A tRNA-acetylating toxin and detoxifying enzyme in Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems allow bacteria to adapt to changing environments without altering gene expression. Despite being overrepresented in Mycobacterium tuberculosis (Mtb), their individual physiological roles remain elusive. We describe a TA system in Mtb which we have named TacAT due to its homology to previously discovered systems in Salmonella. The toxin, TacT, blocks growth by acetylating glycyl-tRNAs and inhibiting translation. Its effects are reversed by the enzyme peptidyl tRNA hydrolase (Pth), which also cleaves peptidyl tRNAs that are prematurely released from stalled ribosomes. Pth is essential in most bacteria and thereby has been proposed as a promising drug target for complex pathogens like Mtb. Transposon sequencing data suggest that the tacAT operon is nonessential for Mtb growth in vitro, and premature stop mutations in this TA system present in some clinical isolates suggest that it is also dispensable in vivo. We assessed whether TacT modulates pth essentiality in Mtb, as drugs targeting Pth might be ineffective if TacAT is disrupted. We find that pth essentiality is unaffected by the absence of tacAT. These results highlight a fundamental aspect of mycobacterial biology and indicate that Pth's essential role hinges on its peptidyl-tRNA hydrolase activity. Our work underscores Pth's potential as a viable target for new antibiotics.

A superefficient ochratoxin A hydrolase with promising potential for industrial applications

As the most seriously controlled mycotoxin produced by Aspergillus spp. and Penicillium spp., ochratoxin A (OTA) results in various toxicological effects and widely contaminates agro-products. Biological detoxification of OTA is the most priority in food and feed industry, but currently available detoxification enzymes are relatively low effectiveness in time and cost. Here we show a superefficient enzyme ADH3 identified from Stenotrophomonas acidaminiphila with a strong ability to transform OTA into non-toxic ochratoxin-α by acting as an amidohydrolase. Recombinant ADH3 (1.2 μg/mL) completely degrades 50 μg/L OTA within 90 seconds, while the availably most efficient OTA hydrolases takes several hours. The kinetic constant showed that rADH3 ( Kcat/Km ) catalytic efficiency was 56.7-35000 times higher than those of previous hydrolases rAfOTase, rOTase and commercial carboxypeptidase A (CPA). Protein structure-based assay suggested that ADH3 has a preference for hydrophobic residues to form a larger hydrophobic area than other detoxifying enzymes at the cavity of the catalytic sites, and this structure makes the OTA easier to access to catalytic sites. In addition, ADH3 shows considerable temperature adaptability to exert hydrolytic function at the temperature down to 0°C or up to 70°C. Collectively, we report a superefficient OTA detoxifying enzyme with promising potential for industrial applications. IMPORTANCE Ochratoxin A (OTA) can result in various toxicological effects and widely contaminates agro-products and feedstuffs. OTA detoxifications by microbial strains and bio-enzymes are significant to food safety. Although previous studies showed OTA could be transformed through several pathways, the ochratoxin-α pathway is recognized as the most effective one. However, the most currently available enzymes are not efficient enough. Here, a superefficient hydrolase ADH3 which can completely transform 50 μg/L OTA into ochratoxin-α within 90 seconds was screened and characterized. The hydrolase ADH3 shows considerable temperature adaptability (0-70°C) to exert the hydrolytic function. Findings of this study supplied an efficient OTA detoxifying enzyme and predicted the superefficient degradation mechanism which lay a foundation for future industrial applications.

Toxicity of Bioactive Molecule Andrographolide against Spodoptera litura Fab and Its Binding Potential with Detoxifying Enzyme Cytochrome P450

Spodoptera litura Fab. is a polyphagous pest causing damage to many agriculture crops leading to yield loss. Recurrent usage of synthetic pesticides to control this pest has resulted in resistance development. Plant-derived diterpenoid compound andrographolide was isolated from the leaves of Andrographis paniculata. It was analysed by gas chromatography-mass spectroscopy and quantified by HPLC. Nutritional indices and digestive enzymatic profile were evaluated. Third, fourth and fifth instar larvae were treated with different concentrations of andrographolide. At 3, 6 and 9 ppm-treated concentrations the larvae showed decreased RGR, RCR, ECI, ECD values with adverse increase in AD. The digestive enzymes were significantly inhibited when compared with control. Conspicuously, andrographolide showed pronounced mortality of S. litura by inhibition of enzyme secretion and intake of food. The binding ability of andrographolide with CYTP450 showed high affinity with low binding energy. Andrographolide has the potential to be exploited as a biocontrol agent against S. litura as an eco-friendly pesticide.

Nuclear hormone receptors promote gut and glia detoxifying enzyme induction and protect C. elegans from the mold P. brevicompactum

Animals encounter microorganisms in their habitats, adapting physiology and behavior accordingly. The nematode Caenorhabditis elegans is found in microbe-rich environments; however, its responses to fungi are not extensively studied. Here we describe interactions of C. elegans and Penicillium brevicompactum, an ecologically-relevant mold. Transcriptome studies reveal that co-culture upregulates stress-response genes, including xenobiotic metabolizing enzymes (XMEs), in C. elegans intestine and AMsh glial cells. The nuclear hormone receptors (NHR) NHR-45 and NHR-156 are key induction regulators, and mutants that cannot induce XMEs in the intestine when exposed to P. brevicompactum experience mitochondrial stress and exhibit developmental defects. Different C. elegans wild isolates harbor sequence polymorphisms in nhr-156, resulting in phenotypic diversity in AMsh glia responses to microbe exposure. We propose that P. brevicompactum mitochondria-targeting mycotoxins are deactivated by intestinal detoxification, allowing tolerance to moldy environments. Our studies support the idea that C. elegans NHR gene expansion/diversification underlies adaptation to microbial environments.

Transcriptome Analysis of Aedes albopictus (Diptera: Culicidae) Larvae Exposed With a Sublethal Dose of Haedoxan A

Aedes albopictus is the vector of arbovirus diseases including yellow fever, dengue, Zika virus, and chikungunya fever, and it poses an enormous threat to human health worldwide. Previous studies have revealed that haedoxan A (HA), which is an insecticidal sesquilignan from Phryma leptostachya L., is a highly effective natural insecticide for managing mosquitoes and houseflies; however, the mechanisms underlying the response of Ae. albopictus after treatment with sublethal concentrations of HA is not clear. Here, high-throughput sequencing was used to analyze the gene expression changes in Ae. albopictus larvae after treatment with the LC30 of HA. In total, 416 differentially expressed genes (DEGs) were identified, including 328 upregulated genes and 88 downregulated genes. Identification and verification of related DEGs were performed by RT-qPCR. The results showed that two P450 unigenes (CYP4C21 and CYP304A1), one carboxylesterase, and one ABC transporter (ABCG1) were induced by HA, which indicated that these detoxifying enzyme genes might play a major role in the metabolic and detoxification processes of HA. Additionally, acetylcholine receptor subunit ɑ2 (AChRα2), AChRα5, AChRα9, and the glutamate receptor ionotropic kainate 2 (GRIK2) were found to be upregulated in HA-treated larvae, suggesting that HA affected the conduction of action potentials and synaptic transmission by disrupting the function of neural receptors. These results provide a foundation for further elucidating the target of HA and the mechanism of detoxification metabolism in Ae. albopictus.