PubMed Health⌕ Search

Biomedical subjects

M Achtman

Publications and source records attributed to M Achtman.

At least 73 records · Page 4Linked to original sources
PubMed1991

Cloning and expression in Escherichia coli of opc, the gene for an unusual class 5 outer membrane protein from Neisseria meningitidis (meningococci/surface antigen).

A genomic library was constructed in a lambda gt11 vector using chromosomal DNA from a meningococcal serogroup A strain and plaques expressing the class 5C protein were recognized by screening with specific monoclonal antibodies. The opc insert was subcloned into a multicopy plasmid which induced expression of that protein in Escherichia coli as a surface-exposed major outer membrane protein. The nucleotide sequence of opc is typical of an outer membrane protein with a promoter and terminator region, a leader peptide which is cleaved during expression and a complete open reading frame. Unlike other meningococcal class 5 proteins or gonococcal P.II proteins, the sequence did not contain any pentanucleotide repeats and the sequence showed little homology to these other functionally related proteins. However, the predicted amino acid sequence of the mature protein for opc showed 27% similarity to that for a second opa gene cloned from the same meningococcal strain. This is the first report of cloning and expression of a functional meningococcal gene encoding a class 5 outer membrane protein in E. coli.

Amino Acid Sequence↗
PubMed1991

Variation in class 5 protein expression by serogroup A meningococci during a meningitis epidemic.

Serogroup A meningococci were isolated from patients and healthy carriers in The Gambia between 1982 and 1988. The class 5 proteins expressed by these bacteria were identified by electrophoretic migration and by serologic tests. Three protein serologic groupings (seroclasses) called A (protein 5a), B (proteins 5b, 5d, or 5e), and C (protein 5c or 5C) were found among 331 bacterial isolates. The number of class 5 proteins expressed per isolate varied from none to four, with a median of two. The class 5 protein composition differed for certain paired isolates obtained from the nasopharynx, blood, and cerebrospinal fluid of diseased patients and for certain pairs of sequential isolates from the nasopharynx of healthy carriers; the medical relevance of this variation remains unclear, although the 5C protein was preferentially isolated from the nasopharynx and the 5a protein from diseased patients. The data show that a large proportion of healthy carriers in The Gambia were exposed to bacteria expressing each of the three seroclasses and that many people were exposed to bacteria expressing each of the three seroclasses and that many people were exposed to two or all three seroclasses during the epidemic of 1982-1983.

Antibodies, Monoclonal↗
PubMed1991

Human hybridomas derived from CD5+ 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.

Adult↗
PubMed1991

Virulence patterns and long-range genetic mapping of extraintestinal Escherichia coli K1, K5, and K100 isolates: use of pulsed-field gel electrophoresis.

A total of 127 extraintestinal Escherichia coli strains of the capsule serotypes K1, K5, and K100 from human and animal sources were analyzed for DNA sequences specific for the genes for various adhesins (P fimbriae [pap] and P-related sequences [prs], S fimbriae [sfa]/F1C fimbriae [foc], and type I fimbriae [fim]), aerobactin (aer), and hemolysin (hly). The expression of corresponding virulence factors was also tested. Twenty-four selected strains were analyzed by long-range DNA mapping to evaluate their genetic relationships. DNA sequences for the adhesins were often found in strains not expressing them, while strains with hemolysin and aerobactin genes usually did express them. Different isolates of the same serotype often expressed different virulence patterns. The use of virulence-associated gene probes for Southern hybridization with genomic DNA fragments separated by pulsed-field gel electrophoresis revealed that a highly heterogeneous restriction fragment length and hybridization pattern existed even within strains of the same serotype. Long-range DNA mapping is therefore useful for the evaluation of genetic relatedness among individual isolates and facilitates the performance of precise molecular epidemiology.

Adhesins, Escherichia coli↗
PubMed1990

Characterization of epidemic and nonepidemic Neisseria meningitidis serogroup A strains from Sudan and Sweden.

