Types Of Outer Membrane Proteins Research Articles

Enteric redmouth disease: Past, present and future: A review

Enteric red mouth disease (also known as Yersiniosis) is one of the most significant bacterial infections in coldwater fish farms that cause significant mortalities and economical losses in the salmonids fish farms, especially in rainbow trout (Oncorhynchus mykiss). ERM is caused by the gram negative pathogen bacteria Yersinia ruckeri that has five O-serotypes (O1, O2, O5, O6 and O7), five outer membrane protein types (OMP types 1–5) and two biotypes 1 and 2. The disease has a wide geographical distribution in various fresh or sea water fish. More than twenty species mainly of Salmonide origin have been affected and this number is likely to rise in the future following the introduction of new species and the increase of aquaculture trade. The disease can affect fish of all age classes but is most acute in small fish up to fingerling size. Affected fish may reveal different clinical symptoms depending on species, age and temperature. The most characteristic and common clinical signs of the disease include lethargic behavior and inactivity, swimming near the surface, anorexia, and darkening of the skin. The reddening of the throat and mouth, caused by subcutaneous haemorrhaging and exophthalmos are commonly present. Different diagnostic methods have been used for Y. ruckeri, including culturing, serological, biochemical tests, histopathological studies and molecular techniques. This review summarizes the past, present and future state of yersiniosis with emphasis on status of this disese in Iran. Also, some criteria in diagnosis, control and prevention of ERM were discussed.

Comparison of molecular and biochemical heterogeneity of Yersinia ruckeri strains isolated from Turkey and the USA

Forty isolates of Yersinia ruckeri from Italy (4), Turkey (24), the USA (10), and two type strains were compared by using biochemical test, pulsed-field gel electrophoresis (PFGE), and lipopolysaccharide (LPS) and outer membrane protein (OMP) profiles. Two biotype 1 and biotype 2 Y. ruckeri were determined based on motility and phospholipase activity. Twenty-two different pulse types were observed by cutting the DNA with SpeI restriction enzyme and running on PFGE. Four major clusters were generated using the UPGMA technique: cluster A contained Italian, Turkish and USA strains; cluster B included all the USA strains; and cluster C and cluster D contained all the Turkish strains. Based on the OMP profile, Y. ruckeri strains were divided into 4 clusters with 30 OMP types. A total of 30 different LPS types were observed. All the Italian, the USA, and most of the Turkish isolates were grouped together to form cluster A that consisted of 26 LPS types. It seems that all the four typing methods are highly discriminatory to distinguish even closely related strains. The overall similarities among the strains were 32.4±6.1%, 58.7±11.1%, and 79.5±3.9% in LPS, PFGE, and OMP profiles, respectively. The PFGE, biochemical, OMP, and LPS profiles of none of the strains were found to be similar. Hence, each typing method showed its own discriminatory characteristics to distinguish between the individual strains. It is apparent that there is sufficient genetic diversity to justify using PFGE for analysis of horizontal transmission of Y. ruckeri in trout rearing facilities. It is possible that diverse environmental conditions resulted in a relatively high degree of genetic diversity in Y. ruckeri.Statement of relevanceWe believe that this manuscript entitled “Comparison of molecular and biochemical heterogeneity of Yersinia ruckeri strains isolated from Turkey and the USA” is relevant to aquaculture because Y. ruckeri is one of the most common bacterial fish diseases in salmonid culture. In the present study, Y. ruckeri strains were compared by using biochemical test, pulsed-field gel electrophoresis (PFGE), and lipopolysaccharide (LPS) and outer membrane protein (OMP) profiles to see which typing method has the most discriminatory power to distinguish strains in order to study epizootiology of yersiniosis.

Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

Aeromonas hydrophila isolates from clinical cases (n=43) were tested against 8 antimicrobial agents and typed by outer membrane protein (OMP) pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin (MICs, ≥16 μg mL−1) and sulfamonomethoxine (MICs, ≥64 μg mL−1), but susceptible to norfloxacin (MICs, ≤0.5 μg mL−1). There was a high incidence of resistance to erythromycin (90.70%) and tylosin (93.02%), while a low incidences of resistance to ciprofloxacin (2.33%), enrofloxacin (2.33%) and florfenicol (4.65%). Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.

