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J B Baseman

Publications and source records attributed to J B Baseman.

At least 19 recordsLinked to original sources
PubMed2001

Peptide methionine sulfoxide reductase (MsrA) is a virulence determinant in Mycoplasma genitalium.

Mycoplasma genitalium is the smallest self-replicating microorganism and is implicated in human diseases, including urogenital and respiratory infections and arthritides. M. genitalium colonizes host cells primarily through adherence mechanisms mediated by a network of surface-associated membrane proteins, including adhesins and cytadherence-related proteins. In this paper, we show that cytadherence in M. genitalium is affected by an unrelated protein known as peptide methionine sulfoxide reductase (MsrA), an antioxidant repair enzyme that catalyzes the reduction of methionine sulfoxide [Met(O)] residues in proteins to methionine. An msrA disruption mutant of M. genitalium, constructed through homologous recombination, displayed markedly reduced adherence to sheep erythrocytes. In addition, the msrA mutant was incapable of growing in hamsters and exhibited hypersensitivity to hydrogen peroxide when compared to wild-type virulent M. genitalium. These results indicate that MsrA plays an important role in M. genitalium pathogenicity, possibly by protecting mycoplasma protein structures from oxidative damage or through alternate virulence-related pathways.

Animals↗
PubMed2000

Intracellular DNA replication and long-term survival of pathogenic mycoplasmas.

We examined intracellular survival and growth of pathogenic mycoplasmas (Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma genitalium) in cultured human cells. By using the eukaryotic nuclear DNA synthesis inhibitor, aphidicolin, we detected the selective synthesis of mycoplasma (My) and mitochondria (Mt) DNA, which could be further differentiated by restriction enzyme analyses. Also, intracellular M. pneumoniae and M. penetrans infectivity of human cells was detected over 6 months using subfractionation of infected cells and determination of mycoIplasma colony forming units (cfu). For M. genitalium, which we failed to re-grow from infected cells, species-specific PCR primers were used to implicate long-term mycoplasma survivability. Data indicated that pathogenic mycoplasmas reside and replicate intracellularly over extended periods in human cells, consistent with the ability of mycoplasmas to circumvent antibiotic therapy and immune surveillance and establish chronic infections.

Aphidicolin↗
PubMed2000

Hemolytic and hemoxidative activities in Mycoplasma penetrans.

Mycoplasma penetrans is a newly isolated Mollicute from the urine of patients infected with human immunodeficiency virus that demonstrates the capacity to adhere to and invade human cells. A previous report, based on assays with mouse red blood cells (RBCs), indicated that M. penetrans lacked hemolytic activity. In our studies, we incubated different isolates of M. penetrans with various RBC species and observed hemolytic zones surrounding individual mycoplasma colonies. All M. penetrans strains displayed hemolysis after 2 to 3 days of incubation. Hemolytic activity diffused from single colonies, eventually causing complete lysis. Hemolysis was most pronounced with sheep RBCs, followed by horse, chicken, and human cells. Furthermore, hemolytic activity was demonstrable in both intact mycoplasma cell preparations and spent culture supernatant. However, unlike intact mycoplasmas, the hemolytic activity in the supernatant was dependent on the reducing agent, cysteine. In addition to hemolysis, a brown precipitate was closely associated with mycoplasma colonies, suggesting oxidation of hemoglobin. Absorption spectra indicated that hemoglobin was oxidized to methemoglobin, and the addition of catalase demonstrated H(2)O(2)-mediated hemoxidation. Other experiments suggested that hemoxidation enhanced total hemolysis, providing the first evidence of both hemolytic and hemoxidative activities in M. penetrans.

Animals↗
PubMed2000

Expression of UGA-containing Mycoplasma genes in Bacillus subtilis.

We used Bacillus subtilis to express UGA-containing Mycoplasma genes encoding the P30 adhesin (one UGA) of Mycoplasma pneumoniae and methionine sulfoxide reductase (two UGAs) of Mycoplasma genitalium. Due to natural UGA suppression, these Mycoplasma genes were expressed as full-length protein products, but at relatively low efficiency, in recombinant wild-type Bacillus. The B. subtilis-expressed Mycoplasma proteins appeared as single bands and not as multiple bands compared to expression in recombinant Escherichia coli. Bacillus mutants carrying mutations in the structural gene (prfB) for release factor 2 markedly enhanced the level of readthrough of UGA-containing Mycoplasma genes.

