The lectin-specific activity of Toxoplasma gondii microneme proteins 1 and 4 binds Toll-like receptor 2 and 4 N-glycans to regulate innate immune priming.

Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-γ in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis

Synthesis, cytotoxicity and in vitro antileishmanial activity of naphthothiazoles

The leishmaniasis is a spectral disease caused by the protozoan Leishmania spp., which threatens millions of people worldwide. Current treatments exhibit high toxicity, and there is no vaccine available. The need for new lead compounds with leishmanicidal activity is urgent. Considering that many lead leishmanicidal compounds contain a quinoidal scaffold and the thiazole heterocyclic ring is found in a number of antimicrobial drugs, we proposed a hybridization approach to generate a diverse set of semi-synthetic heterocycles with antileishmanial activity. We found that almost all synthesized compounds demonstrated potent activity against promastigotes of Leishmania (Viannia) braziliensis and reduced the survival index of Leishmania amastigotes in mammalian macrophages. Furthermore, the compounds were not cytotoxic to macrophages at fivefold higher concentrations than the EC50 for promastigotes. All molecules fulfilled Lipinski's Rule of Five, which predicts efficient orally absorption and permeation through biological membranes, the in silico pharmacokinetic profile confirmed these characteristics. The potent and selective activity of semi-synthetic naphthothiazoles against promastigotes and amastigotes reveals that the 2-amino-naphthothiazole ring may represent a scaffold for the design of compounds with leishmanicidal properties and encourage the development of drug formulation and new compounds for further studies in vivo. © 2013 John Wiley & Sons A/S

The lectin-specific activity of Toxoplasma gondii microneme proteins 1 and 4 binds Toll-like receptor 2 and 4 N-glycans to regulate innate immune priming.

Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-γ in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis

A host defense peptide mimetic, brilacidin, potentiates caspofungin antifungal activity against human pathogenic fungi.

Fungal infections cause more than 1.5 million deaths a year. Due to emerging antifungal drug resistance, novel strategies are urgently needed to combat life-threatening fungal diseases. Here, we identify the host defense peptide mimetic, brilacidin (BRI) as a synergizer with caspofungin (CAS) against CAS-sensitive and CAS-resistant isolates of Aspergillus fumigatus, Candida albicans, C. auris, and CAS-intrinsically resistant Cryptococcus neoformans. BRI also potentiates azoles against A. fumigatus and several Mucorales fungi. BRI acts in A. fumigatus by affecting cell wall integrity pathway and cell membrane potential. BRI combined with CAS significantly clears A. fumigatus lung infection in an immunosuppressed murine model of invasive pulmonary aspergillosis. BRI alone also decreases A. fumigatus fungal burden and ablates disease development in a murine model of fungal keratitis. Our results indicate that combinations of BRI and antifungal drugs in clinical use are likely to improve the treatment outcome of aspergillosis and other fungal infections

Differential Gene Expression and Infection Profiles of Cutaneous and Mucosal <i>Leishmania braziliensis</i> Isolates from the Same Patient

