Mutation of lipoprotein processing pathway gene lspA or inhibition of LspA activity by globomycin increases MRSA resistance to β-lactam antibiotics

The Staphylococcus aureus cell envelope comprises numerous components, including peptidoglycan (PG), wall teichoic acids (WTA), lipoteichoic acids (LTA), targeted by antimicrobial drugs. MRSA resistance to methicillin is mediated by the mecA-encoded β-lactam-resistant transpeptidase, penicillin binding protein 2a (PBP2a). However, PBP2a-dependent β-lactam resistance is also modulated by the activity of pathways involved in the regulation or biosynthesis of PG, WTA or LTA. Here, we report that mutation of the lipoprotein signal peptidase II gene, lspA, from the lipoprotein processing pathway, significantly increased β-lactam resistance in MRSA. Mutation of lgt, which encodes diacylglycerol transferase (Lgt) responsible for synthesis of the LspA substrate did not impact β-lactam susceptibility. Consistent with previous reports, lgt and lspA mutations impaired growth in chemically defined media, but not in complex broth. MRSA exposure to the LspA inhibitor globomycin also increased β-lactam resistance. Mutation of lgt in an lspA background restored β-lactam resistance to wild type. The lspA mutation had no effect on PBP2a expression, PG composition or autolytic activity indicating a potential role for WTA or LTA. The lspA and lgt mutants exhibited marginally increased resistance to the D-alanine pathway inhibitor D-cycloserine. In addition, mutation of lgt and multicopy lspA expression, but not mutation of lspA, significantly increased susceptibility to the lipoteichoic acid synthase inhibitor Congo red revealing complex interplay between lipoprotein processing mutations and the expression/stability of cell surface glycopolymers. These findings indicate that accumulation of the LspA substrate, diacylglyceryl lipoprotein, increases MRSA resistance to β-lactam antibiotics through impacts on cell envelope components other than PG.

BAKING PROPERTIES OF DIFFERENT AMARANTH FLOURS AS WHEAT BREAD INGREDIENTS

The technological properties of full-fat amaranth flour depend on the varietal characteristics of the Ukrainian amaranth grain and differ significantly from amaranth flour from flakes and amaranth flour from groats. In comparison with patent wheat flour, amaranth flour has a lower moisture content, higher water absorption capacity and autolytic activity. The variety of Amaranthus hypochondriacus significantly effects on the whiteness of full-fat flours, the lightest of which is obtained from the grain cultivar Kharkivsky-1. A higher fat, protein, and fibre content makes amaranth flours more acidic. The water absorption capacity of the flours shows positive correlation with their autolytic activity (+0.885). The acidity negatively correlates with the moisture (-0.939) and whiteness (-0.814) of the flours. Using amaranth flour of the different types to replace 5, 15, and 25% of patent wheat flour when making bread increases its specific volume and crumb porosity and decreases its shape stability. The positive correlation of the overall quality of the bread samples under study with their specific volume (+0.540) and the negative correlation with the acidity (- 0.685) are statistically significant. The shape stability negatively correlates with the porosity (-0.598), and the latter positively correlates with the specific volume (+0.533). The use of full-fat amaranth flour increases the specific volume and porosity of bread by 1.1–1.3 and 1.1 times respectively. The use of defatted flour from flakes leads to a 1.3–1.9 -fold increase in the specific volume and to a 1.1–1.2 -old increase in the porosity. Incorporation of amaranth flour from groats allows increasing the specific volume and porosity of bread by 1.3–1.5 and 1.1–1.2 times respectively. The bread samples with 25% of all amaranth flours considered and with 15% of full-fat flour of the Liera variety have the lowest consumer characteristics. It has been proved that using 5–15% of full-fat flour from the amaranth grain of variety Kharkivsky-1 and defatted flour from flakes and groats (by-products of processing amaranth grain into oil) improves the quality and nutritional value of bread.

Mutational analysis of ocriplasmin to reduce proteolytic and autolytic activity in Pichia pastoris

Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases.Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

Inhibition of Streptococcus pneumoniae autolysins highlight distinct differences between chemical and genetic inactivation

Despite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive pathogen Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide fgkc as a narrow spectrum bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 7.8 μM. The diamide inhibited detergent-induced autolysis in a concentration dependent manner indicating peptidoglycan degradation as the mode-of-action. Genetic screening of autolysin mutants suggested LytB, an endo-N-acetylglucosaminidase, involved in cell division as the potential target. Surprisingly, biochemical, and phenotypic analysis contradicted the genetic screen results. Phenotypic studies with the Δlytb strain illustrate the difference between genetic and chemical inactivation of autolysins. These findings suggest that meta-phenotypes including autolytic activity, cell morphology, and genetic screening can be the result of the complex interaction of one or more possible pathways that are connected to cell wall metabolism.

Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia Pastoris

Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activities duration as a result of its autolytic and proteolytic nature after injection within the eye. Moreover, the proteolytic activities can cause photoreceptor damage, which may result in visual impairment in the more serious cases.Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both the mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in Pichia pastoris expression system. The mutant variants analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mix variants. Moreover, in variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated combine mutations at ocriplasmin sequence were more effective compared with single mutations. Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at non-surgical resolution of vitreomacular adhesion.

