Simone Santiago Carvalho de Oliveira

The Calpain Inhibitor MDL28170 Induces the Expression of Apoptotic Markers in Leishmania amazonensis Promastigotes

Background Human cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals. Methodology/Principal Findings In this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM) and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis. Conclusions/Significance The data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the potential clinical utility of calpain inhibitors in the chemotherapy of leishmaniases.

Conidial germination in Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans: influence of growth conditions and antifungal susceptibility profiles

In the present study, we have investigated some growth conditions capable of inducing the conidial germination in Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans. Germination in Sabouraud medium (pH 7.0, 37ºC, 5% CO2) showed to be a typically time-dependent event, reaching ~75% in S. minutisporum and > 90% in S. apiospermum, S. aurantiacum and L. prolificans after 4 h. Similar germination rate was observed when conidia were incubated under different media and pHs. Contrarily, temperature and CO2 tension modulated the germination. The isotropic conidial growth (swelling) and germ tube-like projection were evidenced by microscopy and cytometry. Morphometric parameters augmented in a time-dependent fashion, evidencing changes in size and granularity of fungal cells compared with dormant 0 h conidia. In parallel, a clear increase in the mitochondrial activity was measured during the transformation of conidia-into-germinated conidia. Susceptibility profiles to itraconazole, fluconazole, voriconazole, amphotericin B and caspofungin varied regarding each morphotype and each fungal species. Overall, the minimal inhibitory concentrations for hyphae were higher than conidia and germinated conidia, except for caspofungin. Collectively, our study add new data about the conidia-into-hyphae transformation in Scedosporium and Lomentospora species, which is a relevant biological process of these molds directly connected to their antifungal resistance and pathogenicity mechanisms.

Protective outcomes of low-dose doxycycline on renal function of Wistar rats subjected to acute ischemia/reperfusion injury

Renal ischemia-reperfusion injury (IRI) is a major cause of acute renal failure. Doxycycline (Dc) belongs to the tetracycline-class of antibiotics with demonstrated beneficial molecular effects in the brain and heart, mainly through matrix metalloproteinases inhibition (MMP). However, Dc protection of renal function has not been demonstrated. We determined whether low doses of Dc would prevent decreases in glomerular filtration rate (GFR) and maintain tubular Na+ handling in Wistar rats subjected to kidney I/R. Male Wistar rats underwent bilateral kidney ischemia for 30min followed by 24h reperfusion (I/R). Doxycycline (1, 3, and 10mg/kg, i.p.) was administered 2h before surgery. Untreated I/R rats showed a 250% increase in urine volume and proteinuria, a 60% reduction in GFR, accumulation of urea-nitrogen in the blood, and a 60% decrease in the fractional Na+ excretion due to unbalanced Na+ transporter activity. Treatment with Dc 3mg/kg maintained control levels of urine volume, proteinuria, GFR, blood urea-nitrogen, fractional Na+ excretion, and equilibrated Na+ transporter activities. The Dc protection effects on renal function were associated with kidney structure preservation and prevention of TGFβ and fibronectin deposition. In vitro, total MMP activity was augmented in I/R and inhibited by 25 and 50μM Dc. In vivo, I/R augmented MMP-2 and -9 protein content without changing their activities. Doxycycline treatment downregulated total MMP activity and MMP-2 and -9 protein content. Our results suggest that treatment with low dose Dc protects from IRI, thereby preserving kidney function.

The potent cell permeable calpain inhibitor MDL28170 affects the interaction of Leishmania amazonensis with macrophages and shows anti-amastigote activity

Since the discovery of the28 first drugs used in leishmaniasis treatment up to now, the search for compounds with anti-Leishmania activity without toxic effects and able to overcome the emergency of resistant strains remains a major goal to combat this neglected disease. With this in mind, in the present work, we evaluated the effects of the calpain inhibitor MDL28170 on the interaction process of Leishmania amazonensis promastigote forms with murine peritoneal macrophages and on the intracellular amastigotes. Our results showed that the calpain inhibitor MDL28170 at 15 and 30μM significantly reduced the interaction process of promastigotes with macrophages by 16% and 41%, respectively. The inhibitor was also able to drastically reduce the number of infected macrophages in a time- and dose-dependent manner: after only 24h, MDL28170 was able to significantly diminish the infection rate, presenting an IC50 value of 18.2μM for amastigotes. The treatment with MDL28170 did not alter the nitric oxide production, but the production of TNF-α was significantly raised. Altogether, the results presented here contribute to the search of new proteolytic inhibitors able to act in a selective and effective manner against the diseases caused by trypanosomatids.

