Abstract
Klebsiella oxytoca is a resident of the human gut. However, certain K. oxytoca toxigenic strains exist that secrete the nonribosomal peptide tilivalline (TV) cytotoxin. TV is a pyrrolobenzodiazepine that causes antibiotic-associated hemorrhagic colitis (AAHC). The biosynthesis of TV is driven by enzymes encoded by the aroX and NRPS operons. In this study, we determined the effect of environmental signals such as carbon sources, osmolarity, and divalent cations on the transcription of both TV biosynthetic operons. Gene expression was enhanced when bacteria were cultivated in tryptone lactose broth. Glucose, high osmolarity, and depletion of calcium and magnesium diminished gene expression, whereas glycerol increased transcription of both TV biosynthetic operons. The cAMP receptor protein (CRP) is a major transcriptional regulator in bacteria that plays a key role in metabolic regulation. To investigate the role of CRP on the cytotoxicity of K. oxytoca, we compared levels of expression of TV biosynthetic operons and synthesis of TV in wild-type strain MIT 09-7231 and a Δcrp isogenic mutant. In summary, we found that CRP directly activates the transcription of the aroX and NRPS operons and that the absence of CRP reduced cytotoxicity of K. oxytoca on HeLa cells, due to a significant reduction in TV production. This study highlights the importance of the CRP protein in the regulation of virulence genes in enteric bacteria and broadens our knowledge on the regulatory mechanisms of the TV cytotoxin.
Highlights
The human gut microbiota is a complex community of microbial species that plays a fundamental role in the health and functioning of the human digestive tract
To determine the optimal conditions of expression of the aroX and NRPS operons of K. oxytoca MIT 09-7231, the bacteria were cultivated in different culture media, such as lysogeny broth (LB), tryptone soy broth (TSB), tryptone lactose broth (TLB), and Dulbecco’s modified Eagle’s medium (DMEM) transcription analyzed by RT-qPCR
The conventional LB medium was used as a reference to determine the basic levels of expression of genes encoded by the aroX and NRPS operons
Summary
The human gut microbiota is a complex community of microbial species that plays a fundamental role in the health and functioning of the human digestive tract. The homeostasis of this community provides protection against pathogens (Belkaid and Harrison, 2017; Lin et al, 2021). Toxigenic K. oxytoca strains carry a gene cluster that codes for proteins that synthesize a cytotoxin known as tilivalline (TV), which is largely responsible for this disease (Dornisch et al, 2017). The NRPS operon is a 6.2-kbp region encoding three genes: npsA, thdA, and npsB (Schneditz et al, 2014; Dornisch et al, 2017; Tse et al, 2017). The intestinal barrier function is impaired (Schneditz et al, 2014; Hering et al, 2019)
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