Silver Nanoparticles Functionalized With Antimicrobial Polypeptides: Benefits and Possible Pitfalls of a Novel Anti-infective Tool

Silver nanoparticles (AgNPs) and antimicrobial peptides or proteins (AMPs/APs) are both considered as promising platforms for the development of novel therapeutic agents effective against the growing number of drug-resistant pathogens. The observed synergy of their antibacterial activity suggested the prospect of introducing antimicrobial peptides or small antimicrobial proteins into the gelatinized coating of AgNPs. Conjugates with protegrin-1, indolicidin, protamine, histones, and lysozyme were comparatively tested for their antibacterial properties and compared with unconjugated nanoparticles and antimicrobial polypeptides alone. Their toxic effects were similarly tested against both normal eukaryotic cells (human erythrocytes, peripheral blood mononuclear cells, neutrophils, and dermal fibroblasts) and tumor cells (human erythromyeloid leukemia K562 and human histiocytic lymphoma U937 cell lines). The AMPs/APs retained their ability to enhance the antibacterial activity of AgNPs against both Gram-positive and Gram-negative bacteria, including drug-resistant strains, when conjugated to the AgNP surface. The small, membranolytic protegrin-1 was the most efficient, suggesting that a short, rigid structure is not a limiting factor despite the constraints imposed by binding to the nanoparticle. Some of the conjugated AMPs/APs clearly affected the ability of nanoparticle to permeabilize the outer membrane of Escherichia coli, but none of the conjugated AgNPs acquired the capacity to permeabilize its cytoplasmic membrane, regardless of the membranolytic potency of the bound polypeptide. Low hemolytic activity was also found for all AgNP-AMP/AP conjugates, regardless of the hemolytic activity of the free polypeptides, making conjugation a promising strategy not only to enhance their antimicrobial potential but also to effectively reduce the toxicity of membranolytic AMPs. The observation that metabolic processes and O2 consumption in bacteria were efficiently inhibited by all forms of AgNPs is the most likely explanation for their rapid and bactericidal action. AMP-dependent properties in the activity pattern of various conjugates toward eukaryotic cells suggest that immunomodulatory, wound-healing, and other effects of the polypeptides are at least partially transferred to the nanoparticles, so that functionalization of AgNPs may have effects beyond just modulation of direct antibacterial activity. In addition, some conjugated nanoparticles are selectively toxic to tumor cells. However, caution is required as not all modulatory effects are necessarily beneficial to normal host cells.

Biotechnology for production of recombinant hybrid proteins from plants and microbes with antifungal activity

The principle of obtaining recombinant antimicrobial polypeptides from plant and microbial origins as a part of chimeric proteins with thioredoxin by heterologous expression in a prokaryotic system is presented. The results obtained in terms of their antifungal activity in relation to plant pathogenic micromycetes allow us to consider these compounds as prototypes of some active substances of environmentally friendly biofungicides, as well as possible components of hybrid plant protection products against fungal diseases.

Functional and correlational relationship between indexes of the metabolic activity of neutrophils and α-defensins in animals of different age groups in the dynamics of acute somatic pain

В последние годы все чаще встречаются штаммы микроорганизмов, устойчивые к антибиотикам. Необходим поиск новых средств, обеспечивающих надежный антибактериальный эффект. Актуальным является изучение антимикробных полипептидов. Возникает вопрос, α-дефензины активируются только при микробной атаке или существуют и иные триггеры их вовлечения в иммунологический процесс. Если исходить из того, что любая рана всегда болезненна, но не любая острая боль связана с внешним повреждением тканей, встает вопрос о возможности вовлечения нейтрофилов и α-дефензинов в долорогенный процесс как таковой. Цель работы - выявление кореляционной связи и особенностей вовлечения нейтрофилов и α-дефензинов в острый долорогенный процесс на этапах онтогенеза. Методика. Объект исследования - белые беспородные крысы 3 возрастных групп: месячные (30-35-дневные, n=36), взрослые половозрелые (2-3 месячные, n=36), старые (старше 1 года, n=36).). Каждая группа делилась на контрольную и экспериментальную подгруппы. В экспериментальных подгруппах моделировали острую соматическую боль методом электростимуляции. После болевого воздействия брали материал для исследования через 2, 30-60, 120-180 мин. Содержание α-дефензинов в сыворотке крови определяли методом иммуноферментного анализа. Для оценки микробицидной активности нейтрофилов использовали спонтанный и стимулированный НСТ - тест. Для оценки корреляционной связи между изучаемыми показателями применяли метод ранговой корреляции Спирмена. Результаты. Острая краткосрочная соматическая боль активирует кислородзависимые механизмы микробицидности нейтрофилов и приводит к увеличению уровня α-дефензинов в периферической крови. В онтогенезе реакция имеет свои особенности, но ее стандартный и непродолжительный характер очевидны. Установлена прямая корреляционная зависимость между функциональной активностью нейтрофилов и уровнем α-дефензинов в периферической крови у 30-35-дневных и 2-3-месячных животных. Заключение. Полученные данные имеют значение при разработке препаратов на основе α-дефензинов и их применении пациентами различного возраста при наличии в анамнезе острого болевого синдрома. Microorganism strains resistant to antibiotics have become more common. This fact prompts searching for new means that would provide a reliable antibacterial effect. Studying antimicrobial polypeptides is relevant. A question arises whether α-defensins are activated only during a microbial attack or there are other triggers for their involvement in the immunological process. Since any wound is always painful but acute pain is not always associated with an external injury, the question is whether neutrophils and α-defensins can be involved in the dolorogenic process as such. The aim of the study was to determine correlation and features of neutrophil and α-defensin involvement in an acute dolorogenic process at stages of ontogenesis. Methods. The study was performed on white rats divided into three age groups (30-35-day-old, 2-3-month-old, and older than one year). Each group was divided into control and experimental subgroups. In experimental subgroups, acute somatic pain was modeled by electrostimulation. Material was collected at 2, 30-60, and 120-180 min after electrostimulation. Serum content of α-defensins was measured by enzyme immunoassay. Neutrophil microbicidal activity was evaluated using the spontaneous and stimulated NBT tests. Values were compared using the non-parametric Mann-Whitney U-test, and correlations were analyzed using the Spearman rank correlation test. Results. Acute short-term somatic pain activated oxygen-dependent mechanisms of neutrophil microbicidity and induced increased levels of α-defensins in peripheral blood. This reaction has specific features in ontogenesis but it is obviously standard and short-term. The functional activity of neutrophils was directly correlated with the level of α-defensins in peripheral blood of 30-35-day-old and 2-3-month-old animals. Conclusion. At stages of ontogenesis, oxygen-dependent neutrophil microbicidity and peripheral blood content of α-defensins increase in response to acute, short-term somatic pain. The type of response is determined by age-related features. The study results are important for development of α-defensin-based drugs and prescribing them to patients of different age with a history of acute pain.

