomics of Campylobacter jejuni Shows Lysine Acetylation Regulates CadF Adhesin Processing and Human Fibronectin Binding
Lysine acetylation (KAc) is a reversible post-translational
modification
(PTM) that can alter protein structure and function; however, specific
roles for KAc are largely undefined in bacteria. Acetyl-lysine immunoprecipitation
and LC–MS/MS identified 5567 acetylated lysines on 1026 proteins
from the gastrointestinal pathogen Campylobacter jejuni (∼63% of the predicted proteome). KAc was identified on proteins
from all subcellular locations, including the outer membrane (OM)
and extracellular proteins. Label-based LC–MS/MS identified
proteins and KAc sites during growth in 0.1% sodium deoxycholate (DOC,
a component of gut bile salts). 3410 acetylated peptides were quantified,
and 784 (from 409 proteins) were differentially abundant in DOC growth.
Changes in KAc involved multiple pathways, suggesting a dynamic role
for this PTM in bile resistance. As observed elsewhere, we show KAc
is primarily nonenzymatically mediated via acetyl-phosphate; however,
the deacetylase CobB also contributes to a global elevation of this
modification in DOC. We observed several multiply acetylated OM proteins
and altered DOC abundance of acetylated peptides in the fibronectin
(Fn)-binding adhesin CadF. We show KAc reduces CadF Fn binding and
prevalence of lower mass variants. This study provides the first system-wide
lysine acetylome of C. jejuni and contributes
to our understanding of KAc as an emerging PTM in bacteria
Global Acetylomics of Campylobacter jejuni Shows Lysine Acetylation Regulates CadF Adhesin Processing and Human Fibronectin Binding
Lysine acetylation (KAc) is a reversible post-translational
modification
(PTM) that can alter protein structure and function; however, specific
roles for KAc are largely undefined in bacteria. Acetyl-lysine immunoprecipitation
and LC–MS/MS identified 5567 acetylated lysines on 1026 proteins
from the gastrointestinal pathogen Campylobacter jejuni (∼63% of the predicted proteome). KAc was identified on proteins
from all subcellular locations, including the outer membrane (OM)
and extracellular proteins. Label-based LC–MS/MS identified
proteins and KAc sites during growth in 0.1% sodium deoxycholate (DOC,
a component of gut bile salts). 3410 acetylated peptides were quantified,
and 784 (from 409 proteins) were differentially abundant in DOC growth.
Changes in KAc involved multiple pathways, suggesting a dynamic role
for this PTM in bile resistance. As observed elsewhere, we show KAc
is primarily nonenzymatically mediated via acetyl-phosphate; however,
the deacetylase CobB also contributes to a global elevation of this
modification in DOC. We observed several multiply acetylated OM proteins
and altered DOC abundance of acetylated peptides in the fibronectin
(Fn)-binding adhesin CadF. We show KAc reduces CadF Fn binding and
prevalence of lower mass variants. This study provides the first system-wide
lysine acetylome of C. jejuni and contributes
to our understanding of KAc as an emerging PTM in bacteria
Global Acetylomics of Campylobacter jejuni Shows Lysine Acetylation Regulates CadF Adhesin Processing and Human Fibronectin Binding
Lysine acetylation (KAc) is a reversible post-translational
modification
(PTM) that can alter protein structure and function; however, specific
roles for KAc are largely undefined in bacteria. Acetyl-lysine immunoprecipitation
and LC–MS/MS identified 5567 acetylated lysines on 1026 proteins
from the gastrointestinal pathogen Campylobacter jejuni (∼63% of the predicted proteome). KAc was identified on proteins
from all subcellular locations, including the outer membrane (OM)
and extracellular proteins. Label-based LC–MS/MS identified
proteins and KAc sites during growth in 0.1% sodium deoxycholate (DOC,
a component of gut bile salts). 3410 acetylated peptides were quantified,
and 784 (from 409 proteins) were differentially abundant in DOC growth.
Changes in KAc involved multiple pathways, suggesting a dynamic role
for this PTM in bile resistance. As observed elsewhere, we show KAc
is primarily nonenzymatically mediated via acetyl-phosphate; however,
the deacetylase CobB also contributes to a global elevation of this
modification in DOC. We observed several multiply acetylated OM proteins
and altered DOC abundance of acetylated peptides in the fibronectin
(Fn)-binding adhesin CadF. We show KAc reduces CadF Fn binding and
prevalence of lower mass variants. This study provides the first system-wide
lysine acetylome of C. jejuni and contributes
to our understanding of KAc as an emerging PTM in bacteria
Secretome of Transmissible <i>Pseudomonas aeruginosa</i> AES-1R Grown in a Cystic Fibrosis Lung-Like Environment
<i>Pseudomonas aeruginosa</i> is the predominant cause
of mortality in patients with cystic fibrosis (CF). We examined the
secretome of an acute, transmissible CF <i>P. aeruginosa</i> (Australian epidemic strain 1-R; AES-1R) compared with laboratory-adapted
PAO1. Culture supernatant proteins from rich (LB) and minimal (M9)
media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated
abundance of PasP protease and absence of AprA protease in AES-1R.
