J M Hardham

New York Medical College, New York City, New York, United States

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Publications (23)135.46 Total impact

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    ABSTRACT: The recently identified flu operon of Treponema pallidum contains several genes that encode motilityrelated proteins. We have determined the nucleotide sequences of three genes, designated flhA, flhF, and or/304, that are located immediately downstream of the flhB gene in the fla operon. The flhA gene encodes a 707-amino acid protein that contains five putative membrane spanning domains. FlhA has strong homology with members of a family of proteins that are involved in flagellar biogenesis and regulation/secretion of virulence-related proteins. The flhF gene encodes a 437-amino acid protein that contains three consensus elements that are characteristic of a GTP-binding domain. The or/304 gene encodes a 304-amino acid protein that contains a consensus ATP-binding motif. The order of the flhA, flhF, and or/304 genes is identical to that of corresponding genes in the Bacillus subtilis che/fla operon. Due to the location of the flhA, flhF and or/304 genes in the T. pallidum fla operon, we hypothesize that the FlhA, FlhF, and Orf304 proteins are involved in the biogenesis/assembly of treponemal periplasmic flagella.
    07/2009; 7(2):107-116.
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    ABSTRACT: VlsE is an outer surface lipoprotein of Borrelia burgdorferi that undergoes antigenic variation through an elaborate gene conversion mechanism and is thought to play a major role in the immune response to the Lyme disease borellia. The crystal structure of recombinant variant protein VlsE1 at 2.3-A resolution reveals that the six variable regions form loop structures that constitute most of the membrane distal surface of VlsE, covering the predominantly alpha-helical, invariant regions of the protein. The surface localization of the variable amino acid segments appears to protect the conserved regions from interaction with antibodies and hence may contribute to immune evasion.
    Journal of Biological Chemistry 07/2002; 277(24):21691-6. · 4.65 Impact Factor
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    J M Hardham, E L Rosey
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    ABSTRACT: Until very recently, the pathogenic spirochetes have been refractory to genetic manipulation. This has been due, in part, to difficulties with in vitro growth and the genetic distance that spirochetes are from typical Gram-negative and Gram-positive organisms. Insertional mutagenesis and other genetic techniques are now possible in some of the pathogenic spirochetes such as Borrelia burgdorferi, Brachyspira (Serpulina) hyodysenteriae, Leptospira sp., and Treponema denticola. However, organisms such as Treponema pallidum, which cannot be grown in vitro, are still not amenable to genetic manipulation. These recent advances have paved the way for more detailed genetic studies of transcriptional regulation, protein function, protein localization, metabolic capabilities, motility, and pathogenic nature of this group of spirochetes. This review article will discuss the current repertoire of antibiotic markers that are useful for spirochetal genetic manipulation. Further advances in selectable markers and shuttle vectors will allow researchers to complete Koch's molecular hypothesis for various virulence genes of the pathogenic spirochetes and increase the overall understanding of these challenging bacteria.
    Journal of Molecular Microbiology and Biotechnology 11/2000; 2(4):425-32. · 1.68 Impact Factor
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    ABSTRACT: Whole genome sequences of microbial pathogens present new opportunities for clinical applications. Chief among these are development of antimicrobials, diagnostics, and vaccines. While antimicrobial development is a more difficult, long-term prospect, new diagnostics and vaccines are likely to be the first products of microbial genomics. To take advantage of whole genome sequences, methods for production of gene products in surrogate hosts (heterologous expression) are required that will work for large-scale, high-throughput gene expression. This will allow genomic information from even the most experimentally difficult pathogens to be mined for applications. In addition, screening methods to test gene products for their potential as vaccine candidates are needed for large-scale screening. These areas for technological development should be stimulated by the potential for converting genomic sequence information into applications.
