Unravelling the mechanism of non-ribosomal peptide synthesis by cyclodipeptide synthases.

International audienceCyclodipeptide synthases form cyclodipeptides from two aminoacyl transfer RNAs. They use a ping-pong mechanism that begins with transfer of the aminoacyl moiety of the first aminoacyl tRNA onto a conserved serine, yielding an aminoacyl enzyme. Combining X-ray crystallography, site-directed mutagenesis and affinity labelling of the cyclodipeptide synthase AlbC, we demonstrate that the covalent intermediate reacts with the aminoacyl moiety of the second aminoacyl tRNA, forming a dipeptidyl enzyme, and identify the aminoacyl-binding sites of the aminoacyl tRNAs

Specificity determinants for the two tRNA substrates of the cyclodipeptide synthase AlbC from Streptomyces noursei

International audienceCyclodipeptide synthases (CDPSs) use two aminoacyl-tRNA substrates in a sequential ping-pong mechanism to form a cyclodipeptide. The crystal structures of three CDPSs have been determined and all show a Rossmann-fold domain similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs). Structural features and mutational analyses however suggest that CDPSs and aaRSs interact differently with their tRNA substrates. We used AlbC from Streptomyces noursei that mainly produces cyclo(l-Phe-l-Leu) to investigate the interaction of a CDPS with its substrates. We demonstrate that Phe-tRNA Phe is the first substrate accommodated by AlbC. Its binding to AlbC is dependent on basic residues located in the helix ␣4 that form a basic patch at the surface of the protein. AlbC does not use all of the Leu-tRNA Leu isoacceptors as a second substrate. We show that the G 1-C 72 pair of the acceptor stem is essential for the recognition of the second substrate. Substitution of D163 located in the loop ␣6–␣7 or D205 located in the loop ␤6–␣8 affected Leu-tRNA Leu isoacceptors specificity, suggesting the involvement of these residues in the binding of the second substrate. This is the first demonstration that the two substrates of CDPSs are accommodated in different binding sites

CD4+ T cell epitopes of FliC conserved between strains of Burkholderia: implications for vaccines against melioidosis and cepacia complex in cystic fibrosis.

Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in B. pseudomallei-exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in B. pseudomallei endemic regions as well as CF patients

Study of bicyclomycin biosynthesis in Streptomyces cinnamoneus by genetic and biochemical approaches

International audienceThe 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Bicyclomycin is a clinically-relevant DKP antibiotic that is the first and only member in a class known to target the bacterial transcription termination factor Rho. It derives from cyclo-(L-isoleucyl-L-leucyl) and has an unusual and highly oxidized bicyclic structure that is formed by an ether bridge between the hydroxylated terminal carbon atom of the isoleucine lateral chain and the alpha carbon of the leucine in the diketopiperazine ring. Here, we paired in vivo and in vitro studies to complete the characterization of the bicyclomycin biosynthetic gene cluster. The construction of in-frame deletion mutants in the biosynthetic gene cluster allowed for the accumulation and identification of biosynthetic intermediates. The identity of the intermediates, which were reproduced in vitro using purified enzymes, allowed us to characterize the pathway and corroborate previous reports. Finally, we show that the putative antibiotic transporter was dispensable for the producing strain

The Tumor Antigen Cyclin B1 Hosts Multiple CD4 T Cell Epitopes Differently Recognized by Pre-Existing Naive and Memory Cells in Both Healthy and Cancer Donors

International audienceCyclin B1 (CCNB1) is considered as a potential target for a cancer vaccine, as it is overexpressed in many malignant cells, while being transiently expressed in normal cells. To evaluate the CD4 T cell response to CCNB1, we derived T cell lines by multiple weekly rounds of stimulation with recombinant CCNB1 of T cells collected in healthy donors (long-term T cell assays). T cell lines were specific for 15 immunodominant peptides and derived preferentially from naive T cells. From 74 overlapping peptides, 20 peptides were selected for their broad specificity of binding to HLA class II molecules and included most of the immunodominant epitopes. They primed in vitro a large number of specific CD4 T cell lines in all the donors. Immunodominant epitopes were the most efficacious in long-term T cell assays, both in terms of number of specific T cell lines and number of responding donors. The 20 peptides were also submitted to short-term T cell assays using cells collected in healthy and cancer patients with the aim to evaluate the memory response. The recognized peptides differed from the immunodominant peptides and were part of the best promiscuous peptides. We also observed pre-existing CCNB1-specifc IgG Abs in both healthy and cancer donors. Long-and short-term T cell assays revealed that CCNB1 contained many CD4 T cell epitopes, which are differentially recognized by pre-existing naive and memory CD4 T cells. These observations are of value for the design of cancer vaccines

Analysis of 51 cyclodipeptide synthases reveals the basis for substrate specificity.

International audienceCyclodipeptide synthases (CDPSs) constitute a family of peptide bond-forming enzymes that use aminoacyl-tRNAs for the synthesis of cyclodipeptides. Here, we describe the activity of 41 new CDPSs. We also show that CDPSs can be classified into two main phylogenetically distinct subfamilies characterized by specific functional subsequence signatures, named NYH and XYP. All 11 previously characterized CDPSs belong to the NYH subfamily, suggesting that further special features may be yet to be discovered in the other subfamily. CDPSs synthesize a large diversity of cyclodipeptides made up of 17 proteinogenic amino acids. The identification of several CDPSs having the same specificity led us to determine specificity sequence motifs that, in combination with the phylogenetic distribution of CDPSs, provide a first step toward being able to predict the cyclodipeptides synthesized by newly discovered CDPSs. The determination of the activity of ten more CDPSs with predicted functions constitutes a first experimental validation of this predictive approach