Proteins injected by the bacterial pathogen "Bartonella" subvert eukaryotic cell signaling

Conclusions
What are the substrates of the VirB/VirD4 T4SS of Bartonella
henselae?
Starting point for the first published report (“A bipartite signal mediates the
transfer of type IV secreted substrates of Bartonella henselae into human cells”,
Chapter 3.1) in my Ph.D. thesis was the finding that the VirB/VirD4 T4SS of B.
tribocorum was essential for the pathogen to establish an intra-erythrocytic
infection in an animal model (1). Additionally, at that time, unpublished data
indicated that many phenotypes which were already known for B. henselae
infecting HUVECs, as anti-apoptosis (2), cytoskeletal rearrangements (3) and
pro-inflammatory activation (4), were dependent on a intact VirB/VirD4 T4SS
(5). These findings were suggestive of substrates being translocated through the
VirB/VirD4 T4SS of Bartonella into the host cells.
Sequencing 23 kb downstream of the virB locus of B. henselae revealed a
coupling protein (virD4), and seven genes encoding at least one common Cterminal
domain. The proteins encoded by these seven genes were later termed
Bartonella exported proteins (Beps), the common C-terminal domain they
contain Bartonella intracellular delivery (BID). By constructing a Hidden
Markov Model from these BID domains and querying protein databases, we
found similar domains in the C-terminus of relaxases from conjugative plasmids
in the
α-proteobacteria. We showed exemplarily that the C-terminus of the TraA
relaxase from the AvhB/TraG conjugation system in A. tumefaciens – one of the
top hits in the database search - could still be translocated by the VirB/VirD4
T4SS of B. henselae. Both these findings support that the BID domain evolved
from conjugative relaxases, in parallel with the T4SS of B. henselae. Full-length
relaxases bind covalently to plasmid DNA and mediate its transfer through
T4SSs. This allows the fascinating speculation that the VirB/VirD4 T4SS of
Bartonella could be used to export DNA by those means in vivo into host cells.
To demonstrate exemplarily the translocation of the Beps through the
VirB/VirD4 T4SS, we fused a FLAG-tag to the N-terminus of BepD and could
show that BepD is translocated into infected endothelial cells in a VirB/VirD4
T4SS-dependent manner, whereupon it localizes to the cytoplasm of these cells
and is tyrosine-phosphorylated by host-cell kinases. The precise experimental
delineation of the domain needed for translocation was made possible by the
development of the Cre-recombinase reporter assay for translocation (CRAFT),
which showed the translocation domain to be bipartite. In addition to the BID
domain, a short, positively charged C-terminal amino acid sequence was needed
for an effective delivery of proteins.
Non-polar deletions of all the ORFs encoding the Beps abolished the ability of Bh
to induce a variety of host-cell phenotypes, suggesting that these proteins elicit
biological effects in their eukaryotic target cells. This finding was the fundament
for the two Manuscripts presented in Chapter 3.3, "A translocated protein of the
vascular-tumor inducing pathogen Bartonella protects human vascular
endothelial cells from Apoptosis" and “Subversion of host cell cytoskeletal
function during invasome-mediated uptake of Bartonella henselae into human
endothelial cells”. In each of these two manuscripts, a major phenotype of the
infection of HUVECs by B. henselae is shown to depend on a single Bep. While
BepA inhibits the apoptosis of endothelial cells, BepG does induce massive
cytoskeletal rearrangements. Interestingly, it is the BID domain of BepA which
mediates the anti-apoptotic activity in the host cell as well as the localization of
this protein to the plasma membrane. This involvement of the BID in localization
and function in the host cell holds also true for BepE, as we showed that the two
BID domains of this protein are crucial for its localization (Chapter 3.2), and to
inhibit the fragmentation of infected endothelial cells (Chapter 3.4).
Many interfaces for interactions over phosphotyrosines
Of the seven Bep proteins, three contain putative tyrosine-phosphorylation
motifs in their N-terminus. We showed that both BepD and BepE are tyrosinephoshorylated
by the c-Src kinase. Because of their similar substrate specificities
and their variable expression levels depending on the cell type, any member of
the SFK could potentially be the kinase of BepD and BepE in vivo. Bioinformatics
and preliminary experiments suggest c-Abl as the kinase of BepF. As Crk was
shown to bind BepF, and one of the functions of Crk has been described to
activate c-Abl, one might speculate that a positive activation loop takes place
once BepF has been phosphorylated, which would bind and activate increasing
amounts of the c-Abl kinase to BepF through Crk. Whereas c-Abl has been
implicated in the invasion process of other pathogens (6), the exact function of
BepF in Bartonella still remains to be uncovered.
