Jerrilyn K. Howell

A plasmid-encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild‐type Borrelia. This complementation also restored the growth and host adaptation of lp25–B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re‐establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid‐encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.

Identification and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transport system, an iron-activated repressor protein homolog, and a glycolytic pathway enzyme homolog

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.

Transcriptome of Treponema pallidum: Gene Expression Profile during Experimental Rabbit Infection

RNA transcript levels in the syphilis spirochete Treponema pallidum subsp. pallidum (Nichols) isolated from experimentally infected rabbits were determined by the use of DNA microarray technology. This characterization of the T. pallidum transcriptome during experimental infection provides further insight into the importance of gene expression levels for the survival and pathogenesis of this bacterium.

Genome Scale Identification of Treponema pallidum Antigens

ABSTRACT Antibody responses for 882 of the 1,039 proteins in the proteome of Treponema pallidum were examined. Sera collected from infected rabbits were used to systematically identify 106 antigenic proteins, including 22 previously identified antigens and 84 novel antigens. Additionally, sera collected from rabbits throughout the course of infection demonstrated a progression in the breadth and intensity of humoral immunoreactivity against a representative panel of T. pallidum antigens.

Reactivity of Antibodies from Syphilis Patients to a Protein Array Representing the Treponema pallidum Proteome

ABSTRACT To identify antigens important in the human immune response to syphilis, the serum antibody reactivity of syphilitic patients was examined with 908 of the 1,039 proteins in the proteome of Treponema pallidum subsp. pallidum using a protein array enzyme-linked immunosorbent assay. Thirty-four proteins exhibited significant reactivity when assayed with human sera from patients in the early latent stage of syphilis. A subset of antigens identified were further scrutinized for antibody reactivity at primary, secondary, and latent disease stages, and the results demonstrate that the humoral immune response to individual T. pallidum proteins develops at different rates during the time course of infection.

Genome Differences between Treponema pallidum subsp. pallidum Strain Nichols and T. paraluiscuniculi Strain Cuniculi A

ABSTRACT The genome of Treponema paraluiscuniculi strain Cuniculi A was compared to the genome of the syphilis spirochete Treponema pallidum subsp. pallidum strain Nichols using DNA microarray hybridization, whole-genome fingerprinting, and DNA sequencing. A DNA microarray of T. pallidum subsp. pallidum Nichols containing all 1,039 predicted open reading frame PCR products was used to identify deletions and major sequence changes in the Cuniculi A genome. Using these approaches, deletions, insertions, and prominent sequence changes were found in 38 gene homologs and six intergenic regions of the Cuniculi A genome when it was compared to the genome of T. pallidum subsp. pallidum Nichols. Most of the observed differences were localized in tpr loci and the vicinity of these loci. In addition, 14 other genes were found to contain frameshift mutations resulting in major changes in protein sequences. Analysis of restriction target sites representing 0.34% of the total genome length and DNA sequencing of three PCR products (0.46% of the total genome length) amplified from Cuniculi A chromosomal regions and comparison to the Nichols genome revealed a sequence similarity of 98.6 to 99.3%. These results are consistent with a close genetic relationship among the T. pallidum strains and subspecies and a strong, but relatively divergent connection between the human and rabbit pathogens.

The Thermophilic, Homohexameric Aminopeptidase of Borrelia burgdorferi Is a Member of the M29 Family of Metallopeptidases

ABSTRACT Proteases are implicated in several aspects of the physiology of microorganisms, as well as in host-pathogen interactions. Aminopeptidases are also emerging as novel drug targets in infectious agents. In this study, we have characterized an aminopeptidase from the spirochete Borrelia burgdorferi, the causative agent of Lyme disease. The aminopeptidolytic activity was identified in cell extracts from B. burgdorferi by using the substrate leucine-7-amido-4-methylcoumarin. A protein displaying this activity was purified from B. burgdorferi by a two-step chromatographic procedure, yielding a ∼300-kDa homo-oligomeric enzyme formed by monomers of ∼50 kDa. Gel enzymography experiments showed that enzymatic activity depends on the oligomeric structure of the protease but does not involve interchain disulfide bonds. The enzyme was identified by peptide mass fingerprinting as the putative aminopeptidase II of B. burgdorferi, encoded by the gene BB0069. It shares significant identity to members of the M29/T family of metallopeptidase, is sensitive to bestatin, has a neutral pH optimum, and displays maximal activity at 60°C. Its activity is 1.75-fold higher at the temperature of the mammalian host than at that of the insect host of the pathogen. The activity of this thermophilic aminopeptidase of B. burgdorferi (TAPBb) depends on Zn2+, and temperatures over 70°C promoted its inactivation through a transition from the hexameric state to the monomeric state. Since B. burgdorferi is deficient in pathways for amino acid synthesis, TAPBb could play a role in supplying required amino acids. Alternatively, the enzyme could be involved in peptide and/or protein processing.