Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering

Comparison of proteomic responses as global approach to antibiotic mechanism of action elucidation

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology

Biosynthesis of Oxytetracycline by Streptomyces rimosus: Past, Present and Future Directions in the Development of Tetracycline Antibiotics

Natural tetracycline (TC) antibiotics were the first major class of therapeutics to earn the distinction of ‘broad-spectrum antibiotics’ and they have been used since the 1940s against a wide range of both Gram-positive and Gram-negative pathogens, mycoplasmas, intracellular chlamydiae, rickett siae and protozoan parasites. The second generation of semisynthetic tetracyclines, such as minocycline and doxycycline, with improved antimicrobial potency, were introduced during the 1960s. Despite emerging resistance to TCs erupting during the 1980s, it was not until 2006, more than four decades later, that a third-generation TC, named tigecycline, was launched. In addition, two TC analogues, omadacycline and eravacycline, developed via (semi)synthetic and fully synthetic routes, respectively, are at present under clinical evaluation. Interestingly, despite very productive early work on the isolation of a Streptomyces aureofaciens mutant strain that produced 6-demethyl-7-chlortetracycline, the key intermediate in the production of second- and third-generation TCs, biosynthetic approaches in TC development have not been productive for more than 50 years. Relatively slow and tedious molecular biology approaches for the genetic manipulation of TC-producing actinobacteria, as well as an insufficient understanding of the enzymatic mechanisms involved in TC biosynthesis have significantly contributed to the low success of such biosynthetic engineering efforts. However, new opportunities in TC drug development have arisen thanks to a signifi cant progress in the development of affordable and versatile biosynthetic engineering and synthetic biology approaches, and, importantly, to a much deeper understanding of TC biosynthesis, mostly gained over the last two decades

Heterologous expression of the atypical tetracycline chelocardin reveals the full set of genes required for its biosynthesis.

Background: Chelocardin (CHD) exhibits a broad-spectrum antibiotic activity and showed promising results in a small phase II clinical study conducted on patients with urinary tract infections. Importantly, CHD was shown to be active also against tetracycline-resistant Gram-negative pathogens, which is gaining even more importance in today’s antibiotic crisis. We have demonstrated that modifications of CHD through genetic engineering of its producer, the actinomycete Amycolatopsis sulphurea, are not only possible but yielded even more potent antibiotics than CHD itself, like 2-carboxamido-2-deacetyl-chelocardin (CD-CHD), which is currently in preclinical evaluation. A. sulphurea is difficult to genetically manipulate and therefore manipulation of the chd biosynthetic gene cluster in a genetically amenable heterologous host would be of high importance for further drug-discovery efforts. Results: We report heterologous expression of the CHD biosynthetic gene cluster in the model organism Streptomyces albus del14 strain. Unexpectedly, we found that the originally defined CHD gene cluster fails to provide all genes required for CHD formation, including an additional cyclase and two regulatory genes. Overexpression of the putative pathway-specific streptomyces antibiotic regulatory protein chdB in A. sulphurea resulted in an increase of both, CHD and CD-CHD production. Applying a metabolic-engineering approach, it was also possible to generate the potent CHD analogue, CD-CHD in S. albus. Finally, an additional yield increase was achieved in S. albus del14 by in-trans overexpression of the chdR exporter gene, which provides resistance to CHD and CDCHD. Conclusions: We identified previously unknown genes in the CHD cluster, which were shown to be essential for chelocardin biosynthesis by expression of the full biosynthetic gene cluster in S. albus as heterologous host. When comparing to oxytetracycline biosynthesis, we observed that the CHD gene cluster contains additional enzymes not found in gene clusters encoding the biosynthesis of typical tetracyclines (such as oxytetracycline). This finding probably explains the different chemistries and modes of action, which make CHD/CD-CHD valuable lead structures for clinical candidates. Even though the CHD genes are derived from a rare actinomycete A. sulphurea, the yield of CHD in the heterologous host was very good. The corrected nucleotide sequence of the CHD gene cluster now contains allgene products required for the production of CHD in a genetically amenable heterologous host, thus opening new possibilities towards production of novel and potent tetracycline analogues with a new mode of action

