scholarly journals The Landscape of Pseudomonas aeruginosa Membrane-Associated Proteins

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2421
Author(s):  
Sara Motta ◽  
Davide Vecchietti ◽  
Alessandra M. Martorana ◽  
Pietro Brunetti ◽  
Giovanni Bertoni ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Protein Identification ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
Protein Domains ◽  
Host Cells ◽  
Inner Membrane ◽  
Proteomic Data ◽  
Associated Proteins

Background: Pseudomonas aeruginosa cell envelope-associated proteins play a relevant role in infection mechanisms. They can contribute to the antibiotic resistance of the bacterial cells and be involved in the interaction with host cells. Thus, studies contributing to elucidating these key molecular elements are of great importance to find alternative therapeutics. Methods: Proteins and peptides were extracted by different methods and analyzed by Multidimensional Protein Identification Technology (MudPIT) approach. Proteomic data were processed by Discoverer2.1 software and multivariate statistics, i.e., Linear Discriminant Analysis (LDA), while the Immune Epitope Database (IEDB) resources were used to predict antigenicity and immunogenicity of experimental identified peptides and proteins. Results: The combination of 29 MudPIT runs allowed the identification of 10,611 peptides and 2539 distinct proteins. Following application of extraction methods enriching specific protein domains, about 15% of total identified peptides were classified in trans inner-membrane, inner-membrane exposed, trans outer-membrane and outer-membrane exposed. In this scenario, nine outer membrane proteins (OprE, OprI, OprF, OprD, PagL, OprG, PA1053, PAL and PA0833) were predicted to be highly antigenic. Thus, they were further processed and epitopes target of T cells (MHC Class I and Class II) and B cells were predicted. Conclusion: The present study represents one of the widest characterizations of the P. aeruginosa membrane-associated proteome. The feasibility of our method may facilitates the investigation of other bacterial species whose envelope exposed protein domains are still unknown. Besides, the stepwise prioritization of proteome, by combining experimental proteomic data and reverse vaccinology, may be useful for reducing the number of proteins to be tested in vaccine development.

scholarly journals N-Acetylcysteine Selectively Antagonizes the Activity of Imipenem in Pseudomonas aeruginosa by an OprD-Mediated Mechanism

2015 ◽  
Vol 59 (6) ◽  
pp. 3246-3251 ◽  
Author(s):  
Jerónimo Rodríguez-Beltrán ◽  
Gabriel Cabot ◽  
Estela Ynés Valencia ◽  
Coloma Costas ◽  
German Bou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
Antibiotic Activity ◽  
Simultaneous Treatment ◽  
Content Type ◽  
Sds Page

ABSTRACTThe modulating effect ofN-acetylcysteine (NAC) on the activity of different antibiotics has been studied inPseudomonas aeruginosa. Our results demonstrate that, in contrast to previous reports, only the activity of imipenem is clearly affected by NAC. MIC and checkerboard determinations indicate that the NAC-based modulation of imipenem activity is dependent mainly on OprD. SDS-PAGE of outer membrane proteins (OMPs) after NAC treatments demonstrates that NAC does not modify the expression of OprD, suggesting that NAC competitively inhibits the uptake of imipenem through OprD. Similar effects on imipenem activity were obtained withP. aeruginosaclinical isolates. Our results indicate that imipenem-susceptibleP. aeruginosastrains become resistant upon simultaneous treatment with NAC and imipenem. Moreover, the generality of the observed effects of NAC on antibiotic activity was assessed with two additional bacterial species,Escherichia coliandAcinetobacter baumannii. Caution should be taken during treatments, as the activity of imipenem may be modified by physiologically attainable concentrations of NAC, particularly during intravenous and nebulized regimes.

scholarly journals Effects of SecE Depletion on the Inner and Outer Membrane Proteomes of Escherichia coli

2008 ◽  
Vol 190 (10) ◽  
pp. 3505-3525 ◽  
Author(s):  
Louise Baars ◽  
Samuel Wagner ◽  
David Wickström ◽  
Mirjam Klepsch ◽  
A. Jimmy Ytterberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Protein Identification ◽  
Protein Translocation ◽  
Outer Membrane Proteins ◽  
Specific Model ◽  
Secretory Proteins ◽  
Inner Membrane ◽  
Sec Translocon

