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J E Clark-Curtiss

Publications and source records attributed to J E Clark-Curtiss.

At least 19 recordsLinked to original sources
PubMed1999

Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS).

A widely applicable, positive cDNA selection method was developed to identify RNAs synthesized by Mycobacterium tuberculosis in response to phagocytosis by cultured human primary macrophages. cDNAs for sigE and sigH (alternative sigma factors), aceA (isocitrate lyase), ponA (class I penicillin-binding protein), pks2 (polyketide synthase), uvrA (UvrABC endonuclease), and ctpV (putative cation transporter) were obtained from macrophage-grown bacteria. cDNAs for ORFs Rv3070, Rv3483c, Rv0903c (encoding a putative bacterial two-component transcriptional activator), and Rv0170 of the mce1 virulence operon also were obtained from phagocytized bacilli. cDNAs for these genomic regions were not obtained from approximately 1, 000-fold more bacteria grown in laboratory broth. Methods described here, which have identified M. tuberculosis genes expressed in response to host interaction, will allow the study of gene expression in a variety of microorganisms, including expression resulting from interaction with human tissues in natural disease states.

Bacterial Proteins↗
PubMed1997

Cloning, sequencing, and expression of the mig gene of Mycobacterium avium, which codes for a secreted macrophage-induced protein.

Mycobacterium avium is an intracellular pathogen that has evolved to be a frequent cause of disseminated infection in immunocompromised patients. Although these bacilli are readily phagocytized, they are able to survive and even multiply within human macrophages. The process whereby mycobacteria circumvent the lytic functions of the macrophages is currently not well understood, but this is a key aspect in the pathogenicity of all pathogenic mycobacteria. Previously, we identified a gene in M. avium, designated mig (for macrophage-induced gene), the expression of which is induced when the bacilli grow in human macrophages (G. Plum and J. E. Clark-Curtiss, Infect. Immun. 62:476-483, 1994). In the present study we show that (i) the nucleotide sequence of the mig gene has an open reading frame of 295 amino acids with a strong bias for mycobacterial codon usage, (ii) the mig gene also codes for a putative signal peptide of 19 amino acid residues, (iii) mig is induced by acidity to be expressed as an early-secreted 30-kDa protein, and (iv) the Mig protein exhibits an AMP-binding domain signature. However, beyond this motif which is common to enzymes that activate a large variety of substrates, no homologies to known sequences are found. We also show that (v) Mycobacterium smegmatis strains expressing the Mig protein have a limited advantage for survival in macrophages. These findings may be concordant with a role of the mig gene in the virulence of M. avium.

Amino Acid Sequence↗
PubMed1995

Immunological and functional characterization of Mycobacterium leprae protein antigens: an overview.

A major focus of leprosy research in the last 10 years has been the identification and characterization of antigens of Mycobacterium leprae that interact with antibodies and T cells of the host's immune response. Through the combined efforts of many different laboratories, a substantial number of protein antigens have been identified and characterized. In this MicroReview we present an updated list of M. leprae protein antigens, and, with emphasis on recent developments, summarize what is known regarding their functional and immunological features.

Antigens, Bacterial↗
PubMed1995

A Mycobacterium leprae gene encoding a fibronectin binding protein is used for efficient invasion of epithelial cells and Schwann cells.

Mycobacterium leprae, the causative agent of leprosy, is an obligate intracellular pathogen. M. leprae can infect a variety of cells in vivo, including epithelial cells, muscle cells, and Schwann cells, in addition to macrophages. The ligand-receptor interactions important in the attachment and ingestion of M. leprae by these nonmacrophage cells remains unknown. Fibronectin (FN) significantly enhances both attachment and ingestion of M. leprae by epithelial and Schwann cell lines. We cloned an M. leprae FN binding protein (FN attachment protein [FAP]) distinct from the 85ABC complex which has been shown previously to bind FN. The FAP open reading frame predicts a protein of 29.5 kDa with a 39-amino-acid signal peptide and was previously described as an antigen in leprosy patients. M. leprae FAP has homologies in M. vaccae, M. avium, and M. tuberculosis, as determined by Southern blotting and direct peptide analysis. Both anti-FAP antibodies and an Escherichia coli-expressed recombinant protein significantly blocked M. leprae attachment and internalization by T-24, an epithelial cell line, and JS1, a Schwann cell line. These data suggest that FN can be a bridging opsonic ligand for attachment of mycobacteria to nonphagocytes and that FAP plays an important role in this process. This may be an important step in the initiation of M. leprae infection in vivo.

