Klebsiella oxytoca enterotoxins tilimycin and tilivalline have distinct host DNA-damaging and microtubule-stabilizing activities

Abstract

Establishing causal links between bacterial metabolites and human intestinal disease is a significant challenge. This study reveals the molecular basis of antibiotic-associated hemorrhagic colitis (AAHC) caused by intestinal resident Klebsiella oxytoca. Colitogenic strains produce the nonribosomal peptides tilivalline and tilimycin. Here, we verify that these enterotoxins are present in the human intestine during active colitis and determine their concentrations in a murine disease model. Although both toxins share a pyrrolobenzodiazepine structure, they have distinct molecular targets. Tilimycin acts as a genotoxin. Its interaction with DNA activates damage repair mechanisms in cultured cells and causes DNA strand breakage and an increased lesion burden in cecal enterocytes of colonized mice. In contrast, tilivalline binds tubulin and stabilizes microtubules leading to mitotic arrest. To our knowledge, this activity is unique for microbiota-derived metabolites of the human intestine. The capacity of both toxins to induce apoptosis in intestinal epithelial cells—a hallmark feature of AAHC—by independent modes of action, strengthens our proposal that these metabolites act collectively in the pathogenicity of colitis.

Cytotoxin-producing Klebsiella oxytoca in the preterm gut and its association with necrotizing enterocolitis

“…The three cases where antibiotics were provided distant to the onset of disease suggest that other mechanisms also may be involved. Tilimycin, a cytotoxin produced by K. oxytoca, can mitigate the growth of commensal bacteria [40]. Thus, luminal conditions that enhance tilimycin synthesis could facilitate toxin-mediated reductions in microbial competition allowing K. oxytoca to thrive.…”

Improved molecular characterization of the Klebsiella oxytoca complex reveals the prevalence of the kleboxymycin biosynthetic gene cluster

“…Little is known about the antibiotic-resistance and virulence genes encoded by K. oxytoca and related species. In the course of ongoing Klebsiella-phage work, with three GES-5-positive clinical strains (8) of K. michiganensis, we sought to determine whether widely recognized virulence factors such as enterobactin, yersiniabactin and salmochelin are encoded in the strains' genomes, and the kleboxymycin biosynthetic gene cluster (BGC), as this was until recently a little-studied BGC implicated in non-Clostridioides difficile antibiotic-associated haemorrhagic colitis (AAHC) (9)(10)(11)(12).…”

Klebsiella pneumoniae Redistributes Katanin Severing Proteins and Alters Astral Microtubules during Mitosis

“…Another strain Klebsiella ( K. oxytoca ) secretes a small molecule called tilivalline (Tse et al, ), and this molecule was recently shown to arrest A549 cells at the G2/M stage (Unterhauser et al, ). Microtubule polymerization was enhanced by tilivalline in vitro , and more multipolar spindles were observed in mitotic cells exposed to tilivalline (Unterhauser et al, ). In contrast, K. pneumoniae disassembled microtubule networks of A549 cells (Chua et al, ), and in this study, we did not observe any multipolar spindles.…”

“…In contrast, K. pneumoniae disassembled microtubule networks of A549 cells (Chua et al, ), and in this study, we did not observe any multipolar spindles. Additionally, the astral microtubules were not elongated in tilivalline‐treated mitotic cells (Unterhauser et al, ), suggesting that different mechanisms are employed by K. oxytoca and K. pneumoniae to cause cell cycle arrest. Regardless, cell cycle arrest has become a newly identified phenotype of Klebsiella infections, and through our study, we have shown that K. pneumoniae has devised its own mechanism to interfere with mitosis through the katanin family of proteins.…”