Abstract:
1. Motility of Treponema pallidum is mediated by endoflagella located within the periplasmic space. The endoflagella comprise a core surrounded by a sheath, with the sheath being composed of a 37-kD antigen (FlaA).
2. Treponemal endoflagella were isolated and component proteins were purified by reverse-phase high pressure liquid chromatography (HPLC). N-terminal amino acid sequence analysis was performed using the sheath protein and two core proteins from T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, and T. phagedenis. For all three proteins, the N-terminal sequences found for T. pallidum and T. pertenue were identical but differed from the sequence of T. phagedenis. The sheath proteins from the three treponemes were also compared by HPLC tryptic peptide maps: maps for T. pallidum and T. pertenue were very similar but differed from T. phagedenis.
3. A λgt11 clone, λA34, that expressed a portion of FlaA was selected from a T. pallidum genomic library using a murine monoclonal antibody. The insert from λA34 provided a probe with which a chimeric plasmid, pRI4, encoding all but the nine N-terminal amino acids of FlaA was selected from a pBR322 T. pallidum genomic library.
4. The nucleotide sequence of flaA upstream of amino acid 10 could not be determined by routine cloning strategies. Instead, the remaining sequence was determined using the inverse- and asymmetric-polymerase chain reaction methods. flaA contains a consensus Escherichia coli promoter and a ribosome binding site. A single open reading frame encodes a precursor protein, pre-FlaA, of 350 amino acids with a calculated Mr 38,860. A 20 amino acid signal sequence with a typical signal peptidase I cleavage site immediately precedes the known N-terminus of FlaA. Twenty six per cent (91/350) of the DNA-derived amino acid sequence was confirmed by N-terminal sequence analysis of ten tryptic peptides derived from native FlaA.
5. Polymerase chain reaction-derived constructs lacking the native T. pallidum promoter were cloned downstream of a T7 promoter. T7 polymerase transcription was under the control of a PL λ promoter. When the λ repressor was inactivated at 42°C, FlaA was expressed at relatively low levels in E. coli. Native and recombinant FlaA were identical as assessed by antibody reactivity and electrophoretic mobility in both sodium dodecyl sulphate- and two dimensional-polyacrylamide gels.
6. Although some pre-FlaA is processed to FlaA in E. coli, pre-FlaA is accumulated indicating inefficient processing of pre-FlaA in E. coli. Soluble FlaA was not detected in either the cytoplasmic or the periplasmic fractions of E. coli transformants. Fractionation of E. coli cell envelopes unexpectedly revealed that pre-FlaA and FlaA were associated with both the inner and outer membranes.
7. Two gene fusions, cro-lacZ-flaA and lacZ’-flaA, were constructed in an attempt to increase expression of the recombinant antigen; a bovine protease Xa site was engineered into the hybrid proteins immediately upstream of the N-terminus of FlaA to allow for subsequent proteolytic cleavage of the purified hybrid proteins. The LacZ’-FlaA protein was expressed in E. coli in increased amounts as compared with FlaA, but like recombinant FlaA, associated with both the inner and outer bacterial membranes. LacZ’-FlaA can be released from the membranes by treatment with 1% [wt/vol] sarkosyl, 6 M urea, or 6 M guanidine hydrochloride.