The Functionality of IbpA from Acholeplasma laidlawii Is Governed by Dynamic Rearrangement of Its Globular-Fibrillar Quaternary Structure.

Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In Escherichia coli, fibrillar EcIbpA binds unfolded proteins and keeps them in a folding-competent state. Further, its structural homologue EcIbpB induces the transition of EcIbpA to globules, thereby facilitating the substrate transfer to the HSP70-HSP100 system for refolding. The phytopathogenic Acholeplasma laidlawii possesses only a single sHSP, AlIbpA. Here, we demonstrate non-trivial features of the function and regulation of the chaperone-like activity of AlIbpA according to its interaction with other components of the mycoplasma multi-chaperone network. Our results show that the efficiency of the A. laidlawii multi-chaperone system is driven with the ability of AlIbpA to form both globular and fibrillar structures, thus combining functions of both IbpA and IbpB when transferring the substrate proteins to the HSP70-HSP100 system. In contrast to EcIbpA and EcIbpB, AlIbpA appears as an sHSP, in which the competition between the N- and C-terminal domains regulates the shift of the protein quaternary structure between a fibrillar and globular form, thus representing a molecular mechanism of its functional regulation. While the C-terminus of AlIbpA is responsible for fibrils formation and substrate capture, the N-terminus seems to have a similar function to EcIbpB through facilitating further substrate protein disaggregation using HSP70. Moreover, our results indicate that prior to the final disaggregation process, AlIbpA can directly transfer the substrate to HSP100, thereby representing an alternative mechanism in the HSP interaction network.

Alterations in Antibiotic Susceptibility of Staphylococcus aureus and Klebsiella pneumoniae in Dual Species Biofilms.

In the last decades, it has been shown that biofilm-associated infections in most cases are caused by rather two or even more pathogens than by single microorganisms. Due to intermicrobial interactions in mixed communities, bacteria change their gene expression profile, in turn leading to alterations in the biofilm structure and properties, as well as susceptibility to antimicrobials. Here, we report the alterations of antimicrobials efficiency in mixed biofilms of Staphylococcus aureus-Klebsiella pneumoniae in comparison with mono-species biofilms of each counterpart and discuss possible mechanisms of these alterations. In cell clumps detached from dual-species biofilms, S. aureus became insensitive to vancomycin, ampicillin, and ceftazidime compared to solely S. aureus cell clumps. In turn, the increased efficiency of amikacin and ciprofloxacin against both bacteria could be observed, compared to mono-species biofilms of each counterpart. Scanning electron microscopy and confocal microscopy indicate the porous structure of the dual-species biofilm, and differential fluorescent staining revealed an increased number of polysaccharides in the matrix, in turn leading to more loose structure and thus apparently providing increased permeability of the dual-species biofilm to antimicrobials. The qRT-PCR showed that ica operon in S. aureus became repressed in mixed communities, and polysaccharides are produced mainly by K. pneumoniae. While the molecular trigger of these changes remains undiscovered, detailed knowledge of the alterations in antibiotic susceptibility to given drugs opens doors for treatment correction options for S. aureus-K. pneumoniae biofilm-associated infections.

The Effect of Ficin Immobilized on Carboxymethyl Chitosan on Biofilms of Oral Pathogens.

