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1.  Expression and Functional Characterization of the First Bacteriophage-Encoded Chaperonin 
Journal of Virology  2012;86(18):10103-10111.
Chaperonins promote protein folding in vivo and are ubiquitously found in bacteria, archaea, and eukaryotes. The first viral chaperonin GroEL ortholog, gene product 146 (gp146), whose gene was earlier identified in the genome of bacteriophage EL, has been shown to be synthesized during phage propagation in Pseudomonas aeruginosa cells. The recombinant gp146 has been expressed in Escherichia coli and characterized by different physicochemical methods for the first time. Using serum against the recombinant protein, gp146's native substrate, the phage endolysin gp188, has been immunoprecipitated from the lysate of EL-infected bacteria and identified by mass spectrometry. In vitro experiments have shown that gp146 has a protective effect against endolysin thermal inactivation and aggregation, providing evidence of its chaperonin function. The phage chaperonin has been found to have the architecture and some properties similar to those of GroEL but not to require cochaperonin for its functional activity.
doi:10.1128/JVI.00940-12
PMCID: PMC3446618  PMID: 22787217
2.  A theoretical and experimental proteome map of Pseudomonas aeruginosa PAO1 
MicrobiologyOpen  2012;1(2):169-181.
A total proteome map of the Pseudomonas aeruginosa PAO1 proteome is presented, generated by a combination of two-dimensional gel electrophoresis and protein identification by mass spectrometry. In total, 1128 spots were visualized, and 181 protein spots were characterized, corresponding to 159 different protein entries. In particular, protein chaperones and enzymes important in energy conversion and amino acid biosynthesis were identified. Spot analysis always resulted in the identification of a single protein, suggesting sufficient spot resolution, although the same protein may be detected in two or more neighboring spots, possibly indicating posttranslational modifications. Comparison to the theoretical proteome revealed an underrepresentation of membrane proteins, though the identified proteins cover all predicted subcellular localizations and all functional classes. These data provide a basis for subsequent comparative studies of the biology and metabolism of P. aeruginosa, aimed at unraveling global regulatory networks.
doi:10.1002/mbo3.21
PMCID: PMC3426416  PMID: 22950023
Mass spectrometry (MS); proteomics; two-dimensional gel electrophoresis (2-DE)
3.  Identification of Protein Networks Involved in the Disease Course of Experimental Autoimmune Encephalomyelitis, an Animal Model of Multiple Sclerosis 
PLoS ONE  2012;7(4):e35544.
A more detailed insight into disease mechanisms of multiple sclerosis (MS) is crucial for the development of new and more effective therapies. MS is a chronic inflammatory autoimmune disease of the central nervous system. The aim of this study is to identify novel disease associated proteins involved in the development of inflammatory brain lesions, to help unravel underlying disease processes. Brainstem proteins were obtained from rats with MBP induced acute experimental autoimmune encephalomyelitis (EAE), a well characterized disease model of MS. Samples were collected at different time points: just before onset of symptoms, at the top of the disease and following recovery. To analyze changes in the brainstem proteome during the disease course, a quantitative proteomics study was performed using two-dimensional difference in-gel electrophoresis (2D-DIGE) followed by mass spectrometry. We identified 75 unique proteins in 92 spots with a significant abundance difference between the experimental groups. To find disease-related networks, these regulated proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). The analysis revealed that 70% of these proteins have been described to take part in neurological disease. Furthermore, some focus networks were created by IPA. These networks suggest an integrated regulation of the identified proteins with the addition of some putative regulators. Post-synaptic density protein 95 (DLG4), a key player in neuronal signalling and calcium-activated potassium channel alpha 1 (KCNMA1), involved in neurotransmitter release, are 2 putative regulators connecting 64% of the identified proteins. Functional blocking of the KCNMA1 in macrophages was able to alter myelin phagocytosis, a disease mechanism highly involved in EAE and MS pathology. Quantitative analysis of differentially expressed brainstem proteins in an animal model of MS is a first step to identify disease-associated proteins and networks that warrant further research to study their actual contribution to disease pathology.
doi:10.1371/journal.pone.0035544
PMCID: PMC3328452  PMID: 22530047
4.  Professor Dr. Richard Bruynoghe 
Bacteriophage  2012;2(1):1-4.