    Research in Microbiology 04/2000; 151(2):151-8. · 2.89 Impact Factor
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    ABSTRACT: The vls (variable major protein [VMP]-like sequence) locus of Borrelia burgdorferi encodes an antigenic variation system that closely resembles the VMP system of relapsing fever borreliae. To determine whether vls sequences are present consistently in low-passage, infectious isolates of B. burgdorferi, 22 blood and erythema migrans biopsy isolates from Lyme disease patients in Westchester County, New York, were examined by Southern blot and PCR analysis. Each of the strains contained a single plasmid varying in size from 21 to 38 kb that hybridized strongly with a vlsE probe based on the B. burgdorferi B31 sequence. In contrast, PCR products were obtained with only 10 of the 22 strains when primers corresponding to the 5' and 3' regions of the B31 vlsE sequence outside the variable cassette region were used. Only 2 of 16 B. burgdorferi-infected tick specimens yielded detectable PCR product. Eight of 10 strains that yielded a PCR product under these conditions were type 1 (a genotype with a high rate of dissemination), according to PCR-restriction fragment length polymorphism analysis of intergenic rDNA sequences, whereas the isolates that did not yield vlsE PCR products were either type 2 or type 3. Comparison of the sequences of cloned PCR products from the patient isolates indicated a high degree of identity to the B31 sequence, with most of the differences restricted to the hypervariable regions known to undergo sequence variation. Taken together, these results both reinforce previous evidence that vls sequences are present consistently in low-passage Lyme disease spirochetes and indicate that both highly conserved and heterogeneous subgroups exist with regard to vlsE sequences.
    Infection and Immunity 04/2000; 68(3):1714-8. · 4.07 Impact Factor
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    ABSTRACT: VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the "whole-cell" ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen.
    Journal of Clinical Microbiology 01/2000; 37(12):3997-4004. · 4.07 Impact Factor
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    ABSTRACT: Genome sequence analysis of Treponema pallidum, the causative agent of syphilis, suggests that this bacterium has a limited iron requirement with few, if any, proteins that require iron. Instead, T. pallidum may use manganese-dependent enzymes for metabolic pathways. This strategy apparently alleviates the necessity of T. pallidum to acquire iron from the host, thus overcoming iron limitation, which is a primary host defense. Interestingly, a putative metal-dependent regulatory protein, TroR, which has homology with the diphtheria toxin regulatory protein, DtxR, from Corynebacterium diphtheriae was identified from T. pallidum. We describe here the characterization of TroR, a regulatory protein. Mobility-shift DNA binding and DNase I footprint assays indicated that purified TroR bound to a 22-nt region of dyad symmetry that overlaps the -10 region of the promoter of the tro operon, which contains the genes for a putative metal transport system, the glycolytic enzyme phosphoglycerate mutase, and TroR. Unlike other metal-dependent regulatory proteins like diphtheria toxin regulatory protein and the ferric ion uptake regulator, Fur, which can be activated by divalent metals such as Fe(2+), Mn(2+), Co(2+), Ni(2+), and Zn(2+), TroR is activated only by Mn(2+). The TroR-Mn(2+) complex binds its target sequence and blocks transcription of the troPO/lacZ fusion, suggesting that TroR acts as a metal-dependent repressor in vivo. In addition, TroR exists as a dimer in both its inactive (metal free) and active states as indicated by chemical crosslinking experiments. Based on these data, we propose that TroR represents a unique regulatory system for controlling gene expression in T. pallidum in response to Mn(2+).
    Proceedings of the National Academy of Sciences 10/1999; 96(19):10887-92. · 9.81 Impact Factor
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    ABSTRACT: TnphoA mutagenesis was used to identify genes encoding exported proteins in a genomic DNA library of Treponema pallidum, the syphilis agent. The nucleotide sequence of an open reading frame (tprJ) that encodes a 755-amino acid protein with a predicted molecular mass of 81.1 kDa was determined. The deduced amino acid sequence of TprJ has homology to the major surface protein of Treponema denticola, a periodontal pathogen. Southern hybridization and genomic DNA sequence analysis indicate that tprJ is a member of a polymorphic multigene family. RT-PCR data showed that tprJ is expressed in treponemes during syphilitic infection. A putative tprJ gene was sequenced from T. pertenue, the closely related yaws agent. The deduced amino acid sequence of T. pertenue TprJ is 87.3% identical to that of T. pallidum TprJ. This is the first report of significant sequence differences within homologous genes of T. pallidum and T. pertenue.
