An overview of databases and bioinformatics tools for plant antimicrobial peptides

: Antimicrobial peptides (AMPs) are small, ribosomally synthesized proteins found in nearly all forms of life. In plants, AMPs play a central role in plant defense due to their distinct physicochemical properties. Due to their broad-spectrum antimicrobial activity and rapid killing action, plant AMPs have become important candidates for the development of new drugs to control plant and animal pathogens that are resistant to multiple drugs. Further research is required to explore the potential uses of these natural compounds. Computational strategies have been increasingly used to understand key aspects of antimicrobial peptides. These strategies will help to minimize the time and cost of "wet-lab" experimentation. Researchers have developed various tools and databases to provide updated information on AMPs. However, despite the increased availability of antimicrobial peptide resources in biological databases, finding AMPs from plants can still be a difficult task. The number of plant AMP sequences in current databases is still small and yet often redundant. To facilitate further characterization of plant AMPs, we have summarized information on the location, distribution, and annotations of plant AMPs available in the most relevant databases for AMPs research. We also mapped and categorized the bioinformatics tools available in these databases. We expect that this will allow researchers to advance in the discovery and development of new plant AMPs with potent biological properties. We hope to provide insights to further expand the application of AMPs in the fields of biotechnology, pharmacy, and agriculture.

977Mechanistic within-host modelling to fast-track the selection of new antimalarial combination therapies

Background The efficacy of artemisinin-based combination therapies (ACTs), currently the first-line antimalarial treatments, is declining due to the emergence of resistance of malaria parasites to these drugs. This has led drug development initiatives to search for novel combination therapies to replace the failing ACTs. We developed a biologically informed within-host model, validated against data from volunteer infection studies, to guide critical drug development decisions. Methods A within-host model was developed, linking drug concentrations of two novel antimalarial drugs, OZ439 and DSM265, to their combined killing action and accounting for differential killing of these compounds against stages of the parasite’s lifecycle. Data collected from malaria-infected volunteers treated with OZ439–DSM265 were used to estimate the model parameters in a hierarchical Bayesian framework. Posterior-predictive simulations of the model were used to determine the dosing regimen required to cure >90% patients. Results The results showed that 800 mg of OZ439 combined with 450 mg of DSM265, which are within the safe and tolerable dose range, can provide day 42 cure rates >90%, despite the estimated antagonistic interaction between the drugs. The importance of accommodating parasite age specificity of drug action was demonstrated. Conclusions The dosing regimens for the combination of OZ439-DSM265 determined from our data-informed in silico model suggest this compound may be a suitable candidate to replace failing ACTs. Key messages Assessing various scenarios within a simulation framework allows discovery of robust dosing regimens, accelerating the drug development process and ensuring efficient allocation of resources for phase 2 and 3 clinical trials.

Aerosol-based antimicrobial photoinactivation in the lungs: an action spectrum study

Chronic lung infections are among the most diffused human infections, being often associated with multidrug-resistant bacteria. In this framework, the European project “Light4Lungs” aims at synthesizing and testing an inhalable light source to control lung infections by antimicrobial photoinactivation (aPDI), addressing endogenous photosensitizers only (porphyrins) in the representative case of S. aureus and P. aeruginosa. In the search for the best emission characteristics for the aerosolized light source, this work defines and calculates the photo-killing action spectrum for lung aPDI in the exemplary case of cystic fibrosis. This was obtained by applying a semi-theoretical modelling with Monte Carlo simulations, according to previously published methodology related to stomach infections and applied to the infected trachea, bronchi, bronchioles and alveoli. In each of these regions, the two low and high oxygen concentration cases were considered to account for the variability of in vivo conditions, together with the presence of endogenous porphyrins and other relevant absorbers/diffusers inside the illuminated biofilm/mucous layer. Furthermore, an a priori method to obtain the “best illumination wavelengths” was defined, starting from maximizing porphyrin and light absorption at any depth. The obtained action spectrum is peaked at 394 nm and mostly follows porphyrin extinction coefficient behavior. This is confirmed by the results from the best illumination wavelengths, which reinforces the robustness of our approach. These results can offer important indications for the synthesis of the aerosolized light source and definition of its most effective emission spectrum, suggesting a flexible platform to be considered in further applications.

