Type IV pili (T4P) are extraordinarily widespread surface appendages made by many species of extant
archaea and bacteria as well as by the last universal common ancestor. T4Ps are assembled by a complex,
multi-component machine that consumes energy from ATP hydrolysis. Many human pathogens require T4P for
virulence. Volunteer studies have proven that T4P of enteropathogenic Escherichia coli (EPEC) and Vibrio
cholerae, which make type IVb pili (T4bP) that diverged from other T4P early in evolutionary history, are
required for illness in people. Despite remarkable progress, the molecular details of T4P assembly remain
obscure. The overall structure of the T4P machinery is remarkably conserved, but closer investigation reveals
fundamental differences among systems. Which findings from convenient model systems can be extrapolated
to pathogens remains unclear. For example, we recently solved the structure of the BfpD extension ATPase
from EPEC to 3.0 Å resolution using cryo-EM. In contrast to the two-fold symmetry and complex enzyme
kinetics of the distantly-related extension ATPases from thermophiles, the BfpD ATPase has six-fold symmetry
and simple kinetics, consistent with a concerted mechanism of catalysis rather than the symmetric rotary
mechanism proposed for its counterparts. In the current brief, circumscribed multi-investigator proposal, we
aim to determine to what extent the structure and mechanism of catalysis exhibited by BfpD is representative
of other potentially lethal human pathogens. To do so, we have purified the extension ATPases from four other
pathogens. Since initial (A0) submission of this revised (A1) proposal, we collected cryo-EM images of the PilB
extension ATPase from Pseudomonas aeruginosa. The 2D classes from these images demonstrate
unambiguous two-fold symmetry like the structures obtained from thermophiles with which it is closely related.
Furthermore, the structure of one such enzyme, which was derived from X-ray crystallography, fits well into our
3D PilB model, currently at 3.4 Å resolution. In the first aim of the current proposal, we will use cryo-EM to
determine the structure of the TcpT ATPase from Vibrio cholerae, which is more closely related to EPEC BfpD
than to those from thermophiles or P. aeruginosa. This structure will reveal whether it has two-fold, six-fold, or
perhaps even three-fold symmetry. In Aim 2 we will perform careful enzyme kinetic studies to determine
whether each of the four additional extension ATPases that we purified exhibits classic Michaelis-Menten
monophasic kinetics like BfpD or complex multiphasic kinetics like the extension ATPase described for a
thermophilic bacterium.
Differences among diverse T4P systems indicate that efforts to develop new approaches to prevent and
treat lethal bacterial pathogens will benefit from a better understanding of which essential features of T4P are
specific to a subset of the organisms and which are universal. As the extension ATPase is a potential target for
anti-virulence therapeutics, the efforts described herein will provide important information regarding its
mechanism of action in pathogens.