    FEMS Microbiology Letters 01/1999; 169(1):155-63. · 2.05 Impact Factor
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    ABSTRACT: TnphoA mutagenesis was used to identify genes encoding exported proteins in a genomic DNA library of Treponema pallidum, the syphilis agent. The nucleotide sequence of an open reading frame (tprJ) that encodes a 755-amino acid protein with a predicted molecular mass of 81.1 kDa was determined. The deduced amino acid sequence of TprJ has homology to the major surface protein of Treponema denticola, a periodontal pathogen. Southern hybridization and genomic DNA sequence analysis indicate that tprJ is a member of a polymorphic multigene family. RT-PCR data showed that tprJ is expressed in treponemes during syphilitic infection. A putative tprJ gene was sequenced from T. pertenue, the closely related yaws agent. The deduced amino acid sequence of T. pertenue TprJ is 87.3% identical to that of T. pallidum TprJ. This is the first report of significant sequence differences within homologous genes of T. pallidum and T. pertenue.
    FEMS Microbiology Letters 11/1998; 169(1):155 - 163. · 2.05 Impact Factor
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    ABSTRACT: Treponema pallidum subsp, pallidum, the causative agent of the sexually transmitted disease syphilis, is a fastidious, microaerophilic obligate parasite of humans. This bacterium is one of the few prominent infectious agents that has not been cultured continuously in vitro and consequently relatively little is known about its virulence mechanisms at the molecular level. T. pallidum therefore represented an attractive candidate for genomic sequencing. The complete genome sequence of T. pallidum has now been completed and comprises 1,138,006 base pairs containing 1041 predicted protein coding sequences. An important goal of this project is to identify possible virulence factors. Analysis of the genome indicates a number of potential virulence factors including a family of 12 proteins related to the Msp protein of Treponema denticola, a number of putative hemolysins, as well as several other classes of proteins of interest. The results of this analysis are reviewed in this article and indicate the value of whole genome sequences for rapidly advancing knowledge of infectious agents.
    FEMS Microbiology Reviews 11/1998; 22(4):323-32. · 13.23 Impact Factor
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    [Show abstract] [Hide abstract]
    ABSTRACT: Treponema pallidum subsp. pallidum, the causative agent of the sexually transmitted disease syphilis, is a fastidious, microaerophilic obligate parasite of humans. This bacterium is one of the few prominent infectious agents that has not been cultured continuously in vitro and consequently relatively little is known about its virulence mechanisms at the molecular level. T. pallidum therefore represented an attractive candidate for genomic sequencing. The complete genome sequence of T. pallidum has now been completed and comprises 1 138 006 base pairs containing 1041 predicted protein coding sequences. An important goal of this project is to identify possible virulence factors. Analysis of the genome indicates a number of potential virulence factors including a family of 12 proteins related to the Msp protein of Treponema denticola, a number of putative hemolysins, as well as several other classes of proteins of interest. The results of this analysis are reviewed in this article and indicate the value of whole genome sequences for rapidly advancing knowledge of infectious agents.
    FEMS microbiology reviews 09/1998; 22(4):323 - 332. · 10.96 Impact Factor
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    ABSTRACT: The complete genome sequence of Treponema pallidum was determined and shown to be 1,138,006 base pairs containing 1041 predicted coding sequences (open reading frames). Systems for DNA replication, transcription, translation, and repair are intact, but catabolic and biosynthetic activities are minimized. The number of identifiable transporters is small, and no phosphoenolpyruvate:phosphotransferase carbohydrate transporters were found. Potential virulence factors include a family of 12 potential membrane proteins and several putative hemolysins. Comparison of the T. pallidum genome sequence with that of another pathogenic spirochete, Borrelia burgdorferi, the agent of Lyme disease, identified unique and common genes and substantiates the considerable diversity observed among pathogenic spirochetes.
