Sub-inhibitory Effects of Antimicrobial Peptides

Antimicrobials, and particularly antimicrobial peptides (AMPs), have been thoroughly studied due to their therapeutic potential. The research on their exact mode of action on bacterial cells, especially at under sublethal concentrations, has resulted in a better understanding of the unpredictable nature of bacterial behavior under stress conditions. In this review, we were aiming to gather the wide yet still under-investigated knowledge about various AMPs and their subinhibition effects on cellular and molecular levels. We describe how AMP action is non-linear and unpredictable, also showing that exposure to AMP can lead to antimicrobial resistance via triggering various regulatory systems. Being one of the most known types of antimicrobials, bacteriocins have dual action and can also be utilized by microorganisms as signaling molecules at naturally achievable sub-inhibitory concentrations. The unpredictable nature of AMP action and the pathogenic response triggered by them remains an area of knowledge that requires further investigation

2,4-Diacetylphloroglucinol Modulates <i>Candida albicans</i> Virulence

The dimorphic fungus Candida albicans is one of the most important opportunistic pathogens for humankind. The use of fungicides against Candida could be associated with sub-inhibitory effects, which are referred to as fungal stress responses and are undesirable for the host. In this work, we investigated the antifungal action of 2,4-diacetylphloroglucinol (2,4-DAPG) against Candida albicans ATCC 10231 with a focus on their biofilm-forming ability. We found that 2,4-DAPG was able to reduce the ability of Candida cells to form biofilms, but complete inhibition and eradication effects were not achieved. Furthermore, C. albicans cells in the adherent state were characterized by reduced susceptibility to 2,4-DAPG compared to planktonic cells. The investigation of the mechanisms that could explain the antibiofilm action of 2,4-DAPG revealed a reduction in the cell`s surface hydrophobicity and the inhibition of the yeast-to-hyphae transition. The inhibition of the Candida cells filamentation was accompanied by an increase in the expression of the NRG1 gene, which is a negative regulator of hyphal development. In addition, we microscopically visualized the treated biofilms and revealed numerous channels that were decorated with particles and localized on the hyphae. We assumed that these hyphal structures could be associated with the secretion of aspartyl proteases (Sap). The performed assessments revealed an increase in the activity of Sap, which was accompanied by an increase in the expression of the sap2 and sap4 genes. The antifungal action of 2,4-DAPG is known to be associated with affecting the permeability of cellular structures, which leads to H+ATPase malfunction and the disruption of mitochondrial respiration. The subsequent cytosol acidification and generation of ROS trigger the inhibition of Candida filamentation and activation of Sap production. The introduction of antioxidant Trolox simultaneously with 2,4-DAPG leads to a reduction in Sap production. Collectively, the obtained data indicate new aspects of the interaction of fungal cells with 2,4-DAPG, an antimicrobial metabolite of Pseudomonas spp

Atomic force microscopy reveals a morphological differentiation of chromobacterium violaceum cells associated with biofilm development and directed by N-hexanoyl-L-homoserine lactone.

Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceum's adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceum's cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure

The Multiple Activities and the Plant Beneficial Potential of Bacillus spp. Derived from the Permafrost of Western Siberia

Agents of biological control are an important part of traditional agriculture, as well as organic farming. However, in the climatic conditions of countries that are located in cold and temperate regions, plant protection requires particular biocontrol agents that have adapted to environments with low and unstable temperatures. This work presents the biocontrol potential and plant-promoting activity of Bacillus spp. that was isolated from permafrost sediments in Western Siberia. It was found that all of the studied strains (n = 10) were able to produce indole-3-acetic acid (IAA) and chitinolytic enzymes at low positive temperatures (5 &deg;C). The antifungal activity of cold-tolerant bacilli against Microdochium sp., Fusarium spp., and Alternaria sp was recorded. In greenhouse and field conditions, the selected strains (B. simplex 948P-1 (IAA-producing) and B. megaterium 312 (with antifungal activity)) were assessed in comparison to a commercially available fungicide (tebuconazole) and biofungicide (B.subtilis 26D). It was found that the bacilli in the seed germination assay exhibited low phytotoxicity and there was no significant advantage over the conventional fungicides in the yield stimulation assay. However, the twin consortia of B. megaterium 312 and B. simplex 948P-1 was able to increase winter wheat yields by 50% (compared to the untreated group), and by 70% (compared to the commercial biofungicide-treated group). Moreover, applying the twin consortia of Bacillus spp. significantly reduced the infection rate of Fusarium spp. in first-generation wheat grain