Advanced Killing Potential of Thymol against a Time and Temperature Optimized Attached Listeria monocytogenes Population in Lettuce Broth

Fresh vegetables and salads are increasingly implicated in outbreaks of foodborne infections, such as those caused by Listeria monocytogenes, a dangerous pathogen that can attach to the surfaces of the equipment creating robust biofilms withstanding the killing action of disinfectants. In this study, the antimicrobial efficiency of a natural plant terpenoid (thymol) was evaluated against a sessile population of a multi-strain L. monocytogenes cocktail developed on stainless steel surfaces incubated in lettuce broth, under optimized time and temperature conditions (54 h at 30.6 °C) as those were determined following response surface modeling, and in comparison, to that of an industrial disinfectant (benzalkonium chloride). Prior to disinfection, the minimum bactericidal concentrations (MBCs) of each compound were determined against the planktonic cells of each strain. The results revealed the advanced killing potential of thymol, with a concentration of 625 ppm (= 4 × MBC) leading to almost undetectable viable bacteria (more than 4 logs reduction following a 15-min exposure). For the same degree of killing, benzalkonium chloride needed to be used at a concentration of at least 20 times more than its MBC (70 ppm). Discriminative repetitive sequence-based polymerase chain reaction (rep-PCR) also highlighted the strain variability in both biofilm formation and resistance. In sum, thymol was found to present an effective anti-listeria action under environmental conditions mimicking those encountered in the salad industry and deserves to be further explored to improve the safety of fresh produce.

Human Antimicrobial Peptide Hepcidin 25-Induced Apoptosis in Candida albicans

Hepcidin 25 (hep 25) is a cysteine-rich 25-amino acid antimicrobial peptide containing the amino-terminal Cu(II)/Ni(II)-binding (ATCUN) motif. Upon metal binding, the ATCUN motif is known to be involved in the generation of reactive oxygen species (ROS), especially hydrogen peroxide and hydroxyl radicals, which act against different bacterial species. However, the antifungal activity and its correlation to the Cu(II)-ATCUN complex of Hep 25 are still poorly understood. Here, we found that ROS accumulation plays an important role in the fungicidal activity of hep 25 against Candida albicans. In addition, Annexin V-FITC staining and TUNEL assay results provide clues about the apoptosis induced by hep 25. Moreover, hep 25 also increases the generation of ROS, possibly because of copper binding to the ATCUN motif, which is relevant to its activity against C. albicans. Finally, the C. albicans killing action of hep 25 is an energy- and temperature-dependent process that does not involve targeting the membrane. Taken together, our results provide new insights into the mechanisms of hep 25 against C. albicans cells and the potential use of hep 25 and its derivatives as novel antifungal agents.

A synthetic microbial consortium to detect and kill Vibrio cholerae

Vibrio cholerae is nowadays still problematic in several countries which are exposed to recurrent disease outbreaks. The current disease detection and treatment methods are efficient so this approach focused on the prevention of the disease. Indeed, current solutions are not efficient enough to deal with this situation. As V. cholerae which infects more than a million people each year is usually found in water, a synthetic microbial consortium was designed to detect and kill efficiently the bacteria in water. This work shows that Vibrio harveyi, a non-pathogenic strain to human, can be an efficient detector of V. cholerae. Moreover, it proves that Pichia pastoris, a yeast, can efficiently produce a novel antimicrobial peptide coming from the crocodile Crocodylus siamensis (i.e D-NY15) and that this peptide has a killing action towards V. cholerae. This study also shows that a communication between a prokaryote (Vibrio harveyi) and an eukaryote (Pichia pastoris) may be possible.