ABSTRACT The Sec translocon is a protein-conducting channel that allows polypeptides to be transferred across or integrated into a membrane. Although protein translocation and insertion in Escherichia coli have been studied using only a small set of specific model substrates, it is generally assumed that most secretory proteins and inner membrane proteins use the Sec translocon. Therefore, we have studied the role of the Sec translocon using subproteome analysis of cells depleted of the essential translocon component SecE. The steady-state proteomes and the proteome dynamics were evaluated using one- and two-dimensional gel analysis, followed by mass spectrometry-based protein identification and extensive immunoblotting. The analysis showed that upon SecE depletion (i) secretory proteins aggregated in the cytoplasm and the cytoplasmic σ32 stress response was induced, (ii) the accumulation of outer membrane proteins was reduced, with the exception of OmpA, Pal, and FadL, and (iii) the accumulation of a surprisingly large number of inner membrane proteins appeared to be unaffected or increased. These proteins lacked large translocated domains and/or consisted of only one or two transmembrane segments. Our study suggests that several secretory and inner membrane proteins can use Sec translocon-independent pathways or have superior access to the remaining Sec translocons present in SecE-depleted cells.

scholarly journals Novel Inner Membrane Retention Signals in Pseudomonas aeruginosa Lipoproteins

2008 ◽  
Vol 190 (18) ◽  
pp. 6119-6125 ◽  
Author(s):  
Shawn Lewenza ◽  
Musa M. Mhlanga ◽  
Anthony P. Pugsley
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
Optical Sectioning ◽  
Inner Membrane ◽  
Outer Membrane Lipoprotein ◽  
Associated Proteins ◽  
And Function

ABSTRACT The ultimate membrane localization and function of most of the 185 predicted Pseudomonas aeruginosa PAO1 lipoproteins remain unknown. We constructed a fluorescent lipoprotein, CSFPOmlA-ChFP, by fusing the signal peptide and the first four amino acids of the P. aeruginosa outer membrane lipoprotein OmlA to the monomeric red fluorescent protein mCherry (ChFP). When cells were plasmolyzed with 0.5 M NaCl, the inner membrane separated from the outer membrane and formed plasmolysis bays. This permits the direct observation of fluorescence in either the outer or inner membrane. CSFPOmlA-ChFP was shown to localize in the outer membrane by fluorescence microscopy and immunoblotting analysis of inner and outer membrane fractions. The site-directed substitution of the amino acids at positions +2, +3, and +4 in CSFPOmlA-ChFP was performed to test the effects on lipoprotein localization of a series of amino acid sequences selected from a panel of predicted lipoproteins. We confirmed Asp+2 and Lys+3 Ser+4 function as inner membrane retention signals and identified four novel inner membrane retention signals: CK+2 V+3 E+4, CG+2 G+3 G+4, CG+2 D+3 D+4, and CQ+2 G+3 S+4. These inner membrane retention signals are found in 5% of the 185 predicted P. aeruginosa lipoproteins. Full-length chimeras of predicted lipoproteins PA4370 and PA3262 fused to mCherry were shown to reside in the inner membrane and showed a nonuniform or patchy distribution in the membrane. The optical sectioning of cells producing PA4370CGDD-ChFP and PA3262CDSQ-ChFP by confocal microscopy improved the resolution and indicated a helix-like localization pattern in the inner membrane. The method described here permits the in situ visualization of lipoprotein localization and should work equally well for other membrane-associated proteins.

Genome-wide analysis and literature-based survey of lipoproteins in Pseudomonas aeruginosa

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2597-2607 ◽  
Author(s):  
Kim Remans ◽  
Ken Vercammen ◽  
Josselin Bodilis ◽  
Pierre Cornelis
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Opportunistic Pathogen ◽  
Signal Peptidase ◽  
Major Influence ◽  
Chronic Infections ◽  
Specific Sequence ◽  
Inner Membrane ◽  
Membrane Lipoproteins

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen able to cause acute or chronic infections. Like all other Pseudomonas species, P. aeruginosa has a large genome, >6 Mb, encoding more than 5000 proteins. Many proteins are localized in membranes, among them lipoproteins, which can be found tethered to the inner or the outer membrane. Lipoproteins are translocated from the cytoplasm and their N-terminal signal peptide is cleaved by the signal peptidase II, which recognizes a specific sequence called the lipobox just before the first cysteine of the mature lipoprotein. A majority of lipoproteins are transported to the outer membrane via the LolCDEAB system, while those having an avoidance signal remain in the inner membrane. In Escherichia coli, the presence of an aspartate residue after the cysteine is sufficient to cause the lipoprotein to remain in the inner membrane, while in P. aeruginosa the situation is more complex and involves amino acids at position +3 and +4 after the cysteine. Previous studies indicated that there are 185 lipoproteins in P. aeruginosa, with a minority in the inner membrane. A reanalysis led to a reduction of this number to 175, while new retention signals could be predicted, increasing the percentage of inner-membrane lipoproteins to 20 %. About one-third (62 out of 175) of the lipoprotein genes are present in the 17 Pseudomonas genomes sequenced, meaning that these genes are part of the core genome of the genus. Lipoproteins can be classified into families, including those outer-membrane proteins having a structural role or involved in efflux of antibiotics. Comparison of various microarray data indicates that exposure to epithelial cells or some antibiotics, or conversion to mucoidy, has a major influence on the expression of lipoprotein genes in P. aeruginosa.