The double life of a bacterial lipoprotein

It has been known for many years that the small lipoprotein Lpp, which is the most abundant protein in E. coli, exists in two forms. The 'bound' form of the protein is tethered to the outer membrane (OM) by its N-terminal lipid moiety and covalently attached to the cell wall by its C-terminal lysine residue. The exact location of the 'free' form, however, has never been determined. In this issue of Molecular Microbiology, Cowles et al. demonstrate that the free form of Lpp is an integral OM protein whose C-terminus is exposed on the cell surface. The new study provides the first example of a lipoprotein that has a dual localization and adds to a growing body of evidence that lipoproteins can span the OM despite the lack of an obvious transmembrane segment. Furthermore, the new results raise intriguing questions about the assembly of both lipoproteins and other types of OM proteins.

Outer membrane proteins of wild-type and intimin-deficient enteropathogenic Escherichia coli induce Hep-2 cell death through intrinsic and extrinsic pathways of apoptosis

Enteropathogenic Escherichia coli cause protracted diarrhoea and malnutrition in infants by cytoskeletal depolymerisation and effacement of enterocyte microvilli. In this study, outer membrane proteins of wild-type enteropathogenic E. coli and an intimin-deficient mutant are shown to induce apoptosis by up-regulation of tumour necrosis factor α and activation of c-jun N-terminal kinase. Fluorescence-activated cell sorter analysis revealed apoptosis of cells treated with outer membrane proteins of wild-type and intimin-deficient strains. Proteinase K treatment of outer membrane proteins reduced apoptosis significantly, as did neutralising tumour necrosis factor α with specific antibodies. Elevated tumour necrosis factor receptor 1-associated death domain and caspase-3 expression were also observed on treatment with both types of outer membrane proteins. Furthermore, apoptosis was associated with suppression of Bcl-2 protein expression, up-regulation of Bax mRNA levels and increased cytochrome c release from mitochondria. Elevated phospho-c-jun N-terminal kinase, c-jun mRNA and activator protein-1 expression were observed, and phosphorylation of activator protein-1 was also observed by DNA-binding assays. Inhibition of c-jun N-terminal kinase, but not inhibition of p38 mitogen-activated protein kinase, resulted in reduction of tumour necrosis factor α mRNA levels and caspase-3 protein levels, and a reduction in apoptosis as observed by fluorescence-activated cell sorter analysis. From the host point of view, this study suggests a possible interplay between the death receptor and mitochondrial pathways when cell-free bacterial outer membrane preparations are used to trigger apoptosis.

Human hybridomas derived from CDS+ B lymphocytes of patients with chronic lymphocytic leukemia (B-CLL) produce multi-specific natural IgM (kappa) antibodies

Great numbers of CD5+ B lymphocytes were detected in the peripheral blood of patients with B-CLL. To study the antibody repertoire of this immune cell subpopulation on a monoclonal level, we fused the lymphocytes derived from five different donors to a highly efficient HAT-sensitive heteromyeloma line (CB-F7). A fusion frequency of up to 10(-5) allowed us to analyse hundreds of initial hybridoma lines per fusion. In all culture supernatants in three out of five fusions IgM lambda antibodies were detected, in two experiments only IgM kappa was measured, suggesting monoclonality of the primary hybridoma cell lines. The later fusions resulted in hybridomas producing multi-specific antibodies against both an autoantigen and an infectious agent: (i) dsDNA/influenza virus haemagglutinin; (ii) dsDNA/class V outer membrane protein type C from Neisseria meningitidis. However, no antibodies of the described specificity were detected in blood sera of patients, indicating a 'switch-on' of the immunoglobulin secretion capacity of malignant B cells during fusion to a myeloma partner. We discuss the results as further evidence for the natural multi-reactive antibody repertoire of CD5+ B cells.

Comparative analyses of Pasteurella multocida strains associated with the ovine respiratory and vaginal tracts

Thirty-five isolates of Pasteurella multocida from the vagina and respiratory tract of sheep were compared by analysing their capsular polysaccharide types and outer membrane protein profiles. The phylogenetic relationships of...

Subspecific differentiation of Burkholderia cepacia isolates in cystic fibrosis.

