The study of organic/inorganic molecules with activity against intracellular fungi of the phylum Microsporidia is of critical importance. Here, for the first time, the inactivation of these parasitic fungi by porphyrins is reported. The biological effects of porphyrins (10 µM and 100 µM) on the microsporidian Nosema ceranae was investigated in honeybee hosts using cage experiments. A significant reduction in the number of spores (from 2.6 to 5 fold) was observed in Nosema-infected honeybees with a sucrose-protoporphyrin amide [PP(Asp)2] syrup diet compared to the control honeybees. PP(Asp)2 and the other porphyrin examined in vitro, TMePyP, had a direct impact on the microsporidia. Notably, neither porphyrin requires light excitation to be active against microsporidia. Moreover, microsporidia preincubated with these porphyrins exhibited decreased ability to infect honeybees. In particular, PP(Asp)2, possessing amphiphilic characteristics, exhibited significant inactivation of microsporidia, preventing the development of the microsporidia and diminishing the mortality of infected honeybees. In addition, the porphyrin-treated spores examined by scanning electron microscopy (SEM) showed morphological changes in their exosporium layers, which were distinctly deformed. Thus, we postulate that the mechanism of action of porphyrins on microsporidia is not based on photodynamic inactivation but on the destruction of the cell walls of the spores.

A biomass-bound lipase from psychrophilic Chrysosporium pannorum A-1 is an efficient biocatalyst for direct esterification of β-citronellol and acetic acid in an organic solvent. The biomass is effectively produced by fungal submerged culture at 20 ℃, which results in lower energy consumption during the production of biocatalyst. Supplementation of the culture medium with calcium carbonate together with olive oil contributed to a significant increase in the active biomass of mycelium in one batch culture and increased the efficiency of the biocatalyst. Biomass-bound lipase showed high catalytic activity in a broad temperature range of 30–60 °C and stability up to 70 °C. A maximum molar conversion value of 98% was obtained at 30 °C in n-hexane using a 2:1 alcohol-to-acid molar ratio and 3% w/v of the biocatalyst within 24 h. The high equimolar concentration of the substrates (200 mM) did not have an adverse effect on mycelial biomass activity. Dry mycelium of C. pannorum is a promising biocatalyst for large-scale biosynthesis of citronellyl acetate, given its low-cost production, high activity at low temperatures, and reusability in a minimum of seven 24-h biocatalytic cycles.

Microsporidian infections are dangerous to honeybees due to the absence of an efficient treatment for nosemosis. in the present work, the abilities of several porphyrins to directly inactivate microsporidia derived from Nosema-infected honeybees were studied in vitro. Amide derivatives of protoporphyrin iX (ppiX) conjugated with one and two amino acid moieties were synthesized, and their activities were compared with those of two cationic porphyrins, tMepyp and ttMepp. the most active porphyrins, pp[Lys-Asp] 2 , pp[Lys-tfA] 2 , pp[Asp(ona) 2 ] 2 and pp[Lys-Lys] 2 at concentrations as low as 10-50 µM exerted significant effects on microsporidia, reducing the number of spores by 67-80% compared to the control. Live-cell imaging of the spores treated with porphyrins showed that only 1.6% and 3.0% of spores remained alive after 24 h-incubation with 50 µM PP[Asp(ONa) 2 ] 2 and pp[Lys-Asp] 2 , respectively. The length of the amino acid side chains and their identity in the PPIX molecules affected the bioactivity of the porphyrin. importantly, the irradiation of the porphyrins did not enhance their potency in destroying Nosema spores. We showed that the porphyrins accumulated inside the living spores but not inside dead spores, thus the destruction of the microsporidia by non-metallated porphyrins is not dependent on photosensitization, but is associated with their active transport into the spore cell. When administered to honeybees in vivo, ppiX[Lys-tfA] 2 and ppiX[Lys-Lys] 2 reduced spore loads by 69-76% in infected individuals. They both had no toxic effect on honeybees, in contrast to zinc-coordinated porphyrin. Microsporidia are a large group of eukaryotic obligatory intracellular parasites that form single-cell spores and can complete their life cycle only within an infected host cell. So far, ~ 1400 microsporidian species representing 200 genera have been reported 1. Microsporidia are in the kingdom Fungi, and chitin is a major component of the inner layer of their spore wall. An electron-dense outer exospore comprises the second layer of the cell wall 2-4. Microsporidia are characterized by their unique metabolism and the absence of certain elements common to eukaryotic cells, i.e., mitochondria, peroxisomes, and the classic Golgi apparatus 3-5. The interior of the spore is filled with sporoplasm and a mass of vesicular tubules, which are structurally homologous to the Golgi apparatus. Microsporidia contain a unique invasion apparatus that consists of a polar tube that coils around the sporoplasm and ends at an anchoring disc in the apical part of the spore. A polar tube is required for cell invasion, which occurs via injection of the spore content into the host cell 6. All of these features are associated with adaptation to the parasitic lifestyle 3. Microsporidia are able to survive outside a host cell but can exist only as metabolically inactive spores 7. Microsporidia cause many contagious diseases commonly known as microsporidiosis, and they can infect a wide range of organisms from invertebrates to vertebr...