BepD localizes in immunofluorescence stainings to a cytoplasmic vesicular-like
compartment. Additionally, it localizes upon its tyrosine-phosphorylation to a
Triton X-1oo insoluble fraction, and binds SHP2 and Csk. Lipid rafts are
prominently associated to Triton X-100 insoluble fractions, and future studies
might show BepD interfering with the signaling in these rafts.
I subsequently focused on BepE, for which bioinformatics revealed an intriguing
similarity to inhibitory immune receptors of mammals. This resulted in the
second manuscript “Molecular mimicry of inhibitory immune receptors by the
bacterial pathogen Bartonella” (Chapter 3.2). BepE contains two C-terminal BID
domains which mediate the localization of this protein to the plasma membrane
of HELA cells and to the plasma membrane and cell-cell contacts in endothelial
cells, as shown by a co-localizing immunofluorescence staining with VE-Cadherin
in HUVECs. This peculiar localization is very intriguing, and might modulate the
cell-cell contact strength or the contact inhibition which is crucial in endothelial
cells. Recently, a publication demonstrated that VE-Cadherin contains in its
intracellular domain a binding site for the SH2 domain of Csk, the binding of Csk
to this site being crucial for the contact inhibition of cell growth (7). We
identified a very similar motif in the N-terminus of BepE which did also bind
Csk. The co-localization and a similar tyrosine-phosphorylation motif binding the
same protein makes this finding a good starting point for investigating the
functions of BepE in the endothelium.
BepE contains in addition to this Csk-binding motif two immunotyrosine
inhibitory motif – immunotyrosine-based switch motif (ITIM/ITSM) tandems.
These motifs are commonly found in the intracellular domain of inhibitory
immune receptors. In vitro phosphorylation with the c-Src kinase and
subsequent mapping by mass spectrometry analysis indicated that c-Src
tyrosine-phosphorylates the Csk-binding site and both ITIMs of BepE. No
phosphorylation of an ITSM could be detected, which might be due to the fact
that another kinase phosphorylates these motifs in vivo. Both the inhibitory
immune receptors and BepE do contain ITIMs, ITSMs, Csk-binding sites, localize
to the plasma membrane, are tyrosine-phosphorylated by Src family kinases and
bind SHP2 and Csk. Additionally, BepE is constitutively tyrosine-phosphorylated
in HEK293T cells as well as in the primary HUVECs (Chapter 3.2), in contrast to
most inhibitory immune receptors, which are thought to only become
phosphorylated upon engagement of their extracellular ligands. All these lines of
evidence indicate BepE mimicking inhibitory immune receptors.
Csk contains one SH2 domain, SHP2 two. To elucidate the binding sites of these
two proteins, and also to be able to use mutants lacking these interactions, we
generated a panel of tyrosine-to-phenylalanine exchange mutants in BepE.
While Csk binds to one motif (the one with the similarity to the Csk-binding site
of VE-Cadherin), SHP2 interacts with motifs in the two ITIM-ITSM tandems.
While this interaction study was carried out in HEK293T cells, more potential
ITIM/ITSM-binding proteins such as SHP-1 and SHIP, EAT-2 and SAP might
increase the complexity of the picture in myeloid and lymphoid cells. The two
adapter proteins SAP and EAT-2 have been shown to bind to ITSMs. While some
cells such as NK express both adapters, other cells as for example T-cell do only
express SAP, and other such as DCs only EAT-2. While SAP recruits Fyn and can
lead to an increase in cell activation, EAT-2 recruits phosphatases and Csk,
inhibiting the activity. For BepE, this opens the possibility of switching its mode
of action, depending on the cell type it is translocated into.
Collaborations have been initiated to assess the immunomodulatory potential of
BepE and we are currently studying the impact of this protein in our HUVEC
models.
To summarize, my Ph.D. thesis aimed at investigating the VirB/VirD4 T4SS of
Bartonella henselae for the presence of secreted substrates and signals mediating
this secretion. Additionally, to describe the functions and interaction partners of
these substrates in the host cell, with an emphasis on the putative tyrosinephosphorylated
effectors. The core findings of this thesis are a.) The discovery
of seven modular substrates secreted by the apparatus b.) Description of the BID
domain mediating the secretion of proteins and protein-DNA complexes, c.) Csk
and SHP2 being interaction partners for BepD and BepE on the host cell side d.)
BepE mimicking inhibitory immune receptors.