Protective effects of lactoferrin chimera and bovine lactoferrin in a mouse model of enterohaemorrhagic Escherichia coli O157:H7 infection

Mice orally infected with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 were used to evaluate the activity of bovine lactoferrin (bLF) and the synthetic peptide LFchimera. Groups of BALB/c mice inoculated intragastrically with EHEC O157:H7 showed chronic intestinal infection with the pathogen that persisted over 6 days and resulted in a high mortality rate (90%). LFchimera and kanamycin significantly decreased (40%) this mortality rate (P = 0.028). On the other hand, although mice administered with bLF showed an important reduction in mortality (50%), this was not statistically significant (P = 0.070). In infected and untreated mice, severe tubular necrosis, glomerular lesions, and moderate intratubular hyaline casts were found in the kidney. However, in the bLF and LFchimera groups we found a reduction in the damage and a substantial decrease in the bacterial concentration excreted in feces 48 h after infection. Furthermore, sepsis caused by EHEC was reduced by the treatments, evidenced by the fact that bacteria were not detected in the kidney or liver 72 h after infection. The results suggest the bLF and LFchimera could have potential as therapeutics in EHEC infections.     

Effect of bovine lactoferrin in a therapeutic hamster model of hepatic amoebiasis

Entamoeba histolytica is the causative agent of amoebiasis, a disease that produces dysentery as a result of the perforation of the large intestine. This parasite often invades other organs, primarily the liver, leading to an amoebic liver abscess (ALA), which can cause death. Metronidazole is the drug of choice for the treatment of ALA; however, it produces toxic side effects in patients. Lactoferrin (Lf) is a glycoprotein of the innate immune response that sequesters iron in the mucosae. Lf possesses immune-regulatory properties, such as antiinflammatory and antioxidant activities. Moreover, the microbicidal activity of apoLf, which lacks bound iron, has been shown. In this study, we evaluated the therapeutic effect of bovine Lf (bLf) against ALA in a model of hepatic amoebiasis in hamsters. Interestingly, hamsters treated intragastrically with Lf (2.5 mg/100 g mass) over a period of 8 days showed no clinical signs of disease and ALA was effectively decreased, with only 0.63% detectable lesion, compared with 63% in untreated animals. Furthermore, liver function and blood cells approached normal levels among those receiving bLf treatment. These results suggest that bLf may aid in the therapy of amoebiasis, likely without producing undesirable effects in patients.     

Lactoferrin and lactoferrin chimera inhibit damage caused by enteropathogenic Escherichia coli in HEp-2 cells

Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries. It produces a characteristic intestinal histopathological lesion on enterocytes known as ‘attaching and effacing’ (A/E), and these two steps are mediated by a type-III secretory system. In the present study, we evaluated the effect on the initial host cell attachment step produced by bovine lactoferrin (bLF) and three synthetic peptides: lactoferricin (LFcin), lactoferrampin (LFampin) and LFchimera. A special focus was given to the hemolytic activity and EPEC-induced actin polymerization in HEp-2 cells, as well as to the espA gene expression, which produces the protein responsible for primary contact with the host cells. Results show that EPEC attachment to HEp-2 cells was significantly suppressed by bLF and LFchimera at 125 and 40 μM, respectively. EPEC-mediated actin polymerization was blocked by bLF and LFchimera at 88 and 99%, respectively. LFchimera inhibited the attachment and A/E lesion caused by EPEC in a dose-dependent manner. In the presence of 125 μM bLF, the expression level of the espA gene was decreased by 50% compared to the untreated control. LFchimera at concentrations of 20 μM and 40 μM diminished the level of espA gene expression 100 and 1000 fold, respectively (P < 0.001). Although bLF, LFchimera, LFcin, and LFampin all significantly blocked the hemolysis produced by EPEC (P < 0.001), the two former compounds produced this effect at lower concentrations. These two compounds, bLF and LFchimera, were able to inhibit the first steps of the mechanism of the damage used by EPEC. This data suggests that LFchimera could provide protection against enteropathogens that share this mechanism.     

Endocytic tubules regulated by Rab GTPases 5 and 11 are used for envelopment of herpes simplex virus

Enveloped viruses employ diverse and complex strategies for wrapping at cellular membranes, many of which are poorly understood. Here, an ultrastructural study of herpes simplex virus 1 (HSV1)‐infected cells revealed envelopment in tubular membranes. These tubules were labelled by the fluid phase marker horseradish peroxidase (HRP), and were observed to wrap capsids as early as 2 min after HRP addition, indicating that the envelope had recently cycled from the cell surface. Consistent with this, capsids did not colocalise with either the trans‐Golgi network marker TGN46 or late endosomal markers, but showed coincidence with the transferrin receptor. Virus glycoproteins were retrieved from the plasma membrane (PM) to label wrapping capsids, a process that was dependent on both dynamin and Rab5. Combined depletion of Rab5 and Rab11 reduced virus yield to <1%, resulting in aberrant localisation of capsids. These results suggest that endocytosis from the PM into endocytic tubules provides the main source of membrane for HSV1, and reveal a new mechanism for virus exploitation of the endocytic pathway.     

A safe and complete algorithm for metagenomic assembly

Background

Reconstructing the genome of a species from short fragments is one of the oldest bioinformatics problems. Metagenomic assembly is a variant of the problem asking to reconstruct the circular genomes of all bacterial species present in a sequencing sample. This problem can be naturally formulated as finding a collection of circular walks of a directed graph G that together cover all nodes, or edges, of G.

