Esteban Serra

Molecular Detection of Histoplasma capsulatum var. capsulatum in Human Clinical Samples

ABSTRACT We developed a seminested PCR for the diagnosis of histoplasmosis that amplifies a portion of the Histoplasma capsulatum H antigen gene. This assay is highly sensitive and specific, being able to detect genomic material corresponding to less than 10 yeast cells without cross-reaction against other bacterial or fungal pathogens.

New generic primer system targeting mucosal/genital and cutaneous human papillomaviruses leads to the characterization of HPV 115, a novel Beta-papillomavirus species 3

We explored the cutaneotropic HPV genetic diversity in 71 subjects from Argentina. New generic primers (CUT) targeting 88 mucosal/cutaneous HPV were designed and compared to FAP primers. Overall, 69 different HPV types/putative types were identified, being 17 of them novel putative types. Phylogenetic analysis of partial L1 sequences grouped 2 novel putative types in the Beta-PV, 14 in the Gamma-PV and 1 in the Mu-PV genera. CUT primers showed broader capacity than FAP primers in detecting different genera/species and novel putative types (p<0.01). Using overlapping PCR, the full-length genome of a Beta-PV putative type was amplified and cloned. The new virus, designated HPV 115, encodes five early genes and two late genes. Phylogenetic analysis indicated HPV 115 as the most divergent type within the genus Beta-PV species 3. This report is the first providing data on cutaneous HPVs circulating in South America and expands our knowledge of the Papillomaviridae family.

Cloning, characterization, and subcellular localization of a Trypanosoma cruzi argonaute protein defining a new subfamily distinctive of trypanosomatids

Over the last years an expanding family of small non-coding RNAs (sRNA) has been identified in eukaryotic genomes which behave as sequence-specific triggers for mRNA degradation, translation repression, heterochromatin formation and genome stability. To achieve their effectors functions, sRNAs associate with members of the Argonaute protein family. Argonaute proteins are segregated into three paralogous groups: the AGO-like subfamily, the PIWI-like subfamily, and the WAGO subfamily (for Worm specific AGO). Detailed phylogenetic analysis of the small RNA-related machinery components revealed that they can be traced back to the common ancestor of eukaryotes. However, this machinery seems to be lost or excessively simplified in some unicellular organisms such as Saccharomyces cerevisiae, Trypanosoma cruzi, Leishmania major and Plasmodium falciparum which are unable to utilize dsRNA to trigger degradation of target RNAs. We reported here a unique ORF encoding for an AGO/PIWI protein in T. cruzi which was expressed in all stages of its life cycle at the transcript as well as the protein level. Database search for remote homologues, revealed the presence of a divergent PAZ domain adjacent to the well supported PIWI domain. Our results strongly suggested that this unique AGO/PIWI protein from T. cruzi is a canonical Argonaute in terms of its domain architecture. We propose to reclassify all Argonaute members from trypanosomatids as a distinctive phylogenetic group representing a new subfamily of Argonaute proteins and propose the generic designation of AGO/PIWI-tryp to identify them. Inside the Trypanosomatid-specific node, AGO/PIWI-tryps were clearly segregated into two paralog groups designated as AGO-tryp and PIWI-tryp according to the presence or absence of a functional link with RNAi-related phenomena, respectively.

Trypanocidal Drug Benznidazole Impairs Lipopolysaccharide Induction of Macrophage Nitric Oxide Synthase Gene Transcription Through Inhibition of NF-κB Activation

In murine macrophages, inducible NO synthase II (NOSII) gene expression is promoted at a transcriptional level by LPS and/or IFN-γ with benznidazole (BZL), a trypanocidal drug, acting to down-regulate NOSII gene induction and hence inhibiting NO production. By performing transient transfection experiments, we now report that BZL also inhibited the expression of NOSII gene promoter or multimerized NF-κB binding site controlled reporter genes. By contrast, no effect was observed on the expression of a reporter gene under the control of the NOSII promoter-derived IFN regulatory factor element. EMSAs demonstrated that BZL inhibited the nuclear availability of NF-κB in stimulated macrophages. NF-κB is activated in macrophages by phosphorylation, ubiquitination, and subsequent proteolysis of IκB. Within this setting, Western blot was also performed to show that BZL blocked IκBα degradation. Collectively, these results demonstrate that BZL is able to specifically inhibit macrophage NF-κB activation after LPS plus IFN-γ stimulation.

Benznidazole blocks NF-κB activation but not AP-1 through inhibition of IKK

Previously, we demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and NO release in macrophages by inhibiting NF-kappaB. We now proceeded to elucidate the molecular mechanisms by which BZL exerts its inhibitory action on NF-kappaB. We demonstrated that the inhibitory effect of BZL is not extended to other macrophage responses, since it did not inhibit other typical hallmarks of macrophage activation such as phagocytosis, MHC-II molecules expression or production of reactive oxygen species (ROS) by NADPH oxidase. BZL was able to interfere specifically with the activation of NF-kappaB pathway without affecting AP-1 activation in RAW 264.7 macrophages, not only in LPS-mediated activation, but also for other stimuli, such as pro-inflammatory cytokines (IL-1beta, TNF-alpha), PMA or H(2)O(2). Also, BZL delayed the activation of p38 MAPK, but not that of ERK1/2 and JNK. Finally, treatment with BZL inhibited IkappaBalpha phosporylation and hence its degradation, whereas it did not block IkappaB kinase (IKK) alpha/beta phosphorylation. Collectively, BZL behaves as a broad range specific inhibitor of NF-kappaB activation, independently of the stimuli tested.

