A 9.0-kilobase-pair circular plasmid of Borrelia burgdorferi encodes an exported protein: evidence for expression only during infection.
In this study, we report the cloning, sequencing, and molecular analysis of a gene located on a 9.0-kbp circular plasmid of virulent Borrelia burgdorferi B31 designated eppA (exported plasmid protein A). This gene encodes a precursor protein of 174 amino acids including a signal peptide of 20 amino acids and a type I signal peptidase cleavage site. The mature EppA protein of 154 amino acids has a calculated molecular weight of 17,972. Several lines of evidence suggest that eppA is not expressed by B. burgdorferi B31 during in vitro cultivation. Immunoblot analysis using hyperimmune rabbit antiserum to recombinant EppA (rEppA) did not detect the presence of EppA in B. burgdorferi B31 cultivated in vitro. Northern blot analysis using total RNA isolated from in vitro-cultivated virulent B. burgdorferi B31 failed to detect an eppA transcript. EppA was not detected in culture supernatants of virulent B. burgdorferi B31 in a sensitive antigen-capture enzyme-linked immunosorbent assay. In contrast, evidence for expression of eppA during infection was based on the observation that patients with Lyme disease as well as rabbits experimentally infected with B. burgdorferi B31 produced antibodies that recognized rEppA. Because the cellular location of EppA in B. burgdorferi cannot be determined in vivo because of very small numbers of organisms present in vertebrate infection, we examined the cellular location of rEppA expressed in Escherichia coli. In E. coli, rEppA is targeted to the outer membrane. In addition, purified E. coli outer membranes containing rEppA treated with chaotrophic agents did not result in rEppA release. These findings are consistent with the idea that EppA is not peripherally associated with the outer membrane of E. coli but rather has an integral outer membrane association.
Champion
C I
CI
Department of Microbiology & Immunology, UCLA School of Medicine 90024.
Blanco
D R
DR
Skare
J T
JT
Haake
D A
DA
Giladi
M
M
Foley
D
D
Miller
J N
JN
Lovett
M A
MA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D015748:Borrelia burgdorferi Group / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008193:Lyme Disease / Q000382:microbiology*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; C087866:EppA protein, Borrelia burgdorferi; D011993:Recombinant Fusion Proteins