Jana Bohova

Anti-biofilm Effects of Honey Against Wound Pathogens Proteus mirabilis and Enterobacter cloacae

Biofilm growth and its persistence within wounds have recently been suggested as contributing factors to impaired healing. The goal of this study was to investigate the anti‐biofilm effects of several honey samples of different botanical origin, including manuka honey against Proteus mirabilis and Enterobacter cloacae wound isolates.

Methylglyoxal-induced modifications of significant honeybee proteinous components in manuka honey: Possible therapeutic implications.

Methylglyoxal (MGO) is a major antibacterial component of manuka honey. Another antibacterial component found in Revamil honey, peptide defensin1, was not identified in manuka honey. The primary aim of the study was to evaluate the content of defensin1 in honeys of different botanical origins and to investigate a presumed effect of reactive MGO on defensin1 and a dominant protein of honey MRJP1 in manuka honey. Immunoblotting of honey samples showed that defensin1 was a regular but quantitatively variable component of honeys. One of the reasons of varying contents of defensin1 in different honeys seems to be constitutive but varying defensin1 expression in individual honeybees in bee populations that we documented on samples of nurse and forager bees by RT-PCR. Comparative analyses of honeys revealed a size modification of defensin1, MRJP1 and probably also α-glucosidase in manuka honey. We further showed that (i) the treatment of purified defensin1 in solution containing high amount of MGO caused a time-dependent loss of its antibacterial activity and (ii) increasing MGO concentrations in a non-manuka honey were connected with a gradual increase in the molecular weight of MRJP1. Obtained results demonstrate that MGO abrogates the antibacterial activity of defensin1 and modifies MRJP1 in manuka honey. We assume that MGO could also have negative effects on the structure and function of other proteins/peptides in manuka honey, including glucose oxidase, generating hydrogen peroxide.

Fir honeydew honey flavonoids inhibit TNF-α-induced MMP-9 expression in human keratinocytes: a new action of honey in wound healing

Matrix metalloproteinase-9 (MMP-9) appears to be a major protease responsible for the degradation of matrix and growth-promoting agents in chronic wounds. Honey has been successfully used for treating non-healing wounds associated with infections. However, the mechanisms of its action at the cellular level have remained poorly understood. The aim of this study was to investigate the effect of fir honeydew honey on TNF-α-induced MMP-9 expression and secretion from human keratinocytes (HaCaT) and to identify the honey component(s) responsible for a discovered effect. A C18 solid-phase column was used for preparation of honey aqueous extract (HAE). Expression and production of MMP-9 by HaCaT cells were determined by reverse transcription-PCR, gelatine zymography and Western blot analysis using a polyclonal antibody against MMP-9. We found that HAE inhibited TNF-α-induced production of MMP-9 in keratinocytes in a dose-dependent manner at both the mRNA and protein levels. Apigenin and kaempferol, identified flavonoids in HAE, markedly inhibited MMP-9 production from HaCaT and epidermal keratinocytes. Taken together, fir honeydew honey, which contains certain flavonoids, prevents TNF-α-induced proteolytic activity in cutaneous inflammation. Thus, our findings provide clear evidence that honey may serve as a natural treatment for dermatological problems associated with a persistent inflammation.

Honeydew honey as a potent antibacterial agent in eradication of multi‐drug resistant Stenotrophomonas maltophilia isolates from cancer patients

Multi‐drug resistance in nosocomial pathogens is a continually evolving and alarming problem in health care units. Since ancient times, honey has been used successfully for the treatment of a broad spectrum of infections with no risk of resistance development.

Methylglyoxal May Affect Hydrogen Peroxide Accumulation in Manuka Honey Through the Inhibition of Glucose Oxidase

Although hydrogen peroxide (H₂O₂) is one of the major antibacterial factors in most honeys, it does not accumulate in medical-grade manuka honey. The goal of this study was to investigate the effect of artificially added methylglyoxal (MGO) on H₂O₂ accumulation in natural non-manuka honeys. H₂O₂ concentrations in the honey solutions were determined using a fluorimetric assay. Two, the most potent H₂O₂ producers honeydew honeys were mixed with MGO at final concentrations of 250, 500, and 1000 mg/kg, and incubated for 4 days at 37°C. Subsequently, H₂O₂ concentrations were determined in 50% (wt/vol) MGO supplemented honey solutions. In vitro crosslinking of the enzyme glucose oxidase (GOX) after incubation with MGO was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tested honeys at a concentration of 50% (wt/vol) accumulated up to 495.8±9.1 μM H₂O₂ in 24 h. The most potent producers were the two honeydew honeys, whose 50% solutions accumulated 306.9±6.8 and 495.8±9.1 μM H₂O₂, respectively. Levels of H₂O₂ increased significantly over time in both honey solutions. Contrary to this, the MGO-treated honeys generated significantly lower amounts of H₂O₂ (P<.001), and this reduction was dose dependent. In addition, MGO-treated GOX formed high molecular weight adducts with increasing time of incubation accompanied by loss of its enzymatic activity. High levels of MGO in manuka honey, by modifying the enzyme GOX, might be responsible for suppressing H₂O₂ generation. These data highlight the detrimental effect of MGO on significant proteinaceous components of manuka honey.