A random selection of 25 strains isolated during an epidemic caused by serogroup A Neisseria meningitidis in Sudan (1988), 3 preepidemic meningococcal strains (1985), and 26 serogroup A strains isolated from sporadic cases of meningitis in Sweden (1973 to 1987) were assessed for multilocus enzyme genotypes (ETs), DNA restriction enzyme patterns, outer membrane proteins, lipopolysaccharides, pilus formation, and antibiograms. All of the 25 Sudanese epidemic isolates and 22 of the Swedish strains were of the same or closely related ETs (ETs 3, 4, and 5), corresponding to clone III-1, which has been responsible for two pandemic waves in the last three decades. The earlier pandemic involved Scandinavia, and the last one caused an outbreak during the pilgrimage to Mecca, Saudi Arabia (August 1987), spreading to Sudan, Chad, and Ethiopia. The three Sudanese preepidemic isolates (1985) were clone IV-1 (sulfonamide susceptible), which has been resident in the African meningitis belt for the last 25 years. The uniformity of clone III-1 strains (all sulfonamide resistant) from Sudan and Sweden was confirmed by DNA restriction enzyme patterns. ETs 3, 4, and 5 from Sudan and Sweden had 86 to 100% similarity to a Swedish clone III-1 reference strain, whereas ETs 1, 2, 6, and 7 showed 50 to 80% similarity. Class 1 protein for clone III-1 showed serosubtype antigens P1.9 and P1.x, whereas ET6 strains (clone IV-1) had serosubtype P1.7. Lipopolysaccharides were variable in the Sudanese and Swedish strains. Pili were expressed in all clone III-1 isolates from Sudan and Sweden but in none of the clone IV-1 isolates (Sudan, 1985).

Antigenic Variation↗
PubMed1989

Isolation of rat IgM to IgG hybridoma isotype switch variants and analysis of the efficiency of rat Ig in complement activation.

Sequential sublining was used in combination with enzyme-linked immunosorbent assays to isolate mu----gamma isotype switch variants of the rat IgM secreting mouse-rat B cell hybridoma line BA1.8. Switch variants to all four subclasses of IgG were obtained. The variant antibodies retained the antigen specificity of the parental IgM for the O18 (lipopolysaccharide) antigen of Escherichia coli. In sodium dodecyl sulfate-polyacrylamide gels the apparent molecular mass of the gamma heavy chains decreased in the order gamma 2b greater than gamma 1 greater than gamma 2c greater than gamma 2a. IgM, IgG1, IgG2a, IgG2b and IgG2c of the BA1.8 variant family and IgG2b, IgE and IgA of the previously described BA1.2 family were used for a comparative analysis of the capacity of rat Ig to activate complement. Efficient lysis of sheep erythrocytes coated with the O18 antigen was observed with IgM and all IgG subclasses, but no lysis was triggered by IgE or IgA. One hundred to 1000 IgG molecules were required to mediate the same hemolytic activity as one IgM molecule. The four IgG subclasses were equally efficient at mediating lysis by rat or human complement, while IgG2a was less efficient with guinea pig complement than the other three IgG subclasses. Antibody-triggered binding of C3 to pathogenic O18:K1 E. coli bacteria was measured using serum containing 125I-labeled C3. K1-encapsulated strains did not fix C3 efficiently in the absence of specific antibodies while acapsular mutants fixed C3 via the alternative pathway. IgM and all IgG subclasses triggered C3 binding to the K1 encapsulated bacteria. The capacity of IgM to mediate C3 fixation was not greater than that observed with IgG.

Animals↗
PubMed1989

Clonal and variable properties of Neisseria meningitidis isolated from cases and carriers during and after an epidemic in The Gambia, West Africa.

A representative collection of meningococci was isolated from cases and healthy carriers in The Gambia between 1982 and 1988, during and after an epidemic of meningococcal meningitis. These bacteria were subjected to a clonal analysis. All serogroup A bacteria from both cases and carriers were of one clone (A IV-1). Several unrelated clones were observed among serogroup 29E and serogroup Y carrier strains. The serogroup A strains were uniform for serotype and subtype antigens (serotype 4, subtype P1.7) and antibiotic sensitivity pattern. Occasional strains varied in their lipopolysaccharide (LPS), DNA fingerprint pattern, and/or the quantitative expression of the class 1 protein. A high degree of strain-specific variation was found for the expression of class 5 proteins, pili, and sulfonamide sensitivity. The frequency of strains expressing reduced amounts of the class 1 protein, altered LPS, and/or increased amounts of capsular polysaccharide rose among case strains obtained after the epidemic had ceased. These strains seem to be generally resistant to antibody-mediated bactericidal activity.

Antibodies, Monoclonal↗
PubMed1988

Purification and characterization of eight class 5 outer membrane protein variants from a clone of Neisseria meningitidis serogroup A.

