Biomedical subjects

M F Kim

Publications and source records attributed to M F Kim.

11 recordsLinked to original sources

PubMed1999

Differential posttranslational processing confers intraspecies variation of a major surface lipoprotein and a macrophage-activating lipopeptide of Mycoplasma fermentans.

The malp gene of Mycoplasma fermentans is shown to occur in single copy but to encode two discrete translated forms of lipid-modified surface protein that can be differentially expressed on isolates within this species: MALP-2, a 14-amino-acid (2-kDa) lipopeptide with potent macrophage-stimulatory activity (P. F. Mühlradt, M. Kiess, H. Meyer, R. Süssmuth, and G. Jung, J. Exp. Med. 185:1951-1958, 1997), and MALP-404, an abundant, full-length (404-amino-acid) surface lipoprotein of 41 kDa, previously designated P41 (K. S. Wise, M. F. Kim, P. M. Theiss, and S.-C. Lo, Infect. Immun. 61:3327-3333, 1993). The sequences, transcripts, and translation products of malp were compared between clonal isolates of strains PG18 (known to express P41) and II-29/1 (known to express high levels of MALP-2). Despite conserved malp DNA sequences containing full-length open reading frames and expression of full-length monocistronic transcripts in both isolates, Western blotting using a monoclonal antibody (MAb) to the N-terminal MALP-2 peptide revealed marked differences in the protein products expressed. Whereas PG18 expressed abundant MALP-404 with detectable MALP-2, II-29/1 revealed no MALP-404 even in samples containing a large comparative excess of MALP-2. Colony immunoblots with the MAb showed uniform surface expression of MALP-2 in II-29/1 populations. A second MAb to an epitope of MALP-404 outside the MALP-2 sequence predictably failed to stain II-29/1 colonies but uniformly stained PG18 populations. Collectively, these results provide evidence for novel posttranscriptional (probably posttranslational) processing pathways leading to differential intraspecies expression of a major lipoprotein, and a potent macrophage-activating lipopeptide, on the surface of M. fermentans. In the course of this study, a striking conserved motif (consensus, TD-G--DDKSFNQSAWE--), designated SLA, was identified in MALP-404; this motif is also distributed among selected lipoproteins and species from diverse bacterial genera, including Bacillus, Borrelia, Listeria, Mycoplasma, and Treponema. In addition, malp was shown to flank a chromosomal polymorphism. In eight isolates of M. fermentans examined, malp occurred upstream of an operon encoding the phase-variable P78 ABC transporter; but, in three of these isolates, a newly discovered insertion sequence, IS1630 (of the IS30 class), was located between these genes.

Bacterial Proteins↗

PubMed1997

Mycoplasma infection and rheumatoid arthritis: analysis of their relationship using immunoblotting and an ultrasensitive polymerase chain reaction detection method.

OBJECTIVE: To examine the relationship between infection with Mycoplasma and the development of rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA). METHODS: Immunoblotting of patient synovial fluid and sera on detergent-phase membrane protein extracts of various Mycoplasma species was carried out to learn whether patients exhibited serologic evidence of previous exposure to mycoplasmas. Moreover, an ultrasensitive polymerase chain reaction (PCR) method was developed for assessing whether Mycoplasma DNA could be detected in synovial fluid from patients and controls. RESULTS: Immunoblotting provided serologic evidence of previous Mycoplasma exposure in patients and controls. The genus-specific PCR detected known human Mycoplasma species and could reliably detect <5 copies of Mycoplasma hominis, Mycoplasma fermentans, or a molecular mimic control in synovial fluid. Repeat testing revealed no evidence of Mycoplasma DNA in patient synovial samples. CONCLUSION: This study provided serologic evidence suggesting that, while previous exposure to Mycoplasma was common, there was no detectable persistence of Mycoplasma DNA in the synovial fluid or tissue of patients with RA or JRA.

Adult↗

PubMed1997

Elongated versions of Vlp surface lipoproteins protect Mycoplasma hyorhinis escape variants from growth-inhibiting host antibodies.

