Appraisal of a Leishmania major Strain Stably Expressing mCherry Fluorescent Protein for Both In Vitro and In Vivo Studies of Potential Drugs and Vaccine against Cutaneous Leishmaniasis

Background

Leishmania major cutaneous leishmaniasis is an infectious zoonotic disease. It is produced by a digenetic parasite, which resides in the phagolysosomal compartment of different mammalian macrophage populations. There is an urgent need to develop new therapies (drugs) against this neglected disease that hits developing countries. The main goal of this work is to establish an easier and cheaper tool of choice for real-time monitoring of the establishment and progression of this pathology either in BALB/c mice or in vitro assays. To validate this new technique we vaccinated mice with an attenuated Δhsp70-II strain of Leishmania to assess protection against this disease.

Methodology

We engineered a transgenic L. major strain expressing the mCherry red-fluorescent protein for real-time monitoring of the parasitic load. This is achieved via measurement of fluorescence emission, allowing a weekly record of the footpads over eight weeks after the inoculation of BALB/c mice.

Results

In vitro results show a linear correlation between the number of parasites and fluorescence emission over a range of four logs. The minimum number of parasites (amastigote isolated from lesion) detected by their fluorescent phenotype was 10,000. The effect of antileishmanial drugs against mCherry+L. major infecting peritoneal macrophages were evaluated by direct assay of fluorescence emission, with IC₅₀ values of 0.12, 0.56 and 9.20 µM for amphotericin B, miltefosine and paromomycin, respectively. An experimental vaccination trial based on the protection conferred by an attenuated Δhsp70-II mutant of Leishmania was used to validate the suitability of this technique in vivo.

Conclusions

A Leishmania major strain expressing mCherry red-fluorescent protein enables the monitoring of parasitic load via measurement of fluorescence emission. This approach allows a simpler, faster, non-invasive and cost-effective technique to assess the clinical progression of the infection after drug or vaccine therapy.     

Listeria monocytogenes Triggers the Cell Surface Expression of Gp96 Protein and Interacts with Its N Terminus to Support Cellular Infection

Listeria monocytogenes is an intracellular food-borne pathogen causing listeriosis in humans. This bacterium deploys an arsenal of virulence factors that act in concert to promote cellular infection. Bacterial surface proteins are of primary importance in the process of host cell invasion. They interact with host cellular receptors, inducing/modulating specific cellular responses. We previously identified Vip, a Listeria surface protein covalently attached to the bacterial cell wall acting as a key virulence factor. We have shown that Vip interacts with Gp96 localized at the surface of host cells during invasion and that this interaction is critical for a successful infection in vivo. To better understand the importance of Vip-Gp96 interaction during infection, we aimed to characterize this interaction at the molecular level. Here we demonstrate that, during infection, L. monocytogenes triggers the cellular redistribution of Gp96, inducing its exposure at the cell surface. Upon infection, Gp96 N-terminal domain is exposed to the extracellular milieu in L2071 fibroblasts and interacts with Vip expressed by Listeria. We identified Gp96 (Asp¹–Leu¹⁷⁰) as sufficient to interact with Vip; however, we also showed that the region Tyr¹⁷⁹–Leu³⁹⁰ of Gp96 is important for the interaction. Our findings unravel the Listeria-induced surface expression of Gp96 and the topology of its insertion on the plasma membrane and improve our knowledge on the Vip-Gp96 interaction during Listeria infection.     

Studies on 6-chloro-5-(1-naphthyloxy)-2-(trifluoromethyl)-1H-benzimidazole/2-hydroxypropyl-β-cyclodextrin association: Characterization, molecular modeling studies, and in vivo anthelminthic activity

The purpose of this work is to study the molecular association that occurs between 2-hydroxypropyl-β-cyclodextrin (HPβCD) and 6-chloro-5-(1-naphthyloxy)-2-(trifluoromethyl)-1H-benzimidazole (RCB20), an antiparasitic compound recently found by our research group, with poor aqueous solubility. The complex stability constant and stoichiometric ratio determined by phase-solubility diagram and Job's plot provided evidence that HPβCD enhanced water solubility of RCB20 through inclusion complex formation. Two-dimensional 1H NMR spectroscopy is used to study the molecular arrangement of inclusion complex in solution. These results are further supported using molecular modeling studies. In the solid state, the complexation is confirmed by differential scanning calorimetry, powder X-ray diffraction, and scanning electron microscopy. Finally, RCB20/HPβCD complex has better activity than RCB20 against the adult and muscle larvae phase of Trichinella spiralis.     

