Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca.
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ABSTRACT:
SUBMITTER: Alexander EM
PROVIDER: S-EPMC7354218 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cytotoxin-producing Klebsiella oxytoca is the causative agent of antibiotic-associated hemorrhagic colitis (AAHC). Recently, the cytotoxin associated with AAHC was identified as tilivalline, a known pentacyclic pyrrolobenzodiazepine (PBD) metabolite produced by K. oxytoca Although this assertion of tilivalline's role in AAHC is supported by evidence from animal experiments, some key aspects of this finding appear to be incompatible with toxicity mechanisms of known PBD toxins. We therefore hypothesized that K. oxytoca may produce some other uncharacterized cytotoxins. To address this question, we investigated whether tilivalline alone is indeed necessary and sufficient to induce cytotoxicity or whether K. oxytoca also produces other cytotoxins. LC-MS- and NMR-based metabolomic analyses revealed the presence of an abundant tricyclic PBD, provisionally designated kleboxymycin, in the supernatant of toxigenic K. oxytoca strains. Moreover, by generating multiple mutants with gene deletions affecting tilivalline biosynthesis, we show that a tryptophanase-deficient, tilivalline-negative K. oxytoca mutant induced cytotoxicity in vitro similar to tilivalline-positive K. oxytoca strains. Furthermore, synthetic kleboxymycin exhibited greater than 9-fold higher cytotoxicity than tilivalline in TC50 cell culture assays. We also found that the biosynthetic pathways for kleboxymycin and tilivalline appear to overlap, as tilivalline is an indole derivative of kleboxymycin. In summary, our results indicate that tilivalline is not essential for inducing cytotoxicity observed in K. oxytoca-associated AAHC and that kleboxymycin is a tilivalline-related bacterial metabolite with even higher cytotoxicity.
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Project description:Klebsiella pneumoniae is a major cause of opportunistic healthcare-associated infections, which are increasingly complicated by the presence of extended-spectrum beta-lactamases (ESBLs) and carbapenem resistance. We conducted a year-long prospective surveillance study of K. pneumoniae clinical isolates in hospital patients. Whole-genome sequence (WGS) data reveals a diverse pathogen population, including other species within the K. pneumoniae species complex (18%). Several infections were caused by K. variicola/K. pneumoniae hybrids, one of which shows evidence of nosocomial transmission. A wide range of antimicrobial resistance (AMR) phenotypes are observed, and diverse genetic mechanisms identified (mainly plasmid-borne genes). ESBLs are correlated with presence of other acquired AMR genes (median n = 10). Bacterial genomic features associated with nosocomial onset are ESBLs (OR 2.34, p = 0.015) and rhamnose-positive capsules (OR 3.12, p < 0.001). Virulence plasmid-encoded features (aerobactin, hypermucoidy) are observed at low-prevalence (<3%), mostly in community-onset cases. WGS-confirmed nosocomial transmission is implicated in just 10% of cases, but strongly associated with ESBLs (OR 21, p < 1 × 10-11). We estimate 28% risk of onward nosocomial transmission for ESBL-positive strains vs 1.7% for ESBL-negative strains. These data indicate that K. pneumoniae infections in hospitalised patients are due largely to opportunistic infections with diverse strains, with an additional burden from nosocomially-transmitted AMR strains and community-acquired hypervirulent strains.
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Project description:Purpose: Klebsiella oxytoca M5a1 (previously Klebsiella pneumonia M5a1) is a principle model organism for free-living biological nitrogen fixation. This strain has been used for decades in the characterisation of nitrogenase function and the genetic regulation of nitrogen fixation physiology. Currently it represents a key model for synthetic biology and biotechnology approaches. This project involves a multi-omics approach to modelling nitrogen physiology in Klebsiella oxytoca M5a1, in order to inform rational genetic engineering for improved nitrogen fixation activity. Here we studied the transition from nitrogen replete (high NH4Cl) to nitrogen starved (NH4Cl run-out) conditions, the latter of which induce nif gene expression and synthesis of the nitrogenase enzyme. We mapped RNA-sequencing (Illumina) reads to our assembled M5aI genome in order to compare the transcriptome between nitrogen replete and nitrogen fixing conditions using differential gene expression (DEG) analysis. Methods: Total RNA was extracted from fixed M5a1 cell samples after culturing in nitrogen replete (10 mM NH4Cl; one replicate) or nitrogen starved (3 hours after complete run-out of 0.5 mM NH4Cl; two replicates) growth medium (NFDM). cDNA libraries were prepared for Illumina (NextSeq 500) sequencing, with a read length of 75 nt and a depth of 100 million reads. Following processing and quality control, sequence reads were mapped to a M5a1 genomic template (GenBank WGS accession JAFHKG010000000; BioSample SAMN17288411) and gene count normalisation performed (RPKM). Differential gene expression analysis was performed using the DEseq2 package utilising settings of regularized logarithm (rlog) normalisation of read counts, Wald hypothesis testing and p-value adjustment using a False Discovery Rate threshold of 0.05. Log2 fold changes between conditions were calculated for differentially expressed genes (DEGs) with an adjusted p value of <0.05. Results: Across the three samples, between 22.0-25.1 million reads (81.4-87.7%) were mapped to the genome, including 56.7-68.5% mapped to annotated genes. More than 50% of genes (2694/5279 genes) were differentially expressed (adjusted p-value <0.05) in the nitrogen starved condition with respect to the nitrogen replate condition. 925 genes were upregulated and 1117 genes downregulated by more than 2-fold. Of these, 1515 differentially expressed genes correspond to annotated proteins in the KEGG functional orthology (KO) database. The 20 nitrogen fixation (nif) genes were ranked among the 200 genes with highest calculated expression values (RPKM) in the nitrogen starved condition. Conclusions: Our study provides a detailed insight into the global gene expression profile associated with diazotrophic (nitrogen fixing) growth, revealing a dominant role for nif and other nitrogen regulatory genes in the adaptation to nitrogen stress, in addition to a large array of secondary genes indirectly associated with shifts in metabolism and growth phenotype (e.g. metabolic enzymes, gene expression machinery, transporters).
2021-07-09 | GSE164668 | GEO
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Project description:Klebsiella oxytoca is known to be a pathogen in immunodeficient adults and children. Here we report the first case of a K. oxytoca infection associated with spontaneous arthritis of the knee in a child with no history of immunosuppressive therapy or previous bacterial infections. Despite an initial antibiotic treatment failure, a second treatment led to a cure of the infection with no joint sequelae.
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