Adhesins, Bacterial↗
PubMed1999

Disruption of gene mg218 of Mycoplasma genitalium through homologous recombination leads to an adherence-deficient phenotype.

Although the complete genome of Mycoplasma genitalium has been sequenced, the functional identification of various genes, including those involved in virulence, has not been accomplished. Further compounding these difficulties has been the failure to develop genetic tools in mycoplasmas that permit the assessment of gene and operon function and regulation. To determine whether homologous recombination could be developed as a tool to analyze the function of genes in M. genitalium, a plasmid that replicates in Escherichia coli but not in M. genitalium was constructed to disrupt the cytadherence-related gene mg218 of M. genitalium. The electroporation of this disruption plasmid into wild-type hemadsorption-positive (HA+) M. genitalium cells permitted the isolation of HA- (strain JB1) and partial HA+ (strains JB2 and JB20) transformants. Analysis of the transformants by Southern hybridization indicated that homologous recombination occurred at the mg218 locus by single-crossover events in JB1 and JB2 and by a double-crossover event in JB20. While integration of the disruption construct abolished the expression MG218 in JB1, strains JB2 and JB20 exhibited a truncated MG218 protein (160 kDa), possibly because of in-frame fusion of the disrupted mg218 gene with sequences downstream of the gentamycin-resistance gene present in the disruption construct. Strain JB1, which lacked MG218, displayed a post-translational defect, being unable to maintain the structural integrity of the major adhesin P140 and its operon-related protein P110, in contrast to JB2 and JB20. It appears that MG218 influences the stability of other cytadherence-related proteins in vivo. Thus, targeted gene disruption through homologous recombination will be a powerful and promising tool for investigating the biology and pathogenesis of M. genitalium.

Bacterial Adhesion↗
PubMed1998

Identification of mycoplasmal promoters in Escherichia coli using a promoter probe vector with Green Fluorescent Protein as reporter system.

A promoter probe vector, pGFPUV2, with Green Fluorescent Protein (GFP) as the reporter system was constructed to identify potential mycoplasmal promoter-containing regulatory sequences in E. coli. Libraries of M. pneumoniae and M. genitalium DNA constructed in pGFPUV2 and transformed into E. coli resulted in GFP-expressing clones. Primer extension analysis with E. coli RNA from five M. pneumoniae clones and two M. genitalium clones indicated that transcription originated from the insert DNA fragments of these promoter constructs. Primers based on the insert DNA sequences were used in primer extension reactions with total RNA isolated from M. pneumoniae or M. genitalium. Of the seven primers used, three generated products by primer extension with mycoplasmal RNA. However, only one of the DNAs had a 5' end similar to that obtained in a comparable reaction with E. coli RNA, and the start site of this transcript appeared to originate one base prior to the predicted open reading frame. These results indicate that E. coli can identify mycoplasmal promoters which have transcriptional elements resembling E. coli promoters.

Base Sequence↗
PubMed1996

Correlations between Mycoplasma pneumoniae sensitivity to cyclosporin A and cyclophilin-mediated regulation of mycoplasma cytadherence.

Adhesins and adhesin-related accessory proteins of the bacterial pathogen, Mycoplasma pneumoniae, are proline-rich in composition and mediate successful parasitism of host target cells. A specific class of peptidyl-proyl cis-trans isomerases (PPIs), called cyclophilins (Cyps), activate proline-rich proteins, and this enzymatic activity is inhibited by the drug, cyclosporin A (CsA). This study builds upon the connection between the structural/functional properties of the proline-rich proteins of M. pneumoniae and the mode of action of CsA to demonstrate that CsA reduces cytadherence capabilities of mycoplasmas, affects colony morphology and can be mycoplasmacidal. As a consequence of CsA treatment early passage mycoplasmas lacked the major adhesin, P1, explaining their cytadherence-negative phenotype. Three mycoplasma proteins with molecular masses of 160, 84 and 80 kDa were identified by CsA-affinity chromatography. A PCR cloned partial cyp gene of M. pneumoniae, which exhibited sequence homologies with prokaryotic and eukaryotic cyclophilins, was present in multiple copies. These results implicate the role of PPIs as important regulators of cytadherence, virulence and growth cycle events in mycoplasmas.