In the last decade, Ficin, a proteolytic enzyme extracted from the latex sap of the wild fig tree, has been widely investigated as a promising tool for the treatment of microbial biofilms, wound healing, and oral care. Here we report the antibiofilm properties of the enzyme immobilized on soluble carboxymethyl chitosan (CMCh) and CMCh itself. Ficin was immobilized on CMCh with molecular weights of either 200, 350 or 600 kDa. Among them, the carrier with a molecular weight of 200 kDa bound the maximum amount of enzyme, binding up to 49% of the total protein compared to 19-32% of the total protein bound to other CMChs. Treatment with pure CMCh led to the destruction of biofilms formed by Streptococcus salivarius, Streptococcus gordonii, Streptococcus mutans, and Candida albicans, while no apparent effect on Staphylococcus aureus was observed. A soluble Ficin was less efficient in the destruction of the biofilms formed by Streptococcus sobrinus and S. gordonii. By contrast, treatment with CMCh200-immobilized Ficin led to a significant reduction of the biofilms of the primary colonizers S. gordonii and S. mutans. In model biofilms obtained by the inoculation of swabs from teeth of healthy volunteers, the destruction of the biofilm by both soluble and immobilized Ficin was observed, although the degree of the destruction varied between artificial plaque samples. Nevertheless, combined treatment of oral Streptococci biofilm by enzyme and chlorhexidine for 3 h led to a significant decrease in the viability of biofilm-embedded cells, compared to solely chlorhexidine application. This suggests that the use of either soluble or immobilized Ficin would allow decreasing the amount and/or concentration of the antiseptics required for oral care or improving the efficiency of oral cavity sanitization.

The Novel Chiral 2(5H)-Furanone Sulfones Possessing Terpene Moiety: Synthesis and Biological Activity.

Over the past decades, 2(5H)-furanone derivatives have been extensively studied because of their promising ability to prevent the biofilm formation by various pathogenic bacteria. Here, we report the synthesis of a series of optically active sulfur-containing 2(5H)-furanone derivatives and characterize their biological activity. Novel thioethers were obtained by an interaction of stereochemically pure 5-(l)-menthyloxy- or 5-(l)-bornyloxy-2(5H)-furanones with aromatic thiols under basic conditions. Subsequent thioethers oxidation by an excess of hydrogen peroxide in acetic acid resulted in the formation of the corresponding chiral 2(5H)-furanone sulfones. The structure of synthesized compounds was confirmed by IR and NMR spectroscopy, HRMS, and single crystal X-ray diffraction. The leading compound, 26, possessing the sulfonyl group and l-borneol moiety, exhibited the prominent activity against Staphylococcus aureus and Bacillus subtilis with MICs of 8 µg/mL. Furthermore, at concentrations of 0.4-0.5 µg/mL, the sulfone 26 increased two-fold the efficacy of aminoglycosides gentamicin and amikacin against S. aureus. The treatment of the model-infected skin wound in the rat with a combination of gentamicin and sulfone 26 speeded up the bacterial decontamination and improved the healing of the wound. The presented results provide valuable new insights into the chemistry of 2(5H)-furanone derivatives and associated biological activities.

Amine-Reactive BODIPY Dye: Spectral Properties and Application for Protein Labeling.

A boron-dipyrromethene (BODIPY) derivative reactive towards amino groups of proteins (NHS-Ph-BODIPY) was synthesized. Spectroscopic and photophysical properties of amine-reactive NHS-Ph-BODIPY and its non-reactive precursor (COOH-Ph-BODIPY) in a number of organic solvents were investigated. Both fluorescent dyes were characterized by green absorption (521-532 nm) and fluorescence (538-552 nm) and medium molar absorption coefficients (46,500-118,500 M-1·cm-1) and fluorescence quantum yields (0.32 - 0.73). Solvent polarizability and dipolarity were found to play a crucial role in solvent effects on COOH-Ph-BODIPY and NHS-Ph-BODIPY absorption and emission bands maxima. Quantum-chemical calculations were used to show why solvent polarizability and dipolarity are important as well as to understand how the nature of the substituent affects spectroscopic properties of the fluorescent dyes. NHS-Ph-BODIPY was used for fluorescent labeling of a number of proteins. Conjugation of NHS-Ph-BODIPY with bovine serum albumin (BSA) resulted in bathochromic shifts of absorption and emission bands and noticeable fluorescence quenching (about 1.5 times). It was demonstrated that the sensitivity of BSA detection with NHS-Ph-BODIPY was up to eight times higher than with Coomassie brilliant blue while the sensitivity of PII-like protein PotN (PotN) detection with NHS-Ph-BODIPY and Coomassie brilliant blue was almost the same. On the basis of the molecular docking results, the most probable binding sites of NHS-Ph-BODIPY in BSA and PotN and the corresponding binding free energies were estimated.