Hydrodynamic cavitation efficiently inactivates potato virus Y in water

Waterborne plant viruses can destroy entire crops, leading not only to high financial losses but also to food shortages. Potato virus Y (PVY) is the most important potato viral pathogen that can also affect other valuable crops. Recently, it has been confirmed that this virus is capable of infecting host plants via water, emphasizing the relevance of using proper strategies to treat recycled water in order to prevent the spread of the infectious agents. Emerging environmentally friendly methods such as hydrodynamic cavitation (HC) provide a great alternative for treating recycled water used for irrigation. In the experiments conducted in this study, laboratory HC based on Venturi constriction with a sample volume of 1 L was used to treat water samples spiked with purified PVY virions. The ability of the virus to infect plants was abolished after 500 HC passes, corresponding to 50 min of treatment under pressure difference of 7 bar. In some cases, shorter treatments of 125 or 250 passes were also sufficient for virus inactivation. The HC treatment disrupted the integrity of viral particles, which also led to a minor damage of viral RNA. Reactive species, including singlet oxygen, hydroxyl radicals, and hydrogen peroxide, were not primarily responsible for PVY inactivation during HC treatment, suggesting that mechanical effects are likely the driving force of virus inactivation. This pioneering study, the first to investigate eukaryotic virus inactivation by HC, will inspire additional research in this field enabling further improvement of HC as a water decontamination technology

Minimum performance parameters to select tests for validation and selection of laboratories for TPS

The aim of deliverable 1.1. is to prepare criteria to select tests for validation and to select laboratories for TPS (test performance study). Criteria for selection of tests for the TPS for each pest have been set (see Tables 7-12). These criteria have been divided in five groups: 1) validation data, 2) applicability, 3) protocols, 4) chemicals and 5) equipment. For selection of participants for the TPS selection criteria have also been set (see Table 13). Amongst the most important criteria for selection for participants of TPS are technical expertise for the pest group and the method, authorization to work with the specific pest and that the participating laboratory has quality assurance in place. These criteria enable evaluation of whether participants are proficient to perform the tests, have the necessary equipment and a permit to work with viable regulated organism. The scope of the testing for specific pests was set and common rules for each selection process was defined

TPS reports with description of the method, materials and software used, as well as the data analysis - Round 2, Version 1.0

The aim of the deliverable 1.4. is to present a summary of the results obtained in the Round 1 of the test performance studies (TPS) organized by WP1 on six prioritized pests. Tests selection for each TPS was conducted following the “Common rules for selection of tests for TPS” and based on the “Weighted criteria for selection of tests for TPS”, both described in deliverable D1.1, while the list of selected tests for each TPS is available and explained in deliverable D1.2. TPS participants were selected following the “Common rules for selection of participants for TPS” and based on the “Criteria for selection of participants of TPS”, also both described in deliverable D1.1. For each of the six TPSs, the methodology used to perform the tests, the results of preliminary studies to select the tests, the results of the TPS and their thorough analysis and interpretation are described in corresponding TPS reports (supplementary information available upon request under confidentiality agreement). The validation data obtained during the six TPSs will be available in the validation section of the EPPO database on the diagnostic expertise. Main outcomes for each of the TPSs are highlighted as well as difficulties noticed during the organization process, which will improve organization of the following studies in the Round 2

List of tests for validation

We prepared a list of methods and tests for validation in test performance study (TPS) Round 1, both for laboratory and on-site use, for 6 selected pests: Erwinia amylovora, Pantoea stewartiisubsp. stewartii, citrus tristeza virus, plum pox virus, Fusarium circinatum and Bursaphelenchus xylophilus. The listed tests were first validated in preliminary studies by TPS organizers in order to select the final tests for TPS, based on the scope and criteria prepared in D1.1