CF lung-like artificial sputum medium (ASMDM) contains serum and mucin
that generally preclude proteomics of secreted proteins. ASMDM culture
supernatants were subjected to 2DLC-MS/MS, which allowed the identification
of 57 <i>P. aeruginosa</i> proteins, and qualitative spectral
counting was used to estimate relative abundance. AES-1R-specific
AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants,
while AprA could only be identified in PAO1. Relative quantitation
was performed using selected reaction monitoring. Significantly elevated
levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were
identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated <i>pasP</i> in AES-1R during early (18 h) ASMDM growth, while no
evidence of <i>aprA</i> expression could be observed. Genomic
screening of CF isolates revealed <i>aes_7139</i> was present
in all AES-1 and one pair of sequential nonepidemic isolates. Secreted
proteins may be crucial in aiding CF-associated <i>P. aeruginosa</i> to establish infection and for adaptation to the CF lung
Secretome of Transmissible <i>Pseudomonas aeruginosa</i> AES-1R Grown in a Cystic Fibrosis Lung-Like Environment
<i>Pseudomonas aeruginosa</i> is the predominant cause
of mortality in patients with cystic fibrosis (CF). We examined the
secretome of an acute, transmissible CF <i>P. aeruginosa</i> (Australian epidemic strain 1-R; AES-1R) compared with laboratory-adapted
PAO1. Culture supernatant proteins from rich (LB) and minimal (M9)
media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated
abundance of PasP protease and absence of AprA protease in AES-1R.
CF lung-like artificial sputum medium (ASMDM) contains serum and mucin
that generally preclude proteomics of secreted proteins. ASMDM culture
supernatants were subjected to 2DLC-MS/MS, which allowed the identification
of 57 <i>P. aeruginosa</i> proteins, and qualitative spectral
counting was used to estimate relative abundance. AES-1R-specific
AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants,
while AprA could only be identified in PAO1. Relative quantitation
was performed using selected reaction monitoring. Significantly elevated
levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were
identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated <i>pasP</i> in AES-1R during early (18 h) ASMDM growth, while no
evidence of <i>aprA</i> expression could be observed. Genomic
screening of CF isolates revealed <i>aes_7139</i> was present
in all AES-1 and one pair of sequential nonepidemic isolates. Secreted
proteins may be crucial in aiding CF-associated <i>P. aeruginosa</i> to establish infection and for adaptation to the CF lung
<i>Staphylococcus aureus</i> Surface Proteins Involved in Adaptation to Oxacillin Identified Using a Novel Cell Shaving Approach
Staphylococcus aureus is a Gram-positive
pathogen responsible for a variety of infections, and some strains
are resistant to virtually all classes of antibiotics. Cell shaving
proteomics using a novel probability scoring algorithm to compare
the surfaceomes of the methicillin-resistant, laboratory-adapted S. aureus COL strain with a COL strain in vitro adapted
to high levels of oxacillin (APT). APT displayed altered cell morphology
compared with COL and increased aggregation in biofilm assays. Increased
resistance to β-lactam antibiotics was observed, but adaptation
to oxacillin did not confer multidrug resistance. Analysis of the S. aureus COL and APT surfaceomes identified 150
proteins at a threshold determined by the scoring algorithm. Proteins
unique to APT included the LytR-CpsA-Psr (LCP) domain-containing MsrR
and SACOL2302. Quantitative RT-PCR showed increased expression of <i>sacol2302</i> in APT grown with oxacillin (>6-fold compared
with COL). Overexpression of <i>sacol2302</i> in COL to
levels consistent with APT (+ oxacillin) did not influence biofilm
formation or β-lactam resistance. Proteomics using iTRAQ and
LC–MS/MS identified 1323 proteins (∼50% of the theoretical S. aureus proteome), and cluster analysis demonstrated
elevated APT abundances of LCP proteins, capsule and peptidoglycan
biosynthesis proteins, and proteins involved in wall remodelling.
Adaptation to oxacillin also induced urease proteins, which maintained
culture pH compared to COL. These results show that S. aureus modifies surface architecture in response
to antibiotic adaptation
Large-Scale Capture of Peptides Containing Reversibly Oxidized Cysteines by Thiol-Disulfide Exchange Applied to the Myocardial Redox Proteome
Redox
regulation is emerging as an important post-translational
modification in cell signaling and pathogenesis. Cysteine (Cys) is
the most redox active of the commonly coded amino acids and is thus
an important target for redox-based modifications. Reactions that
oxidize the Cys sulfur atom to low oxidation states (e.g., disulfide)
are reversible, while further reactions to higher oxidation states
(e.g., sulfonic acid) may be irreversible under biological conditions.