In 1921, Richard Bruynoghe and his student Joséph Maisin published on the first use of bacteriophages in a phage therapy context. At that time, Bruynoghe (a medical doctor) was affiliated as a professor at the KU Leuven (Belgium) for just over a decade, within the Bacteriological Institute which he founded and led. After a distinguished career (he was acting mayor of the city of Leuven-Belgium during the second World War), he received a special medical award in 1951 just before his retirement in 1952. In this perspective, he was asked to provide an overview of his research for a lay-audience within the local University magazine: Onze Alma Mater (Our alma mater). We, as current affiliates of the KU Leuven are honored to present some of his legacy, which to date has been largely overlooked in historical accounts.
doi:10.4161/bact.20024
PMCID: PMC3357380  PMID: 22666651
bacteria; bacteriophages; historical overview; phage biology; phage therapy
5.  Detection of a Lysozyme Inhibitor in Proteus mirabilis by a New Reverse Zymogram Method▿  
Applied and Environmental Microbiology  2008;74(15):4978-4981.
A reverse zymogram method for the detection of bacterial lysozyme inhibitors was developed. This method was validated by using a periplasmic protein extract of Escherichia coli containing a known inhibitor and subsequently led to the detection of a new proteinaceous hen egg white lysozyme inhibitor in Proteus mirabilis.
doi:10.1128/AEM.00219-08
PMCID: PMC2519368  PMID: 18539812
6.  The Genome and Structural Proteome of YuA, a New Pseudomonas aeruginosa Phage Resembling M6▿ †  
Journal of Bacteriology  2007;190(4):1429-1435.
Pseudomonas aeruginosa phage YuA (Siphoviridae) was isolated from a pond near Moscow, Russia. It has an elongated head, encapsulating a circularly permuted genome of 58,663 bp, and a flexible, noncontractile tail, which is terminally and subterminally decorated with short fibers. The YuA genome is neither Mu- nor λ-like and encodes 78 gene products that cluster in three major regions involved in (i) DNA metabolism and replication, (ii) host interaction, and (iii) phage particle formation and host lysis. At the protein level, YuA displays significant homology with phages M6, φJL001, 73, B3, DMS3, and D3112. Eighteen YuA proteins were identified as part of the phage particle by mass spectrometry analysis. Five different bacterial promoters were experimentally identified using a promoter trap assay, three of which have a σ54-specific binding site and regulate transcription in the genome region involved in phage particle formation and host lysis. The dependency of these promoters on the host σ54 factor was confirmed by analysis of an rpoN mutant strain of P. aeruginosa PAO1. At the DNA level, YuA is 91% identical to the recently (July 2007) annotated phage M6 of the Lindberg typing set. Despite this level of DNA homology throughout the genome, both phages combined have 15 unique genes that do not occur in the other phage. The genome organization of both phages differs substantially from those of the other known Pseudomonas-infecting Siphoviridae, delineating them as a distinct genus within this family.
doi:10.1128/JB.01441-07
PMCID: PMC2238194  PMID: 18065532
7.  A New Family of Lysozyme Inhibitors Contributing to Lysozyme Tolerance in Gram-Negative Bacteria 
PLoS Pathogens  2008;4(3):e1000019.
Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in gram-negative bacteria. First, a lysozyme inhibitor was isolated by affinity chromatography from a periplasmic extract of Salmonella Enteritidis, identified by mass spectrometry and correspondingly designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme). A pliC knock-out mutant no longer produced lysozyme inhibitory activity and showed increased lysozyme sensitivity in the presence of the outer membrane permeabilizing protein lactoferrin. PliC lacks similarity with the previously described Escherichia coli lysozyme inhibitor Ivy, but is related to a group of proteins with a common conserved COG3895 domain, some of them predicted to be lipoproteins. No function has yet been assigned to these proteins, although they are widely spread among the Proteobacteria. We demonstrate that at least two representatives of this group, MliC (membrane bound lysozyme inhibitor of c-type lysozyme) of E. coli and Pseudomonas aeruginosa, also possess lysozyme inhibitory activity and confer increased lysozyme tolerance upon expression in E. coli. Interestingly, mliC of Salmonella Typhi was picked up earlier in a screen for genes induced during residence in macrophages, and knockout of mliC was shown to reduce macrophage survival of S. Typhi. Based on these observations, we suggest that the COG3895 domain is a common feature of a novel and widespread family of bacterial lysozyme inhibitors in gram-negative bacteria that may function as colonization or virulence factors in bacteria interacting with an animal host.
