High alcohol-producing <i>Klebsiella pneumoniae</i> causes fatty liver disease through 2,3-butanediol fermentation pathway <i>in vivo</i>

Abstract

High alcohol-producing <i>Klebsiella pneumoniae</i> (HiAlc <i>Kpn</i>) in the gut microbiota had been demonstrated to be the causative agent of fatty liver disease (FLD). However, the catabolic pathways for alcohol production <i>in vivo</i> remain unclear. Here, we characterized the genome of HiAlc and medium alcohol-producing (MedAlc) <i>Kpn</i> and constructed an <i>adh</i> (an essential gene encoding alcohol dehydrogenase) knock-out HiAlc <i>Kpn</i> W14 strain (W14<i>Δadh</i>) using CRISPR-Cas9 system. Subsequently, we established the mouse model <i>via</i> gavage administration of HiAlc <i>Kpn</i> W14 and W14 <i>Δadh</i> strains, respectively. Proteome and metabolome analysis showed that 10 proteins and six major metabolites involved in the 2,3-butanediol fermentation pathway exhibited at least a three-fold change or greater during intestinal growth. Compared with HiAlc <i>Kpn</i> W14-fed mice, W14<i>Δadh</i>-fed mice with weak alcohol-producing ability did not show apparent pathological changes at 4 weeks, although some steatotic hepatocytes were observed at 12 weeks. Our data demonstrated that carbohydrate substances are catabolized to produce alcohol and 2,3-butanediol <i>via</i> the 2,3-butanediol fermentation pathway in HiAlc <i>Kpn</i>, which could be a promising clinical diagnostic marker. The production of high amounts of endogenous alcohol is responsible for the observed steatosis effects in hepatocytes <i>in vivo</i>.