Molecular characterization of Clostridioides difficile ribotype 027 in a major Chinese hospital.

BACKGROUND: The rapid spread of C. difficile 027 has become one of the leading threats of healthcare-associated infections wordwild. However, C. difficile 027 infections have rarely been reported in China. The objective of this study was to strengthen the understanding of the molecular characterizations of C. difficile 027 in China. METHODS: In this study, stool specimens from 176 suspected CDI cases were collected from 1 Jan 2018 to 30 Jun 2019. These specimens were measured by GeneXpert test and C.difficile colonies were identified and analyzed. RESULTS: There were five samples positive for tcdA, tcdB, binary toxin genes and had deletions in tcdC gene. These five Clostridioides difficile isolates belonged to ST1 and confirmed as Clostridioides difficile 027 strains by PCR ribotyping. Through using whole genome sequencing, , we found that these five strains were closely clustered into the same predominant evolutionary branch and were highly similar to C. difficile 027 strain R20291. Antimicrobial susceptibility testing result showed they were highly resistant to fluoroquinolones. CONCLUSIONS: In Our study, five C. difficile 027 isolates were identified and characterized using MLST, PCR ribotyping and whole genome sequencing. We proposed that C. difficile 027 infections are probably neglected in China. Further epidemiological studies across the country together with the introduction of routine diagnostic testing and multi-center or national level surveillance are needed to ascertain the size of this potentially significant problem.

Uncovering the functional residues of Arabidopsis isoprenoid biosynthesis enzyme HDS.

The methylerythritol phosphate (MEP) pathway is responsible for producing isoprenoids, metabolites with essential functions in the bacterial kingdom and plastid-bearing organisms including plants and Apicomplexa. Additionally, the MEP-pathway intermediate methylerythritol cyclodiphosphate (MEcPP) serves as a plastid-to-nucleus retrograde signal. A suppressor screen of the high MEcPP accumulating mutant plant (ceh1) led to the isolation of 3 revertants (designated Rceh1-3) resulting from independent intragenic substitutions of conserved amino acids in the penultimate MEP-pathway enzyme, hydroxymethylbutenyl diphosphate synthase (HDS). The revertants accumulate varying MEcPP levels, lower than that of ceh1, and exhibit partial or full recovery of MEcPP-mediated phenotypes, including stunted growth and induced expression of stress response genes and the corresponding metabolites. Structural modeling of HDS and ligand docking spatially position the substituted residues at the MEcPP binding pocket and cofactor binding domain of the enzyme. Complementation assays confirm the role of these residues in suppressing the ceh1 mutant phenotypes, albeit to different degrees. In vitro enzyme assays of wild type and HDS variants exhibit differential activities and reveal an unanticipated mismatch between enzyme kinetics and the in vivo MEcPP levels in the corresponding Rceh lines. Additional analyses attribute the mismatch, in part, to the abundance of the first and rate-limiting MEP-pathway enzyme, DXS, and further suggest MEcPP as a rheostat for abundance of the upstream enzyme instrumental in fine-tuning of the pathway flux. Collectively, this study identifies critical residues of a key MEP-pathway enzyme, HDS, valuable for synthetic engineering of isoprenoids, and as potential targets for rational design of antiinfective drugs.

Evolution of the Cholesterol Biosynthesis Pathway in Animals.

Cholesterol plays essential roles in animal development and disease progression. Here, we characterize the evolutionary pattern of the canonical cholesterol biosynthesis pathway (CBP) in the animal kingdom using both genome-wide analyses and functional experiments. CBP genes in the basal metazoans were inherited from their last common eukaryotic ancestor and evolutionarily conserved for cholesterol biosynthesis. The genomes of both the basal metazoans and deuterostomes retain almost the full set of CBP genes, while Cnidaria and many protostomes have independently experienced multiple massive losses of CBP genes that might be due to the geologic events during the Ediacaran period, such as the appearance of an exogenous sterol supply and the frequent perturbation of ocean oxygenation. Meanwhile, the indispensable utilization processes of cholesterol potentially strengthened the maintenance of the complete set of CBP genes in vertebrates. These results strengthen both biotic and abiotic roles in the macroevolution of a biosynthesis pathway in animals.

Insights into Pipecolic Acid Biosynthesis in Huperzia serrata.

For the biosynthesis of Pip in Huperzia serrata, the mechanistic studies were evaluated. Through a series of biochemical analyses, Pip is biosynthesized through a two-step cascade reaction. Three intermediates possibly exist simultaneously as an equilibrium matter in the first-step reaction catalyzed by HsAld1, while HsSard4 performs as a ketimine reductase and chemoselectively and stereoselectively takes 1,2-dehydropipecolic acid as the preferred substrate in vitro.

Construction of an octosyl acid backbone catalyzed by a radical S-adenosylmethionine enzyme and a phosphatase in the biosynthesis of high-carbon sugar nucleoside antibiotics.

Unique bicyclic octosyl uronic acid nucleosides include ezomycin, malayamycin, and octosyl acid (OA). They are structurally characterized by OA, an unusual 8-carbon furanosyl nucleoside core proposed to be the precursor to polyoxin and nikkomycin. Despite the well-known bioactivity of these nucleoside antibiotics, the biosynthesis of OA has not been elucidated yet. Here we report the two pivotal enzymatic steps in the polyoxin biosynthetic pathway leading to the identification of OA as a key intermediate. Our data suggest that this intermediate is formed via a free radical reaction catalyzed by the radical S-adenosylmethionine (SAM) enzyme, PolH, and using 3'-enolpyruvyl uridine 5'-monophosphate (3'-EUMP) as a substrate. Subsequent dephosphorylation catalyzed by phosphatase PolJ converts the resulting octosyl acid 5'-phosphate (OAP) to OA. These results provide, for the first time, significant in vitro evidence for the biosynthetic origins of the C8 backbone of OA.

Antioxidant and free radical-scavenging activity of the extracellular death factor in Escherichia coli.

The extracellular death factor (EDF) is a newly discovered linear pentapeptide with the sequence of H-Asn-Asn-Trp-Asn-Asn-OH (NNWNN). It could act as a quorum-sensing molecule and participate in the mazEF-mediated cell death in Escherichia coli. In the present study, we firstly studied the antioxidant and free radical-scavenging activities of EDF. EDF could scavenge hydroxyl radicals in vitro and protect protein, lipid and DNA from being damaged by hydroxyl radicals. Our results indicated that this extracellular death factor might have dual effects during the programmed cell death process of Escherichia coli.