Structural design and antimicrobial properties of polypeptides and saccharide–polypeptide conjugates

The development and progress of antimicrobial polypeptides and saccharide–polypeptide conjugates in regards to their structural design, biological functions and antimicrobial mechanism.

Bacterial Virus Lambda Gpd-Fusions to Cathelicidins, α- and β-Defensins, and Disease-Specific Epitopes Evaluated for Antimicrobial Toxicity and Ability to Support Phage Display

We showed that antimicrobial polypeptides, when translated as gene fusions to the bacteriophage lambda capsid decoration protein gpD, formed highly toxic molecules within E. coli, suggesting that they can retain their antimicrobial activity conformation when fused to gpD. These include gpD-fusions to human and porcine cathelicidins LL37 and PR39, β-defensins HBD3 and DEFB126-Δ (deleted for its many COOH-terminal glycosylation sites), and α-defensin HD5. Antimicrobial toxicity was only observed when the peptides were displayed from the COOH-terminal, and not the NH2-terminal end, of gpD. This suggests that COOH-terminal displayed polypeptides of gpD-fusions can more readily form an active-state conformation than when they are displayed from the NH2-terminal end of gpD. The high toxicity of the COOH-displayed gpD-defensins suggests either that the fused defensin peptides can be oxidized, forming three correct intramolecular disulfide bonds within the cytosol of bacterial cells, or that the versions without disulfide bonds are highly toxigenic. We showed the high efficiency of displaying single epitope 17 amino-acid fusions to gpD on LDP (lambda display particles), even when the gpD-fusion protein was toxic. The efficient formation of high display density LDP, displaying a single disease specific epitope (DSE), suggests the utility of LDP-DSE constructs for use as single epitope vaccines (SEV).

Selective killing of Helicobacter pylori with pH-responsive helix–coil conformation transitionable antimicrobial polypeptides

Current clinical treatment of Helicobacter pylori infection, the main etiological factor in the development of gastritis, gastric ulcers, and gastric carcinoma, requires a combination of at least two antibiotics and one proton pump inhibitor. However, such triple therapy suffers from progressively decreased therapeutic efficacy due to the drug resistance and undesired killing of the commensal bacteria due to poor selectivity. Here, we report the development of antimicrobial polypeptide-based monotherapy, which can specifically kill H. pylori under acidic pH in the stomach while inducing minimal toxicity to commensal bacteria under physiological pH. Specifically, we designed a class of pH-sensitive, helix–coil conformation transitionable antimicrobial polypeptides (HCT-AMPs) (PGA)m-r-(PHLG-MHH)n, bearing randomly distributed negatively charged glutamic acid and positively charged poly(γ-6-N-(methyldihexylammonium)hexyl-l-glutamate) (PHLG-MHH) residues. The HCT-AMPs showed unappreciable toxicity at physiological pH when they adopted random coiled conformation. Under acidic condition in the stomach, they transformed to the helical structure and exhibited potent antibacterial activity against H. pylori, including clinically isolated drug-resistant strains. After oral gavage, the HCT-AMPs afforded comparable H. pylori killing efficacy to the triple-therapy approach while inducing minimal toxicity against normal tissues and commensal bacteria, in comparison with the remarkable killing of commensal bacteria by 65% and 86% in the ileal contents and feces, respectively, following triple therapy. This strategy renders an effective approach to specifically target and kill H. pylori in the stomach while not harming the commensal bacteria/normal tissues.