Methods published for the purification of P.II proteins from Neisseria gonorrhoea have been modified to allow the purification of class 5 proteins from Neisseria meningitidis serogroup A bacteria. The five class 5 protein electrophoretic variants detected within an epidemic in the Gambia (a, b, c, d, and e) and three other variants (f, g, and h) found within other isolates of the same clone in West Africa have been purified with yields of 6-28 mg. The NH2-terminal amino acid sequence for variant c differs from those of the other class 5 proteins, whereas the latter are very similar to the sequence predicted for two class 5 proteins from DNA analyses of serogroup C meningococci and determined for 8 P.II proteins from gonococci. Numerous other regulatory, chemical, and serological differences were found between the c protein and the other class 5 proteins such that we recommend that the class 5 proteins be subdivided into two subclasses. mAbs have been isolated that distinguish between these two protein subclasses and Western blotting with these antibodies enabled us to conclude that both protein subclasses were found in bacteria isolated from different epidemics and pandemics of the last 50 yr.

Amino Acid Sequence↗
PubMed1988

Two monoclonal antibodies specific for serotype 4 antigen of Neisseria meningitidis.

Two new monoclonal antibodies (MN14G21.17 and MN14E9.15) specific for the serotype 4 antigen of meningococci were isolated. The antibodies were raised against a previously non-typable serogroup B strain from the Netherlands and were shown to react with the serotype antigen of the prototype reference strains for serotype 4 and serogroup A as well as with that of the homologous strain. Further screening of 290 serogroup A and B case isolates with the monoclonal antibodies indicated that the serotype 4 epitope was present on all 100 serogroup A strains tested, including representative isolates from 28 epidemics, and on most isolates from a recent serogroup B epidemic (1981-1983) in Cuba. In addition, 68 isolates from recent sporadic cases (1980-1986) and/or clusters of cases of serogroup B disease in many parts of Europe, the USA, the USSR and Australia were also of this serotype.

Antibodies, Monoclonal↗
PubMed1988

Comparison of clonal analysis and DNA restriction analysis for typing of Neisseria meningitidis.

We have formerly described a clonal analysis of 423 serogroup A isolates of Neisseria meningitidis which distinguished 21 clones and 34 electrophoretic types. The clones were found to be uniform within distinct epidemiological situations and distinct between different sets of epidemics. Strains representative of the 34 electrophoretic types have now been analyzed by restriction endonuclease digestion of their genomic DNA's. The results correlated partially with the clonal assignments; the discrepancies were epidemiologically more consistent with the interpretation of the clonal analysis than with that of the restriction endonuclease analysis.

Bacterial Typing Techniques↗
PubMed1987

A clonal analysis of Neisseria meningitidis serogroup A.

A typing scheme has recently been developed for Neisseria meningitidis serogroup A based on the clonal population structure of these bacteria. An international strain collection consisting of 423 group A strains isolated from 23 epidemics or outbreaks since 1963, as well as from older epidemics and numerous non-epidemic situations was used in the analysis. Strains were first segregated into electrophoretic types, depending on the combined score for the electrophoretic mobilities of 7 cytoplasmic isoenzymes resolved by starch gel electrophoresis and of 2 outer membrane proteins resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The bacteria were subsequently assigned to one of 21 clones after numerical analysis of their electrophoretic types. The epidemiological value of the typing scheme was assessed by examining case and carrier strains isolated during (1982-83) and subsequent to (1984-85) an epidemic in the Gambia, West Africa. The case isolates, all of which were serogroup A, were of a single clonal type. All serogroup A carrier isolates were also of this clone, while carrier strains of other serogroups showed greater clonal diversity. These results indicate that case strains during an epidemic show little clonal diversity and thus that the typing scheme is of value in distinguishing the etiology of epidemics. A retrospective epidemiological analysis of the strains in the international collection showed that most serogroup A epidemics were associated with a single or predominant clone, although some epidemics were of mixed etiology.(ABSTRACT TRUNCATED AT 250 WORDS)

Africa↗
PubMed1986

Clonal analysis of Escherichia coli O2:K1 isolated from diseased humans and animals.

