The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile.

Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacteria that is one of the leading causes of antibiotic-associated diarrhea. The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. We constructed a cwp66 gene deletion mutant using Clustered Regularly Interspaced Short Palindromic Repeats Cpf1 (CRISPR-Cpf1) system. The phenotypic and transcriptomic changes of the Δcwp66 mutant compared with the wild-type (WT) strain were studied. The deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the Δcwp66 mutant is more sensitive than the WT strain to clindamycin, ampicillin, and erythromycin but more resistant than the latter to vancomycin and metronidazole. Moreover, mannitol utilization capability in the Δcwp66 mutant was lost. Comparative transcriptomic analyses indicated that (i) 22.90-fold upregulation of cwpV gene and unable to express gpr gene were prominent in the Δcwp66 mutant; (ii) the cwp66 gene was involved in vancomycin resistance of C. difficile by influencing the expression of d-Alanine-d-Alanine ligase; and (iii) the mannose/fructose/sorbose IIC and IID components were upregulated in Δcwp66 mutant. The present work deepens our understanding of the contribution of the cwp66 gene to cell adhesion, stress tolerance, antibiotic resistance, and mannitol transportation of C. difficile. IMPORTANCE The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. The current study provides direct evidence that the cwp66 gene serves as a major adhesion in C. difficile, and also suggested that deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the antibiotic resistance and carbon source utilization profiles of the Δcwp66 mutant were significantly changed. These phenotypes were detrimental to the survival and pathogenesis of C. difficile in the human gut and may shed light on preventing C. difficile infection.

Species and sex differences in the blood clearance and immunogenicity of PEGylated uricase: A comparative 26-week toxicity study in rats and monkeys.

Low response rates and high immunogenicity were observed after repeated injections of pegloticase (Krystexxa) into gout patients during clinical trials. However, related research had not been reported in preclinical animal experiments, which has limited the development of this drug. In this study, the toxicity of mPEG-UHC was studied in rats and monkeys over a 26-week period of repeated intravenous dosing. There were no obvious toxic reactions in the tested animals, with the exception of mPEG-UHC blood clearance and immunogenicity. After repeated injections of mPEG-UHC, rapid loss of uricolytic activity (RLA) was not detected in rats, whereas RLA was observed in 44.4% of drug-treated monkeys. In these monkeys, RLA was observed in 11.1% of males and 77.8% of females, and such incidences increased with higher dosing. High titres of anti-uricase IgG antibodies were associated with RLA but did not result in any toxicity. Remission and recurrence of RLA occurred in one female monkey in the high-dose group because of suppressed and altered immune responses in this animal. The predicted incidence of RLA after repeated injections of mPEG-UHC in gout patients may be lower than that of pegloticase. In this study, the no-observed-adverse-effect levels (NOAELs) of mPEG-UHC in rats and monkeys were 32.0 mg/kg and 20.0 mg/kg, respectively. Therefore, the results showed that rats and monkeys could tolerate long-term and high-dose administrations of mPEG-UHC, and mPEG-UHC blood clearance and immunogenicity showed obvious species and sex differences. These findings will provide valuable information to direct the clinical use of mPEG-UHC.

Doxorubicin - chitosan - hydroxyapatite composite coatings on titanium alloy for localized cancer therapy.

The doxorubicin-chitosan composite is deposited electrochemically on the Ti alloy post hydroxyapatite coated for reducing the side effects by sustaining release of drugs localized near the tumor to achieve the inhibition or apoptosis of cancer. The possibility of danger in case of exfoliation of medicine composite and HA agglomerates from the alloy surface due to the dynamic erosion of blood flow could be overcome with the additional surface modification by the electrochemical deposition way. The cathodic polarization tests coupled with electrochemical reactions were analyzed to speculate the deposition mechanism of doxorubicin, spectrophotometer (UV visible spectrometer) to measure doxorubicin loading and release, field emission scanning electron microscope (FESEM) to observe surface morphology, Fourier transform infrared (FTIR) spectroscopy for chemical bonding of composites, and X-ray diffractometry (XRD) for crystal structure. The cell culture was carried out to analyze the drug efficacy on cell viability. It is concluded that doxorubicin-chitosan composites can be successfully deposited on the uncoated and hydroxyapatite-coated titanium specimen alloy by electrochemical methods. Both have revealed the sustaining drug release for a month and the latter with high porosity can enhance the drug loading to 37.46 µg/cm2, revealing this electrochemical method is a practical way to load doxorubicin cancer drug releasing locally to significantly reduce the amount of medication needed for future clinical applications.