Amino Acid Isomerases↗
PubMed1996

Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium.

Cytadherence and subsequent parasitism of host cells by the human pathogens, Mycoplasma pneumoniae and Mycoplasma genitalium, are mediated by adhesins and adherence-related accessory proteins. In this report we demonstrate the use of transposon Tn4001 to generate Tn-induced transformants displaying cytadherence-deficient characteristics. Mycoplasma pneumoniae Tn-generated transformant, designated 8R, lacked the high-molecular weight adherence-accessory proteins HMW1/4 and was deficient in hemadsorption and cytadherence capabilities. In transformant 8R, Tn4001 was not localized in or near the hmw1 gene or in the upstream adhesin (p30/hmw3) locus, suggesting an alternate site associated with the regulation of hmw1 gene expression. Sequence analysis identified the transposon insertion site at the crl locus previously reported, although the protein characteristics of transformant 8R differed from the earlier described transformants. The M. genitalium Tn-transformant, designated G26, was also defective in hemadsorption and cytadherence. However, transformant G26 synthesized adhesins P140 and P32 suggesting that Tn4001 transposed into a new gene or site previously unlinked to cytadherence, namely ORF MG032. This study demonstrates the utility of Tn4001 mutagenesis for both M. pneumoniae and M. genitalium which, in the latter case, has special relevance in light of the recent complete characterization of its continuous total genomic sequence.

Adhesins, Bacterial↗
PubMed1996

Adherence, fibronectin binding, and induction of cytoskeleton reorganization in cultured human cells by Mycoplasma penetrans.

Mycoplasma penetrans adhered to cultured human cells, forming clusters that localized to specific areas of the host cell surface. Adherence and cluster formation were inhibited by anti-M. penetrans antibodies, suggesting the involvement of specific adhesin-receptor interactions. Ultrastructural studies showed that after 2 h of infection, mycoplasmas attach to and penetrate the host cell surface. M. penetrans bound selectively to immobilized fibronectin, an interaction which was not inhibited by a 70-kDa fragment containing a heparin-gelatin-binding domain of fibronectin, other matrix glycoproteins, or an RGD tripeptide, suggesting the recognition of other specific binding sites on the fibronectin molecule. A ca. 65-kDa fibronectin-binding protein of M. penetrans was eluted following Sepharose-fibronectin affinity chromatography. Confocal, light, and immunofluorescence microscopy demonstrated that the interaction of M. penetrans with target cells triggers a signal that causes recruitment of several cytoskeletal components, including tubulin and alpha-actinin, and aggregation of phosphorylated proteins. Detergent-soluble mycoplasma proteins with apparent molecular masses of 18, 28, 32, 36, 39, and 41 kDa selectively bound to glutaraldehyde-fixed HEp-2 cells. Our findings offer new insights into understanding the interaction of this human mycoplasma with host target cells.

Adhesins, Bacterial↗
PubMed1996

Biofunctional domains of the Mycoplasma pneumoniae P30 adhesin.

