Intestinal carcinogenicity screening of environmental pollutants using organoid-based cell transformation assay.

The high incidence of colorectal cancer (CRC) is closely associated with environmental pollutant exposure. To identify potential intestinal carcinogens, we developed a cell transformation assay (CTA) using mouse adult stem cell-derived intestinal organoids (mASC-IOs) and assessed the transformation potential on 14 representative chemicals, including Cd, iPb, Cr-VI, iAs-III, Zn, Cu, PFOS, BPA, MEHP, AOM, DMH, MNNG, aspirin, and metformin. We optimized the experimental protocol based on cytotoxicity, amplification, and colony formation of chemical-treated mASC-IOs. In addition, we assessed the accuracy of in vitro study and the human tumor relevance through characterizing interdependence between cell-cell and cell-matrix adhesions, tumorigenicity, pathological feature of subcutaneous tumors, and CRC-related molecular signatures. Remarkably, the results of cell transformation in 14 chemicals showed a strong concordance with epidemiological findings (8/10) and in vivo mouse studies (12/14). In addition, we found that the increase in anchorage-independent growth was positively correlated with the tumorigenicity of tested chemicals. Through analyzing the dose-response relationship of anchorage-independent growth by benchmark dose (BMD) modeling, the potent intestinal carcinogens were identified, with their carcinogenic potency ranked from high to low as AOM, Cd, MEHP, Cr-VI, iAs-III, and DMH. Importantly, the activity of chemical-transformed mASC-IOs was associated with the degree of cellular differentiation of subcutaneous tumors, altered transcription of oncogenic genes, and activated pathways related to CRC development, including Apc, Trp53, Kras, Pik3ca, Smad4 genes, as well as WNT and BMP signaling pathways. Taken together, we successfully developed a mASC-IO-based CTA, which might serve as a potential alternative for intestinal carcinogenicity screening of chemicals.

Improved genetic transformation by disarmament of type II Restriction-Modification system in Streptococcus zooepidemicus.

Streptococcus zooepidemicus, group C Streptococci, is currently used for the industrial production of hyaluronic acid (HA). However, genetic manipulation of S. zooepidemicus is severely limited by its low transformation efficiency, which might be in part due to the Restriction-Modification (R-M) systems. The complete genome sequence of S. zooepidemicus ATCC39920 revealed the presence of two putative R-M systems, type I and type II. The putative type I R-M system is encoded by three closely linked genes: hsdR (SeseC_01315), hsdS, hsdM (SeseC_01318), and the putative type II R-M system consists of two closely linked genes: SeseC_02360 and yhdJ (SeseC_02362). Inactivation of hsdR, encoding the restriction endonuclease (REase) of the type I R-M system, showed no apparent effects on transformation efficiency, implying that disarmament of the type I R-M system alone is not sufficient for increasing transformation efficiency. However, inactivation of SeseC_02360, encoding the REase of the type II R-M system, improved transformation efficiency by 4.97 folds, indicating that type II R-M system is the major barrier that restricts genetic transformation in S. zooepidemicus. Furthermore, S. zooepidemicus strains lacking either of the two R-M systems are phenotypically indistinguishable from the wild-type in terms of cell growth and HA production. In summary, our study revealed that the type II R-M system is the main barrier to genetic transformation in S. zooepidemicus ATCC39920, and that the deletion of the type II R-M system renders S. zooepidemicus more transformable, thus facilitating metabolic engineering of this industrially important microorganism. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03227-x.

Is hyaluronic acid production transcriptionally regulated? A transcriptional repressor gene deletion study in Streptococcus zooepidemicus.

Hyaluronic acid (HA) is a multiple-function biopolymer that is widely used in food, cosmetic, and biomedical fields. In group C streptococci, the major workhorse of HA production in industry, the HA biosynthetic pathway has been proposed, while how HA synthesis is regulated is unclear. In this study, we identified twenty-five putative transcriptional repressors in S. zooepidemicus and studied whether they regulate HA synthesis or not. The individual gene deletion strain was firstly constructed, and the phenotypic changes of the corresponding deletion strains in stress tolerance and HA production were detected. The hrcA deletion strain is more sensitive to high temperature, and the rex deletion strain is more resistant to the oxidative stress. Three transcriptional repressor deletions resulted significantly decreased transcriptional levels of hasA, among which the scrR deletion strain shows most dramatical decrease in HA production. The regulatory mechanism of how ScrR affects the production of HA was further explored by transcriptional expression analysis of scrA and scrB, two direct target genes of ScrR regulon. Our results indicates that the deficiency of ScrR results in the unbalanced expression of scrA and scrB, which might also partly account for the decreasing production of HA. In agreement with the speculation, overexpression of scrB in ΔscrR genetic background results in 80% improvement in HA production. Taken together, the systemic genetic study of transcriptional repressors expands our understanding for the physiological regulation process of S. zooepidemicus and should help in the development of high-performance industrial strains for the efficient production of HA. KEY POINTS: • Twenty-two transcriptional repressor genes in S. zooepidemicus were deleted individually, and the phenotypes of corresponding mutants on a variety of conditions were characterized. • HrcA deficiency showed inferior cell tolerance to high temperature, and Rex deficiency showed superior cell tolerance to reactive oxygen stress, and four repressors deficiency showed inferior hyaluronic acid synthesis, among which the transcriptional levels of hasA of three mutants decreased significantly. • Optimizing sucrose metabolic flux can enhance hyaluronic acid synthesis significantly.