Adhesins, Bacterial↗
PubMed1994

Leprosy vaccine.

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Bacterial Proteins↗
PubMed1994

Induction of Mycobacterium avium gene expression following phagocytosis by human macrophages.

Little is known about the bacterial factors that enable pathogenic mycobacteria to survive and multiply within the macrophages of the infected host. By preparing cDNA from Mycobacterium avium bacilli grown in human-derived macrophages and in broth culture and using subtractive hybridization to remove commonly expressed genes, a procedure was developed to identify genes of M. avium that are specifically expressed when the bacilli are growing within macrophages. Total RNA was isolated from M. avium recovered 5 days after infection of human macrophages and from bacilli grown in vitro in broth. Mycobacterial mRNAs were converted to cDNA by reverse transcription. Biotin-modified cDNAs prepared from M. avium grown in broth culture were used to subtract the housekeeping genes from the cDNAs of the macrophage-derived M. avium. After each round of subtraction, a sample of the unsubtracted cDNA was amplified, labeled, and hybridized to cosmid clones of M. avium DNA. After three rounds of subtraction, the amplified DNA hybridized to approximately 1% of the cosmid clones under stringent conditions. Although the majority of the genes that are induced in phagocytized M. avium cells are expressed in the broth-grown bacilli, one DNA fragment that was identified coded for an mRNA that is highly specific for M. avium in phagosomes. This procedure will be especially useful for identifying genes that are expressed in response to growth in specific environments from organisms with genetic systems that are not well characterized.

Adaptation, Physiological↗
PubMed1993

A Mycobacterium leprae-specific gene encoding an immunologically recognized 45 kDa protein.

By screening a Mycobacterium leprae lambda gt11 expression library with a serum from an Ethiopian lepromatous leprosy (LL) patient a clone was isolated (LL4) belonging to hybridization group III of a panel of previously isolated M. leprae clones. Members of this hybridization group encode a serologically recognized 45 kDa protein. The complete DNA sequences of the partially overlapping clones LL4 and L1 (hybridization group III) are presented and these revealed the presence of an open reading frame (ORF) predicting a protein with a molecular size of 42,448 Da. Southern hybridizations on total genomic DNA of M. leprae, Mycobacterium tuberculosis and eight atypical mycobacteria showed that the LL4 DNA fragment is specific for M. leprae DNA even under low-stringency conditions. The M. leprae specificity of LL4 DNA was further confirmed by the polymerase chain reaction using four different sets of primers. Western blotting analyses showed that the M. leprae 45 kDa protein is frequently recognized by antibodies from leprosy patients and that this recognition is specific since no antibodies could be detected in sera of tuberculosis patients. T-cell proliferation assays also demonstrated T-cell recognition by leprosy patients and healthy contacts of the M. leprae 45 kDa protein. The specificity of the LL4 DNA region and the 45 kDa antigen that is encoded by hybridization group III could provide unique tools for the development of M. leprae-specific immunological and DNA reagents.

Amino Acid Sequence↗
PubMed1993

Escherichia coli heat-labile toxin subunit B fusions with Streptococcus sobrinus antigens expressed by Salmonella typhimurium oral vaccine strains: importance of the linker for antigenicity and biological activities of the hybrid proteins.