The P30 adhesin genes of spontaneous, hemadsorption-negative (HA-) class II Mycoplasma pneumoniae mutants that displayed P30 adhesin-deficient protein profiles were analyzed. One subclass of P30-deficient mutants possessed the entire p3O structural gene without alterations (825 nucleotides, encoding 275 amino acids with a predicted molecular mass of 29,743 Da [S. F. Dallo, A. Chavoya, and J. B. Baseman, Infect. Immun. 58:4163-4165, 1990]). However, the second mutant subclass contained a deletion in p3O resulting in the expression of a 25-kDa peptide (681 nucleotides, encoding 227 amino acids with a calculated molecular mass of 24,823 Da). This P25-truncated peptide lacked 8 of the 13 proline-rich amino acid repeat sequences at the carboxy terminus. Whole-cell radioimmunoprecipitation of M. pneumoniae with antibodies directed against the proline-rich repeat sequences located in the carboxy terminus demonstrated their surface accessibility. In contrast, antibodies generated against N-terminal amino acid sequences upstream of the repeats did not bind to intact mycoplasmas. The amino acid sequence homologies exhibited by the P30 adhesin and eucaryotic structural proteins were corroborated by cross-reactive epitopes shared between the P30 adhesin and fibrinogen, keratin, and myosin. These data reinforce the importance of the P30 protein in cytadherence and virulence and provide a molecular basis for postinfectious autoimmunity associated with M. pneumoniae-mediated pathologies.

Adhesins, Bacterial↗
PubMed1996

Interplay between mycoplasma surface proteins, airway cells, and the protean manifestations of mycoplasma-mediated human infections.

Adherence of mycoplasmas to specific tissue surfaces is a crucial step in the establishment of infection. Several pathogenic mycoplasmas are flask-shaped and possess specialized tips that permit a highly oriented surface parasitism of host target cells. Mycoplasma pneumoniae, which causes primary atypical pneumonia in humans, requires a network of interactive adhesins and accessory proteins to cytadhere. The adhesins must cluster at the mycoplasma tip organelle in close association with cytadherence-related accessory proteins and a naplike structure, which together appear to comprise a primitive cytoskeleton-like system. Proline-rich regions associated with these proteins play critical roles in the maintenance of the structural and functional integrity of the tip. Mycoplasma genitalium, originally isolated from the human urogenital tract of patients with nongonococcal urethritis, also colonizes airway cells along with M. pneumoniae. The molecular basis for cytadherence of these mycoplasmas is discussed in terms of the identification, cloning, and sequencing of the implicated mycoplasma genes, their common DNA and amino acid homologies and structural and functional domains, and the organizational similarities in their cytadherence-related operons. In addition, the multiorgan protean manifestations of mycoplasma infection are discussed in terms of the role that mycoplasma adhesins may play in molecular mimicry, postinfectious autoimmunity, and immune-mediated damage.

Adhesins, Bacterial↗
PubMed1995

Interplay between mycoplasmas and host target cells.

The infectious pattern of mycoplasmas (Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma genitalium) in mammalian cells was examined using confocal microscopy and flow cytometry combined with cell fractionation and mycoplasma viability determinations. Within 2 h postinfection mycoplasmas parasitize cell surfaces, enter the intracellular spaces and locate throughout the cytoplasmic and perinuclear regions. These mycoplasmas can be cultivated from cytoplasmic and nuclear fractions 96 h later and continue to persist intracellularly for at least 7 days, suggesting a much more active intracellular role for mycoplasmas than had been considered previously.

Animals↗
PubMed1995

Molecular cloning and characterization of an adherence-related operon of Mycoplasma genitalium.

Adhesins and adhesin-related accessory proteins of pathogenic mycoplasmas are required for cytadherence and the subsequent development of disease pathology. The classic example has been Mycoplasma pneumoniae, which causes primary atypical pneumonia in humans. Mutants of M. pneumoniae defective in adhesins (P1 and P30) or in adherence-accessory proteins (HMW1 through HMW4) are unable to colonize host tissues and are avirulent. Mycoplasma genitalium, implicated in nongonococcal, nonchlamydial urethritis, pneumonia, arthritis, and AIDS progression, was found to encode a 140-kDa adhesin that shared both DNA and protein sequence similarities with P1, a major adhesin of M. pneumoniae. In this report, we show that M. genitalium possesses additional homolog sequences to well-characterized adherence-related genes and proteins of M. pneumoniae. The M. genitalium homologs are designated P32 and P69 and correspond to P30 and HMW3 of M. pneumoniae, respectively (J. B. Baseman, p. 243-259, in S. Rottem and I. Kahane, ed., Subcellular biochemistry, vol. 20. Mycoplasma cell membranes, 1993, and D. C. Krause, D. K. Leith, R. M. Wilson, and J. B. Baseman, Infect. Immun. 35:809-817, 1982). Interestingly, the operon-like organizations of P32 and P69 in the M. genitalium genome are similar to the organizations of P30 and HMW3 genes of M. pneumoniae, suggesting that the conservation of these adherence-related genes and proteins might have occurred through horizontal gene transfer events originating from an ancestral gene family.