Synthesis and Biological Activity of Unsymmetrical Monoterpenylhetaryl Disulfides.

New unsymmetrical monoterpenylhetaryl disulfides based on heterocyclic disulfides and monoterpene thiols were synthesized for the first time in 48-88% yields. Hydrolysis of disulfides with fragments of methyl esters of 2-mercaptonicotinic acid was carried out in 73-95% yields. The obtained compounds were evaluated for antioxidant, antibacterial, antifungal activity, cytotoxicity and mutagenicity.

The Bovhyaluronidase Azoximer (Longidaza®) Disrupts Candida albicans and Candida albicans-Bacterial Mixed Biofilms and Increases the Efficacy of Antifungals.

Background and Objectives: Candida albicans causes various diseases ranging from superficial mycoses to life-threatening systemic infections often associated with biofilm formation, including mixed fungal−bacterial consortia. The biofilm matrix protects cells, making Candida extremely resistant to treatment. Here, we show that the bovhyaluronidase azoximer (Longidaza®) in vitro destroys the biofilm formed by either C. albicans alone or mixed with bacteria, this way decreasing the concentrations of antimicrobials required for the pathogen's eradication. Materials and Methods: Bovhyaluronidase azoximer, Longidaza® was obtained from NPO Petrovax Pharm Ltd., Moscow, Russia as lyophilized powder. The antifungal activity was assessed by microdilution assay and CFUs counting. Antibiofilm activity was evaluated via biofilms staining and scanning electron microscopy. Results: Thus, treatment with Longidaza® reduced the biofilm biomass of nine C. albicans clinical isolates by 30−60%, while mixed biofilms of C. albicans with various bacteria were destroyed by 30−40%. Furthermore, the concentration of fluconazole required to achieve a similar reduction of the residual respiratory activity of detached cell clumps of four C. albicans isolates has been reduced four-fold when combined with Longidaza®. While in the biofilm, two of four isolates became significantly more susceptible to fluconazole in combination with Longidaza®. Conclusion: Taken together, our data indicate that Longidaza® is capable of suppression of tissues and artificial surfaces biofouling by C. albicans biofilms, as well as facilitating drug penetration into the cell clumps, this way decreasing the effective MIC of antifungals.

Biochemical Properties and Anti-Biofilm Activity of Chitosan-Immobilized Papain.

Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine protease (EC: 3.4.22.2) from Carica papaya latex immobilized on the matrixes of medium molecular (200 kDa) and high molecular (350 kDa) weight chitosans exhibits anti-biofilm activity and increases the antimicrobials efficiency against biofilm-embedded bacteria. Immobilization in glycine buffer (pH 9.0) allowed adsorption up to 30% of the total protein (mg g chitosan-1) and specific activity (U mg protein-1), leading to the preservation of more than 90% of the initial total activity (U mL-1). While optimal pH and temperature of the immobilized papain did not change, the immobilized enzyme exhibited elevated thermal stability and 6-7-fold longer half-life time in comparison with the soluble papain. While one-half of the total enzyme dissociates from both carriers in 24 h, this property could be used for wound-dressing materials design with dosed release of the enzyme to overcome the relatively high cytotoxicity of soluble papain. Our results indicate that both soluble and immobilized papain efficiently destroy biofilms formed by Staphylococcus aureus and Staphylococcus epidermidis. As a consequence, papain, both soluble and immobilized on medium molecular weight chitosan, is capable of potentiating the efficacy of antimicrobials against biofilm-embedded Staphylococci. Thus, papain immobilized on medium molecular weight chitosan appears a presumably beneficial agent for outer wound treatment for biofilms destruction, increasing antimicrobial treatment effectiveness.

