New insights into the antimicrobial action and protective therapeutic effect of tirapazamine towards Escherichia coli-infected mice.

OBJECTIVES: Escherichia coli is an important pathogen responsible for numerous cases of diarrhoea worldwide. The bioreductive agent tirapazamine (TPZ), which was clinically used to treat various types of cancers, has obvious antibacterial activity against E. coli strains. In the present study, we aimed to evaluate the protective therapeutic effects of TPZ in E. coli-infected mice and provide insights into its antimicrobial action mechanism. METHODS: The MIC and MBC tests, drug sensitivity test, crystal violet assay and proteomic analysis were used to detect the in vitro antibacterial activity of TPZ. The clinical symptoms of infected mice, tissue bacteria load, histopathological features and gut microbiota changes were regarded as indicators to evaluation the efficacy of TPZ in vivo. RESULTS: Interestingly, TPZ-induced the reversal of drug resistance in E. coli by regulating the expression of resistance-related genes, which may have an auxiliary role in the clinical treatment of drug-resistant bacterial infections. More importantly, the proteomics analysis showed that TPZ upregulated 53 proteins and downregulated 47 proteins in E. coli. Among these, the bacterial defence response-related proteins colicin M and colicin B, SOS response-related proteins RecA, UvrABC system protein A, and Holliday junction ATP-dependent DNA helicase RuvB were all significantly upregulated. The quorum sensing-related protein glutamate decarboxylase, ABC transporter-related protein glycerol-3-phosphate transporter polar-binding protein, and ABC transporter polar-binding protein YtfQ were significantly downregulated. The oxidoreductase activity-related proteins pyridine nucleotide-disulfide oxidoreductase, glutaredoxin 2 (Grx2), NAD(+)-dependent aldehyde reductase, and acetaldehyde dehydrogenase, which participate in the elimination of harmful oxygen free radicals in the oxidation-reduction process pathway, were also significantly downregulated. Moreover, TPZ improved the survival rate of infected mice; significantly reduced the bacteria load in the liver, spleen, and colon; and alleviated E. coli-associated pathological damages. The gut microbiota also changed in TPZ-treated mice, and these genera were considerably differentiated: Candidatus Arthromitus, Eubacterium coprostanoligenes group, Prevotellaceae UCG-001, Actinospica, and Bifidobacterium. CONCLUSIONS: TPZ may represent an effective and promising lead molecule for the development of antimicrobial agents for the treatment of E. coli infections.

Rapid Detection of Staphylococcus aureus in Milk and Pork via Immunomagnetic Separation and Recombinase Polymerase Amplification.

Separation processes using immunomagnetic beads (IMBs) are advantageous for the rapid detection of Staphylococcus aureus (S. aureus). Herein, a novel method, based on immunomagnetic separation using IMBs and recombinase polymerase amplification (RPA), was employed to detect S. aureus strains in milk and pork. IMBs were formed by the carbon diimide method using rabbit anti-S. aureus polyclonal antibodies and superparamagnetic carboxyl-Fe3O4 MBs. The average capture efficiency for 2.5 to 2.5 × 105 (CFU)/mL gradient dilution of S. aureus with 6 mg of IMBs within 60 min were a range of 62.74 to 92.75%. The detection sensitivity of the IMBs-RPA method in artificially contaminated samples was 2.5 × 101 CFU/mL. The entire detection process was completed within 2.5 h, including bacteria capture, DNA extraction, amplification, and electrophoresis. Among 20 actual samples, one case of raw milk sample and two cases of pork samples were tested positive using the established IMBs-RPA method, which were verified by the standard S. aureus inspection procedure. Therefore, the novel method shows potential for food safety supervision owing to its short detection time, higher sensitivity, and high specificity. IMPORTANCE Our study established IMBs-RPA method, which simplified the steps of bacteria separation, shortened the detection time, and realized the convenient detection of S. aureus in milk and pork samples. IMBs-RPA method was also suitable for the detection of other pathogens, providing a new method for food safety monitoring and a favorable basis for rapid and early diagnosis of diseases.

The RNA helicase Ski2 in the fungal pathogen Cryptococcus neoformans highlights key roles in azoles resistance and stress tolerance.

The yeast SKI (superkiller) complex was originally identified from cells that were infected by the M 'killer' virus. Ski2, as the core of the SKI complex, is a cytoplasmic cofactor and regulator of RNA-degrading exosome. The putative RNA helicase Ski2 was highly conserved from yeast to animals and has been demonstrated to play a key role in the regulation of RNA surveillance, temperature sensitivity, and growth in several yeasts but not yet in Cryptococcus neoformans (C. neoformans). Here, we report the identification of a gene encoding an equivalent Ski2 protein, named SKI2, in the fungal pathogen C. neoformans. To obtain insights into the function of Ski2, we created a mutant strain, ski2Δ, with the CRISPR-Cas9 editing tool. Disruption of SKI2 impaired cell wall integrity. Further investigations revealed the defects of the ski2Δ mutant in resistance to osmotic stresses and extreme growth temperatures. However, significantly, the ability to undergo invasive growth under nutrient-depleted conditions was increased in the ski2Δ mutant. More importantly, our results showed that the ski2Δ mutant exhibited slightly lower virulence and severe susceptibility to anti-ribosomal drugs by comparison to the wild type, but it developed multidrug resistance to azoles and flucytosine. By constructing the double deletion strain ski2Δafr1Δ, we verified that increased Afr1 in ski2Δ contributed to the azole resistance, which might be influenced by nonclassical small interfering RNA. Our work suggests that Ski2 plays critical roles in drug resistance and regulation of gene transcription in the yeast pathogen C. neoformans.

Involvement of the Noncanonical Polyadenylation Polymerase Cid14 in Fungal Azole Resistance in the Pathogen Cryptococcus neoformans.

The yeast noncanonical polyadenylation polymerase Cid14 was originally identified from fission yeast and plays a critical role in the TRAMP complex. This protein is a cytoplasmic cofactor and regulator of RNA-degrading exosomes. Cid14 is highly conserved from yeast to animals and has been demonstrated to play key roles in the regulation of RNA surveillance, nutrition metabolism, and growth in model organisms, but not yet in Cryptococcus neoformans (C. neoformans). Here, we report the identification of a gene encoding an equivalent Cid14 protein, named CID14, in the fungal pathogen C. neoformans. To obtain insights into the function of Cid14, we created a mutant strain, cid14Δ, with the CRISPR-Cas9 editing tool. Disruption of CID14 impaired cell membrane stability. Further investigations revealed the defects of the cid14Δ mutant in resistance to low carbohydrate levels. Meanwhile, significantly, the ability to grow under flucytosine stress was decreased in the cid14Δ mutant. More importantly, our results showed that the cid14Δ mutant does not affect yeast virulence but exhibits multidrug resistance to azole. Our work is the first to suggest that Cid14 plays critical roles in azole resistance by affecting Afr1, which is chiefly responsible for azole excretion in the ABC (ATP-binding cassette) transporter.