Adhesins, Bacterial↗
PubMed1995

Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate.

A mycoplasma cultured from synovial fluid specimens from a patient with pneumonia and subsequent polyarthritis was identified initially as Mycoplasma pneumoniae. In retrospective studies, the culture was shown also to contain Mycoplasma genitalium. In this paper, the laboratory techniques employed in the identification and separation of the two species are presented, and evidence to implicate postinfectious autoimmunity is provided. An increasing number of reports of M. genitalium in human tissue sites and difficulties in isolation and identification of the organism in the clinical laboratory suggest the need for more extensive application of rapid and specific detection systems for both M. genitalium and M. pneumoniae in the clinical laboratory.

Animals↗
PubMed1994

Identification of lactoferrin-binding proteins from Treponema pallidum subspecies pallidum and Treponema denticola.

Lactoferrin-binding or -associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent-solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40 kDa and a broad band from 29-34 kDa exhibited association with human apo- and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin-associated proteins from T. pallidum and T. denticola in competitive-binding assays. However, the T. denticola proteins dissociated from a lactoferrin-affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29-34 kDa band of T. pallidum suggesting that the polypeptide was lipid-modified. Each of the lactoferrin-binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin-binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29-34 kDa protein.

Antibodies, Bacterial↗
PubMed1993

Properties of adhering and nonadhering populations of Mycoplasma genitalium.

The interaction between Mycoplasma genitalium and human lung fibroblasts (HLFs) was studied with the use of wild-type and hemadsorption-negative (HA-) mycoplasmas. [35S]-methionine-labeled M. genitalium adhered to HLFs by first-order kinetics, with maximal interaction occurring at approximately 2 hours. Electron microscopy of chemically fixed cells revealed an almost immediate association of mycoplasmas with the HLF plasma membrane that was mediated by the mycoplasma tip and a nap-like layer, which appeared to extend from the tip around much of the mycoplasmal unit membrane. Following cytadherence, M. genitalium appeared capable of invading the intracellular spaces of targeted HLF cells, possibly by receptor-mediated endocytosis. Spontaneously arising HA- variants of M. genitalium strain G37 failed to adhere to HLF cells and were distinguished on the basis of their protein profiles. SDS-PAGE analysis of the class I (lacking the 140-kd protein but containing a polypeptide doublet at approximately 140-kd) and class II (lacking the 140-kd protein and doublet) variants of M. genitalium revealed that class I variants contain a doublet protein in the 140-kd region, which reacted with a monoclonal antibody generated to the adhesin-implicated 140-kd protein (P140) of wild-type M. genitalium. Class II variants completely lacked the 140-kd protein or immunologically related peptides.

Bacterial Adhesion↗
PubMed1993

Possible origin of sequence divergence in the P1 cytadhesin gene of Mycoplasma pneumoniae.

Specific regions of the P1 adhesin structural gene of Mycoplasma pneumoniae hybridize to various parts of the mycoplasma genome, indicating their multiple-copy nature. In addition, restriction fragment length polymorphisms and sequence divergence have been observed in the P1 gene, permitting the classification of clinical isolates of M. pneumoniae into two groups, I and II. These data suggest that the observed P1 gene diversity may be explained by homologous recombination between similar but not identical multicopy P1-related sequences and the P1 structural gene. We used oligonucleotide probes specific to the diverged regions of the group I and group II P1 structural genes to clone and sequence multicopy P1-related DNA segments. We detected sequences in group I M. pneumoniae isolates that were homologous not only to the group I P1 structural gene but also to the diverged regions of the group II P1 structural gene. Likewise, sequences in group II clinical isolates that were homologous both to the group II P1 structural gene and the diverged regions of the group I P1 structural gene were detected.

Adhesins, Bacterial↗