The mechanism of prion inhibition by HET-S.

HET-S (97% identical to HET-s) has an N-terminal globular domain that exerts a prion-inhibitory effect in cis on its own prion-forming domain (PFD) and in trans on HET-s prion propagation. We show that HET-S fails to form fibrils in vitro and that it inhibits HET-s PFD fibrillization in trans. In vivo analyses indicate that beta-structuring of the HET-S PFD is required for HET-S activity. The crystal structures of the globular domains of HET-s and HET-S are highly similar, comprising a helical fold, while NMR-based characterizations revealed no differences in the conformations of the PFDs. We conclude that prion inhibition is not encoded by structure but rather in stability and oligomerization properties: when HET-S forms a prion seed or is incorporated into a HET-s fibril via its PFD, the beta-structuring in this domain induces a change in its globular domain, generating a molecular species that is incompetent for fibril growth.

Contribution of specific residues of the ß-solenoid fold to HET-s prion function, amyloid structure and stability.

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific ß-solenoid fold with two rungs of ß-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-ß core, an observation that might be relevant to other amyloid models.

Identification of a novel nanoRNase in Bartonella.

In Escherichia coli, only one essential oligoribonuclease (Orn) can degrade oligoribonucleotides of five residues and shorter in length (nanoRNA). In Bacillus subtilis, NrnA and NrnB, which do not show any sequence similarity to Orn, have been identified as functional analogues of Orn. Sequence comparisons did not identify orn, nrnA or nrnB homologues in the genomes of the Chlamydia/Cyanobacteria and Alphaproteobacteria family members. Screening a genomic library from Bartonella birtlesii, a member of the Alphaproteobacteria, for genes that can complement a conditional orn mutant in E. coli, we identified BA0969 (NrnC) as a functional analogue of Orn. NrnC is highly conserved (more than 80 % identity) in the Bartonella genomes sequenced to date. Biochemical characterization showed that this protein exhibits oligo RNA degradation activity (nanoRNase activity). Like Orn from E. coli, NrnC is inhibited by micromolar amounts of 3'-phosphoadenosine 5'-phosphate in vitro. NrnC homologues are widely present in genomes of Alphaproteobacteria. Knock down of nrnC decreases the growth ability of Bartonella henselae, demonstrating the importance of nanoRNase activity in this bacterium.

The Trw type IV secretion system of Bartonella mediates host-specific adhesion to erythrocytes.

Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The alpha-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS) Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage to facilitate host-restricted adhesion to erythrocytes in a wide range of mammals.

Probing the in vivo dynamics of type I protein secretion complex association through sensitivity to detergents.

The Serratia marcescens hemophore is secreted by a type I secretion system consisting of three proteins: a membrane ABC protein, an adaptor protein, and the TolC-like outer membrane protein. Assembly of these proteins is induced by substrate binding to the ABC protein. Here we show that a hemophore mutant lacking the last 14 C-terminal amino acids is not secreted but rather interacts with the ABC protein and promotes a stable multiprotein complex. Strains expressing the transporter and the mutant protein are sensitive to detergents (sodium dodecyl sulfate [SDS]). TolC is trapped in the transporter jammed by the truncated substrate and therefore is not present at sufficient concentrations to allow the efflux pumps to expel detergents. Using an SDS sensitivity assay, we showed that the hemophore interacts with the ABC protein via two nonoverlapping sites. We also demonstrated that the C-terminal peptide, which functions as an intramolecular signal sequence in the complete substrate, may also have intermolecular activity and triggers complex dissociation in vivo when it is provided as a distinct peptide. The SDS sensitivity test on plates enables workers to study type I secretion protein association and dissociation independent of the secretion process itself.