Pathogenicity Biofilm formation of Enterococcus faecalis

Biofilm formation is closely related to the pathogenicity of E. faecalis in persistent root canal infections. Limited information is available about the ability and characteristics of E. faecalis biofilm-forming in the persistent pathogenicity of root canal infections. Based on these problems, the purpose of this paper is to improve the molecular understanding of E. faecalis on virulence factors associated with biofilm formation against persistent root canal infections to support laboratory diagnosis and therapy of oral E. faecalis. In conclusion, the release of cytokines triggers the dlt gene of LTA to improving: homeostasis, autolytic activity, and bacterial envelope properties. The role of cytolysin activated by the cylLL and cylLS genes improving the survival ability of E. Faecalis. Hyaluronidase will facilitate other bacteria to migrate from the root canal to periapical lesions resulting in the triggering of toxin production, which will increase damage and inflammation in the host. Enterococcus faecalis, through dentine matrix formation, hydrolyze collagen and induce apatite deposition in developing biofilms. Besides, these bacteria can also increase their tolerance to antimicrobials by blocking the inflammatory response's acid reaction. Alkaline conditions will neutralize the lactic acid secreted by osteoclasts to absorb hard tissue

Pathogenicity Biofilm formation of Enterococcus faecalis

Biofilm formation is closely related to the pathogenicity of E. faecalis in persistent root canal infections. Limited information is available about the ability and characteristics of E. faecalis biofilm-forming in the persistent pathogenicity of root canal infections. Based on these problems, the purpose of this paper is to improve the molecular understanding of E. faecalis on virulence factors associated with biofilm formation against persistent root canal infections to support laboratory diagnosis and therapy of oral E. faecalis. In conclusion, the release of cytokines triggers the dlt gene of LTA to improving: homeostasis, autolytic activity, and bacterial envelope properties. The role of cytolysin activated by the cylLL and cylLS genes improving the survival ability of E. Faecalis. Hyaluronidase will facilitate other bacteria to migrate from the root canal to periapical lesions resulting in the triggering of toxin production, which will increase damage and inflammation in the host. Enterococcus faecalis, through dentine matrix formation, hydrolyze collagen and induce apatite deposition in developing biofilms. Besides, these bacteria can also increase their tolerance to antimicrobials by blocking the inflammatory response's acid reaction. Alkaline conditions will neutralize the lactic acid secreted by osteoclasts to absorb hard tissue

Pathogenicity Biofilm formation of Enterococcus faecalis

Biofilm formation is closely related to the pathogenicity of E. faecalis in persistent root canal infections. Limited information is available about the ability and characteristics of E. faecalis biofilm-forming in the persistent pathogenicity of root canal infections. Based on these problems, the purpose of this paper is to improve the molecular understanding of E. faecalis on virulence factors associated with biofilm formation against persistent root canal infections to support laboratory diagnosis and therapy of oral E. faecalis. In conclusion, the release of cytokines triggers the dlt gene of LTA to improving: homeostasis, autolytic activity, and bacterial envelope properties. The role of cytolysin activated by the cylLL and cylLS genes improving the survival ability of E. Faecalis. Hyaluronidase will facilitate other bacteria to migrate from the root canal to periapical lesions resulting in the triggering of toxin production, which will increase damage and inflammation in the host. Enterococcus faecalis, through dentine matrix formation, hydrolyze collagen and induce apatite deposition in developing biofilms. Besides, these bacteria can also increase their tolerance to antimicrobials by blocking the inflammatory response's acid reaction. Alkaline conditions will neutralize the lactic acid secreted by osteoclasts to absorb hard tissue

APPLICATION OF THE PUMPKIN POMACE POWDER IN ENRICHED CRACKER TECHNOLOGY

Рассмотрены аспекты применения вторичного продукта сокового производства – выжимок из тыквы, полученных конвективным вакуумным высушиванием при температуре, не превышающей 50°С, в качестве обогащающего пищевыми волокнами сырьевого ингредиента рецептуры крекера. Дана характеристика высушенных выжимок из тыквы сорта Мускатная. Приведены результаты исследования влияния порошка из выжимок тыквы дисперсностью от 63 до 125 мкм на хлебопекарные свойства муки – количество и качество клейковины, автолитическую активность. Обоснование рецептуры крекера с повышенным содержанием пищевых волокон и β-каротина проведено методом математического планирования – полный факторный эксперимент 22 с последующей оптимизацией методом ридж-анализа. Разработана производственная рецептура. Апробация разработанной рецептуры показала, что крекер с выжимками из тыквы в количестве 11,34 г/100 г муки имеет отличительные органолептические характеристики, обусловленные привкусом и ароматом тыквы, по физико-химическим показателям соответствует требованиям межгосударственного стандарта, по составу, г/100 г, пищевых волокон (3,4) и β-каротина (2,9) может быть отнесен к продукту – источнику пищевых волокон с высоким содержанием β-каротина. The aspects of the use of the secondary product of juice production – pumpkin pomace obtained by convection vacuum drying at a temperature not exceeding 50°C, as an enriching raw ingredient of the cracker formulation with food fibers are considered. The characteristics of dried pumpkin pomace of Muscatnaya variety are given. The results of the study of the influence of pumpkin pomace powder dispersion from 63 to 125 microns on the baking properties of flour – the quantity and quality of gluten, autolytic activity are adduced. The substantiation of the cracker formulation with high content of dietary fibers and β-carotene was carried out by the method of mathematical planning – a complete factorial experiment 22 with subsequent optimization method by ridge analysis. The production formulation is developed. Approbation of the developed formulation showed that the cracker with pumpkin extracts – 11,34 g/100 g of flour has distinctive organoleptic characteristics due to the taste and aroma of pumpkin, on physical and chemical indicators meets the requirements of the interstate standard, by composition, g/100 g, dietary fiber (3,4) and β-carotene (2,9) can be attributed to the product – a source of dietary fiber with a high content of β-carotene.