Peptide Extracts from Seven Medicinal Plants Discovered to Inhibit Oomycete Phytophthora infestans, a Causative Agent of Potato Late Blight Disease

We report the inhibitory effect of peptide extracts obtained from seven medicinal plants against a causative agent of late blight disease Phytophthora infestans. We find that all the extracts possess inhibitory activity toward the zoospores output, zoosporangium germination, and the development of P. infestans on potato disc tubers at different quantitative levels. Based on the biological effects detected, an extract of common horsetail (Equisetum arvense) biomass is recognized as the most effective and is selected for further structural analysis. We perform a combination of amino acid analysis and MALDI-TOF mass spectrometry, which reveal the presence of Asn/Asp- and Gln/Glu-rich short peptides with molecular masses in the range of 500&ndash;900 Da and not exceeding 1500 Da as the maximum. Analytical anion-exchange HPLC is successfully applied for separation of the peptide extract from common horsetail (E. arvense). We collect nine dominant components that are combined in two groups with differences in retention times. The N-terminal amino acid sequence of the prevalent compounds after analytical ion-exchange HPLC allows us to identify them as peptide fragments of functionally active proteins associated with photosynthesis, aquatic transport, and chitin binding. The anti-oomycete effects may be associated with the conversion of ribulose-1,5-bisphosphate carboxylase/oxygenase to produce a number of biologically active anionic peptides with possible regulatory functions. These data inform our knowledge regarding biologically active peptide fragments; they are the components of programmed or induced proteolysis of plant proteins and can realize secondary antimicrobial functions

Toxicity of Graphene Shells, Graphene Oxide, and Graphene Oxide Paper Evaluated with Escherichia coli Biotests

The plate-like graphene shells (GS) produced by an original methane pyrolysis method and their derivatives graphene oxide (GO) and graphene oxide paper (GO-P) were evaluated with luminescent Escherichia coli biotests and additional bacterial-based assays which together revealed the graphene-family nanomaterials’ toxicity and bioactivity mechanisms. Bioluminescence inhibition assay, fluorescent two-component staining to evaluate cell membrane permeability, and atomic force microscopy data showed GO expressed bioactivity in aqueous suspension, whereas GS suspensions and the GO-P surface were assessed as nontoxic materials. The mechanism of toxicity of GO was shown not to be associated with oxidative stress in the targeted soxS::lux and katG::lux reporter cells; also, GO did not lead to significant mechanical disruption of treated bacteria with the release of intracellular DNA contents into the environment. The well-coordinated time- and dose-dependent surface charge neutralization and transport and energetic disorders in the Escherichia coli cells suggest direct membrane interaction, internalization, and perturbation (i.e., “membrane stress”) as a clue to graphene oxide’s mechanism of toxicity

Atomic force microscopy recognition of protein A on Staphylococcus aureus cell surfaces by labelling with IgG–Au conjugates

The labelling of functional molecules on the surface of bacterial cells is one way to recognize the bacteria. In this work, we have developed a method for the selective labelling of protein A on the cell surfaces of Staphylococcus aureus by using nanosized immunogold conjugates as cell-surface markers for atomic force microscopy (AFM). The use of 30-nm size Au nanoparticles conjugated with immunoglobulin G (IgG) allowed the visualization, localization and distribution of protein A–IgG complexes on the surface of S. aureus. The selectivity of the labelling method was confirmed in mixtures of S. aureus with Bacillus licheniformis cells, which differed by size and shape and had no IgG receptors on the surface. A preferential binding of the IgG–Au conjugates to S. aureus was obtained. Thus, this novel approach allows the identification of protein A and other IgG receptor-bearing bacteria, which is useful for AFM indication of pathogenic microorganisms in poly-component associations