<div><p>Background</p><p>Leishmaniasis is a complex disease in which clinical outcome depends on factors such as parasite species, host genetics and immunity and vector species. In Brazil, <i>Leishmania</i> (<i>Viannia) braziliensis</i> is a major etiological agent of cutaneous (CL) and mucosal leishmaniasis (MCL), a disfiguring form of the disease, which occurs in ~10% of <i>L</i>. <i>braziliensis</i>-infected patients. Thus, clinical isolates from patients with CL and MCL may be a relevant source of information to uncover parasite factors contributing to pathogenesis. In this study, we investigated two pairs of <i>L</i>. <i>(V</i>.<i>) braziliensis</i> isolates from mucosal (LbrM) and cutaneous (LbrC) sites of the same patient to identify factors distinguishing parasites that migrate from those that remain at the primary site of infection.</p><p>Methodology/Principal Findings</p><p>We observed no major genomic divergences among the clinical isolates by molecular karyotype and genomic sequencing. RT-PCR revealed that the isolates lacked <i>Leishmania</i> RNA virus (LRV). However, the isolates exhibited distinct <i>in vivo</i> pathogenesis in BALB/c mice; the LbrC isolates were more virulent than the LbrM isolates. Metabolomic analysis revealed significantly increased levels of 14 metabolites in LbrC parasites and 31 metabolites in LbrM parasites that were mainly related to inflammation and chemotaxis. A proteome comparative analysis revealed the overexpression of <i>Lbr</i>PGF2S (prostaglandin f2-alpha synthase) and HSP70 in both LbrC isolates. Overexpression of <i>Lbr</i>PGF2S in LbrC and LbrM promastigotes led to an increase in infected macrophages and the number of amastigotes per cell at 24–48 h post-infection (p.i.).</p><p>Conclusions/Significance</p><p>Despite sharing high similarity at the genome structure and ploidy levels, the parasites exhibited divergent expressed genomes. The proteome and metabolome results indicated differential profiles between the cutaneous and mucosal isolates, primarily related to inflammation and chemotaxis. BALB/c infection revealed that the cutaneous isolates were more virulent than the mucosal parasites. Furthermore, our data suggest that the <i>Lbr</i>PGF2S protein is a candidate to contribute to parasite virulence profiles in the mammalian host.</p></div

Hierarchical Clustering Heatmap of biologically relevant metabolites in LbrM and LbrC.

<p>The color code indicates the metabolites abundance. To enable the comparison of data obtained from HPLC-MS, CE-MS and GC-MS the metabolite abundance was normalized. The MetaboAnalyst (v. 2.0) website was used to normalize the data and to generate the figure. The normalization procedure consisted of mean-centering and division by the standard deviation of each variable. The lines in the heatmap represent the relative abundance of metabolites across the samples of the two compared groups, LbrC and LbrM; each metabolite is indicated on the right side of the figure. The columns corresponding to the LbrM and LbrC groups are indicated at the bottom. Each of the seven columns corresponds to one biological replicate (seven per group). To the left-hand side of the figure, a scale indicates the color code relative to the normalized metabolite abundance (ranging from 0.5 up to 4.0).</p

Chromosome somy of the LbrC and LbrM isolates.

<p>The median coverage for a haploid allele of a chromosome was calculated. The median coverage of each chromosome was divided by the haploid chromosome coverage to obtain the somy of the individual chromosomes [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004018#pntd.0004018.ref031" target="_blank">31</a>].</p

LbrC and LbrM isolates exhibit distinct virulence in BALB/c mice.

<p><b>(A)</b> The panel represents the lesion development in hamsters (<i>Mesocricetus auratus</i>) after infection with the isolates. Ear thickness was measured with a Mitutuyo digital caliper for six weeks. Each point represents the mean lesion size (±SEM) of five animals per group. <b>(B)</b> The graph represents the parasite load in the ear of BALB/c mice after a four-week infection with the isolates by limiting dilution assay. Three or five mice were used per group. The <i>lower panel</i> shows representative images of ear lesions after 4 weeks of infection. <b>(C)</b> Quantification of IFN-γ and IL-4 cytokines released by lymph node and spleen cells after one month of infection with the LbrC² and LbrM² isolates following stimulation with SLA for 72 h. Three mice were included in each group. *p<0.05 (two-tailed t-test).</p

Ectopic overexpression of <i>Lbr</i>PGF2S increases the infection index <i>in vitro</i>.

<p><b>(A)</b> Peritoneal macrophages from BALB/c mice were infected with Lbrc and LbrM transfectants and the wild type strain. At 0 h, 24 h and 48 h post-infection, the cells were stained, and 600 cells were counted. Each bar represents the average and SD of three replicates. *p<0.05 (Student's t test). <b>(B)</b> Number of amastigotes in the macrophages.</p