Two‐dimensional electrophoretic analysis of the protein family at 90 kDa of abortifacient<i>Chlamydia psittaci</i>

Giannikopoulou, Panagiota; Bini, Luca; Simitsek, Phaedra D.; Pallini, Vitaliano; Vretou, Evangelia

doi:10.1002/elps.1150181137

Cited by 19 publications

(23 citation statements)

References 15 publications

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“…The large sizes of proteins 1 to 6 suggest that they may be members of the POMP family previously identified in C. trachomatis serovar L2, C. pneumoniae, and ovine strains of C. psittaci (6,12,13,19,26,27,30,37). All of the putative POMPs were also found in whole EBs and were labeled with [ 125 I]TID (Fig.…”

Section: Resultsmentioning

confidence: 94%

“…It is present throughout the chlamydial developmental cycle and is disulfide cross-linked in EBs but not in logarithmically dividing RBs (16,17,33). A third class of proteins, variously referred to as polymorphic OM proteins (POMPs) and polymorphic membrane proteins, was identified in the Sarkosyl COMC of ovine abortion strains of Chlamydia psittaci by Cevenini et al (6) and others (12,13,27,37). Genes encoding 9 and 21 potential POMPs are present in the genomes of Chlamydia trachomatis and Chlamydia pneumoniae, respectively (18,35,38).…”

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confidence: 99%

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Identification of Polymorphic Outer Membrane Proteins ofChlamydia psittaci6BC

2001

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The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine-and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.The cell envelope structure of Chlamydia is similar to that of other gram-negative bacteria, with an outer membrane (OM) containing lipopolysaccharide, a periplasm, and an inner membrane. However, two envelope features are unique to chlamydiae: an apparent lack of or deficiency in peptidoglycan and the presence of disulfide-bond-cross-linked proteins in the OM and the periplasm (reviewed in reference 15). Nevertheless, the infectious elementary body (EB) form of chlamydiae, but not the dividing reticulate body (RB) form, is osmotically stable, and chlamydiae are sensitive to ␤-lactams and D-cycloserine. The sensitivity of chlamydiae to peptidoglycan synthesis-inhibiting drugs in the possible absence of peptidoglycan has been termed the chlamydial anomaly by Moulder (29). The scope of the anomaly has been expanded by the recent sequencing of the genomes of several chlamydial strains (18,35,38), revealing the presence of what has been thought to be all of the genes required for peptidoglycan synthesis (7). Ghuysen and Goffin (11) proposed a solution to the anomaly, suggesting that chlamydiae use their peptidoglycan genes to synthesize a glycanless wall polymer whose synthesis is penicillin sensitive. Three key points of their proposal are as follows: (i) the predicted amino acid sequence of the three chlamydial penicillinbinding proteins suggests that they are capable of carrying out cross-linking transpeptidase reactions but are incapable of transglycosylating N-acetylglucosamine-N-acetylmuramic acid disaccharides into a glycan polymer; (ii) potential chlamydial N-acetylmuramoyl-L-alanine amidases cleave cross-linked peptidyl polymers from the disaccharide subunits; and (iii) the disaccharide subunits, as part of lipid II, serve strictly as carriers and therefore do not accumulate in stoichiometric amounts. Ghuysen and Goffin (11) further speculated that the glycanless polymer may be covalently linked to lipoproteins in the inner membrane or OM or to a highly dis...

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Section: Resultsmentioning

confidence: 94%

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confidence: 99%

Identification of Polymorphic Outer Membrane Proteins ofChlamydia psittaci6BC

2001

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“…In initial studies, this strain was propagated in cycloheximide-treated McCoy cell monolayers, and EBs were purified by centrifugation through a discontinuous gradient of Gastrografin (Schering, Berlin, Germany) as previously described for proteomic studies. Highresolution two-dimensional gel electrophoresis of EB proteins was performed using the Immobiline-polyacrylamide system (4,10). Approximately 1 mg of total EB was pelleted by centrifugation and resuspended in 8 M urea-4% CHAPS {3-[(3-cholamidopropyl) dimethylammonium]-1-propanesulfate}-40 mM Tris base-65 mM dithioerythritol-bromophenol blue.…”

Section: Methodsmentioning

confidence: 99%

Molecular Characterization of a Bacteriophage (Chp2) from Chlamydia psittaci

Liu

Everson

Fane

et al. 2000

J Virol

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Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C. psittaci RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the X174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein.

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“…Several proteins encoded by these genes had previously been investigated in C. psittaci (6,11,12,20,25). Nine genes are found in C. trachomatis (pmpA to pmpI) (26).…”

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Polymorphic Membrane Protein H Has Evolved in Parallel with the Three Disease-Causing Groups ofChlamydia trachomatis

2003

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Chlamydia trachomatis is a human pathogen causing trachoma, urogenital disease, and lymphogranuloma venereum (LGV). A family of nine polymorphic membrane protein genes (pmpA to pmpI), resembling autotransporter proteins, has recently been discovered in C. trachomatis. pmp genes are large and predicted to be outer membrane proteins. We hypothesized that they would contain useful nucleotide sequence variability for epidemiologic studies. Since sequence information is available only for serovars D and L2, we sought to determine the amount of diversity within an individual pmp gene among serovars. We used restriction fragment length polymorphism (RFLP) analysis as a primary screen to assess the amount of sequence divergence among the pmp genes for serovars A to L3 of C. trachomatis. RFLP analysis showed little variation for some of the genes, such as pmpA, but substantial variation in others, such as pmpI. pmpH and pmpE yielded RFLP patterns that clustered the 15 serovars into ocular, urogenital, and LGV groups, and both proteins have been localized to the outer membrane. Therefore, we chose to sequence pmpE, pmpH, and pmpI from each of the 15 serovars. Evolutionary analysis showed three distinct divergence patterns. PmpI was least variable, resulting in an ambiguous evolutionary pattern. PmpE showed a high degree of diversity in the ocular strains compared to the other strains. Finally, the evolution of PmpH shows three groups that reflect disease groups, suggesting this protein may play a role in pathogenesis.

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Two‐dimensional electrophoretic analysis of the protein family at 90 kDa of abortifacientChlamydia psittaci

Cited by 19 publications

References 15 publications

Identification of Polymorphic Outer Membrane Proteins ofChlamydia psittaci6BC

Identification of Polymorphic Outer Membrane Proteins ofChlamydia psittaci6BC

Molecular Characterization of a Bacteriophage (Chp2) from Chlamydia psittaci

Polymorphic Membrane Protein H Has Evolved in Parallel with the Three Disease-Causing Groups ofChlamydia trachomatis

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