Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli.

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodes ricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of (1)H-(15)N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in (13)C/(15)N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.

Prospective multicentre study on antimicrobial resistance of Helicobacter pylori in Germany.

OBJECTIVES: Antimicrobial resistance of Helicobacter pylori endangers the successful eradication of the bacteria. The aim of this prospective surveillance study (ResiNet) is to continuously keep antimicrobial resistance of H. pylori in Germany under surveillance and to identify risk factors for its development. METHODS: From July 2001 until December 2012, we tested the antimicrobial susceptibility of H. pylori strains isolated from 1651 prospectively enrolled patients. We analysed clinical and epidemiological data and identified risk factors for the development of resistance. RESULTS: Average primary resistances were 29.4% for metronidazole, 6.7% for clarithromycin and 3.1% for both antimicrobials. Prior unsuccessful eradication treatments, female sex and country or continent of origin were identified as independent risk factors for development of resistance. CONCLUSIONS: H. pylori-positive patients without prior eradication therapy can be treated empirically; antimicrobial susceptibility testing is recommended in previously unsuccessfully treated patients and in patients who have received antimicrobial chemotherapies due to unrelated bacterial infections.

Internal core protein cleavage leaves the hepatitis B virus capsid intact and enhances its capacity for surface display of heterologous whole chain proteins.

Virus capsids find increasing use as nanoparticulate platforms for the surface display of heterologous ligands, including as multivalent vaccine carriers. Presentation on the icosahedral hepatitis B virus capsid (HBcAg) is known to strongly enhance immunogenicity of foreign sequences, most efficiently if they are inserted into the dominant c/e1 B cell epitope, a surface-exposed loop in the center of the constituent core protein primary sequence. Even some complete proteins were successfully inserted but others, e.g. the outer surface protein A (OspA) of the Lyme disease agent Borrelia burgdorferi, impaired formation of capsid-like particles (CLPs). This difference can be rationalized by the requirement for the termini of the insert to fit into the predetermined geometry of the two acceptor sites in the carrier. We reasoned that cleavage of one of the two bonds connecting insert and carrier should relieve these constraints, provided the cleaved protein fragments remain competent to support the particle structure. Indeed, HBcAg CLPs containing a recognition site for tobacco etch virus (TEV) protease in the c/e1 loop remained intact after cleavage, as did CLPs carrying a 65-residue peptide insertion. Most importantly, in situ cleavage of a core-OspA fusion protein by coexpressed TEV protease strongly enhanced CLP formation compared with the uncleaved protein. These data attest to the high structural stability of the HBcAg CLP and they significantly widen its applicability as a carrier for heterologous proteins. This approach should be adaptable to any protein-based particle with surface-exposed yet sequence-internal loops.

Monoclonal antibodies providing topological information on the duck hepatitis B virus core protein and avihepadnaviral nucleocapsid structure.

The icosahedral capsid of duck hepatitis B virus (DHBV) is formed by a single core protein species (DHBc). DHBc is much larger than HBc from human HBV, and no high-resolution structure is available. In an accompanying study (M. Nassal, I. Leifer, I. Wingert, K. Dallmeier, S. Prinz, and J. Vorreiter, J. Virol. 81:13218-13229, 2007), we used extensive mutagenesis to derive a structural model for DHBc. For independent validation, we here mapped the epitopes of seven anti-DHBc monoclonal antibodies. Using numerous recombinant DHBc proteins and authentic nucleocapsids from different avihepadnaviruses as test antigens, plus a panel of complementary assays, particle-specific and exposed plus buried linear epitopes were revealed. These data fully support key features of the model.

A structural model for duck hepatitis B virus core protein derived by extensive mutagenesis.

Duck hepatitis B virus (DHBV) shares many fundamental features with human HBV. However, the DHBV core protein (DHBc), forming the nucleocapsid shell, is much larger than that of HBV (HBc) and, in contrast to HBc, there is little direct information on its structure. Here we applied an efficient expression system for recombinant DHBc particles to the biochemical analysis of a large panel of mutant DHBc proteins. By combining these data with primary sequence alignments, secondary structure prediction, and three-dimensional modeling, we propose a model for the fold of DHBc. Its major features are a HBc-like two-domain structure with an assembly domain comprising the first about 185 amino acids and a C-terminal nucleic acid binding domain (CTD), connected by a morphogenic linker region that is longer than in HBc and extends into the CTD. The assembly domain shares with HBc a framework of four major alpha-helices but is decorated at its tip with an extra element that contains at least one helix and that is made up only in part by the previously predicted insertion sequence. All subelements are interconnected, such that structural changes at one site are transmitted to others, resulting in an unexpected variability of particle morphologies. Key features of the model are independently supported by the accompanying epitope mapping study. These data should be valuable for functional studies on the impact of core protein structure on virus replication, and some of the mutant proteins may be particularly suitable for higher-resolution structural investigations.

Quaternary structure is critical for protein display on capsid-like particles (CLPs): efficient generation of hepatitis B virus CLPs presenting monomeric but not dimeric and tetrameric fluorescent proteins.

Self-organizing assemblies such as viral capsids may be used as symmetrical molecular platforms for the display of heterologous sequences, with applications ranging from vaccines to structural studies. The 183-amino-acid hepatitis B virus (HBV) core protein assembles spontaneously into icosahedral capsid-like particles (CLPs). The most exposed, and most immunogenic, substructure on the CLPs is a small loop that connects two long antiparallel alpha-helices which act as dimerization interface. Ninety (90) or 120 dimers multimerize into the capsid; the four-helix bundles formed by the dimers protrude as spikes from the surface. We recently demonstrated that the entire enhanced green fluorescent protein (eGFP) can be inserted into this loop, yielding CLPs that natively displayed eGFP on their surface. The central location of the insertion site requires that any insert be fixed to the carrier via both termini, with corresponding restrictions regarding insert size and structure. eGFP obviously satisfied these criteria but, surprisingly, all attempts to produce CLPs with the isostructural red fluorescent proteins DsRed1, DsRed2, and HcRed failed. Suspecting their oligomerization tendency to be responsible, we generated fusions containing instead monomeric yellow, cyan, and red fluorescent proteins (mYFP, mCFP and mRFP1). This strongly increased the yields of YFP and CFP-CLPs, and it allowed for the first time to efficiently generate red fluorescent CLPs. Hence insert quaternary structure is a highly critical factor for CLP assembly. These data have important implications for the rational design of self-assembling fusion proteins.