Author Summary
Lysozyme is an ancient bactericidal enzyme that is part of the antibacterial defense system of vertebrate and invertebrate animals. Bacteria colonizing or infecting an animal host have developed various ways to overcome lysozyme action, a recently proposed mechanism being the production of lysozyme inhibitors. However, the only high affinity bacterial lysozyme inhibitor known thus far is produced only in few bacteria, and this raised questions about their wider relevance in bacteria–host interactions. We here report the discovery of a novel and distinct family of bacterial lysozyme inhibitors that is widely distributed among the Proteobacteria, including several major pathogens. The family comprises periplasmic as well as membrane-bound inhibitors, and both types contribute to lysozyme tolerance of bacterial cells, as we experimentally demonstrate for the periplasmic inhibitor from Salmonella Typhimurium and the membrane-bound inhibitors from Escherichia coli and Pseudomonas aeruginosa. Interestingly, a gene encoding one of the newly identified inhibitors has been previously found to promote macrophage survival of Salmonella Typhi. The widespread occurrence of lysozyme inhibitors in bacteria is likely to reflect their functional importance in a wide range of bacteria–host interactions. As such, they are also attractive novel targets for antibacterial drug development.
doi:10.1371/journal.ppat.1000019
PMCID: PMC2267010  PMID: 18369469
8.  Mutational Analysis of Endoxylanases XylA and XylB from the Phytopathogen Fusarium graminearum Reveals Comprehensive Insights into Their Inhibitor Insensitivity▿  
Applied and Environmental Microbiology  2007;73(14):4602-4608.
Endo-β-1,4-xylanases (EC 3.2.1.8; endoxylanases), key enzymes in the degradation of xylan, are considered to play an important role in phytopathogenesis, as they occupy a prominent position in the arsenal of hydrolytic enzymes secreted by phytopathogens to breach the cell wall and invade the plant tissue. Plant endoxylanase inhibitors are increasingly being pinpointed as part of a counterattack mechanism. To understand the surprising XIP-type endoxylanase inhibitor insensitivity of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum, an extensive mutational study of these enzymes was performed. Using combinatorial and site-directed mutagenesis, the XIP insensitivity of XylA as well as XylB was proven to be solely due to amino acid sequence adaptations in the “thumb” structural region. While XylB residues Cys141, Asp148, and Cys149 were shown to prevent XIP interaction, the XIP insensitivity of XylA could be ascribed to the occurrence of only one aberrant residue, i.e., Val151. This study, in addition to providing a thorough explanation for the XIP insensitivity of both F. graminearum endoxylanases at the molecular level, generated XylA and XylB mutants with altered inhibition specificities and pH optima. As this is the first experimental elucidation of the molecular determinants dictating the specificity of the interaction between endoxylanases of phytopathogenic origin and a plant inhibitor, this work sheds more light on the ongoing evolutionary arms race between plants and phytopathogenic fungi involving recognition of endoxylanases.
