Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.

In this study, the effect of epinephrine on the biofilm formation of Micrococcus luteus C01 isolated from human skin was investigated in depth for the first time. This hormone has a complex effect on biofilms in various systems. In a system with polytetrafluoroethylene (PTFE) cubes, treatment with epinephrine at a physiological concentration of 4.9 × 10 −9 M increased the total amount of 72-h biofilm biomass stained with crystal violet and increased the metabolic activity of biofilms, but at higher and lower concentrations, the treatment had no significant effect. On glass fiber filters, treatment with the hormone decreased the number of colony forming units (CFUs) and changed the aggregation but did not affect the metabolic activity of biofilm cells. In glass bottom plates examined by confocal microscopy, epinephrine notably inhibited the growth of biofilms. RNA-seq analysis and RT–PCR demonstrated reproducible upregulation of genes encoding Fe–S cluster assembly factors and cyanide detoxification sulfurtransferase, whereas genes encoding the co-chaperone GroES, the LysE superfamily of lysine exporters, short-chain alcohol dehydrogenase and the potential c-di-GMP phosphotransferase were downregulated. Our results suggest that epinephrine may stimulate matrix synthesis in M. luteus biofilms, thereby increasing the activity of NAD(H) oxidoreductases. Potential c-di-GMP pathway proteins are essential in these processes.

The first evidence of the atrial natriuretic peptide (ANP) effect on mono-species and dual-species biofilms of skin commensals Cutibacterium acnes and Staphylococcus epidermidis was obtained in different model systems. Elucidation of the mechanism of action of hormones on the microbial communities of human skin is an important physiological and medical aspect. Under anaerobic conditions, ANP at a concentration of 6.5 × 10−10 M inhibits the growth of S. epidermidis biofilms and stimulates the growth of C. acnes biofilms, and a lesser effect has been demonstrated on planktonic cultures. In biofilms, ANP stimulates aggregation in C. acnes and aggregate dispersion of S. epidermidis, while in S. epidermidis, ANP also stimulates the metabolic activity of cells. Analysis of dual-species biofilms has shown the dominance of S. epidermidis, while ANP increases the ratio of C. acnes biomass in the community. ANP decreases the growth rate of S. epidermidis biofilms and increases that of C. acnes. The effect of ANP is not dependent on the surface type and probably affects other targets in microbial cells. Thus, the potential regulatory effect of human ANP on skin microbe dual-species communities has been shown, and its potential has been demonstrated to change microbiota homeostasis on the skin.

The impact of steroid hormones, and particularly estradiol, on human microbiota could be recognized as a substantial part of human-microbiota interactions. However, an area that remains poorly investigated is that of the skin and vaginal microbial communities and biofilms, which contain non-pathogenic bacteria of phyla Firmicutes and Actinobacteria, especially probiotic bacteria of the genus Lactobacillus and the widespread, safe skin genus, Micrococcus. Experiments with Lactobacillus paracasei AK508 and Micrococcus luteus C01 biofilms on PTFE cubes showed dose-dependent effects of estradiol at concentrations of 0.22 nM and 22 nM. The hormone mostly inhibits L. paracasei growth and stimulates M. luteus. The presented studies of colony-forming unit (CFU) amountsand cell aggregation in biofilms on glass fiber filters showed the same general tendencies. Estradiol generally increased the aggregation of cells in monospecies communities and potentially changed the synthesis of antibacterial metabolites in L. paracasei. The balance between two bacteria in mixed-species biofilms depended on the initial adhesion stage, and when this stage was reduced, micrococci were more resistant to the antagonistic action of L. paracasei. Moreover, in mixed-species biofilms, the effect of estradiol on lactobacilli altered from inhibition to stimulation, potentially due to the presence of M. luteus. At the same time, ethanol as a solvent for estradiol at the concentration 0.6% acted mostly as an antagonist of the hormone and had an opposite effect on bacteria; nevertheless, the overlapping of ethanol and estradiol effects was shown to be minimal. The data obtained prove the complexity of microbial interactions and the regulatory effect of estradiol on commensal bacteria biofilms.

The effect of C-type natriuretic peptide in a concentration closer to the normal level in human blood plasma was studied on the mono-species and dual-species biofilms of the skin commensal bacteria Cutibacterium acnes HL043PA2 and Staphylococcus epidermidis ATCC14990. Despite the marginal effect of the hormone on cutibacteria in mono-species biofilms, the presence of staphylococci in the community resulted in a global shift of the CNP effect, which appeared to increase the competitive properties of C. acnes, its proliferation and the metabolic activity of the community. S. epidermidis was mostly inhibited in the presence of CNP. Both bacteria had a significant impact on the gene expression levels revealed by RNA-seq. CNP did not affect the gene expression levels in mono-species cutibacterial biofilms; however, in the presence of staphylococci, five genes were differentially expressed in the presence of the hormone, including two ribosomal proteins and metal ABC transporter permease. In staphylococci, the Na-translocating system protein MpsB NADH-quinone oxidoreductase subunit L was downregulated in the dual-species biofilms in the presence of CNP, while in mono-species biofilms, two proteins of unknown function were downregulated. Hypothetically, at least one of the CNP mechanisms of action is via the competition for zinc, at least on cutibacteria.