Crystal Structure of Inhibitor-Bound Bacterial Oligopeptidase B in the Closed State: Similarity and Difference between Protozoan and Bacterial Enzymes.

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the ß-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the ß-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations.

Splice-site variant in the RPS7 5'-UTR leads to a decrease in the mRNA level and development of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by erythroid aplasia. Pathogenic variants in ribosomal protein (RP) genes, GATA1, TSR2, and EPO, are considered to be the etiology of DBA. Variants in 5'-untranslated regions (UTRs) of these genes are poorly studied and can complicate the variant interpretation. We investigated the functional consequences NM_001011.4:c.-19 + 1G > T variant in the donor splice-site of the RPS7 5'-UTR. This variant was found in a family where two sons with DBA were carriers. Father, who also had this variant, developed myelodysplastic syndrome, which caused his death. Search for candidate causal variants and copy number variations in DBA-associated genes left RPS7 variant as the best candidate. Trio whole exome sequencing analysis revealed no pathogenic variants in other genes. Functional analysis using luciferase expression system revealed that this variant leads to disruption of splicing. Also, a decrease in the levels of mRNA and protein expression was detected. In conclusion, the established consequences of 5'-UTR splice-site variant c.-19 + 1G > T in the RPS7 gene provide evidence that it is likely pathogenic.

Mega-plasmid found worldwide confers multiple antimicrobial resistance in Salmonella Infantis of broiler origin in Russia.

Plasmids which are the mobile part of the bacterial genome can acquire and carry over genes conferring antimicrobial resistance, thus contributing to rapid adaptation of bacterial community to human-defined environment. In 2014, Israeli scientists have reported a large conjugative mega-plasmid pESI (plasmid for emerging S. Infantis) that provides multiple drug resistance (MDR) of Salmonella Infantis isolated from broilers. Later, very similar pESI-like plasmids have been found in Salmonella isolated from poultry in the United States, Italy, Switzerland, Hungary, and Japan. Here we report detection of pESI-like plasmids in Salmonella Infantis isolated from chicken food products in Russia. Whole genome sequencing of three MDR isolates revealed pESI-like plasmids in all three cases. These plasmids have such typical pESI features as a locus for siderophore yersiniabactin, a cluster of IncI1 conjugative genes, a cluster of type IV pilus genes, and three toxin-antitoxin modules. The pESI-like plasmids carry from two to five resistance genes in each isolate. In total, we observed six antimicrobial resistance genes associated with pESI-like plasmids (aadA1, blaCTX-M-14, dfrA14, sul1, tetA/tetR, tetM). Besides plasmid genes of antimicrobial resistance, all three MDR isolates of S. Infantis harbor a mutation in chromosomal gene gyrA (p.S83Y or p.D87Y) that is associated with resistance to fluoroquinolones. In addition, we performed a comparative bioinformatics meta-analysis of 25 pESI-like plasmids hosted by S. Infantis from the USA, Europe, Latin America, Israel, and Japan. This analysis identified a 173 kB sequence that is common for all pESI-like plasmids and carries virulence operons and toxin-antitoxin modules.