scholarly journals Phylogenetic Distribution, Ultrastructure, and Function of Bacterial Flagellar Sheaths

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 363 ◽  
Author(s):  
Joshua Chu ◽  
Jun Liu ◽  
Timothy R. Hoover
Keyword(s):  
Immune Response ◽  
Outer Membrane ◽  
Electron Tomography ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
Wall Layer ◽  
Host Cells ◽  
Outer Cell ◽  
Flagellar Filament ◽  
And Function

A number of Gram-negative bacteria have a membrane surrounding their flagella, referred to as the flagellar sheath, which is continuous with the outer membrane. The flagellar sheath was initially described in Vibrio metschnikovii in the early 1950s as an extension of the outer cell wall layer that completely surrounded the flagellar filament. Subsequent studies identified other bacteria that possess flagellar sheaths, most of which are restricted to a few genera of the phylum Proteobacteria. Biochemical analysis of the flagellar sheaths from a few bacterial species revealed the presence of lipopolysaccharide, phospholipids, and outer membrane proteins in the sheath. Some proteins localize preferentially to the flagellar sheath, indicating mechanisms exist for protein partitioning to the sheath. Recent cryo-electron tomography studies have yielded high resolution images of the flagellar sheath and other structures closely associated with the sheath, which has generated insights and new hypotheses for how the flagellar sheath is synthesized. Various functions have been proposed for the flagellar sheath, including preventing disassociation of the flagellin subunits in the presence of gastric acid, avoiding activation of the host innate immune response by flagellin, activating the host immune response, adherence to host cells, and protecting the bacterium from bacteriophages.

scholarly journals The Conservation of Low Complexity Regions in Bacterial Proteins Depends on the Pathogenicity of the Strain and Subcellular Location of the Protein

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 451
Author(s):  
Pablo Mier ◽  
Miguel A. Andrade-Navarro
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
Subcellular Location ◽  
Low Complexity ◽  
Extracellular Proteins ◽  
Bacterial Strains ◽  
Bacterial Proteins ◽  
Protein Subcellular Location

Low complexity regions (LCRs) in proteins are characterized by amino acid frequencies that differ from the average. These regions evolve faster and tend to be less conserved between homologs than globular domains. They are not common in bacteria, as compared to their prevalence in eukaryotes. Studying their conservation could help provide hypotheses about their function. To obtain the appropriate evolutionary focus for this rapidly evolving feature, here we study the conservation of LCRs in bacterial strains and compare their high variability to the closeness of the strains. For this, we selected 20 taxonomically diverse bacterial species and obtained the completely sequenced proteomes of two strains per species. We calculated all orthologous pairs for each of the 20 strain pairs. Per orthologous pair, we computed the conservation of two types of LCRs: compositionally biased regions (CBRs) and homorepeats (polyX). Our results show that, in bacteria, Q-rich CBRs are the most conserved, while A-rich CBRs and polyA are the most variable. LCRs have generally higher conservation when comparing pathogenic strains. However, this result depends on protein subcellular location: LCRs accumulate in extracellular and outer membrane proteins, with conservation increased in the extracellular proteins of pathogens, and decreased for polyX in the outer membrane proteins of pathogens. We conclude that these dependencies support the functional importance of LCRs in host–pathogen interactions.

scholarly journals Anti-Pseudomonas aeruginosa antibody detection in patients with bronchiectasis without cystic fibrosis

Thorax ◽  
2001 ◽  
Vol 56 (9) ◽  
pp. 669-674
Author(s):  
E Caballero ◽  
M-E Drobnic ◽  
M-T Pérez ◽  
J-M Manresa ◽  
A Ferrer ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Antibody Detection ◽  
Individual Bands ◽  
Diagnostic Usefulness ◽  
Group A ◽  
Group B

