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Mass spectrometry imaging and single-cell transcriptome reveal metabolic profiles of the postauricular glands of the Bufo gargarizans, and hypothesize Bufotoxin biosynthesis pathways

ABSTRACT: bufotoxin, which contain alkaloids, small peptides and bufadienolides (BDS), are neurotoxic and cardiotoxic, they are promising medical drugs, but the unclear specifics of bufotoxin in the postauricular glands (PG) and the biosynthetic routes of BDS are unknown. We used MALDI/MS to visualize 1872 components of PG, and established the expression profiles of 9316 cells by single-cell sequencing, which were classified into nine clusters by marker genes. We analyzed the expression differences of key genes for alkaloid, terpene backbone and steroid synthesis, identified optimal and silenced genes involved in key enzymes encoding bufotoxin biosynthesis, and hypothesized the full route of BDS synthesis via by analyzing the cholesterol metabolic pathway. BDS was enriched in EP, FB, and GG2 cells, and genes were expressed mainly in EP and FB cells. Combined with the bulk-RNA transcriptome we suggest that BDS synergistically synthesized by the liver and PG. metabolic mapping of toxin components in the PG of the toad, as demonstrated by our data.

ORGANISM(S): Bufo gargarizans

PROVIDER: GSE261560 | GEO | 2024/03/20

REPOSITORIES: GEO

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Project description:Streptococcus pneumoniae is a human commensal bacterium that causes serious diseases. Peptidoglycan (PG) is the critical component of bacteria that maintains cell shape, size, chaining, and turgor resistance. Because of its surface exposure and eubacterial-specific mechanism of biosynthesis, PG biosynthesis remains an outstanding target for discovery of new antibiotics to resistant “superbugs,” like S. pneumoniae. Pneumococcal PG biosynthesis is carried out by a balance of septal and peripheral (side-wall-like) PG synthesis mediated by bPBP2x and bPBP2b, respectively, that emanates from midcell regions. We selected for additional suppressor mutations, besides mltG, that eliminate the requirement for essential bPBP2b in peripheral PG synthesis. We found that mutations that inactivate a putative RNA binding protein, designated KhpA, suppressed the requirement for some, but not all of the essential proteins required for peripheral PG synthesis. KhpA was used as bait in co-IP experiments to demonstrate interactions with a second RNA binding protein, designated KhpB, in cells. Mutations in khpA or khpB phenocopy each other and result in slower growth rates, smaller cell size with normal aspect ratios, and induction of cell wall stress responses. RIP-Seq analyses confirmed KhpAB as RNA binding protein in cells. Another suppressor of Δpbp2b implicated induction of a cell division operon as a way to bypass the requirement for bPBP2b. We show that increased expression of this cell division operon occurs in ΔkhpAB mutants and that this induction is necessary and sufficient to account for suppression of Δpbp2b. Comparisons of relative transcript amounts and protein levels indicate that induction of this cell division operon in ΔkhpAB mutants occurs primarily at the post-transcriptional level. Together, our results show that KhpAB, which are virulence factors in S. pneumoniae and are conserved in other Gram-positive pathogens, acts as a new class of RNA binding protein, with properties analogous to Hfq in Gram-negative bacteria.

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Mass spectrometry imaging and single-cell transcriptome reveal metabolic profiles of the postauricular glands of the Bufo gargarizans, and hypothesize Bufotoxin biosynthesis pathways

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