Susceptibility of Phytomonas serpens to calpain inhibitors in vitro: interference on the proliferation, ultrastructure, cysteine peptidase expression and interaction with the invertebrate host

A pleiotropic response to the calpain inhibitor MDL28170 was detected in the tomato parasite Phytomonas serpens. Ultrastructural studies revealed that MDL28170 caused mitochondrial swelling, shortening of flagellum and disruption of trans Golgi network. This effect was correlated to the inhibition in processing of cruzipain-like molecules, which presented an increase in expression paralleled by decreased proteolytic activity. Concomitantly, a calcium-dependent cysteine peptidase was detected in the parasite extract, the activity of which was repressed by pre-incubation of parasites with MDL28170. Flow cytometry and Western blotting analyses revealed the differential expression of calpain-like proteins (CALPs) in response to the pre-incubation of parasites with the MDL28170, and confocal fluorescence microscopy confirmed their surface location. The interaction of promastigotes with explanted salivary glands of the insect Oncopeltus fasciatus was reduced when parasites were pre-treated with MDL28170, which was correlated to reduced levels of surface cruzipain-like and gp63-like molecules. Treatment of parasites with anti-Drosophila melanogaster (Dm) calpain antibody also decreased the adhesion process. Additionally, parasites recovered from the interaction process presented higher levels of surface cruzipain-like and gp63-like molecules, with similar levels of CALPs cross-reactive to anti-Dm-calpain antibody. The results confirm the importance of exploring the use of calpain inhibitors in studying parasites’ physiology.

Surface properties, adhesion and biofilm formation on different surfaces by Scedosporium spp. and Lomentospora prolificans

Abstract In the present work, some surface properties of the fungi Scedosporium apiospermum, S. aurantiacum, S. minutisporum, and Lomentospora prolificans and their capability to adhere to and form a biofilm on diverse surfaces were evaluated. All four species had high conidial surface hydrophobicity and elevated electronegative zeta potentials. Abundant quantities of melanin were detected at the conidial surface, whereas sialic acid was absent. The numbers of non-germinated and germinated conidia adhered to poly-L-lysine-covered slides was higher than on glass after 4 h of fungi–surface contact. Additionally, after 72 h of interaction a typical biofilm structure had formed. Mature biofilms were also observed after 72 h on a nasogastric catheter (made from polyvinyl chloride), a late bladder catheter (siliconized latex), and a nasoenteric catheter (polyurethane). Interestingly, biofilm biomass increased considerably when the catheters had previously been incubated with serum. These results confirm that Scedosporium/Lomentospora spp. are capable of forming biofilms on diverse abiotic surfaces.

Susceptibility of promastigotes and intracellular amastigotes from distinct Leishmania species to the calpain inhibitor MDL28170

Despite the available drug options, leishmaniasis treatment remains unsatisfactory. The repurposing of calpain inhibitors originally developed for human diseases became an interesting alternative, since Leishmania cells express calpain-related proteins. The susceptibility of six Leishmania species (L. amazonensis, L. braziliensis, L. major, L. mexicana, L. chagasi, and L. donovani) to the calpain inhibitor MDL28170 was determined. Promastigote and intracellular amastigote viability in the presence of MDL28170 was evaluated. MDL28170 was able to reduce promastigote proliferation in a dose-dependent manner for all the parasites. A significant reduction on the general parasite metabolism was detected, as judged by resazurin assay, as well as induced important morphological alterations, including rounding promastigotes and loss of the flagellum. MDL28170 was also able to reduce the number of intracellular amastigotes in RAW macrophages. The susceptibility of both parasite stages (promastigotes and amastigotes) to MDL28170 was similar for all Leishmania species tested. MDL28170 showed a much higher toxicity to Leishmania amastigotes when compared with mammalian macrophages, displaying selectivity index values varying from 13.1 to 39.8. These results suggest that the development of calpain inhibitors may represent an interesting alternative in the treatment of leishmaniasis.