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 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 Insights into the Assembly of the Alginate Biosynthesis Machinery in Pseudomonas aeruginosa

2013 ◽  
Vol 79 (10) ◽  
pp. 3264-3272 ◽  
Author(s):  
Zahid U. Rehman ◽  
Yajie Wang ◽  
M. Fata Moradali ◽  
Iain D. Hay ◽  
Bernd H. A. Rehm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stability Analysis ◽  
Experimental Evidence ◽  
Outer Membrane ◽  
Opportunistic Pathogen ◽  
Multiprotein Complex ◽  
Inner Membrane ◽  
Content Type ◽  
Alginate Production ◽  
The Impact

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen of particular significance to cystic fibrosis patients. This bacterium produces the exopolysaccharide alginate, which is an indicator of poor prognosis for these patients. The proteins required for alginate polymerization and secretion are encoded by genes organized in a single operon; however, the existence of internal promoters has been reported. It has been proposed that these proteins form a multiprotein complex which extends from the inner to outer membrane. Here, experimental evidence supporting such a multiprotein complex was obtained via mutual stability analysis, pulldown assays, and coimmunoprecipitation. The impact of the absence of single proteins or subunits on this multiprotein complex, i.e., on the stability of potentially interacting proteins, as well as on alginate production was investigated. Deletion ofalgKin an alginate-overproducing strain, PDO300, interfered with the polymerization of alginate, suggesting that in the absence of AlgK, the polymerase and copolymerase subunits, Alg8 and Alg44, are destabilized. Based on mutual stability analysis, interactions between AlgE (outer membrane), AlgK (periplasm), AlgX (periplasm), Alg44 (inner membrane), Alg8 (inner membrane), and AlgG (periplasm) were proposed. Coimmunoprecipitation using a FLAG-tagged variant of AlgE further demonstrated its interaction with AlgK. Pulldown assays using histidine-tagged AlgK showed that AlgK interacts with AlgX, which in turn was also copurified with histidine-tagged Alg44. Detection of AlgG and AlgE in PAO1 supported the existence of internal promoters controlling expression of the respective genes. Overall experimental evidence was provided for the existence of a multiprotein complex required for alginate polymerization and secretion.

scholarly journals Role of Pseudomonas aeruginosa Peptidoglycan-Associated Outer Membrane Proteins in Vesicle Formation

2012 ◽  
Vol 195 (2) ◽  
pp. 213-219 ◽  
Author(s):  
Aimee K. Wessel ◽  
Jean Liew ◽  
Taejoon Kwon ◽  
Edward M. Marcotte ◽  
Marvin Whiteley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Opportunistic Pathogen ◽  
Outer Membrane Vesicles ◽  
Gram Negative Bacteria ◽  
Content Type

ABSTRACTGram-negative bacteria produce outer membrane vesicles (OMVs) that package and deliver proteins, small molecules, and DNA to prokaryotic and eukaryotic cells. The molecular details of OMV biogenesis have not been fully elucidated, but peptidoglycan-associated outer membrane proteins that tether the outer membrane to the underlying peptidoglycan have been shown to be critical for OMV formation in multipleEnterobacteriaceae. In this study, we demonstrate that the peptidoglycan-associated outer membrane proteins OprF and OprI, but not OprL, impact production of OMVs by the opportunistic pathogenPseudomonas aeruginosa. Interestingly, OprF does not appear to be important for tethering the outer membrane to peptidoglycan but instead impacts OMV formation through modulation of the levels of thePseudomonasquinolone signal (PQS), a quorum signal previously shown by our laboratory to be critical for OMV formation. Thus, the mechanism by which OprF impacts OMV formation is distinct from that for other peptidoglycan-associated outer membrane proteins, including OprI.