The effect of two protoporphyrin IX derivatives conjugated with single (PP[Lys(TFA)-OH)]2) or double (PP[Lys(TFA)-Lys(TFA)-OH]2) lysine moieties on the infectious capacity of Nosema ceranae spores was examined, and their efficacies were compared with those of a cationic porphyrin (H2TTMePP). Honeybees were inoculated with spores preincubated with porphyrins or with untreated spores (control). A significantly lower level of infection was observed in the bees infected with the porphyrin-treated spores than in the infected control. Porphyrins 1 and 2 reduced the infectious capability of microsporidia more efficiently than porphyrin 3, with bee mortality declining to almost 50%. Confocal analysis of the midguts of infected bees revealed distinct differences in the number of spores between the control group and the group infected with PP[Lys(TFA)-Lys(TFA)-OH]2-treated spores. Notably, bees with a reduced level of infection consumed less sucrose syrup than the control bees, indicating a reduction in digestive disorders and an improvement in food absorption.

The intracellular microsporidian parasite Nosema ceranae is known to compromise bee health by induction of energetic stress and downregulation of the immune system. Porphyrins are candidate therapeutic agents for controlling Nosema infection without adverse effects on honeybees. In the present work, the impact of two protoporphyrin IX derivatives, i.e. PP[Asp]2 and PP[Lys]2, on Apis mellifera humoral immune response has been investigated in laboratory conditions in non-infected and N. ceranae-infected honeybees. Fluorescence spectroscopy analysis of hemolymph showed for the first time that porphyrin molecules penetrate into the hemocoel of honeybees. Phenoloxidase (PO) activity and the expression of genes encoding antimicrobial peptides (AMPs: abaecin, defensin, and hymenoptaecin) were assessed. Porphyrins significantly increased the phenoloxidase activity in healthy honeybees but did not increase the expression of AMP genes. Compared with the control bees, the hemolymph of non-infected bees treated with porphyrins had an 11.3- and 6.1-fold higher level of PO activity after the 24- and 48-h porphyrin administration, respectively. Notably, there was a significant inverse correlation between the PO activity and the AMP gene expression level (r =  − 0.61696, p = 0.0143). The PO activity profile in the infected bees was completely opposite to that in the healthy bees (r =  − 0.5118, p = 0.000), which was related to the changing load of N. ceranae spores in the porphyrin treated-bees. On day 12 post-infection, the spore loads in the infected porphyrin-fed individuals significantly decreased by 74%, compared with the control bees. Our findings show involvement of the honeybee immune system in the porphyrin-based control of Nosema infection. This allows the infected bees to improve their lifespan considerably by choosing an optimal PO activity/AMP expression variant to cope with the varying level of N. ceranae infection.