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Evaluation of procedures for outer membrane isolation from Campylobacter jejuni.

ABSTRACT: Although infection with Campylobacter jejuni is one of the leading causes of gastroenteritis worldwide, relatively little is known about the factors that are required to elicit a protective immune response. The need for a vaccine against this pathogen is well recognized and a number of vaccine candidates have been tested with varying degrees of success; however, there is still a lack of a suitable vaccine. To gain a better understanding of the outer-membrane protein components of this organism, a 'gold standard' method to purify the outer membrane is needed. Therefore, we attempted to develop a robust and reliable method which resulted in a pure outer-membrane fraction. A total of nine methodologies were examined and analysed by SDS-PAGE and immunoblotting using subcellular markers for the cytoplasm, cytoplasmic membrane and outer membrane. We found that glycine extraction, differential detergent extraction using Triton X-100, serial extraction using 1 M Tris pH 7, spheroplasting by lysozyme and sonication, and carbonate extraction did not produce pure outer-membrane preparations. However, we identified three methods that provided outer-membrane fractions free from subcellular contamination. Isopycnic centrifugation using a 30-60 % sucrose gradient produced seven fractions free from cytoplasmic or cytoplasmic membrane contamination; however, these fractions did not correspond as well as expected with the typical outer-membrane-associated peak (e.g. Escherichia coli or Salmonella). The spheroplast method using lysozyme alone also resulted in pure outer-membrane fraction, as did carbonate washing of this sample. The extraction of outer membranes using N-lauroylsarcosine (Sarkosyl) produced the purest and most reproducible sample. These outer-membrane preparations will be useful for future studies aimed at identifying C. jejuni surface proteins as vaccine components.

SUBMITTER: Hobb RI

PROVIDER: S-EPMC2763183 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Evaluation of procedures for outer membrane isolation from Campylobacter jejuni.

Hobb Rhonda I RI Fields Joshua A JA Burns Christopher M CM Thompson Stuart A SA

Microbiology (Reading, England) 20090301 Pt 3

Although infection with Campylobacter jejuni is one of the leading causes of gastroenteritis worldwide, relatively little is known about the factors that are required to elicit a protective immune response. The need for a vaccine against this pathogen is well recognized and a number of vaccine candidates have been tested with varying degrees of success; however, there is still a lack of a suitable vaccine. To gain a better understanding of the outer-membrane protein components of this organism, ...[more]

PMID: 19246768

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Project description:Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C. jejuni NCTC11168 were isolated from culture supernatants then characterized using electron microscopy and dynamic light scattering revealing spherical OMVs of an average diameter of 50nm. We then identified 134 vesicular proteins using high-resolution LTQ-Orbitrap mass spectrometry. Subsequent functional analysis of the genes revealed the relationships of the vesicular proteins. Furthermore, known N-glycoproteins were identified from the list of the vesicular proteome, implying the potential role of the OMVs as a delivery means for biologically relevant bacterial glycoproteins. These results enabled us to elucidate the overall proteome profile of pathogenic bacterium C. jejuni and to speculate on the function of OMVs in bacterial infections and communication.Biological significanceThis work demonstrates the importance of understanding vesicular proteomes from a human pathogen Campylobacter jejuni. From the secreted outer membrane vesicles (OMVs) of C. jejuni NCTC11168, we found a variety of virulence factors and essential proteins for bacterial survival. Bioinformatics analysis of these proteins predicted functional enrichment and localization. The most highly enriched were redox enzymes, which are considered to be essential for survival in oxygen-limiting environments and are predicted to be on the twin-arginine translocation (Tat) pathway suggesting a role for this pathway in the biogenesis of OMVs. This study additionally implicates a biological role for N-linked glycoproteins in OMVs. These approaches allow for a better understanding of the physiology of this important human pathogen.

Project description:We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428. The nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed. The amino acid sequence had the typical features of a leader sequence and a signal peptidase II cleavage site at the N-terminal part of Omp18. Moreover, the sequence had a high degree of similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of Haemophilus influenzae and the peptidoglycan-associated lipoprotein PAL of E. coli. Southern blot analysis in which the cloned gene was used as a probe revealed genes similar to that encoding Omp18 in all species of the thermophilic group of campylobacters as well as Campylobacter sputorum. All campylobacters tested expressed a protein with a molecular mass identical to that of Omp18. The protein reacted immunologically with polyclonal antibodies directed against Omp18 from C. jejuni. PCR amplification of the gene encoding Omp18 with specific primers and subsequent restriction enzyme analysis of the amplified DNA fragments showed that the gene for Omp18 is highly conserved in C. jejuni strains isolated from humans, dogs, cats, calves, and chickens but is different in other Campylobacter species. In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacing the signal sequence with a DNA segment encoding six adjacent histidine residues. Expression of this construct in E. coli allowed purification of the modified protein (Omp18-6xHis) by metal chelation chromatography. Sera from patients with past C. jejuni infection reacted positively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen. Omp18, which is an outer membrane protein belonging to the family of PALs is well conserved in C. jejuni and is highly immunogenic. It is therefore a good candidate as an antigen for the serological diagnosis of past C. jejuni infections.

Project description:The proteomes of outer membrane vesicles (OMVs) secreted by C. jejuni 81-176 strain, which was exposed to oxygen or antibiotic stress (polymyxin B), were characterized. We also assessed the OMVs production and their content in two mutated strains - ∆dsbI and ∆htrA. OMVs production was significantly increased under the polymyxin B stress and remained unaltered in all other variants. Interestingly, the qualitative load of OMVs was constant regardless of the stress conditions or genetic background. However, certain proteins exhibited notable quantitative changes, ranging from 4-fold decrease to 10-fold increase. Up- or downregulated proteins (e.g. major outer membrane protein porA, iron ABC transporter, serine protease- htrA, 60 kDa chaperonin-groL, enolase) represented various cell compartments (cytoplasm, periplasm, and membrane) and exhibited various functions; nevertheless, one common group was noted that consisted of components of flagellar apparatus, i.e., FlaA/B, FlgC/E, which were mostly upregulated. Some of these proteins are the putative substrates of DsbI protein. Further investigation of the regulation of C. jejuni OMVs composition and their role in virulence will allow a better understanding of the infectious process of C. jejuni.The proteomes of outer membrane vesicles (OMVs) secreted by C. jejuni 81–176 strain, which was exposed to oxygen or antibiotic stress (polymyxin B), were characterized. We also assessed the OMVs production and their content in two mutated strains – ∆dsbI and ∆htrA. OMVs production was significantly increased under the polymyxin B stress and remained unaltered in all other variants. Interestingly, the qualitative load of OMVs was constant regardless of the stress conditions or genetic background. However, certain proteins exhibited notable quantitative changes, ranging from 4-fold decrease to 10-fold increase. Up- or downregulated proteins (e.g. major outer membrane protein porA, iron ABC transporter, serine protease- htrA, 60 kDa chaperonin-groL, enolase) represented various cell compartments (cytoplasm, periplasm, and membrane) and exhibited various functions; nevertheless, one common group was noted that consisted of components of flagellar apparatus, i.e., FlaA/B, FlgC/E, which were mostly upregulated. Some of these proteins are the putative substrates of DsbI protein. Further investigation of the regulation of C. jejuni OMVs composition and their role in virulence will allow a better understanding of the infectious process of C. jejuni.

Dataset Information

Evaluation of procedures for outer membrane isolation from Campylobacter jejuni.

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Evaluation of procedures for outer membrane isolation from Campylobacter jejuni.

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