Reversible modifications are particularly interesting as they mediate
redox signaling and regulation of proteins under physiological conditions
and during adaptation to oxidant stress. An enrichment method that
relied on rapid and specific alkylation of free Cys, followed by thiol-based
reduction and resin capture by thiol-disulfide exchange chemistry
was applied to isolate reversibly modified Cys-containing peptides.
Chromatographic conditions were optimized to provide increased specificity
by removal of noncovalent interactions. The technique was highly efficient,
based on near equimolar reactions with the resin, reproducible and
linear for peptide elution, as quantified by label-free mass spectrometry.
The method was applied to a complex protein lysate generated from
rat myocardial tissue and 6559 unique Cys-containing peptides from
2694 proteins were identified. Comparison with the rat database and
previous studies showed effective enrichment of proteins modified
by S-nitrosylation, disulfide formation, and Cys-sulfenic acid. Analysis
of amino acid sequence features indicated a preference for acidic
residues and increased hydrophilicity in the regions immediately up-
or downstream of the reactive Cys. This technique is ideally suited
for the enrichment and profiling of reversible Cys modifications on
a proteome-wide scale
<i>Staphylococcus aureus</i> Surface Proteins Involved in Adaptation to Oxacillin Identified Using a Novel Cell Shaving Approach
Staphylococcus aureus is a Gram-positive
pathogen responsible for a variety of infections, and some strains
are resistant to virtually all classes of antibiotics. Cell shaving
proteomics using a novel probability scoring algorithm to compare
the surfaceomes of the methicillin-resistant, laboratory-adapted S. aureus COL strain with a COL strain in vitro adapted
to high levels of oxacillin (APT). APT displayed altered cell morphology
compared with COL and increased aggregation in biofilm assays. Increased
resistance to β-lactam antibiotics was observed, but adaptation
to oxacillin did not confer multidrug resistance. Analysis of the S. aureus COL and APT surfaceomes identified 150
proteins at a threshold determined by the scoring algorithm. Proteins
unique to APT included the LytR-CpsA-Psr (LCP) domain-containing MsrR
and SACOL2302. Quantitative RT-PCR showed increased expression of <i>sacol2302</i> in APT grown with oxacillin (>6-fold compared
with COL). Overexpression of <i>sacol2302</i> in COL to
levels consistent with APT (+ oxacillin) did not influence biofilm
formation or β-lactam resistance. Proteomics using iTRAQ and
LC–MS/MS identified 1323 proteins (∼50% of the theoretical S. aureus proteome), and cluster analysis demonstrated
elevated APT abundances of LCP proteins, capsule and peptidoglycan
biosynthesis proteins, and proteins involved in wall remodelling.
Adaptation to oxacillin also induced urease proteins, which maintained
culture pH compared to COL. These results show that S. aureus modifies surface architecture in response
to antibiotic adaptation
Large-Scale Capture of Peptides Containing Reversibly Oxidized Cysteines by Thiol-Disulfide Exchange Applied to the Myocardial Redox Proteome
Redox
regulation is emerging as an important post-translational
modification in cell signaling and pathogenesis. Cysteine (Cys) is
the most redox active of the commonly coded amino acids and is thus
an important target for redox-based modifications. Reactions that
oxidize the Cys sulfur atom to low oxidation states (e.g., disulfide)
are reversible, while further reactions to higher oxidation states
(e.g., sulfonic acid) may be irreversible under biological conditions.
Reversible modifications are particularly interesting as they mediate
redox signaling and regulation of proteins under physiological conditions
and during adaptation to oxidant stress. An enrichment method that
relied on rapid and specific alkylation of free Cys, followed by thiol-based
reduction and resin capture by thiol-disulfide exchange chemistry
was applied to isolate reversibly modified Cys-containing peptides.
Chromatographic conditions were optimized to provide increased specificity
by removal of noncovalent interactions. The technique was highly efficient,
based on near equimolar reactions with the resin, reproducible and
linear for peptide elution, as quantified by label-free mass spectrometry.
The method was applied to a complex protein lysate generated from
rat myocardial tissue and 6559 unique Cys-containing peptides from
2694 proteins were identified. Comparison with the rat database and
previous studies showed effective enrichment of proteins modified
by S-nitrosylation, disulfide formation, and Cys-sulfenic acid. Analysis
of amino acid sequence features indicated a preference for acidic
residues and increased hydrophilicity in the regions immediately up-
or downstream of the reactive Cys. This technique is ideally suited
for the enrichment and profiling of reversible Cys modifications on
a proteome-wide scale