Variation in Vlp surface proteins of Mycoplasma hyorhinis was evaluated in terms of its role in determining susceptibility of organisms to growth inhibition by host antibodies (Abs). High-frequency switching of Vlp surface lipoproteins has been studied in isogenic lineages of M. hyorhinis SK76. In these lineages, the products of three genes, vlpA, vlpB, and vlpC, are subject to phase and size variation in vitro, which occur through distinct mutator elements that independently govern the expression of each vlp gene (promoter mutations) or the size of the vlp gene product (by intragenic expansion or contraction of a 3' region containing tandem repeats). Isogenic clonal variants of M. hyorhinis SK76 expressing distinct profiles of Vlp products were assessed for their susceptibility to complement-independent growth inhibition by serum Abs of swine experimentally infected with the arthritigenic SK76 strain. Invariably, variants expressing longer versions of VlpA, VlpB, or VlpC (each expressed individually) were completely resistant to host immune serum Abs, whereas variants expressing shorter allelic versions of each Vlp were susceptible. The target of growth-inhibiting Abs was not the Vlp products, since removal of anti-Vlp Abs had no effect on the inhibitory activity of the host immune serum on susceptible variants. Escape variant populations derived by propagating susceptible variants in an immune (versus control) host serum revealed a strong selection for the long-Vlp phenotype, irrespective of the identity of the Vlp expressed. Apparent mutational pathways of acquiring the protective phenotype included mutational switches to express long vlp genes that had been transcriptionally silent or switches to elongate expressed vlp genes. These results suggest that a major function of the Vlp system is to shield the wall-less mycoplasma surface from host Abs capable of binding vital (and as-yet-unidentified) surface antigens of this organism.

Alleles↗

PubMed1995

Identification of mycoplasma membrane proteins by systematic Tn phoA mutagenesis of a recombinant library.

Wall-less prokaryotes in the genus Mycoplasma include over 90 species of infectious agents whose pathogenicity for humans and other animals is currently being assessed. Molecular characterization of surface proteins is critical in this regard but is hampered by the lack of genetic systems in these organisms. We used TnphoA transposition to systematically mutagenize, in Escherichia coli, a genomic plasmid library constructed from Mycoplasma fermentans, a potential human pathogen. The strategy circumvented problems of expressing mycoplasma genes containing UGA (Trp) codons and relied on the construction of the vector pG7ZCW, designed to reduce TnphoA transposition into vector sequences. Functional phoA gene fusions directly identified genes encoding 19 putative membrane-associated proteins of M. fermentans. Sequences of fusion constructs defined three types of export sequence: (1) non-cleavable, membrane-spanning sequences, (2) signal peptides with signal peptidase (SPase) I-like cleavage sites, and (3) signal peptides with SPase II-like lipoprotein-cleavage sites which, like most other mycoplasmal lipoprotein signals analysed to date, differed from those in several Gram-negative and Gram-positive eubacteria in their lack of a Leu residue at the -3 position. Antibodies to synthetic peptides that were deduced from two fusions to predicted lipoproteins, identified corresponding amphiphilic membrane proteins of 57 kDa and 78 kDa expressed in the mycoplasma. The P57 sequence contained a proline-rich N-terminal region analogous to an adhesin of Mycoplasma gallisepticum. The P78 protein was identical to a serologically defined phase-variant surface lipoprotein. TnphoA mutagenesis provides an efficient means of systematically characterizing functionally diverse lipoproteins and other exported proteins in mycoplasmas.

Alkaline Phosphatase↗

PubMed1994

Processing and surface presentation of the Mycoplasma hyorhinis variant lipoprotein VlpC.

The variant surface lipoprotein VlpC of Mycoplasma hyorhinis was shown to be processed by cleavage of a characteristic prokaryotic prolipoprotein signal peptide. In addition, a vlpC::phoA fusion protein expressed and translocated in Escherichia coli was recognized by surface-binding monoclonal antibodies, which identified the characteristic region II of Vlps, containing divergent external sequences proximal to the membrane, as an exposed portion of these surface proteins subject to immune recognition and selection.

Amino Acid Sequence↗

PubMed1993

Differential protein expression and surface presentation generate high-frequency antigenic variation in Mycoplasma fermentans.