Targeting pathogenic fungi, bacteria and fungal-bacterial biofilms by newly synthesized quaternary ammonium derivative of pyridoxine and terbinafine with dual action profile.

Many pathogenic bacteria and microscopic fungi form rigid polymicrobial biofilms this way enhancing their resistant to treatment. A series of novel pyridoxine-based quaternary ammonium derivatives of terbinafine characterized by both antifungal and antibacterial activities was designed. The leading compound named KFU-127 exhibits promising antifungal and antibacterial activities against various bacteria and micromycetes in both planktonic and biofilm-embedded forms demonstrating MIC values comparable with those of conventional antifungals and antimicrobials. Similar to other antiseptics like benzalkonium chloride and miramistin, KFU-127 is considerably toxic for eukaryotic cells that limits is application to topical treatment options. On the other hand, KFU-127 reduces the number of viable biofilm-embedded bacteria and C. albicans by 3 orders of magnitude at concentrations 2-4 times lower than those of reference drugs and successfully eradicates S. aureus-C. albicans mixed biofilms. The mechanism of antimicrobial action of KFU-127 is bimodal including both membrane integrity damage and pyridoxal-dependent enzymes targeting. We expect that this bilateral mechanism would result in lower rates of resistance development in both fungal and bacterial pathogens. Taken together, our data suggest KFU-127 as a new promising broad spectrum topical antimicrobial capable of one-shot targeting of bacterial and fungal-bacterial biofilms.

Complete Genome Sequence of Lactobacillus hilgardii LMG 7934, Carrying the Gene Encoding for the Novel PII-Like Protein PotN.

Lactic acid bacteria are widespread in various ecological niches with the excess of nutrients and have reduced capabilities to adapt to starvation. Among more than 280 Lactobacillus species known to the date, only five, including Lactobacillus hilgardii, carry in their genome the gene encoding for PII-like protein, one of the central regulators of cellular metabolism generally responding to energy- and carbon-nitrogen status in many free-living Bacteria, Archaea and in plant chloroplasts. In contrast to the classical PII encoding genes, in L. hilgardii genome the gene for PII homologue is located within the potABCD operon, encoding the ABC transporter for polyamines. Based on the unique genetic context and low sequence identity with genes of any other so-far characterized PII subfamilies, we termed this gene potN (Pot-protein, Nucleotide-binding). The second specific feature of L. hilgardii genome is that many genes encoding the proteins with similar function are present in two copies, while with low mutual identity. Thus, L. hilgardii LMG 7934 genome carries two genes of glutamine synthetase with 55% identity. One gene is located within classical glnRA operon with the gene of GlnR-like transcriptional regulator, while the second is monocistronic. Together with the relative large genome of L. hilgardii as compared to other Lactobacilli (2.771.862 bp vs ~ 2.2 Mbp in median), these data suggest significant re-arrangements of the genome and a wider range of adaptive capabilities of L. hilgardii in comparison to other bacteria of the genus Lactobacillus.

Increasing Susceptibility of Drug-Resistant Candida albicans to Fluconazole and Terbinafine by 2(5H)-Furanone Derivative.

The frequency of mycoses caused by drug-resistant fungal pathogen Candida albicans has increased drastically over the last two decades. The spread of drug-resistant strains, along with the limitations of currently available antifungals, complicates the management of fungal infections, thereby representing great challenges for clinical healthcare. Among various antimicrobial pharmacophores, 2(5H)-furanone derivatives have demonstrated antimicrobial, antifungal, and antibiofilm activities. In this study, we report the antifungal activity of the 2(5H)-furanone derivative F105, consisting of three pharmacophores, namely chlorinated 2(5H)-furanone, sulfonyl group, and l-menthol moiety. Although exhibiting moderate antifungal activity alone with the minimum inhibitory concentration (MIC) values of 32-256 µg/mL, F105 potentiates the activity of fluconazole and terbinafine with fractional inhibitory concentration index (FICI) values of 0.27-0.50. Thus, 16 µg/mL of F105 reduced the MICs of these antifungals against fluconazole-resistant C. albicans isolates four-fold, achieving similar values as for the intermediately susceptible phenotype. Confocal laser scanning microscopy revealed that the fluorescent 2(5H)-furanone derivative F145 was also able to penetrate through biofilms formed by C. albicans. Indeed, in the presence of F105, even sub-MIC concentrations of both fluconazole and terbinafine led to significant reduction of C. albicans CFUs in the mature biofilm. Thus, F105 appears to be a promising candidate for the development of novel antifungal agents as well as enhancers of current antifungal agents, particularly for the treatment of drug-resistant C. albicans infections.