A Novel Highly Divergent Protein Family Identified from a Viviparous Insect by RNA-seq Analysis: A Potential Target for Tsetse Fly-Specific Abortifacients

In tsetse flies, nutrients for intrauterine larval development are synthesized by the modified accessory gland (milk gland) and provided in mothers milk during lactation. Interference with at least two milk proteins has been shown to extend larval development and reduce fecundity. The goal of this study was to perform a comprehensive characterization of tsetse milk proteins using lactation-specific transcriptome/milk proteome analyses and to define functional role(s) for the milk proteins during lactation. Differential analysis of RNA-seq data from lactating and dry (non-lactating) females revealed enrichment of transcripts coding for protein synthesis machinery, lipid metabolism and secretory proteins during lactation. Among the genes induced during lactation were those encoding the previously identified milk proteins (milk gland proteins 1–3, transferrin and acid sphingomyelinase 1) and seven new genes (mgp4–10). The genes encoding mgp2–10 are organized on a 40 kb syntenic block in the tsetse genome, have similar exon-intron arrangements, and share regions of amino acid sequence similarity. Expression of mgp2–10 is female-specific and high during milk secretion. While knockdown of a single mgp failed to reduce fecundity, simultaneous knockdown of multiple variants reduced milk protein levels and lowered fecundity. The genomic localization, gene structure similarities, and functional redundancy of MGP2–10 suggest that they constitute a novel highly divergent protein family. Our data indicates that MGP2–10 function both as the primary amino acid resource for the developing larva and in the maintenance of milk homeostasis, similar to the function of the mammalian casein family of milk proteins. This study underscores the dynamic nature of the lactation cycle and identifies a novel family of lactation-specific proteins, unique to Glossina sp., that are essential to larval development. The specificity of MGP2–10 to tsetse and their critical role during lactation suggests that these proteins may be an excellent target for tsetse-specific population control approaches.

Sphingomyelinase Activity in Mother's Milk Is Essential for Juvenile Development: A Case from Lactating Tsetse Flies

ABSTRACT Sphingosine is a structural component of sphingolipids. The metabolism of phosphoethanolamine ceramide (sphingomyelin) by sphingomyelinase (SMase), followed by the breakdown of ceramide by ceramidase (CDase) yields sphingosine. Female tsetse fly is viviparous and generates a single progeny within her uterus during each gonotrophic cycle. The mother provides her offspring with nutrients required for development solely via intrauterine lactation. Quantitative PCR showed that acid smase1 (asmase1) increases in mothers milk gland during lactation. aSMase1 was detected in the milk gland and larval gut, indicating this protein is generated during lactation and consumed by the larva. The higher levels of SMase activity in larval gut contents indicate that this enzyme is activated by the low gut pH. In addition, cdase is expressed at high levels in the larval gut. Breakdown of the resulting ceramide is likely accomplished by the larval gut-secreted CDase, which allows absorption of sphingosine. We used the tsetse system to understand the critical role(s) of SMase and CDase during pregnancy and lactation and their downstream effects on adult progeny fitness. Reduction of asmase1 by short interfering RNA negatively impacted pregnancy and progeny performance, resulting in a 4–5-day extension in pregnancy, 10%–15% reduction in pupal mass, lower pupal hatch rates, impaired heat tolerance, reduced symbiont levels, and reduced fecundity of adult progeny. This study suggests that the SMase activity associated with tsetse lactation and larval digestion is similar in function to that of mammalian lactation and represents a critical process for juvenile development, with important effects on the health of progeny during their adulthood.

Selective Antibiofilm Effects of Lucilia sericata Larvae Secretions/Excretions against Wound Pathogens

Background. Maggot debridement therapy (MDT), using Lucilia sericata larvae, represents efficient, simple, and low-cost therapy for the treatment of chronic wounds. Aim. The aim was to investigate the antibiofilm activity of maggot excretions/secretions (ES) against biofilm of wound isolates Staphylococcus aureus (S. aureus), Enterobacter cloacae (E. cloacae), and Proteus mirabilis (P. mirabilis). Methods. Quantification of biofilm formation, was carried out using a microtiter plate assay. Proteolytic activity of maggot ES was performed using skim milk agar plates. A solid phase extraction and reverse phase HPLC C18 chromatography were employed to the isolate of maggot ES antibiofilm compounds. Results. Maggot ES at 100 mg/mL concentration significantly reduced biofilm formation thus disrupting established biofilm of E. cloacae. Heat-treated ES did not show any antibiofilm activity towards E. cloacae. Similar results were obtained in the case of S. aureus; however, the heat-treatment of maggot ES did not affect its antibiofilm activity. Moreover, a compound with molecular weight of 25 kDa exhibiting antibiofilm activity was identified in maggot ES. On the other hand, maggot ES protected and even stimulated P. mirabilis biofilm formation. Conclusions. Our results suggest that maggot ES may act selectively against different bacterial strain.

Antibacterial properties of lucifensin in Lucilia sericata maggots after septic injury.

OBJECTIVE To investigate the antibacterial properties of lucifensin in maggots of Lucilia sericata after septic injury. METHODS In our preliminary study we have shown that injuring the maggots with a needle soaked in lipopolysaccharide solution induced within 24 h lucifensin expression in the fat body and in the grease coupler of the salivary glands. It is assumed that lucifensin is secreted solely from this tissue into the haemolymph (similar to other insect defensins) and not into secreted/excreted products. We used high-performance liquid chromatography fractionation and radial diffusion assay to investigate the antibacterial properties of haemolymph extracted from larvae after septic injury. RESULTS After septic injury, production of lucifensin in the haemolymph is increased. This led to higher antibacterial activity of such haemolymph in comparison to non-stimulated larvae. COCLUSIONS These results suggest that beside the previously demonstrated role of lucifensin in the debridement therapy, lucifensin is simultaneously important as a part of the systematic immune response.