Fungal killing by mammalian phagocytic cells

Phagocytes are considered the most important effector cells in the immune response against fungal infections. To exert their role, they must recognize the invading fungi, internalise, and kill them within the phagosome. Major advances in the field have elucidated the roles of pattern-recognition receptors in the innate immunity sensing and the importance of reactive oxygen and nitrogen species in intracellular killing of fungi. Surprising exit mechanisms for intracellular pathogens and extracellular traps have also been discovered. These and several other recent breakthroughs in our understanding of the mechanisms used by phagocytes to kill fungal pathogens are reviewed in this work.     

Cloning and characterization of the pH 2.5 acid phosphatase gene, appA: Cyclic AMP mediated negative regulation

Summary The structural gene for an acid phosphatase coded for by the gene appA of Escherichia coli K12 was cloned from a cosmid library into pBR322 and the restriction map determined. Several appA deletion plasmids and a smaller appA ⁺ plasmid were constructed by in vitro recombination techniques and tested for their ability to complement an appA1 mutation. The appA gene was localized within a 2.1 kb segment. Its orientation was determined by construction of a hybrid plasmid carrying an appA-lacZ fusion. -galactosidase synthesized from the appA promoter was negatively regulated by cyclic AMP.     

Crosstalk between Helicobacter pylori and Gastric Epithelial Cells Is Impaired by Docosahexaenoic Acid

H. pylori colonizes half of the world''s population leading to gastritis, ulcers and gastric cancer. H. pylori strains resistant to antibiotics are increasing which raises the need for alternative therapeutic approaches. Docosahexaenoic acid (DHA) has been shown to decrease H. pylori growth and its associated-inflammation through mechanisms poorly characterized. We aimed to explore DHA action on H. pylori-mediated inflammation and adhesion to gastric epithelial cells (AGS) and also to identify bacterial structures affected by DHA. H. pylori growth and metabolism was assessed in liquid cultures. Bacterial adhesion to AGS cells was visualized by transmission electron microscopy and quantified by an Enzyme Linked Immunosorbent Assay. Inflammatory proteins were assessed by immunoblotting in infected AGS cells, previously treated with DHA. Bacterial total and outer membrane protein composition was analyzed by 2-dimensional gel electrophoresis. Concentrations of 100 µM of DHA decreased H. pylori growth, whereas concentrations higher than 250 µM irreversibly inhibited bacteria survival. DHA reduced ATP production and adhesion to AGS cells. AGS cells infected with DHA pre-treated H. pylori showed a 3-fold reduction in Interleukin-8 (IL-8) production and a decrease of COX2 and iNOS. 2D electrophoresis analysis revealed that DHA changed the expression of H. pylori outer membrane proteins associated with stress response and metabolism and modified bacterial lipopolysaccharide phenotype. As conclusions our results show that DHA anti-H. pylori effects are associated with changes of bacteria morphology and metabolism, and with alteration of outer membrane proteins composition, that ultimately reduce the adhesion of bacteria and the burden of H. pylori-related inflammation.     

Identification and recombinant expression of a novel chymotrypsin from Spodoptera exigua

A novel chymotrypsin which is expressed in the midgut of the lepidopteran insect Spodoptera exigua is described. This enzyme, referred to as SeCT34, represents a novel class of chymotrypsins. Its amino-acid sequence shares common features of gut chymotrpysins, but can be clearly distinguished from other serine proteinases that are expressed in the insect gut. Most notable, SeCT34 contains a chymotrypsin activation site and the highly conserved motive DSGGP in the catalytic domain around the active-site serine is changed to DSGSA. Recombinant expression of SeCT34 was achieved in Sf21 insect cells using a special baculovirus vector, which has been engineered for optimized protein production. This is the first example of recombinant expression of an active serine proteinase which functions in the lepidopteran digestive tract. Purified recombinant SeCT34 enzyme was characterized by its ability to hydrolyze various synthetic substrates and its susceptibility to proteinase inhibitors. It appeared to be highly selective for substrates carrying a phenylalanine residue at the cleavage site. SeCT34 showed a pH-dependence and sensitivity to inhibitors, which is characteristic for semi-purified lepidopteran gut proteinases. Expression analysis revealed that SeCT34 was only expressed in the midgut of larvae at the end of their last instar, just before the onset of pupation. This suggests a possible role of this protein in the proteolytic remodelling that occurs in the gut during the larval to pupal molt.     

Immobilization of Botryococcus braunii in alginate: influence on chlorophyll content,photosynthetic activity and degeneration during batch cultures

Summary Immobilization of the hydrocarbonrich microalga Botryococcus braunii in calcium alginate beads results in a large increase in chlorophyll content and chlorophyll photosynthetic activity, relative to free cells, at any stage of standard batch cultures. Immobilization exerts a protective influence on ageing cultures both under standard and air lift conditions. Decreases in chlorophyll content and photosynthetic activity are delayed and slowed down; the organization of protein-chlorophyll complexes is stabilized. These positive effects are related to protection of entrapped cells against photoinhibition owing to gel screening and self-shadowing.Entrapped cells show also, immediately after immobilization, a higher photosynthetic activity than free controls. The unusually high activity yield thus achieved probably results from increase in ionic concentration in the microenvironment of B. braunii.     

Glyphosate inhibits melanization and increases susceptibility to infection in insects

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin’s broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate—the most widely used herbicide globally—inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate’s mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation–reduction balance. Overall, these findings suggest that glyphosate’s environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.

Glyphosate, the most commonly used herbicide in the world, inhibits the production of melanin. Melanin is an important pigment and a key component of the insect immune system; this study shows that glyphosate weakens insects’ melanin-based immune system and makes them more vulnerable to infections, including with the human malaria parasite Plasmodium falciparum.     

Effects of disrupting the polyketide synthase gene WdPKS1 in Wangiella [Exophiala] dermatitidis on melanin production and resistance to killing by antifungal compounds, enzymatic degradation, and extremes in temperature

Background  

Wangiella dermatitidis is a human pathogenic fungus that is an etiologic agent of phaeohyphomycosis. W. dermatitidis produces a black pigment that has been identified as a dihydroxynaphthalene melanin and the production of this pigment is associated with its virulence. Cell wall pigmentation in W. dermatitidis depends on the WdPKS1 gene, which encodes a polyketide synthase required for generating the key precursor for dihydroxynaphthalene melanin biosynthesis.     

The ovarian morphology of Scorpaena notata shows a specialized mode of oviparity

Scorpaena notata is an oviparous species with external fertilization that deposits its eggs in a gelatinous matrix. The internal epithelium of the ovarian wall is chiefly responsible for the production of this matrix, which is particularly abundant and viscous during the spawning period. The oocytes lack lipid droplets, so flotation and transport of the eggs is probably accomplished by means of the matrix that surrounds them. The ovarian stroma is situated along the centre of the gonad and the developing oocytes are connected to it by peduncles. The paucity and small size of the cortical alveoli of the oocytes are notable, as is the thinness of the zona radiata. These are characteristics that would be typical of viviparous species. The histological and ultrastructural observations lead to the conclusion that this species presents a type of oviparity more highly specialized than that of the majority of teleosts.