Spectroscopic and In Vitro Investigations of Boron(III) Complex with Meso-4-Methoxycarbonylpropylsubstituted Dipyrromethene for Fluorescence Bioimaging Applications.

This study focuses on the behavior of a new fluorescent marker for labeling individual biomolecules and staining cell organelles developed on a meso-substituted BODIPY platform. Boron(III) complex with meso-4-methoxycarbonylpropylsubstituted 3,3',5,5'-tetramethyl-2,2'-dipyrromethene has been synthesized and identified via visible, UV-, NMR- and MS-spectra X-ray. The behavior of fluorophore in solutions has been studied with various experimental techniques. It has been found that luminophore exhibits a high quantum yield (almost ~100-75%) in the blue-green region (513-520 nm) and has high photostability. In addition, biological analysis indicates that the fluorophore exhibits a tendency to effectively penetrate into cell membranes. On the other hand, the proposed BODIPY can be used to study the significant differences among a large number of pathogens of mycotic infections, as well as to visualize structural changes in the plasma membrane, which is necessary for the clearance of mammalian cells undergoing apoptotic cell death.

Novel Bis-Ammonium Salts of Pyridoxine: Synthesis and Antimicrobial Properties.

A series of 108 novel quaternary bis-ammonium pyridoxine derivatives carrying various substituents at the quaternary nitrogen's and acetal carbon was synthesized. Thirteen compounds exhibited antibacterial and antifungal activity (minimum inhibitory concentration (MIC) 0.25-16 µg/mL) comparable or superior than miramistin, benzalkonium chloride, and chlorhexidine. A strong correlation between the lipophilicity and antibacterial activity was found. The most active compounds had logP values in the range of 1-3, while compounds with logP > 6 and logP < 0 were almost inactive. All active compounds demonstrated cytotoxicity comparable with miramistin and chlorhexidine on HEK-293 cells and were three-fold less toxic when compared to benzalkonium chloride. The antibacterial activity of leading compound 5c12 on biofilm-embedded Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli or Pseudomonas aeruginosa was comparable or even higher than that of the benzalkonium chloride. In vivo 5c12was considerably less toxic (LD50 1705 mg/kg) than benzalkonium chloride, miramistine, and chlorhexidine at oral administration on CD-1 mice. An aqueous solution of 5c12 (0.2%) was shown to be comparable to reference drugs efficiency on the rat's skin model. The molecular target of 5c12 seems to be a cellular membrane as other quaternary ammonium salts. The obtained results make the described quaternary bis-ammonium pyridoxine derivatives promising and lead molecules in the development of the new antiseptics with a broad spectrum of antimicrobial activity.

Newly isolated lactic acid bacteria from silage targeting biofilms of foodborne pathogens during milk fermentation.

