Background: Treponema pallidum is predicted to express approximately 1000 proteins (the T. pallidum proteome), yet expression of only 69% of these proteins has been experimentally confirmed. In the current study our goals were to improve treponemal proteome coverage and identify proteins that contain post-translational modifications (PTMs; modifications added after protein biosynthesis that change protein properties). Achieving these goals will provide novel insight into T. pallidum pathogenesis and will inform syphilis vaccine development.
Methods: To improve T. pallidum proteome coverage, we developed a workflow comprised of four key steps: (1) use of both in vivo- and in vitro-grown T. pallidum; (2) method optimization for lysis of T. pallidum and protein isolation; (3) enhanced sample preparation and processing to simplify mass spectrometry analyses; and (4) efficient data analyses to improve identification of proteins and PTMs.
Results: Our workflow detected 94% of predicted T. pallidum proteins. When combined with previous studies, our workflow increased the T. pallidum proteome coverage from 69% to 95% (26% increase). The expression of 11 predicted outer membrane proteins, several of which have been identified as potential syphilis vaccine candidates, was detected for the first time. Several predicted proteins deleted from the most current proteome annotations were also identified, indicating current proteome annotations contain inaccuracies and may be missing important proteins. PTMs including glycosylations, methylations, acetylations and unknown modifications were identified throughout the T. pallidum proteome, a consideration that must be taken into account with vaccine design.
Conclusion: Our results provide deep coverage of the T. pallidum proteome, at the protein expression and PTM levels, and to the author’s best knowledge represent the highest coverage of a bacterial proteome to date. We present a robust pipeline for determining T. pallidum protein expression profiles, an essential early consideration in vaccine design. These findings will guide syphilis vaccine design by: (1) confirming expression of T. pallidum vaccine candidates; (2) identifying PTMs on vaccine candidates, enabling engineering of subunit vaccines to closely resemble the natural protein forms presented to the host during infection; and (3) identifying novel syphilis vaccine candidates via the detection of proteins missed in the proteome annotation.
