Design and Synthesis of Novel Amino and Acetamidoaurones with Antimicrobial Activities.

The development of new and effective antimicrobial compounds is urgent due to the emergence of resistant bacteria. Natural plant flavonoids are known to be effective molecules, but their activity and selectivity have to be increased. Based on previous aurone potency, we designed new aurone derivatives bearing acetamido and amino groups at the position 5 of the A ring and managing various monosubstitutions at the B ring. A series of 31 new aurone derivatives were first evaluated for their antimicrobial activity with five derivatives being the most active (compounds 10, 12, 15, 16, and 20). The evaluation of their cytotoxicity on human cells and of their therapeutic index (TI) showed that compounds 10 and 20 had the highest TI. Finally, screening against a large panel of pathogens confirmed that compounds 10 and 20 possess large spectrum antimicrobial activity, including on bioweapon BSL3 strains, with MIC values as low as 0.78 µM. These results demonstrate that 5-acetamidoaurones are far more active and safer compared with 5-aminoaurones, and that benzyloxy and isopropyl substitutions at the B ring are the most promising strategy in the exploration of new antimicrobial aurones.

APOBEC3F Is a Mutational Driver of the Human Monkeypox Virus Identified in the 2022 Outbreak.

BACKGROUND: On May 6, 2022, a powerful outbreak of monkeypox virus (MPXV) had been reported outside of Africa, with many continuing new cases being reported around the world. Analysis of mutations among the 2 different lineages present in the 2021 and 2022 outbreaks revealed the presence of G->A mutations occurring in the 5'GpA context, indicative of APOBEC3 cytidine deaminase activity. METHODS: By using a sensitive polymerase chain reaction (differential DNA denaturation PCR) method allowing differential amplification of AT-rich DNA, we analyzed the level of APOBEC3-induced MPXV editing in infected cells and in patients. RESULTS: We demonstrate that G->A hypermutated MPXV genomes can be recovered experimentally from APOBEC3 transfection followed by MPXV infection. Here, among the 7 human APOBEC3 cytidine deaminases (A3A-A3C, A3DE, A3F-A3H), only APOBEC3F was capable of extensively deaminating cytidine residues in MPXV genomes. Hyperedited genomes were also recovered in ∼42% of analyzed patients. Moreover, we demonstrate that substantial repair of these mutations occurs. Upon selection, corrected G->A mutations escaping drift loss contribute to the MPXV evolution observed in the current epidemic. CONCLUSIONS: Stochastic or transient overexpression of the APOBEC3F gene exposes the MPXV genome to a broad spectrum of mutations that may be modeling the mutational landscape after multiple cycles of viral replication.

Genomic and RT-qPCR analysis of trimethoprim-sulfamethoxazole and meropenem resistance in Burkholderia pseudomallei clinical isolates.

BACKGROUND: Melioidosis is an endemic disease in southeast Asia and northern Australia caused by the saprophytic bacteria Burkholderia pseudomallei, with a high mortality rate. The clinical presentation is multifaceted, with symptoms ranging from acute septicemia to multiple chronic abscesses. Here, we report a chronic case of melioidosis in a patient who lived in Malaysia in the 70s and was suspected of contracting tuberculosis. Approximately 40 years later, in 2014, he was diagnosed with pauci-symptomatic melioidosis during a routine examination. Four strains were isolated from a single sample. They showed divergent morphotypes and divergent antibiotic susceptibility, with some strains showing resistance to trimethoprim-sulfamethoxazole and fluoroquinolones. In 2016, clinical samples were still positive for B. pseudomallei, and only one type of strain, showing atypical resistance to meropenem, was isolated. PRINCIPAL FINDINGS: We performed whole genome sequencing and RT-qPCR analysis on the strains isolated during this study to gain further insights into their differences. We thus identified two types of resistance mechanisms in these clinical strains. The first one was an adaptive and transient mechanism that disappeared during the course of laboratory sub-cultures; the second was a mutation in the efflux pump regulator amrR, associated with the overexpression of the related transporter. CONCLUSION: The development of such mechanisms may have a clinical impact on antibiotic treatment. Indeed, their transient nature could lead to an undiagnosed resistance. Efflux overexpression due to mutation leads to an important multiple resistance, reducing the effectiveness of antibiotics during treatment.

Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a very sensitive widespread technique considered as the gold standard to explore transcriptional variations. While a particular methodology has to be followed to provide accurate results many published studies are likely to misinterpret results due to lack of minimal quality requirements. Yersinia pestis is a highly pathogenic bacterium responsible for plague. It has been used to propose a ready-to-use and complete approach to mitigate the risk of technical biases in transcriptomic studies. The selection of suitable reference genes (RGs) among 29 candidates was performed using four different methods (GeNorm, NormFinder, BestKeeper and the Delta-Ct method). An overall comprehensive ranking revealed that 12 following candidate RGs are suitable for accurate normalization: gmk, proC, fabD, rpoD, nadB, rho, thrA, ribD, mutL, rpoB, adk and tmk. Some frequently used genes like 16S RNA had even been found as unsuitable to study Y. pestis. This methodology allowed us to demonstrate, under different temperatures and states of growth, significant transcriptional changes of six efflux pumps genes involved in physiological aspects as antimicrobial resistance or virulence. Previous transcriptomic studies done under comparable conditions had not been able to highlight these transcriptional modifications. These results highlight the importance of validating RGs prior to the normalization of transcriptional expression levels of targeted genes. This accurate methodology can be extended to any gene of interest in Y. pestis. More generally, the same workflow can be applied to identify and validate appropriate RGs in other bacteria to study transcriptional variations.

Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets.

The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding. Here, we show that a semi-rational approach can be used to derive hundreds of novel proteins from I-CreI, a homing endonuclease from the LAGLIDADG family. These novel endonucleases display a wide range of cleavage patterns in yeast and mammalian cells that in most cases are highly specific and distinct from I-CreI. Second, rules for protein/DNA interaction can be inferred from statistical analysis. Third, novel endonucleases can be combined to create heterodimeric protein species, thereby greatly enhancing the number of potential targets. These results describe a straightforward approach for engineering novel endonucleases with tailored specificities, while preserving the activity and specificity of natural homing endonucleases, and thereby deliver new tools for genome engineering.

In vivo selection of engineered homing endonucleases using double-strand break induced homologous recombination.

Homing endonucleases, endonucleases capable of recognizing long DNA sequences, have been shown to be a tool of choice for precise and efficient genome engineering. Consequently, the possibility to engineer novel endonucleases with tailored specificities is under strong investigation. In this report, we present a simple and efficient method to select meganucleases from libraries of variants, based on their cleavage properties. The method has the advantage of directly selecting for the ability to induce double-strand break induced homologous recombination in a eukaryotic environment. Model selections demonstrated high levels of enrichments. Moreover, this method compared favorably with phage display for enrichment of active mutants from a mutant library. This approach makes possible the exploration of large sequence spaces and thereby represents a valuable tool for genome engineering.