Identification of Novel Serodiagnostic Signatures of Typhoid Fever Using a Salmonella Proteome Array.

Current diagnostic tests for typhoid fever, the disease caused by Salmonella Typhi, are poor. We aimed to identify serodiagnostic signatures of typhoid fever by assessing microarray signals to 4,445 S. Typhi antigens in sera from 41 participants challenged with oral S. Typhi. We found broad, heterogeneous antibody responses with increasing IgM/IgA signals at diagnosis. In down-selected 250-antigen arrays we validated responses in a second challenge cohort (n = 30), and selected diagnostic signatures using machine learning and multivariable modeling. In four models containing responses to antigens including flagellin, OmpA, HlyE, sipC, and LPS, multi-antigen signatures discriminated typhoid (n = 100) from other febrile bacteremia (n = 52) in Nepal. These models contained combinatorial IgM, IgA, and IgG responses to 5 antigens (ROC AUC, 0.67 and 0.71) or 3 antigens (0.87), although IgA responses to LPS also performed well (0.88). Using a novel systematic approach we have identified and validated optimal serological diagnostic signatures of typhoid fever.

Ribosome Induces a Closed to Open Conformational Change in Release Factor 1.

Bacterial translation termination is triggered when a stop codon arrives at the ribosomal A site. Stop codons are recognized by class I release factors (RF1 and RF2 in Escherichia coli), which bind to the ribosome and catalyze the release of the newly synthesized protein. Crystal structures showed that RF1 and RF2 are in an open conformation when bound to the ribosome but are in a closed conformation when not bound to the ribosome. It is not clear whether only the open form of RF1 and RF2 binds to the ribosome. Alternatively, the closed form of RF1 and RF2 may bind to the ribosome and undergo a conformational change to the open state upon binding. We used transition metal ion fluorescence resonance energy transfer experiments to monitor precisely the conformation of RF1 in the absence and presence of the ribosome. Our results indicate that RF1 undergoes a large conformational change from a closed to an open form upon binding to the ribosome. Our results are consistent with the mechanism, in which high termination fidelity is achieved by linking stop codon recognition by RF1 to the change in conformation from closed to open state.

Thermodynamic and kinetic insights into stop codon recognition by release factor 1.

Stop codon recognition is a crucial event during translation termination and is performed by class I release factors (RF1 and RF2 in bacterial cells). Recent crystal structures showed that stop codon recognition is achieved mainly through a network of hydrogen bonds and stacking interactions between the stop codon and conserved residues in domain II of RF1/RF2. Additionally, previous studies suggested that recognition of stop codons is coupled to proper positioning of RF1 on the ribosome, which is essential for triggering peptide release. In this study we mutated four conserved residues in Escherichia coli RF1 (Gln185, Arg186, Thr190, and Thr198) that are proposed to be critical for discriminating stop codons from sense codons. Our thermodynamic and kinetic analysis of these RF1 mutants showed that the mutations inhibited the binding of RF1 to the ribosome. However, the mutations in RF1 did not affect the rate of peptide release, showing that imperfect recognition of the stop codon does not affect the proper positioning of RF1 on the ribosome.

Deoxyribozyme-based, semisynthetic access to stable peptidyl-tRNAs exemplified by tRNAVal carrying a macrolide antibiotic resistance peptide.

We present a protocol for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. The approach is exemplified by tRNA(Val)-3'-NH-VFLVM-NH(2) and relies on commercially available Escherichia coli tRNA(Val). This tRNA was cleaved site-specifically within the TΨC loop using a 10-23 type DNA enzyme to obtain a 58 nt tRNA 5'-fragment which contained the modifications. After cleavage of the 2',3'-cyclophosphate moiety from the 5'-fragment, it was ligated to the 18 nt RNA-pentapeptide conjugate which had been chemically synthesized. By this methodology, tRNA(Val)-3'-NH-VFLVM-NH(2) is accessible in efficient manner. Furthermore, we point out that the approach is applicable to other types of tRNA.

The ribosome: a molecular machine powered by RNA.

Metal ions are the salt in the soup of essentially every biological system. Also in the ribosome, the largest natural ribozyme that produces all proteins in every living cell, metal ions have been found contributing significantly to the highly dynamic and accurate process of translation. The ribosome is considered a molecular fossil of the 'RNA world' and it could be shown that the evolutionarily oldest parts of the particle, which build the catalytic center and surrounding domains, are densely packed with divalent metal ions. Nevertheless, metal ions do not seem to directly participate in ribosomal catalysis, their important roles in the ribosome, however, cannot be denied. It is probable that mono- and divalent metal ions primarily promote the functionally competent architecture of the ribosomal RNAs, but more direct roles in mRNA decoding and reading frame maintenance are likely. Decades of biochemical studies and the recent high resolution crystallographic structures of the ribosome strongly indicate that metal ions are involved in essentially every phase of the ribosomal elongation cycle, thus contributing significantly to the precise translation of the genetic code.

Platelets enhance activity of antimycotic substances against non-Aspergillus fumigatus Aspergillus species in vitro.

Platelets are known to be part of haemostasis but they are also players in innate host defense. Recently, we observed that platelets attenuate the virulence of Aspergillus spp. in vitro. However, little is known about the antifungal effects of platelets in the presence of antimycotics against non-A. fumigatus Aspergillus species. We therefore investigated whether platelets increase the in vitro activity of amphotericin B, voriconazole, posaconazole and caspofungin against two clinical isolates each of Aspergillus flavus, Aspergillus terreus and Aspergillus niger. The antifungal activity was evaluated by assessing germination percentages, hyphal elongation and hyphal damage by use of XTT. The combination of platelets plus amphotericin B significantly (P < 0.05) enhanced the reduction of germination percentage compared to either substance alone. Among triazoles, voriconazole exhibited significant effects with platelets for all tested aspergilli. Overall, these findings suggest that among the tested antimycotic substances, amphotericin B in combination with platelets has enhancing effects in reducing germination and hyphal elongation in the tested non-A. fumigatus Aspergillus species. These data indicate that platelets act beneficially with antimycotics in an early stage of fungal growth by blocking and/or delaying fungal germination and hyphal elongation; both crucial mechanisms in the development of invasive fungal disease.

Reliable semi-synthesis of hydrolysis-resistant 3'-peptidyl-tRNA conjugates containing genuine tRNA modifications.

The 3'-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3'-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5'-fragments carrying the modifications. After dephosphorylation of the 2',3'-cyclophosphate moieties from these fragments, they are ligated to the respective 3'-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3'-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner.

Non-hydrolyzable RNA-peptide conjugates: a powerful advance in the synthesis of mimics for 3'-peptidyl tRNA termini.

Translation of specific small peptides on the ribosome can confer resistance to macrolide antibiotics. To reveal the molecular details of this and related phenomena, stable RNA-peptide conjugates that mimic peptidyl-tRNA would be desirable, especially for ribosome structural biology. A flexible solid-phase synthesis strategy now allows efficient access to these highly requested derivatives without restriction on the RNA and peptide sequences.