Functional and structural analysis of the "Yersinia enterocolitica" type III secretion translocon

Many pathogenic bacteria use type III secretion systems (TTSS) to deliver effector
proteins into the cytosol of target cells to subvert host cell functions. The actual secretion
apparatus, called injectisome, consist of a basal body embedded in the bacterial membranes
and a needle. The needle is thought to serve as a conduit for protein secretion. However, to
cross the target cell membrane an additional translocation step is necessary. This
translocation involves the formation of a pore in the target cell membrane, which is
presumably connected to the needle. Three proteins are required for the assembly of this
pore. In Yersinia, the three “translocators” are YopB and YopD, two putative membrane
proteins, and LcrV a hydrophilic protein. LcrV is also known, since the mid-fifties, to represent
the major protective antigen against plague infections. The aim of my thesis was to
characterize the structure and function of the translocators.
Infection of erythrocytes with wildtype Yersiniae causes hemolysis due to the formation
of the translocation pore in the red blood cell membrane. We showed that the isolated
membranes of these erythrocytes contain the hydrophobic translocators YopB and YopD, but
not LcrV. Bacteria deprived of LcrV did not assemble a functional pore, but were still able to
insert reduced amounts of YopB and YopD into the target cell membrane. This is in
agreement with reports showing that purified YopB and YopD can oligomerize and insert into
artificial membranes independently of LcrV. We showed further that polyclonal antibodies
directed against LcrV interfere with the formation of a functional translocation pore by live
bacteria. Based on these results, we hypothesized that LcrV acts as a platform or scaffold
onto which the YopBD translocation pore assembles (Goure, Broz et al. 2005, Journal of
Infectious Diseases 192:218-25).
We purified needles and detected LcrV as well as YscF, the needle subunit, in these
preparations. In parallel we analyzed these purified needles by STEM (scanning transmission
electron microscopy) and found that the needle ends with a defined tip complex, that
comprises a head, a neck and a base. We then showed that the tip complex is missing in lcrV
mutant bacteria and can be restored after the mutation is complemented in trans. These
results indicated that LcrV is involved in the formation of the tip complex. In addition,
crosslinking of purified needles indicated that LcrV and YscF interact and thus the V-antigen
might form the tip complex. Immunolabelling of wildtype needles with anti-LcrV antibodies
showed a strong binding to the tip complex, anti-YscF antibodies bound to the bottom of the
needle. Together these results demonstrate that LcrV forms the observed tip complex and
explain why anti-LcrV antibodies can inhibit pore formation. In addition, these data reinforce
the assembly platform hypothesis (Mueller, Broz et al. 2005, Science 310: 674-676).
P. aeruginosa and A. salmonicida possess an injectisome closely related to that of
Yersinia. Their respective LcrV orthologs, PcrV (32.3 kDa) and AcrV (40.2 kDa) are slightly
different in size to LcrV (37.6 kDa). We demonstrated that PcrV as well as AcrV can
functionally complement a lcrV deletion in Y. enterocolitica . The needles exhibited distinct tip
complexes similar to those of wildtype needles but they were smaller in the case of PcrV and
larger with AcrV (Mueller, Broz et al. 2005, Science 310: 674-676). Hybrids between the three
proteins LcrV, PcrV and AcrV could complement an lcrV deletion in Y. enterocolitica in the
hemolysis assay, but the level of complementation varied. The amino-terminus seemed to
play an important role in the function of the protein. STEM analysis of tip complexes formed by
different hybrid proteins allowed us to show that the aminoterminal domain of LcrV forms the
base while the second globular domain forms the head of the tip complex. In addition we
determined the stoichiometry of YscF and LcrV in purified needles and found that between
three to six molecules of LcrV form the tip complex. Together, these results allowed us to
propose an atomic modeling of the LcrV tip complex on top of the injectisome needle.