Potato Lectin: A Cell-Wall Glycoprotein

The activity and the amount of potato lectin were measured inpotato tuber slices (Solanum tuberosum cv. Huinkul) aeratedfor 48 h. Lectin was found in a soluble form, liberated to themedium and associated with insoluble structures. Polyacrylamidegel electrophoresis in denaturating conditions and immunologicaltechniques indicated that the lectins associated to cell wall,soluble or liberated to the medium, were identical. The cell-wallfraction was found to contain 65% of total lectin in the tuber.The possible role of potato lectin in tubers was discussed. (Received June 5, 1985; Accepted September 3, 1985)     

Using antagonistic soil bacteria and their cell‐free filtrates to control the black rot pathogen Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris (Xcc) is a phytopathogenic bacteria, and it is the causative agent of black rot in crucifers. Recent studies have shown that Bacillus species have strong biological control on Xanthomonas. One of the mechanisms of this control is secondary metabolites production. A collection of 257 bacteria isolated from a suppressive soil was evaluated for in vitro antagonistic activity against X. campestris, and 92 isolates (44.6%) were able to inhibit its growth. Among the 92 isolates evaluated in the double‐layer technique, 51 (55.43%) inhibited Xcc growth on the inhibition tests with cell‐free filtrates (CFF) in liquid medium. Thirteen of these isolates presented 50% or more growth inhibition, and five isolates presented 100% growth inhibition of Xcc. The CFF of the isolate TCDT‐08, which belongs to the Paenibacillus genus, was used for in vivo tests with kale crops. The artificial inoculation of kale with Xcc‐629IBSBF pretreated with CFF from the isolate TCDT‐08 demonstrated that the bacterium loses the ability of colonizing kale and of causing black rot. A Paenibacillus sp. isolate has strong inhibitory activity against X. campestris pv. campestris, and further studies can result in the use of this isolate to protect kale from Xcc infection.     

Unraveling the plant diversity of the Amazonian canga through DNA barcoding

The canga of the Serra dos Carajás, in Eastern Amazon, is home to a unique open plant community, harboring several endemic and rare species. Although a complete flora survey has been recently published, scarce to no genetic information is available for most plant species of the ironstone outcrops of the Serra dos Carajás. In this scenario, DNA barcoding appears as a fast and effective approach to assess the genetic diversity of the Serra dos Carajás flora, considering the growing need for robust biodiversity conservation planning in such an area with industrial mining activities. Thus, after testing eight different DNA barcode markers (matK, rbcL, rpoB, rpoC1, atpF‐atpH, psbK‐psbI, trnH‐psbA, and ITS2), we chose rbcL and ITS2 as the most suitable markers for a broad application in the regional flora. Here we describe DNA barcodes for 1,130 specimens of 538 species, 323 genera, and 115 families of vascular plants from a highly diverse flora in the Amazon basin, with a total of 344 species being barcoded for the first time. In addition, we assessed the potential of using DNA metabarcoding of bulk samples for surveying plant diversity in the canga. Upon achieving the first comprehensive DNA barcoding effort directed to a complete flora in the Brazilian Amazon, we discuss the relevance of our results to guide future conservation measures in the Serra dos Carajás.     

Genome-wide association study of multiple sclerosis confirms a novel locus at 5p13.1

Multiple Sclerosis (MS) is the most common progressive and disabling neurological condition affecting young adults in the world today. From a genetic point of view, MS is a complex disorder resulting from the combination of genetic and non-genetic factors. We aimed to identify previously unidentified loci conducting a new GWAS of Multiple Sclerosis (MS) in a sample of 296 MS cases and 801 controls from the Spanish population. Meta-analysis of our data in combination with previous GWAS was done. A total of 17 GWAS-significant SNPs, corresponding to three different loci were identified:HLA, IL2RA, and 5p13.1. All three have been previously reported as GWAS-significant. We confirmed our observation in 5p13.1 for rs9292777 using two additional independent Spanish samples to make a total of 4912 MS cases and 7498 controls (ORpooled = 0.84; 95%CI: 0.80-0.89; p = 1.36 × 10-9). This SNP differs from the one reported within this locus in a recent GWAS. Although it is unclear whether both signals are tapping the same genetic association, it seems clear that this locus plays an important role in the pathogenesis of MS.