Mycoplasma fermentans, a wall-less prokaryote, is currently under investigation as a potential human pathogen. Recently, several surface lipoproteins have been shown to vary in expression between M. fermentans strains. Using specific antibodies to these lipoproteins, we investigated the extent and nature of antigenic variation within this species. Immunoscreening of type strain PG18 agar-grown colonies revealed marked heterogeneity in expression of distinct surface lipoproteins. Subsequent isolation and propagation of clonal isolates established isogenic lineages which displayed high-frequency (10(-2) to 10(-5) per generation) antigenic phase variation. [35S]cysteine-labeled protein profiles and Western immunoblots of phase-variant clones showed that several distinct integral membrane proteins undergo noncoordinate variation in expression. In addition to differential expression of epitope-bearing lipoproteins, differential accessibility of epitopes to antibodies was also documented as a mechanism generating surface phenotypic variation. Examination of one strain-variant antigen showed high-frequency phase variation to underlie previously observed antigenic differences between strains of this species. Thus, M. fermentans has a complex system capable of creating rapid changes in surface mosaics. This may profoundly affect mycoplasma-host interactions and may limit the methods by which populations of M. fermentans may be studied in vivo.

Antibodies, Monoclonal↗

PubMed1993

A family of strain-variant surface lipoproteins of Mycoplasma fermentans.

The wall-less procaryote Mycoplasma fermentans is currently being examined as an agent potentially associated with human disease, including infectious processes affecting immunocompromised individuals. To delineate and understand the interactions of M. fermentans with its host, specific membrane surface components were characterized as markers for detecting the organism and for assessing heterogeneity in antigenic surface architecture within this mycoplasma species. Detergent phase fractionation of metabolically labeled organisms of type strain PG18 identified a family of prominent integral membrane proteins; several of these labeled with 35S-cysteine and 3H-palmitate, which are characteristics of procaryotic lipoproteins. Specific monoclonal and polyclonal antibodies raised to strain PG18 components further distinguished seven of these membrane proteins, which were localized on the organism's surface by monitoring their selective susceptibility during trypsin treatment of intact cells. With these antibodies, Western immunoblot profiles of surface membrane antigens expressed on strain PG18 were compared with those expressed on the recently identified Incognitus strain of M. fermentans, as well as with several other human and animal mycoplasma species. While the antibodies were specific for M. fermentans, marked differences were observed between the strains in the size of one surface lipoprotein and in the apparent levels of several antigens expressed in the cultured populations analyzed. Some monoclonal antibodies to strain PG18 and a previously described monoclonal antibody to strain Incognitus showed apparent selectivity for the strain used for immunization. Monoclonal antibodies developed here recognize stable epitopes defining a family of surface lipoproteins and provide critical tools to determine the basis of surface variation in this mycoplasma species and to assess the location and antigenic phenotypes of organisms in the human host.

Animals↗

PubMed1990

Identification and mapping of an immunogenic region of Mycoplasma hyopneumoniae p65 surface lipoprotein expressed in Escherichia coli from a cloned genomic fragment.

A previously characterized lipid-modified amphiphilic surface protein of Mycoplasma hyopneumoniae, p65, has been defined by its reaction with a surface-binding monoclonal antibody (MAb) and by its exclusive partitioning into the detergent phase during Triton X-114 phase fractionation (K. S. Wise and M. F. Kim, J. Bacteriol. 169:5546-5555, 1987). In the current study, polyclonal mouse antibody (PAb) to gel-purified p65 was used to identify recombinant phage plaques expressing p65-related epitopes. Several characteristic partial tryptic fragments of p65 were recognized by both PAb and p65 and MAb to p65, but the PAb population specifically eluted from recombinant phage plaques bound only epitopes restricted to the largest of these fragments. Graded carboxypeptidase-Y digestion of intact M. hyopneumoniae generated C terminally truncated peptides that were recognized by PAb to p65 and MAb to p65, indicating that the C terminus and much of the adjoining region of p65 were present and accessible on the external face of the membrane. However, antibody eluted from recombinant phage plaques bound only to the largest truncated polypeptide, suggesting that a recombinant product corresponding to the C-terminal region of p65 was expressed in Escherichia coli. A 19-kilodalton recombinant protein (p19), which was recognized by PAb to p65 but not by MAb to p65, was detected in recombinant phage lysates. Serum antibodies from swine taken after, but not before, experimentally induced M. hyopneumoniae pneumonia preferentially recognized the native, amphiphilic p65 lipoprotein and also bound specifically to the p19 recombinant product. This confirmed that the p65 lipoprotein is a major immunogen of M. hyopneumoniae recognized during disease and identified its C-terminal region as an immunogenic domain.