A set of vectors possessing the genes for aspartate semialdehyde dehydrogenase (asd) and the B subunit of the heat-labile enterotoxin of Escherichia coli (LT-B) has been developed. These vectors allow operon or gene fusions of foreign gene epitopes at the C-terminal end of LT-B. Two groups of vectors have been constructed with and without leader sequences to facilitate placing of the foreign antigen in different cell compartments. Two Streptococcus sobrinus genes coding for principal colonization factors, surface protein antigen A (SpaA), and dextranase (Dex), have been fused into the 3' end of the LT-B gene. Resulting protein fusions of approximately 120 to 130 kDa are extremely well recognized by antibodies directed against both SpaA and Dex as well as against LT-B domains and retain the enzymatic activity of dextranase and the biological activity of LT-B in that they bind to GM1 gangliosides. Maximum antigenicity was obtained with the vector possessing an intervening linker of at least six amino acids with two proline residues. Some of the fusion proteins also exhibited another property of LT-B in that they were exported into the periplasm where they oligomerized. LT-B-SpaA and LT-B-Dex hybrid proteins are expressed stably and at a high level in avirulent Salmonella typhimurium vaccine strains which are being used to investigate their immunogenicity and types of induced immune responses. The fusion vectors will also be useful for production and purification of LT-B fusion antigens to be used and evaluated in other vaccine compositions.

Amino Acid Sequence↗
PubMed1992

Localization of the steroid 1-dehydrogenase in Rhodococcus erythropolis IMET 7030 by immunoelectron microscopy.

The steroid 1-dehydrogenase of Rhodococcus erythropolis IMET 7030, an active steroid-transforming strain, was localized by immunogold labelling both in cells induced with 17-alpha-methyl-testosterone and in noninduced cells. The labelling intensity was much higher in induced cells than in noninduced cells, indicating increased enzyme production in the case of induction. Using the postembedding procedure, the main portion of the enzyme was found in the peripheral region of the cytoplasm. A considerable amount was bound in clusters to the inner side of the cytoplasmic membrane. The enzyme was also detected in channels connecting the cytoplasm with the cell surface. By means of the preembedding labelling, a few gold clusters could be detected on the cell surface. The significance of this observation was discussed.

Cytoplasm↗
PubMed1992

Overexpression of a Rhodococcus erythropolis protein in Escherichia coli with immunological identity to the Rhodococcus steroid 1-dehydrogenase. Immunoelectron microscopic localization and electrophoretic studies.

The recombinant Escherichia coli K-12 strain chi 6060 harbouring the plasmid pYA 1201 with a gene from Rhodococcus erythropolis IMET 7030 overexpressed a protein which reacts with a monospecific antiserum against the steroid 1-dehydrogenase (Sdh) from the same Rhodococcus strain. It was shown previously that this recombinant protein exhibits no enzymatic activity. By immunogold labelling the protein was localized on ultrathin sections of the recombinant E. coli strain. After cultivation at 37 degrees C it was found within large cytoplasmic compartments (inclusion bodies). The inclusion bodies occupied 40% to 75% of the sectioned cell area. The highest amount of protein was observed after induction of the culture with isopropyl-beta-D-thiogalactopyranoside. Approximately 20% of the induced cells became enlarged (up to 5-fold of the normal size) and deformed; multiplication of the Rhodococcus protein producing cells was inhibited. After ultrasonic cell disintegration the inclusion bodies were found only in the fraction of the sedimented cell debries and did still react with anti-Sdh. When recombinant E. coli cells were cultivated at 28 degrees C, inclusion bodies appeared very seldom and the immunoreactive protein was distributed throughout the whole cytoplasm.

Animals↗
PubMed1992

Molecular and immunological analysis of a fibronectin-binding protein antigen secreted by Mycobacterium leprae.