    Science 08/1998; 281(5375):375-88. · 31.03 Impact Factor
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    ABSTRACT: The nucleotide sequence of a DNA adenine methyltransferase gene (dam) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The dam gene encodes a 303 amino acid protein whose deduced sequence has significant homology with DNA (N6-adenine) methyltransferases. T. pallidum Dam can be assigned to group alpha DNA amino methyltransferases based on the order of nine conserved motifs that are present in the protein. Digests of T. pallidum chromosomal DNA performed with isoschizomer restriction endonucleases (Sau3AI, DpnI, and MboI) confirmed the presence of methylated adenine residues in GATC sequences (Dam+ phenotype).
    FEMS Microbiology Letters 11/1997; 155(1):115-9. · 2.05 Impact Factor
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    ABSTRACT: We have characterized a 5.2-kilobase (kb) putative transport related operon (tro) locus of Treponema pallidum subsp. pallidum (Nichols strain) (Tp) encoding six proteins: TroA, TroB, TroC, TroD, TroR and Phosphoglycerate mutase (Pgm). Four of these gene products (TroA-TroD) are homologous to members of the ATP-Binding Cassette (ABC) superfamily of bacterial transport proteins. TroA (previously identified as Tromp1) has significant sequence similarity to a family of Gram-negative periplasmic substrate-binding proteins and to a family of streptococcal proteins that may have dual roles as substrate binding proteins and adhesins. TroB is homologous to the ATP-binding protein component, whereas TroC and TroD are related to the hydrophobic membrane protein components of ABC transport systems. TroR is similar to Gram-positive iron-activated repressor proteins (DesR, DtxR, IdeR, and SirR). The last open reading frame (ORF) of the tro operon encodes a protein that is highly homologous to the glycolytic pathway enzyme, Pgm. Primer extension results demonstrated that the tro operon is transcribed from a sigma 70-type promoter element. Northern analysis and reverse transcriptase-polymerase chain reactions provided evidence for the presence of a primary 1-kb troA transcript and a secondary, less abundant, troA-pgm transcript. The tro operon is flanked by a Holliday structure DNA helicase homolog (upstream) and two ORFs representing a purine nucleoside phosphorylase homolog and tpp15, a previously characterized gene encoding a membrane lipoprotein (downstream). The presence of a complex operon containing a putative ABC transport system and a DtxR homolog indicates a possible linkage between transport and gene regulation in Tp.
    Gene 10/1997; 197(1-2):47-64. · 2.20 Impact Factor
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    ABSTRACT: We have identified and characterized an elaborate genetic system in the Lyme disease spirochete Borrelia burgdorferi that promotes extensive antigenic variation of a surface-exposed lipoprotein, VlsE. A 28 kb linear plasmid of B. burgdorferi B31 (lp28-1) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for antigenic variation of relapsing fever organisms. Portions of several of the 15 nonexpressed (silent) vls cassette sequences located upstream of vlsE recombined into the central vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. This combinatorial variation could potentially produce millions of antigenic variants in the mammalian host.
    Cell 05/1997; 89(2):275-85. · 31.96 Impact Factor
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    ABSTRACT: Treponema pallidum, the agent of syphilis, is a pathogenic spirochete that has no known mechanisms of genetic exchange and cannot be continuously cultivated in vitro. A probe based on the nucleotide sequence of the T. pallidum cheA gene was used to screen a T. pallidum genomic DNA library. A treponemal DNA region containing four open reading frames (orfs) was identified. The proteins encoded by these orfs have significant homology with proteins involved in bacterial chemotaxis. The orfs have been designated cheA, cheW, cheX, and cheY. The cheA, cheW, and cheY genes were individually-cloned and expressed in vitro. The observed molecular mass of each protein correlated well with its predicted molecular mass. Reverse transcriptase-PCR data indicate that cheA through cheY are co-transcribed. The organization of these genes suggests that they comprise an operon. We hypothesize that the ability to sense and respond to nutrient gradients is important for the survival and dissemination of T. pallidum in vivo. The presence of a putative che operon strongly suggests that T. pallidum has the potential for a chemotactic response.