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 Pseudomonas aeruginosa las and rhl quorum-sensing systems are important for infection and inflammation in a rat prostatitis model

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2612-2619 ◽  
Author(s):  
Lisa K. Nelson ◽  
Genevieve H. D'Amours ◽  
Kimberley M. Sproule-Willoughby ◽  
Douglas W. Morck ◽  
Howard Ceri
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Tissue Damage ◽  
Inflammatory Markers ◽  
Bacterial Colonization ◽  
Opportunistic Pathogen ◽  
Infection Model ◽  
Chronic Infections ◽  
Strain Pao1 ◽  
Rat Prostate

Pseudomonas aeruginosa frequently acts as an opportunistic pathogen of mucosal surfaces; yet, despite causing aggressive prostatitis in some men, its role as a pathogen in the prostate has not been investigated. Consequently, we developed a Ps. aeruginosa infection model in the rat prostate by instilling wild-type (WT) Ps. aeruginosa strain PAO1 into the rat prostate. It was found that Ps. aeruginosa produced acute and chronic infections in this mucosal tissue as determined by bacterial colonization, gross morphology, tissue damage and inflammatory markers. WT strain PAO1 and its isogenic mutant PAO-JP2, in which both the lasI and rhlI quorum-sensing signal systems have been silenced, were compared during both acute and chronic prostate infections. In acute infections, bacterial numbers and inflammatory markers were comparable between WT PA01 and PAO-JP2; however, considerably less tissue damage occurred in infections with PAO-JP2. Chronic infections with PAO-JP2 resulted in reduced bacterial colonization, tissue damage and inflammation as compared to WT PAO1 infections. Therefore, the quorum-sensing lasI and rhlI genes in Ps. aeruginosa affect acute prostate infections, but play a considerably more important role in maintaining chronic infections. We have thus developed a highly reproducible model for the study of Ps. aeruginosa virulence in the prostate.

scholarly journals Heterogenous response to R-pyocin killing in populations of Pseudomonas aeruginosa sourced from cystic fibrosis lungs

2020 ◽  
Author(s):  
Madeline Mei ◽  
Jacob Thomas ◽  
Stephen P. Diggle
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Genotypic Diversity ◽  
Opportunistic Pathogen ◽  
Chronic Infections ◽  
Bacterial Populations ◽  
Heterogeneous Populations ◽  
Lung Infections ◽  
The Impact ◽  
Lps Binding

AbstractBacteriocins are proteinaceous antimicrobials produced by bacteria which are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa. Due to their anti-pseudomonal activity, R-pyocins have potential as therapeutics in infection. P. aeruginosa is a Gram-negative opportunistic pathogen and is particularly problematic for individuals with cystic fibrosis (CF). P. aeruginosa from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. P. aeruginosa populations become phenotypically and genotypically diverse during infection, however little is known of the efficacy of R-pyocins against heterogeneous populations. R-pyocins vary by subtype (R1-R5), distinguished by binding to different residues on the lipopolysaccharide (LPS). Each type varies in killing spectrum, and each strain produces only one R-type. To evaluate the prevalence of different R-types, we screened P. aeruginosa strains from the International Pseudomonas Consortium Database (IPCD) and from our biobank of CF strains. We found that (i) R1-types were the most prevalent R-type among strains from respiratory sources and (ii) isolates collected from the same patient have the same R-type. We then assessed the impact of diversity on R-pyocin susceptibility and found a heterogenous response to R-pyocins within populations, likely due to differences in the LPS core. Our work reveals that heterogeneous populations of microbes exhibit variable susceptibility to R-pyocins and highlights that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage.ImportanceR-pyocins have potential as alternative therapeutics against Pseudomonas aeruginosa in chronic infection, however little is known about the efficacy of R-pyocins in heterogeneous bacterial populations. P. aeruginosa is known to become resistant to multiple antibiotics, as well as evolve phenotypic and genotypic diversity over time; thus it is particularly difficult to eradicate in chronic cystic fibrosis (CF) lung infections. In this study, we found that P. aeruginosa populations from CF lungs maintain the same R-pyocin genotype but exhibit heterogeneity in susceptibility to R-pyocins from other strains. Our findings suggest there is likely heterogeneity in response to other types of LPS-binding antimicrobials, such as phage, highlighting the necessity of further studying the potential of LPS-binding antimicrobial particles as alternative therapies in chronic infections.

scholarly journals Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

2008 ◽  
Vol 190 (11) ◽  
pp. 4001-4016 ◽  
Author(s):  
Wallace A. Kaserer ◽  
Xiaoxu Jiang ◽  
Qiaobin Xiao ◽  
Daniel C. Scott ◽  
Matthew Bauler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Binding Motif ◽  
Inner Membrane ◽  
E Coli ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

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