Thirty clinical isolates of Burkholderia cepacia from cystic fibrosis (CF) patients in the UK and Denmark were characterised, together with other clinical isolates and laboratory strains of B. cepacia, B. gladioli and B. vietnamiensis. Outer-membrane protein (OMP) profiles were determined, and the organisms were typed genotypically by pulsed-field gel electrophoresis after DNA restriction analyses with Xbal and DraI. This latter method revealed four clusters among the clinical isolates studied; one of these contained isolates of the UK and intercontinental CF epidemic lineage ET12, a cluster which appeared to contain three subtypes. Each of the four clusters appeared less closely related to laboratory strains of B. cepacia than were laboratory strains of B. vietnamiensis, but more closely related to both these species than to B. gladioli. Two types of OMP profile were distinguished among the clinical isolates and strains, and were designated A and B. In type A isolates the major proteins had mol.wts of 39, 27 and 18 kDa. Type B strains additionally contained a group of proteins in the size range 80-90 kDa, although detection of these depended upon the conditions for sample denaturation. In most cases, the OMP type correlated with the genotype, suggesting that examination of OMPs might be of value in the initial characterisation of isolates.

Intra-specific diversity within Pasteurella trehalosi based on variation of capsular polysaccharide, lipopolysaccharide and outer-membrane proteins.

Intra-specific diversity within Pasteurella trehalosi was investigated by analysis of variation of capsular polysaccharide, and lipopolysaccharide (LPS) and outer-membrane protein (OMP) profiles. Sixty isolates of P. trehalosi, from diverse geographical locations within the UK, were examined. Capsular polysaccharide serotypes were determined by indirect haemagglutination assay; LPS and OMP profiles were compared by SDS-PAGE analysis. Capsular serotyping identified three isolates of serotype T3, 18 isolates each of serotypes T4, T10 and T15, and three untypable (UT) isolates. Analysis of LPS and OMP profiles identified six smooth LPS types and four OMP types among the 60 isolates. Forty-five (75%) of the isolates belonged to a single OMP type whereas 52 (87%) of the isolates possessed one of three LPS types. Each typing method, by itself, was not very discriminating but when the data from the three methods were combined, the 60 isolates could be separated into 14 distinct subgroups containing from one to 16 isolates as follows: serotype T3, two subgroups; serotype T4, four subgroups; serotype T10, two subgroups; serotype T15, five subgroups; UT isolates, one subgroup. Certain subgroups were associated with only one serotype whereas other subgroups were common to two or more serotypes. The subgroupings were capable of differentiating between isolates of the same serotype from the same and different geographical origins. Based on their LPS and OMP profiles, isolates of serotypes T4 and T15 were more closely related to each other than to isolates of serotype T10; serotype T4 and T15 isolates were also more heterogeneous than those of serotype T10. Certain isolates of serotype T10, recovered from a wide geographical area, were characterized by the possession of a unique capsule/LPS/OMP combination and represented a single clonal group which was responsible for a large proportion (31%) of recent disease outbreaks. Overall, a combination of capsular serotyping, and LPS and OMP typing, was found to be extremely useful for assessing diversity within P. trehalosi and should be of value for epidemiological and virulence studies.

Major subtypes of invasive Haemophilus influenzae from 1983 to 1985 in Atlanta, Ga

We compared outer membrane protein (OMP) patterns of Haemophilus influenzae isolated in metropolitan Atlanta, Ga., from July 1983 to June 1985. Of 74 randomly selected H. influenzae serotype b, biotype I, isolates (24% of the total number of H. influenzae, and 32% of the total number of H. influenzae serotype b, biotype I, isolates), 66 (89.2%) had the same OMP pattern. Of the remaining eight, five (6.7%) had an identical OMP pattern. The other three isolates had separate and distinct patterns. A greater diversity of OMP patterns was found with H. influenzae serotype b, biotype II, and nonserotypeable H. influenzae. Of the 18 H. influenzae serotype b, biotype II, isolates (5.8% of the total number of H. influenzae isolates), 1 had an OMP pattern similar to that of the predominate biotype I OMP type, 6 (33% of the biotype II) had the same pattern, and 11 had heterogeneous patterns. Of the 19 recoverable, nonserotypeable biotype II isolates (6.8% of the total number of H. influenzae), 18 had different OMP patterns, and no pattern was similar to those observed with serotype b. These findings indicate that most H. influenzae strains isolated during this 2-year period were indistinguishable by serotype, biotype, or OMP patterns.