By screening a Mycobacterium leprae lambda gt11 genomic DNA library with leprosy-patient sera we have previously identified 50 recombinant clones that expressed novel M. leprae antigens (Sathish et al., 1990). In this study, we show by DNA sequencing and immunoblot analysis that three of these clones express a M. leprae homologue of the fibronectin-binding antigen 85 complex of mycobacteria. The complete gene was characterized and it encodes a 327-amino-acid polypeptide, consisting of a consensus signal sequence of 38 amino acids followed by a mature protein of 289 amino acids. This is the first sequence of a member of the M. leprae antigen 85 complex, and Southern blotting analysis indicated the presence of multiple genes of the 85 complex in the genome of M. leprae. The amino acid sequence displays 75-85% sequence identity with components of the antigen 85 complex from M. tuberculosis, M. bovis BCG and M. kansasii. Furthermore, antibodies to the antigen 85 complex of M. tuberculosis and M. bovis BCG reacted with two fusion proteins containing the amino acid regions 55-266 and 266-327 of the M. leprae protein. The M. leprae 30/31 kDa protein induces strong humoral and cellular responses, as judged by Western blot analysis with patient sera and proliferation of T cells derived from healthy individuals and leprosy patients. Amino acid regions 55-266 and 265-327 both were shown to bind to fibronectin, indicating the presence of at least two fibronectin-binding sites on the M. leprae protein. These data indicate that this 30/31 kDa protein is not only important in the immune response against M. leprae, but may also have a biological role in the interaction of this bacillus with the human host.

Amino Acid Sequence↗
PubMed1991

Cloning and characterization of the Mycobacterium leprae putative ribosomal RNA promoter in Escherichia coli.

The putative promoter region of the 16S ribosomal RNA-encoding gene (rRNA) of Mycobacterium leprae was cloned and characterized in Escherichia coli. A 932-bp HaeIII restriction fragment, containing the 5' end of the 16S rRNA gene and flanking upstream region, was cloned in front of a promoterless reporter gene in the shuttle vector, pMH109, to generate the plasmid, pYA1101. This clone exhibits promoter activity both in Gram-(E. coli) and Gram+ (Bacillus subtilis) bacteria. Sequence analysis and primer extension experiments with mRNA derived from the M. leprae clone were used to determine the structure and the location of the promoter, as well as the transcription start point in E. coli. The promoter region contains sequences that resemble the -35 and -10 consensus sequences found in many bacteria. A region located 34 bp distal to the promoter is a putative rRNA processing signal, based on sequence homology with processing signals involved in the maturation of the rRNA precursor in B. subtilis and several Mycoplasma species.

Base Sequence↗
PubMed1991

Identification of Mycobacterium leprae antigens from a cosmid library: characterization of a 15-kilodalton antigen that is recognized by both the humoral and cellular immune systems in leprosy patients.

Screening of the Mycobacterium leprae cosmid library with pooled sera from lepromatous leprosy (LL) patients by a colony immunoblot technique resulted in the identification of about 100 colonies that produced immunologically reactive proteins. Twenty-four of these clones were purified, analyzed, and found to comprise two groups according to the reactivity of the recombinant proteins with LL sera and to the DNA restriction patterns of the recombinant plasmids and cosmids. Proteins specified by clones from group I reacted strongly with LL patients' sera on a Western blot (immunoblot), demonstrating a 15-kDa protein band designated A15. The A15 antigen also reacted with pooled sera from patients with tuberculoid leprosy from the United States and Brazil. Clones from group II did not show any reactive protein band on a Western blot, when reacted with patients' sera. DNAs from cosmids of group II all contain a 10-kb PstI fragment that hybridized to the unique repetitive M. leprae DNA. Sequence analysis of a 1.2-kb fragment containing the entire coding sequence of A15 revealed three open reading frames (ORFs), only one of which (ORF II) contains sufficient genetic information to encode for A15. Part of the A15 gene was found to exist also in a group of lambda gt11:M. leprae clones previously isolated in our laboratory by immunological screening with LL patients' sera. One of the lambda gt11 clones (L8) expresses a beta-galactosidase fusion protein with 89 amino acids from the C terminus of A15. An important result was that the fusion protein was clearly recognized by T cells from leprosy patients. Interestingly, Mycobacterium tuberculosis-stimulated T cells from M. leprae nonresponder (LL as well as borderline tuberculoid) patients were able to respond to the isolated recombinant M. leprae antigen, indicating that nonresponsiveness to M. leprae antigens can be reversible. The sequence of the M. leprae DNA fused to the beta-galactosidase gene of lambda gt11 clone L8 was identical to that of a lambda gt11:M. leprae clone isolated recently that expresses an immunologically reactive fusion protein (S. Laal, Y. D. Sharma, H. K. Prasad, A. Murtaza, S. Singh, S. Tangri, R. Misra, and I. Nath, Proc. Natl. Acad. Sci. USA 88:1054-1058, 1991). Besides the complete sequence of the A15 gene, sequencing data of two flanking ORFs are presented. Downstream from ORF II (A15), ORF III has a high degree of similarity to the genes for tomato ATP-dependent proteases that are members of a larger class of highly conserved proteases ubiquitous among prokaryotes and eukaryotes.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Identification and characterization of antigenic determinants of Mycobacterium leprae that react with antibodies in sera of leprosy patients.