Forty-six Escherichia coli isolates of serotype O2:K1 from human urinary tract infections, chicken sepsis, and bovine mastitis were obtained from laboratories in England, Denmark, Sweden, and Finland. The bacteria were compared for outer membrane protein (OMP) pattern, lipopolysaccharide pattern, electrophoretic mobilities of enzymes, and flagellar serotype and were tested for fimbriation, biotype, hydroxamate production, hemolysin production, antibiotic resistance, plasmid content, colicin production, and virulence in neonatal rats. Isolates from humans were assigned to two clonal groups; poultry isolates belonged to one of these clonal groups, whereas bovine isolates belonged to the other. Poultry and human isolates of the same clonal group could be distinguished only by their plasmid content. Strains within this group were heterogeneous with respect to biotype, fimbriation, virulence, and flagellar serotype. Human and bovine isolates of the second clonal group were distinguished by a minor change in OMP pattern and by their plasmid content. It is concluded that meaningful clonal groupings are best recognized by the combination of OMP and electrophoretic enzyme patterns. The O:K serotype can aid in the recognition of important subclones, whereas the other microbiological properties tested can vary widely within clonal groupings. Furthermore, we conclude that certain O:K serotypes can contain very different clonal groupings having little genetic relatedness.

Animals↗
PubMed1986

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and monoclonal antibodies as tools for the subgrouping of Escherichia coli lipopolysaccharide O18 and O23 antigens.

The lipopolysaccharide (LPS) of Escherichia coli O18 isolated from a wide variety of sources was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four different LPS types, designated O18A, O18A1, O18B, and O18B1, were identified. Most O18 strains possess O18A, O18A1, or O18B LPS types, and these types are clonally associated. A reference test strain with the classical O18ab designation possessed O18B LPS, while two reference O18ac strains possessed O18A and O18A1 LPS, respectively. A panel of 15 anti-O18A B-cell hybridomas was isolated. Enzyme-linked immunosorbent assays revealed that some of the monoclonal antibodies produced by these cells recognize different epitopes. Four of these antibodies suffice to distinguish the four O18 types. Numerous strains whose LPS had been typed by SDS-PAGE were tested by agglutination with seven monoclonal antibodies whose specificities had been determined by enzyme-linked immunosorbent assays. The results indicated a perfect correlation between the two methods. Rabbit antisera raised against O18A bacteria agglutinated boiled bacteria of each of the O18 LPS types efficiently. The antisera were adsorbed with bacteria possessing each of the LPS types. The adsorbed sera only distinguished between two groups: O18A and O18A1 versus O18B and O18B1, as shown by agglutination assays and Western blotting. E. coli O4 and O23 and Serratia marcescens O8 antigens, which are reputed to cross-react with O18, were also analyzed. One O4, one O8, and four O23 strains were tested. All made an LPS which was distinguishable from O18 LPS types by SDS-PAGE. Each O23 strain synthesized a different LPS, and three of them synthesized only few short chains. Some of the monoclonal antibodies reacted with O4, O8, and O23A LPSs. The results are interpreted as indicating that numerous E. coli O serogroups will prove to be chemically heterogeneous and that future analyses of subgroup heterogeneity should be guided by results from SDS-PAGE and rely preferentially on monoclonal antibodies as opposed to rabbit hyperimmune sera.

Antibodies, Bacterial↗
PubMed1986

A reexamination of the O1 lipopolysaccharide antigen group of Escherichia coli.

A total of 64 Escherichia coli strains of the O1 serogroup were tested for the migration pattern of their lipopolysaccharides (LPS) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. O1:K1 and O1:K51 strains of the OMP5 outer membrane protein pattern possessed LPS with a doublet pattern (O1A1) or the lowermost band of the O1A1 doublet (O1A2). O1:K1 strains of the OMP9 pattern possessed LPS referred to as O1A, which corresponded to the uppermost band of the O1A1 doublet pattern. A few O1:K? strains possessed LPS of different migration patterns (O1B and O1C). O1A and O1A1 LPS were indistinguishable by chemical techniques, and both reacted with each of 10 different monoclonal antibodies tested. However, O1A1 had an additional epitope within the additional band in each doublet, as demonstrated by adsorption experiments with hyperimmune rabbit sera followed by Western blotting. Furthermore, purified polysaccharide from O1A bacteria was incapable of inhibition in enzyme-linked immunosorbent assays performed with O1A1 LPS as antigen and adsorbed, specific anti-O1A1 antibodies, whereas O1A1 polysaccharide inhibited this reaction. O1B and O1C LPS differed in all respects tested, including chemical composition, from O1A and O1A1 LPS.

Animals↗