Characterization of DprE1-Mediated Benzothiazinone Resistance in Mycobacterium tuberculosis

ABSTRACTBenzothiazinones (BTZs) are a class of compounds found to be extremely potent against both drug-susceptible and drug-resistantMycobacterium tuberculosisstrains. The potency of BTZs is explained by their specificity for their target decaprenylphosphoryl-d-ribose oxidase (DprE1), in particular by covalent binding of the activated form of the compound to the critical cysteine 387 residue of the enzyme. To probe the role of C387, we used promiscuous site-directed mutagenesis to introduce other codons at this position intodprE1ofM. tuberculosis. The resultant viable BTZ-resistant mutants were characterizedin vitro,ex vivo, and biochemically to gain insight into the effects of these mutations on DprE1 function and onM. tuberculosis. Five different mutations (C387G, C387A, C387S, C387N, and C387T) conferred various levels of resistance to BTZ and exhibited different phenotypes. The C387G and C387N mutations resulted in a lower growth rate of the mycobacterium on solid medium, which could be attributed to the significant decrease in the catalytic efficiency of the DprE1 enzyme. All five mutations rendered the mycobacterium less cytotoxic to macrophages. Finally, differences in the potencies of covalent and noncovalent DprE1 inhibitors in the presence of C387 mutations were revealed by enzymatic assays. As expected from the mechanism of action, the covalent inhibitor PBTZ169 only partially inhibited the mutant DprE1 enzymes compared to the near-complete inhibition with a noncovalent DprE1 inhibitor, Ty38c. This study emphasizes the importance of the C387 residue for DprE1 activity and for the killing action of covalent inhibitors such as BTZs and other recently identified nitroaromatic inhibitors.

N-Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils

Brucella abortusis an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover,B. abortuscauses the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils withB. abortus. In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smoothB. abortusbacteria at early stages of infection. Murine normal serum components do not opsonize smoothBrucellastrains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing roughBrucellamutants. Despite this, neutrophils still fail to killBrucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonizeYersinia enterocoliticaO:9, a bacterium displaying the same surface polysaccharide antigen as smoothB. abortus. Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for theBrucellacamouflage areN-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution.

Combating Antimicrobial Resistance in Foodborne Microorganisms

ABSTRACT This review addresses an important public health hazard affecting food safety. Antimicrobial agents are used in foods to reduce or eliminate microorganisms that cause disease. Many traditional organic compounds, novel synthetic organic agents, natural products, peptides, and proteins have been extensively studied for their effectiveness as antimicrobial agents against foodborne Campylobacter spp., Escherichia coli, Listeria spp. and Salmonella. However, antimicrobial resistance can develop in microorganisms, enhancing their ability to withstand the inhibiting or killing action of antimicrobial agents. Knowledge gaps still exist with regard to the actual chemical and microbiological mechanisms that must be identified to facilitate the search for new antimicrobial agents. Technical implementation of antimicrobial active packing films and coatings against target microorganisms must also be improved for extended product shelf life. Recent advances in antimicrobial susceptibility testing can provide researchers with new momentum to pursue their quest for a resistance panacea.

The Production of Reactive Oxygen Species Is a Universal Action Mechanism of Amphotericin B against Pathogenic Yeasts and Contributes to the Fungicidal Effect of This Drug

ABSTRACTAmphotericin B (AMB) is an antifungal drug that binds to ergosterol and forms pores at the cell membrane, causing the loss of ions. In addition, AMB induces the accumulation of reactive oxygen species (ROS), and although these molecules have multiple deleterious effects on fungal cells, their specific role in the action mechanism of AMB remains unknown. In this work, we studied the role of ROS in the action mechanism of AMB. We determined the intracellular induction of ROS in 44 isolates of different pathogenic yeast species (Candida albicans,Candida parapsilosis,Candida glabrata,Candida tropicalis,Candida krusei,Cryptococcus neoformans, andCryptococcus gattii). We also characterized the production of ROS in AMB-resistant isolates. We found that AMB induces the formation of ROS in all the species tested. The inhibition of the mitochondrial respiratory chain by rotenone blocked the induction of ROS by AMB and provided protection from the killing action of the antifungal. Moreover, this phenomenon was absent in strains that displayed resistance to AMB. These strains showed an alteration in the respiration rate and mitochondrial membrane potential and also had higher catalase activity than that of the AMB-susceptible strains. Consistently, AMB failed to induce protein carbonylation in the resistant strains. Our data demonstrate that the production of ROS by AMB is a universal and important action mechanism that is correlated with the fungicidal effect and might explain the low rate of resistance to the molecule. Finally, these data provide an opportunity to design new strategies to improve the efficacy of this antifungal.