Typing of Aeromonas strains by DNA restriction endonuclease analysis and polyacrylamide gel electrophoresis of cell envelopes

The outer membrane protein (OMP) composition (OMP typing) of 46 fecal Aeromonas strains from hybridization groups (HGs) 1 (A. hydrophila; n = 10), 4 (A. caviae; n = 16), and 8 (A. veronii; n = 20) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a phenotypic typing method. Almost every isolate of HG-1 and HG-8 had a unique OMP profile, in contrast to isolates of HG-4, which were separated into five different OMP types. It was possible to recognize HGs 1, 4, and 8 by OMP profiles. Twenty-three Aeromonas strains from HGs 1 (n = 5), 4 (n = 10), and 8 (n = 8) were tested by whole-cell DNA restriction endonuclease analysis (REA) as a genetic typing method. All strains tested by REA (with SmaI) had different DNA digestion patterns. Although additional DNA-rRNA hybridization analyses with SmaI and 16S and 23S rRNAs from Escherichia coli showed a reduction in the number of restriction bands to 8 to 13 hybridized fragments, the discriminative value was less when compared with that obtained by REA. The individual differences found by REA were used to analyze whether patients remained colonized by the same Aeromonas strain. Of 11 patients with diarrhea, 2 had a different isolate on repeat culture. In addition, one of nine tested fecal samples contained two Aeromonas isolates with different REA patterns. These results indicate that during diarrheal disease the intestinal tract may be colonized simultaneously with different Aeromonas isolates.

Haemophilus influenzae serotype a: outer membrane protein classification and correlation with DNA polymorphism at the cap locus.

Fifty-four temporally and geographically distinct isolates of Haemophilus influenzae serotype a were characterized by (i) outer membrane protein composition and (ii) DNA polymorphism at the cap locus. In 53 of 54 cases, knowledge of the outer membrane protein type was accurately predictive of the cap locus polymorphism, suggesting that the majority of H. influenzae (type a) isolates have evolved from a limited number of clones.

Antimicrobial susceptibility and characterization of outer membrane proteins of Haemophilus ducreyi isolated in Thailand.

One hundred strains of Haemophilus ducreyi isolated in Thailand from patients with chancroid were tested by the agar dilution method against 10 antimicrobial agents and typed by outer membrane protein pattern by using sodium dodecyl sulfate gel electrophoresis. All strains produced beta-lactamase and were resistant to tetracycline, kanamycin, and sulfonamides. Most had a decreased susceptibility to trimethoprim (MIC for 50% of the strains [MIC50], 0.5 micrograms/ml) and chloramphenicol (MIC50, 8 micrograms/ml). Strains were susceptible to ciprofloxacin (MIC90, 0.001 micrograms/ml), ceftriaxone (MIC50, 0.0015 micrograms/ml), erythromycin (MIC50, 0.015 micrograms/ml), rosoxacin (MIC50, 0.03 micrograms/ml), and spectinomycin (MIC50, 8 micrograms/ml). The degree of antimicrobial resistance found in Thailand is higher than that reported for H. ducreyi isolated in other regions. Five different outer membrane protein patterns were found by analyzing proteins in the range of 29 to 61 kilodaltons, but 98% of the Thai strains fell into three patterns which did not differ greatly. Outer membrane protein patterns of Thai strains were also seen in strains from other geographic areas. A new outer membrane protein type was found among nine strains isolated in Singapore.

The Clonal Nature of Enteropathogenic Escherichia coli Strains

Enteropathogenic Escherichia coli belonging to O groups O111 and O55 and isolated from cases of infantile diarrhea in 26 countries all over the world during 1950-1960 were examined for their outer-membrane protein (OMP) patterns; nine O:H serotypes of O111 and 11 serotypes of O55 were represented. Characteristic biotypes have earlier been described as closely associated with the most common O:H serotypes: O111:H2, O111:H12, O55:H6, and O55:H7. Different OMP patterns characterized each of the common O:H serobiotypes. The OMP patterns can be looked upon as another set of stable phenotypic characters in addition to the serotype antigens and the fermentative characters. The description of geographically widely spread, stable sero-/bio-/OMP types supports the concept that the typical enteropathogenic E coli strains also have a clonal connection.