BACKGROUNDPseudomonas aeruginosa is a frequent cause of infection in patients with bronchiectasis. Differentiation between non-infected patients and those with different degrees of P aeruginosainfection could influence the management and prognosis of these patients. The diagnostic usefulness of serum IgG antibodies againstP aeruginosa outer membrane proteins was determined in patients with bronchiectasis without cystic fibrosis.METHODSFifty six patients were classified according to sputum culture into three groups: group A (n=18) with no P aeruginosain any sample; group B (n=18) with P aeruginosa alternating with other microorganisms; and group C (n=20) with P aeruginosa in all sputum samples. Each patient had at least three sputum cultures in the 6 months prior to serum collection. Detection of antibodies was performed by Western blot and their presence against 20 protein bands (10–121 kd) was assessed.RESULTSAntibodies to more than four bands in total or to five individual bands (36, 26, 22, 20 or 18 kd) differentiated group B from group A, while antibodies to a total of more than eight bands or to 10 individual bands (104, 69, 63, 56, 50, 44, 30, 25, 22, 13 kd) differentiated group C from group B. When discordant results between the total number of bands and the frequency of P aeruginosa isolation were obtained, the follow up of patients suggested that the former, in most cases, predicted chronic P aeruginosacolonisation.CONCLUSIONIn patients with bronchiectasis the degree of P aeruginosa infection can be determined by the number and type of outer membrane protein bands indicating which serum antibodies are present.

scholarly journals Beyond Slam: the DUF560 family outer membrane protein superfamily SPAM has distinct network subclusters that suggest a role in non-lipidated substrate transport and bacterial environmental adaptation

2020 ◽  
Author(s):  
Terra J. Mauer ◽  
Alex S. Grossman ◽  
Katrina T. Forest ◽  
Heidi Goodrich-Blair
Keyword(s):  
Host Range ◽  
Outer Membrane ◽  
Binding Protein ◽  
Sequence Similarity ◽  
Outer Membrane Proteins ◽  
Environmental Adaptation ◽  
Host Cells ◽  
Xenorhabdus Nematophila ◽  
Associated Bacteria ◽  
Host Phylogeny

AbstractIn host-associated bacteria, surface and secreted proteins mediate acquisition of nutrients, interactions with host cells, and specificity of host-range and tissue-localization. In Gram-negative bacteria, the mechanism by which many proteins cross, become embedded within, or become tethered to the outer membrane remains unclear. The domain of unknown function (DUF)560 occurs in outer membrane proteins found throughout and beyond the proteobacteria. Functionally characterized DUF560 representatives include NilB, a host-range specificity determinant of the nematode-mutualist Xenorhabdus nematophila and the surface lipoprotein assembly modulators (Slam), Slam1 and Slam2 which facilitate surface exposure of lipoproteins in the human pathogen Neisseria meningitidis. Through network analysis of protein sequence similarity we show that DUF560 subclusters exist and correspond with organism lifestyle rather than with taxonomy, suggesting a role for these proteins in environmental adaptation. Cluster 1 had the greatest number of representative proteins, was dominated by homologs from animal-associated symbionts, and was composed of subclusters: 1A (containing NilB, Slam1, and Slam2), 1B, and 1C. Genome neighborhood networks revealed that Cluster 1A DUF560 members are strongly associated with TonB, TonB-dependent receptors, and predicted co-receptors such as the Slam1 lipoprotein substrates transferrin binding protein and lactoferrin binding protein. The genome neighborhood network of Cluster 1B sequences are similarly dominated by TonB loci, but typically the associated co-receptors (the presumed DUF560 substrates) are predicted to be non-lipidated. We suggest that these subclusters within the DUF560 protein family indicate distinctive activities and that Slam activity may be characteristic of Cluster 1A members but not all DUF560 homologs. For Cluster 1 DUF560 homologs we propose the name SPAM (Surface/Secreted Protein Associated Outer Membrane Proteins) to accommodate the potential for non-lipoprotein substrates or different activities. We show that the repertoire of SPAM proteins in Xenorhabdus correlates with host phylogeny, suggesting that the host environment drives the evolution of these symbiont-encoded proteins. This pattern of selection for specific sequences based on host physiology and/or environmental factors may extend to other clusters of the DUF560 family.

scholarly journals Proteomic Characterization of the Whole Secretome of Legionella pneumophila and Functional Analysis of Outer Membrane Vesicles

2008 ◽  
Vol 76 (5) ◽  
pp. 1825-1836 ◽  
Author(s):  
Frank Galka ◽  
Sun Nyunt Wai ◽  
Harald Kusch ◽  
Susanne Engelmann ◽  
Michael Hecker ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Legionella Pneumophila ◽  
Protein Identification ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Laser ◽  
Lipolytic Enzyme ◽  
New Findings

ABSTRACT Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (soluble supernatant proteins [SSPs]) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here we present comprehensive proteome reference maps for both SSPs and OMVs. Protein identification and assignment analyses revealed a total of 181 supernatant proteins, 107 of which were specific to the SSP fraction and 33 of which were specific to OMVs. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that the SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill host cells but specifically modulate their cytokine response. Binding of immunofluorescently stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggested that there is delivery of a large and complex group of proteins and lipids in the infected tissue in association with OMVs. On the basis of these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors, as well as environmental aspects of the vesicle-mediated secretion.

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