    DNA Sequence 02/1997; 7(5):267-84. · 0.75 Impact Factor
  • J M Hardham, J G Frye, N R Young, L V Stamm
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    ABSTRACT: The recently identified fla operon of Treponema pallidum contains several genes that encode motility-related proteins. We have determined the nucleotide sequences of three genes, designated flhA, flhF, and orf304, that are located immediately downstream of the flhB gene in the fla operon. The flhA gene encodes a 707-amino acid protein that contains five putative membrane spanning domains. FlhA has strong homology with members of a family of proteins that are involved in flagellar biogenesis and regulation/secretion of virulence-related proteins. The flhF gene encodes a 437-amino acid protein that contains three consensus elements that are characteristic of a GTP-binding domain. The orf304 gene encodes a 304-amino acid protein that contains a consensus ATP-binding motif. The order of the flhA, flhF, and orf304 genes is identical to that of corresponding genes in the Bacillus subtilis che/fla operon. Due to the location of the flhA, flhF and orf304 genes in the T. pallidum fla operon, we hypothesize that the FlhA, FlhF, and Orf304 proteins are involved in the biogenesis/assembly of treponemal periplasmic flagella.
    DNA Sequence 02/1997; 7(2):107-16. · 0.75 Impact Factor
  • L V Stamm, N R Young, J G Frye, J M Hardham
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    ABSTRACT: Treponema pallidum, the agent of syphilis, cannot be continuously cultivated in vitro. To identify treponemal genes encoding exported proteins, we performed TnphoA mutagenesis of a T. pallidum genomic DNA library in Escherichia coli. Clone 6D2 was chosen for further study based on partial nucleotide sequence obtained from p6D2 containing a TnphoA insertion. A complete open reading frame (orf1) and a truncated orf (orf2) were identified in the treponemal DNA of p6D2. Orf1 encodes a hydrophobic protein of 531 amino acids with a calculated M(r) of 57,882 Da. The deduced amino acid sequence of Orf1 has homology to the MglC proteins of E. coli, Haemophilus influenzae, and Salmonella typhimurium. T. pallidum Orf1 (MglC) contains a conserved motif that is found in integral cytoplasmic membrane proteins of ATP-binding cassette (ABC) transport systems. T. pallidum orf2 encodes a protein of 496 amino acids with a calculated M(r) of 55,547 Da. The deduced amino acid sequence of Orf2 has homology to the MglA proteins of S. typhimurium, E. coli, H. influenzae, and Mycoplasma genitalium. Orf2 (MglA) contains two consensus ATP-binding motifs. T. pallidum mglA and mglC are located downstream of mglB, consistent with the gene order of previously identified mgl operons. The putative T. pallidum mgl operon encodes the first high-affinity ABC transport system identified in this spirochete.
    DNA Sequence 02/1996; 6(5):293-8. · 0.75 Impact Factor
  • L V Stamm, J M Hardham, J G Frye
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    ABSTRACT: An Escherichia coli clone containing recombinant plasmid C19 was identified from a Treponema pallidum genomic DNA library by in situ immunoassay. E. coli maxicells containing pC19 synthesized a treponemal protein doublet of 39.2 and 38.2 kDa, designated TpN38(b). Pulse-chase and protein processing studies showed that TpN38(b) is synthesized with a cleavable amino-terminal signal peptide. A 2.0-kb fragment of pC19 containing the tpn38(b) gene was subcloned and sequenced. The tpn38(b) gene is 1029 nucleotides long and encodes a protein of 343 amino acids with a calculated molecular mass of 37.9 kDa. The deduced amino acid sequence of TpN38(b) has homology with the T. pallidum TpN35 lipoprotein and the Borrelia burgdorferi BmpA, BmpB, BmpC, and BmpD proteins.
    FEMS Microbiology Letters 02/1996; 135(1):57-63. · 2.05 Impact Factor

Publication Stats

1k Citations
135.46 Total Impact Points

Institutions

  • 2000
    • New York Medical College
      New York City, New York, United States
    • Pfizer Inc.
      • Pfizer Global Research & Development
      New York City, New York, United States
  • 1997–2000
    • University of Texas Medical School
      • • Department of Pathology & Laboratory Medicine
      • • Department of Microbiology and Molecular Genetics
      Houston, Texas, United States
  • 1994–1997
    • University of North Carolina at Chapel Hill
      • • Department of Epidemiology
      • • Department of Microbiology and Immunology
      Chapel Hill, NC, United States