doi:10.1128/AEM.00442-07
PMCID: PMC1932832  PMID: 17513587
9.  The Genome of the Kinetoplastid Parasite, Leishmania major 
Ivens, Alasdair C. | Peacock, Christopher S. | Worthey, Elizabeth A. | Murphy, Lee | Aggarwal, Gautam | Berriman, Matthew | Sisk, Ellen | Rajandream, Marie-Adele | Adlem, Ellen | Aert, Rita | Anupama, Atashi | Apostolou, Zina | Attipoe, Philip | Bason, Nathalie | Bauser, Christopher | Beck, Alfred | Beverley, Stephen M. | Bianchettin, Gabriella | Borzym, Katja | Bothe, Gordana | Bruschi, Carlo V. | Collins, Matt | Cadag, Eithon | Ciarloni, Laura | Clayton, Christine | Coulson, Richard M. R. | Cronin, Ann | Cruz, Angela K. | Davies, Robert M. | Gaudenzi, Javier De | Dobson, Deborah E. | Duesterhoeft, Andreas | Fazelina, Gholam | Fosker, Nigel | Frasch, Alberto Carlos | Fraser, Audrey | Fuchs, Monika | Gabel, Claudia | Goble, Arlette | Goffeau, André | Harris, David | Hertz-Fowler, Christiane | Hilbert, Helmut | Horn, David | Huang, Yiting | Klages, Sven | Knights, Andrew | Kube, Michael | Larke, Natasha | Litvin, Lyudmila | Lord, Angela | Louie, Tin | Marra, Marco | Masuy, David | Matthews, Keith | Michaeli, Shulamit | Mottram, Jeremy C. | Müller-Auer, Silke | Munden, Heather | Nelson, Siri | Norbertczak, Halina | Oliver, Karen | O'Neil, Susan | Pentony, Martin | Pohl, Thomas M. | Price, Claire | Purnelle, Bénédicte | Quail, Michael A. | Rabbinowitsch, Ester | Reinhardt, Richard | Rieger, Michael | Rinta, Joel | Robben, Johan | Robertson, Laura | Ruiz, Jeronimo C. | Rutter, Simon | Saunders, David | Schäfer, Melanie | Schein, Jacquie | Schwartz, David C. | Seeger, Kathy | Seyler, Amber | Sharp, Sarah | Shin, Heesun | Sivam, Dhileep | Squares, Rob | Squares, Steve | Tosato, Valentina | Vogt, Christy | Volckaert, Guido | Wambutt, Rolf | Warren, Tim | Wedler, Holger | Woodward, John | Zhou, Shiguo | Zimmermann, Wolfgang | Smith, Deborah F. | Blackwell, Jenefer M. | Stuart, Kenneth D. | Barrell, Bart | Myler, Peter J.
Science (New York, N.Y.)  2005;309(5733):436-442.
doi:10.1126/science.1112680
PMCID: PMC1470643  PMID: 16020728
10.  Genomic Analysis of Pseudomonas aeruginosa Phages LKD16 and LKA1: Establishment of the φKMV Subgroup within the T7 Supergroup†  
Journal of Bacteriology  2006;188(19):6924-6931.
Lytic Pseudomonas aeruginosa phages LKD16 and LKA1 were locally isolated and morphologically classified as Podoviridae. While LKD16 adsorbs weakly to its host, LKA1 shows efficient adsorption (ka = 3.9 × 10−9 ml min−1). LKA1, however, displays a narrow host range on clinical P. aeruginosa strains compared to LKD16. Genome analysis of LKD16 (43,200 bp) and LKA1 (41,593 bp) revealed that both phages have linear double-stranded DNA genomes with direct terminal repeats of 428 and 298 bp and encode 54 and 56 genes, respectively. The majority of the predicted structural proteins were experimentally confirmed as part of the phage particle using mass spectrometry. Phage LKD16 is closely related to bacteriophage φKMV (83% overall DNA homology), allowing a more thoughtful gene annotation of both genomes. In contrast, LKA1 is more distantly related, lacking significant DNA homology and showing protein similarity to φKMV in 48% of its gene products. The early region of the LKA1 genome has diverged strongly from φKMV and LKD16, and intriguing differences in tail fiber genes of LKD16 and LKA1 likely reflect the observed discrepancy in infection-related properties. Nonetheless, general genome organization is clearly conserved among φKMV, LKD16, and LKA1. The three phages carry a single-subunit RNA polymerase gene adjacent to the structural genome region, a feature which distinguishes them from other members of the T7 supergroup. Therefore, we propose that φKMV represents an independent and widespread group of lytic P. aeruginosa phages within the T7 supergroup.
doi:10.1128/JB.00831-06
PMCID: PMC1595506  PMID: 16980495

Results 1-10 (10)