BACKGROUND: Raw milk, meat and plant materials are subjected to high risks of contamination by various pathogenic bacteria and thus their growth prevention is a great challenge in the food industry. Food fermentation by lactic acid bacteria (LAB) besides changing its organoleptic characteristics also helps to eliminate unfavorable microflora and represses growth of pathogens. To the date only few LABs has been reported to exhibit activity against bacteria embedded in the biofilms characterized by extreme resistance to antimicrobials, high exchange rate with resistance genes and represent high risk factor for foodborne disease development. RESULTS: Six novel LAB strains isolated from the clover silage exhibited pronounced antibacterial activity against biofilm embedded pathogens. We show explicitly that these strains demonstrate high acidification rate, completely repress the growth of E. coli, S. aureus and to a lesser extent P. aeruginosa as well as exhibit appropriate probiotic and milk-fermenting properties. Moreover, in contrast to the approved probiotic strain Lactobacillus plantarum 8PA3, the new isolates were able to efficiently eradicate preformed biofilms of these pathogens and prevent bacterial spreading originating from the biofilm. We suggest these strains as potential additives to the pre-cultures of conventional LAB strains as efficient tools targeting foodborne pathogens in order to prevent food contamination from either seeded raw material or biofilm-fouled equipment. CONCLUSIONS: The AG10 strain identified as L. plantarum demonstrate attractive probiotic and milk fermentation properties as well as high resistance to simulated gastric conditions thus appearing perspective as a starter culture for the prevention of bacterial contamination originating from fouled equipment during milk fermentation.

Targeting Bacillus cereus cells: increasing efficiency of antimicrobials by the bornylpossessing 2(5Н)-furanone derivative.

Among a variety of antimicrobial compounds, the derivatives of 2(5H)-furanone exhibit different effects on Firmicutes and Proteobacteria. While inhibiting quorum-dependent biofilm formation and virulence factor expression by Gram-negative bacteria through specific interference with the AI-2 signaling pathways, these compounds demonstrate bactericidal effects against Gram-positive bacteria. Here we report that 3,4-dichloro-5(S)-[(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yloxy]-2(5H)-furanone designed as F123 inhibits growth and biofilm formation by the food-poisoning bacterium Bacillus cereus at 8 µg/ ml and kills bacteria at 16 µg/ml. While the growth of Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis were also inhibited at 8-16 µg/ml of F123, no bactericidal effect on these strains was observed at concentrations up to 128 µg/ml, suggesting pronounced specificity of F123 for B. cereus. In a checker-board assay F123 increased the efficacy of amikacin, gentamicin and benzalkonium chloride against B. cereus with medians of fractional inhibitory concentration index of 0.38, 0.56 and 0.56, respectively. Moreover, the number of viable B. cereus cells in biofilm was reduced by more than 3 orders of magnitude at 64 µg/ml of F123, suggesting its chemotype as a promising enhancer for specific treatment of B. cereus-associated topical infections, including biofilm-embedded bacteria.

Unraveling the Molecular Mechanism of Selective Antimicrobial Activity of 2(5H)-Furanone Derivative against Staphylococcus aureus.

Staphylococcus aureus causes various infectious diseases, from skin impetigo to life-threatening bacteremia and sepsis, thus appearing an important target for antimicrobial therapeutics. In turn, the rapid development of antibiotic resistance and biofilm formation makes it extremely robust against treatment. Here, we unravel the molecular mechanism of the antimicrobial activity of the recently unveiled F105 consisting of three pharmacophores: chlorinated 2(5H)-furanone, sulfone, and l-menthol moieties. F105 demonstrates highly selective activity against Gram-positive bacteria and biofilm-embedded S. aureus and exhibits low risk of resistance development. We show explicitly that the fluorescent analogue of F105 rapidly penetrates into Gram-positive bacteria independently of their cell integrity and viability and accumulates there. By contrast, Gram-negative bacteria remain impermeable and, therefore, insusceptible to F105. Apparently, in bacterial cells, F105 induces reactive oxygen species (ROS) formation and nonspecifically interacts with a number of proteins, including ROS-utilizing ones. Using native and 2D PAGE, we confirm that F105 changes the charge of some proteins by either oxidation or direct interaction with them. Therefore, it seems justified to conclude that being simultaneously a ROS inducer and damaging proteins responsible for ROS utilization, F105 impairs the cellular anti-ROS defense representing a prospective ROS-inducing antibacterial agent.

The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis.

TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine, and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active but unable to interact with TnrA. This conformation was stabilized by phosphorylated L-methionine sulfoximine (MSX), fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive, and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no longer able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. Surface plasmon resonance analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state, whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size exclusion and multiangle light scattering analysis, the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA.

Direct inhibition of oncogenic KRAS by Bacillus pumilus ribonuclease (binase).

RAS proteins function as molecular switches that transmit signals from cell surface receptors into specific cellular responses via activation of defined signaling pathways (Fang, 2015). Aberrant constitutive RAS activation occurs with high incidence in different types of cancer (Bos, 1989). Thus, inhibition of RAS-mediated signaling is extremely important for therapeutic approaches against cancer. Here we showed that the ribonuclease (RNase) binase, directly interacts with endogenous KRAS. Further, molecular structure models suggested an inhibitory nature of binase-RAS interaction involving regions of RAS that are important for different aspects of its function. Consistent with these models, phosphorylation analysis of effectors of RAS-mediated signaling revealed that binase inhibits the MAPK/ERK signaling pathway. Interestingly, RAS activation assays using a non-hydrolysable GTP analog (GTPγS) demonstrated that binase interferes with the exchange of GDP by GTP. Furthermore, we showed that binase reduced the interaction of RAS with the guanine nucleotide exchange factor (GEF), SOS1. Our data support a model in which binase-KRAS interaction interferes with the function of GEFs and stabilizes the inactive GDP-bound conformation of RAS thereby inhibiting MAPK/ERK signaling. This model plausibly explains the previously reported, antitumor-effect of binase specific towards RAS-transformed cells and suggests the development of anticancer therapies based on this ribonuclease.

Antibacterial effects of quaternary bis-phosphonium and ammonium salts of pyridoxine on Staphylococcus aureus cells: A single base hitting two distinct targets?

We studied the effects of quaternary bis-phosphonium and bis-ammonium salts of pyridoxine with lipophilic substituents on the survival and morphology of Staphylococcus aureus cells. We found that, while originating from the same base, they exhibit considerably different antimicrobial mechanisms. In the presence of Ca(2+) ions the MIC and MBC values of ammonium salt increased 100-fold, suggesting that Ca(2+) ions can successfully impede the membrane Ca(2+) ions exchange required for ammonium salt incorporation. In contrast, in the presence of quaternary phosphonium salt, the artificial capsular-like material was formed around the cells and the filamentous and chain-like growth of the cells was observed suggesting the disruption of the cell division mechanisms. Altogether, both pyridoxine derivatives successfully inhibited the growth of gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis) and Escherichia coli considerably, while demonstrated nearly no effect against Klebsiella pneumoniae and Pseudomonas aeruginosa. We suggest that due to their effects on distinct and likely complementary targets the derivatives of pyridoxine represent potentially perspective antibacterials with complicated adaptation and thus with lower risk of drug resistance development.

Two-Year Study on the Intra-Individual Dynamics of Gut Microbiota and Short-Chain Fatty Acids Profiles in Healthy Adults.

While the gut microbiome has been intensively investigated for more than twenty years already, its role in various disorders remains to be unraveled. At the same time, questions about what changes in the gut microbiota can be considered as normal or pathological and whether communities are able to recover after exposure to negative factors (diseases, medications, environmental factors) are still unclear. Here, we describe changes in the gut microbiota composition and the content of short-chain fatty acids in adult healthy volunteers (n = 15) over a 24 month-period. Intraindividual variability in gut microbial composition was 40%, whereas the short chain fatty acids profile remained relatively stable (2-year variability 20%, inter-individual 26%). The changes tend to accumulate over time. Nevertheless, both short-term and long-term changes in the gut microbiome composition were significantly smaller within individuals than interindividual differences (two-year interindividual variability was 75%). Seasonal changes in gut microbiota were found more often in autumn and spring involving the content of minor representatives (less than 1.5% of the community in average) in the phyla Actinobacteriota, Firmicutes and Proteobacteria.