Antigenic determinants of Mycobacterium leprae were identified by screening a lambda gt11::M. leprae genomic library with two separate pools of sera from leprosy patients. A total of 45 recombinant clones were detected with pooled sera from 21 lepromatous (LL) leprosy patients and 5 additional clones specified polypeptides that reacted with antibodies in pooled sera from 30 borderline tuberculoid or tuberculoid leprosy patients. The recombinant clones that specified antigenic determinants that reacted with sera from LL patients were condensed into eight groups on the basis of DNA hybridization experiments among the M. leprae DNA insert fragments. In addition, 11 of the 45 recombinant clones did not hybridize to members of the eight groups nor to one another; these represent unique recombinant clones. None of the recombinant clones identified by screening with sera from tuberculoid leprosy patients hybridized to each other or to any of the 45 LL recombinant clones. The polypeptides specified by the recombinant clones were usually fusion proteins with beta-galactosidase, ranging in size from 117 to 175 kilodaltons (kDa). Members of hybridization group III specified nonfusion proteins of 45 kDa. Only members of hybridization group I reacted with any of 30 monoclonal antibodies prepared against M. leprae proteins; recombinant proteins from these clones reacted with a single monoclonal antibody directed against the M. leprae 65-kDa protein. Thus, at least 22 new antigenic determinants of M. leprae have been identified on the basis of their reactivity to antibodies in sera from LL patients or sera from tuberculoid leprosy patients or both.

Antibodies, Bacterial↗
PubMed1989

A species-specific repetitive sequence in Mycobacterium leprae DNA.

A 2.2-kilobase Mycobacterium leprae DNA insert fragment from a recombinant genomic library (pYA1065) was found to hybridize to at least 19 fragments of chromosomal M. leprae DNA by Southern hybridizations. The probe hybridized to identical fragments of chromosomal DNA from four M. leprae isolates (two from patients with leprosy, one from a naturally infected armadillo, and one from a naturally infected Mangabey monkey) whether the chromosomal DNA was digested with BamHI, BstEII, PstI, or SacI. The pYA1065 probe is specific for M. leprae; it did not hybridize to chromosomal DNA from 14 cultivable slow- and fast-growing mycobacterial species. Dot-blot hybridizations between pYA1065 and purified M. leprae chromosomal DNA indicate that the probe can detect DNA equivalent to 4 x 10(3) M. leprae cells in a spot. The probe can also hybridize to DNA in M. leprae cells spotted on a filter from homogenized skin biopsy specimens from patients with lepromatous leprosy.

Animals↗
PubMed1989

Genetic relationships among Mycobacterium leprae, Mycobacterium tuberculosis, and candidate leprosy vaccine strains determined by DNA hybridization: identification of an M. leprae-specific repetitive sequence.

Comparative DNA hybridization studies of genomic DNA indicated that, while different isolates of armadillo-derived Mycobacterium leprae have a high degree of homology, binding of M. leprae genomic DNA to DNA of other species of mycobacteria or to corynebacteria was low, establishing that M. leprae is only remotely genetically related to any of the species examined. Several candidate leprosy vaccine mycobacterial strains were similarly found to have little genetic similarity to M. leprae. In contrast, the DNAs of the slow-growing mycobacteria M. tuberculosis, M. africanum, M. bovis, and M. microti were found to be very closely related. In the course of these studies, an M. leprae-specific repetitive sequence, greater than 15-fold per genome equivalent, was identified that might be useful for diagnostic and epidemiological studies.

Antigens, Bacterial↗