Schistosoma mansoni ferredoxin NADP(H) oxidoreductase and its role in detoxification

Ferredoxin NADP(H) oxidoreductases (FNR) are flavoenzymes that catalyze the electron transfer between NADP(H) and a wide range of compounds including ferredoxins and bacterial flavodoxins. FNRs are classified into two major groups: plant- and vertebrate-type. Plant-type FNRs are implicated in photosynthesis and nitrogen fixation in plastids and photosynthetic bacteria, and were recently implicated in cell protection against reactive oxygen species (ROS). Vertebrate-type FNRs are mitochondrial enzymes implicated in steroid hormone biosynthesis in mammals and in Fe(+) uptake and metabolism in yeasts. We have cloned and sequenced a cDNA coding for the vertebrate-type Schistosoma mansoni FNR. Gel diaphorase activity and western blot assays demonstrated that SmFNR represented the major diaphorase activity of adult worms. An active recombinant SmFNR was expressed in Escherichia coli that made the bacteria tolerant to oxygen peroxide, cumene hydroperoxide and the superoxide-generating herbicide, methyl viologen (MV).

Benznidazole treatment attenuates liver NF-κB activity and MAPK in a cecal ligation and puncture model of sepsis.

Recent studies have shown that Benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and nitric oxide (NO) release in activated macrophages by blocking NF-κB through inhibition of IKK in vitro. As so far, little is known about the mechanism by which BZL provokes the inhibition of inflammatory response in sepsis in vivo, we aimed to delineate the possible role of BZL as a modulator in liver inflammation in mice with sepsis induced by cecal ligation and puncture (CLP). Specifically, we analyzed leukocytes, liver production of TNF-α and NO and the intracellular pathways modulated by these mediators, including NF-κB and MAPKs, in the liver of mice 24 h post-CLP. Our results show that BZL reduces leukocytes in peripheral blood accompanied by an increase in peritoneal macrophages 24h after CLP. In the liver of these septic mice, BZL decreased expression of mRNA and protein for TNF-α and NOS-2 by inhibition of NF-κB and MAPK (p-38 and ERK). The body of evidence suggests that the immunomodulatory effects of BZL could act selectively, as it is able to decrease the systemic inflammatory reaction and the hepatic response but it can increase the number of cells in the site of infection.

One-tube nested Polymerase Chain Reaction for detection of Chlamydia trachomatis

Here we present a one-tube nested PCR test, which allows the detection of minimal quantities of Chlamydia trachomatis in human fluids. This assay includes the use of an internal control to avoid false negative results due to the presence of inhibitors. The results obtained show that this assay is robust enough to be used for clinical diagnosis.

Overexpression of cytoplasmic TcSIR2RP1 and mitochondrial TcSIR2RP3 impacts on Trypanosoma cruzi growth and cell invasion.

Background Trypanosoma cruzi is a protozoan pathogen responsible for Chagas disease. Current therapies are inadequate because of their severe host toxicity and numerous side effects. The identification of new biotargets is essential for the development of more efficient therapeutic alternatives. Inhibition of sirtuins from Trypanosoma brucei and Leishmania ssp. showed promising results, indicating that these enzymes may be considered as targets for drug discovery in parasite infection. Here, we report the first characterization of the two sirtuins present in T. cruzi. Methodology Dm28c epimastigotes that inducibly overexpress TcSIR2RP1 and TcSIR2RP3 were constructed and used to determine their localizations and functions. These transfected lines were tested regarding their acetylation levels, proliferation and metacyclogenesis rate, viability when treated with sirtuin inhibitors and in vitro infectivity. Conclusion TcSIR2RP1 and TcSIR2RP3 are cytosolic and mitochondrial proteins respectively. Our data suggest that sirtuin activity is important for the proliferation of T. cruzi replicative forms, for the host cell-parasite interplay, and for differentiation among life-cycle stages; but each one performs different roles in most of these processes. Our results increase the knowledge on the localization and function of these enzymes, and the overexpressing T. cruzi strains we obtained can be useful tools for experimental screening of trypanosomatid sirtuin inhibitors.

Lysine Acetylation: Elucidating the Components of an Emerging Global Signaling Pathway in Trypanosomes

In the past ten years the number of acetylated proteins reported in literature grew exponentially. Several authors have proposed that acetylation might be a key component in most eukaryotic signaling pathways, as important as phosphorylation. The enzymes involved in this process are starting to emerge; acetyltransferases and deacetylases are found inside and outside the nuclear compartment and have different regulatory functions. In trypanosomatids several of these enzymes have been described and are postulated to be novel antiparasitic targets for the rational design of drugs. In this paper we overview the most important known acetylated proteins and the advances made in the identification of new acetylated proteins using high-resolution mass spectrometry. Also, we summarize what is known so far about the acetyltransferases and deacetylases in eukaryotes, focusing on trypanosomes and their potential use as chemotherapeutic targets.