Animals↗

PubMed1987

Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid.

Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125I-labeled proteins with a series of monoclonal antibodies (MAbs). Surface proteins p70, p65, p50, and p44 were shown to be integral membrane components by selective partitioning into the hydrophobic phase during Triton X-114 (TX-114)-phase fractionation, whereas p41 was concomitantly identified as a surface protein exclusively partitioning into the aqueous phase. Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [35S]methionine, 14C-amino acids, or [3H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Covalent lipid attachment was established by high-performance liquid chromatography identification of [3H]methyl palmitate after acid methanolysis of delipidated proteins. An additional, unidentified methanolysis product suggested conversion of palmitate to another form of lipid also attached to these proteins. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a larger p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11. These studies establish that a highly restricted set of distinct, lipid-modified hydrophobic membrane proteins are major surface antigens of M. hyopneumoniae and that structural variants of surface antigens occur within this species.

Animals↗

PubMed1987

Identification of intrinsic and extrinsic membrane proteins bearing surface epitopes of Mycoplasma hyopneumoniae.

A library of monoclonal antibodies (McAbs) raised against Mycoplasma hyopneumoniae was screened to select McAbs that bound both to surface epitopes on intact organisms and to corresponding proteins identified by immunoblot analysis. Four proteins (indicated by MW in kilodaltons) were established as surface antigens: p65, p50, p44 and p41. Triton X-114 detergent phase fractionation of whole organisms clearly distinguished p65, p50 and p44 as hydrophobic integral membrane proteins, whereas p41 was identified as a hydrophilic surface protein apparently extrinsic to the membrane. This technique also provided a rapid and efficient method for isolating the relatively small number of hydrophobic proteins associated with M. hyopneumoniae. Preliminary evidence suggests that p65, p50 and p44 are linked covalently to lipids, which may be important in dictating their interaction with the membrane. Immunoblot analysis of antibodies from swine immunized with M. hyopneumoniae also suggests that p65 and p44 may be "immunodominant" antigens in this host.

Antibodies, Bacterial↗

PubMed1983

Differentiation of B lineage cells from liver of neonatal mice: generation of immunoglobulin isotype diversity in vitro.

The development and differentiation of B cells expressing different immunoglobulin (Ig) isotypes was studied in cultures of murine neonatal liver cells. Before culture, 5 to 15% of the liver cells were mu + pre-B cells; 1 to 3% had surface IgM and less than 0.1% had slgG. During 4 days in culture the number of pre-B cells declined, whereas the number of IgM B cells increased greater than 20-fold; IgG B cells also increased in number. Of the four subclasses, IgG3+ and IgG2b+ cells predominated, each representing 3 to 10% of the total B cells at day 4. IgG1+ and IgG2a+ cells were present in lower numbers, representing 1 to 5% and 0.3 to 2.5% of B cells, respectively. Most IgG+ cells also expressed sIgM. Only a minority (less than 10%) of the sIgM+ cells were sIgD+, and most sIgG+ cells were sIgD-. Few T cells were present in these cultures (less than 0.5% in newborn liver), and sIgG+ cells were generated in normal frequencies in cultures of cells from nude mice. The numbers of B cells expressing each IgG subclass were similar in cultures from athymic nu/nu mice, nu/+ heterozygous littermates, and normal BALB/c mice. Plasmablasts and plasma cells appeared over a 14-day culture interval, and these expressed cytoplasmic IgM, IgG3, IgG1, IgG2b, IgG2a, and IgA. Measurable amounts of the first four isotypes were detected in the culture supernatants by radioimmunoassay. These results indicate that neonatal B cells can undergo isotype switching in the absence of T cell help, and that the expression of sIgD may not be a prerequisite for cells to switch Ig isotypes.

Animals↗