Approach

We address this problem with the “safe and complete” framework of Tomescu and Medvedev (Research in computational Molecular biology—20th annual conference, RECOMB 9649:152–163, 2016). An algorithm is called safe if it returns only those walks (also called safe) that appear as subwalk in all metagenomic assembly solutions for G. A safe algorithm is called complete if it returns all safe walks of G.

Results

We give graph-theoretic characterizations of the safe walks of G, and a safe and complete algorithm finding all safe walks of G. In the node-covering case, our algorithm runs in time \(O(m^2 + n^3)\), and in the edge-covering case it runs in time \(O(m^2n)\); n and m denote the number of nodes and edges, respectively, of G. This algorithm constitutes the first theoretical tight upper bound on what can be safely assembled from metagenomic reads using this problem formulation.

     

Guianin: A neolignan from Aniba guianensis

The wood of Aniba guianensis Aubl. (Lauraceae) contains benzyl benzoate, benzyl salicylate, sitosterol, O-methyleugenol, O-methylisoeugenol and the neolignan guianin for which the structure of 1-allyl-8-hydroxy-3-methoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene (VI) is proposed.     

Genomic methods reveal independent demographic histories despite strong morphological conservatism in fish species

Human overexploitation of natural resources has placed conservation and management as one of the most pressing challenges in modern societies, especially in regards to highly vulnerable marine ecosystems. In this context, cryptic species are particularly challenging to conserve because they are hard to distinguish based on morphology alone, and thus it is often unclear how many species coexist in sympatry, what are their phylogenetic relationships and their demographic history. We answer these questions using morphologically similar species of the genus Mugil that are sympatric in the largest coastal Marine Protected Area in the Tropical Southwestern Atlantic marine province. Using a sub-representation of the genome, we show that individuals are assigned to five highly differentiated genetic clusters that are coincident with five mitochondrial lineages, but discordant with morphological information, supporting the existence of five species with conserved morphology in this region. A lack of admixed individuals is consistent with strong genetic isolation between sympatric species, but the most likely species tree suggests that in one case speciation has occurred in the presence of interspecific gene flow. Patterns of genetic diversity within species suggest that effective population sizes differ up to two-fold, probably reflecting differences in the magnitude of population expansions since species formation. Together, our results show that strong morphologic conservatism in marine environments can lead to species that are difficult to distinguish morphologically but that are characterized by an independent evolutionary history, and thus that deserve species-specific management strategies.Subject terms: Genetic variation, Phylogenetics, Speciation     

Entamoeba histolytica uses ferritin as an iron source and internalises this protein by means of clathrin-coated vesicles

Entamoeba histolytica is a parasitic protozoan that produces dysentery and often reaches the liver, leading to abscess formation. Ferritin is an iron-storage protein that is mainly found in liver and spleen in mammals. The liver contains a plentiful source of iron for amoebae multiplying in that organ, making it a prime target for infection since iron is essential for the growth of this parasite. The aim of this study was to determine whether trophozoites are able to take up ferritin and internalise this protein for their growth in axenic culture. Interaction between the amoebae and ferritin was studied by flow cytometry, confocal laser-scanning microscopy and transmission electron microscopy. Amoebae were viable in iron supplied by ferritin. Trophozoites quickly internalised ferritin via clathrin-coated vesicles, a process that was initiated within the first 2 min of incubation. In 30 min, ferritin was found colocalizing with the LAMP-2 protein at vesicles in the cytosol. The uptake of ferritin was time- temperature- and concentration-dependent, specific and saturated at 46 nM of ferritin. Haemoglobin and holo-transferrin did not compete with ferritin for binding to amoebae. Amoebae cleaved ferritin leading to the production of several different sized fragments. Cysteine proteases of 100, 75 and 50 kDa from amoeba extracts were observed in gels copolymerised with ferritin. For a pathogen such as E. histolytica, the capacity to utilise ferritin as an iron source may well explain its high pathogenic potential in the liver.     

Infectivity of Leishmania mexicana Is Associated with Differential Expression of Protein Kinase C-Like Triggered during a Cell-Cell Contact

Mammalian host cell invasion by Leishmania is a complex process in which various parasite and host cell components interact, triggering the activation of signaling cascades in both cells. Little is known regarding PKC biological functions in Leishmania sp. during parasite-macrophage interaction. PKC-like enzyme was first identified in homogenates and membrane fraction of L. mexicana stationary promastigotes by immunoblot. PKC-like enzyme activity was then detected in cell homogenates but also on intact promastigotes showing for the first time the presence of an ecto-PKC dependent on Ca²⁺/phosphatidylserine for activation. This ecto-PKC was activated with phorbol myristate acetate (PMA) and inhibited by RO-32-0432, a selective PKCαβ_Iε bisindolylmaleimide inhibitor. Interestingly, the Leishmania PKC- activity was higher in the infective stationary than in non-infective logarithmic stage. Then, promastigotes at different stages of time proliferation curve were used in order to identify the role of PKC-like during macrophage invasion. After attachment to macrophages, PKC-like is over-expressed in promastigotes at the 6^th culture day but also at the 4^th day of culture corresponding to the maximal infection capacity. An antibody microarray for MAPK and PKC corroborate the Leishmania PKC-like over-expression during contact with macrophages. Pretreatment with RO-32-0432 inhibitor reduced the number of infected macrophages and the parasite burden. These data suggest for the first time a direct link between PKC expression level and infectivity, and provide evidence that PKC-like plays a critical role in attachment and in the internalization steps involved in the invasion process.