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

Abstract

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.

In vitroselection ofPhytomonas serpenscells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps

“…Phytomonas serpens WT promastigotes were initially cultured in the presence of 5 µ m MDL28170, a sub-lethal concentration of the drug corresponding to a sixth of the IC 50 value (50% inhibitory concentration) to the WT cells (30·9 µ m ) (Oliveira et al 2017). A stepwise increase in drug concentration was undertaken only when the drug-exposed promastigotes had a growth rate similar to those of the WT culture.…”

“…After fixation, cells were washed in cacodylate buffer and post-fixed with a solution of 1% OsO 4 , 0·8% potassium ferrocyanide and 5 m m CaCl 2 in the same buffer for 20 min at 25 °C. Cells were dehydrated in graded series of acetone (30–100%) and then dried by the critical point method, mounted on stubs, coated with gold (20–30 nm) and observed in a Jeol JSM 6490LV scanning electron microscope (Massachusets, USA) (Oliveira et al 2017). For transmission electron microscopy observation, cells over coverslips were fixed and post-fixed as described above, dehydrated in an ascending acetone series and embedded in PolyBed 812 resin.…”

“…In this sense, our group has demonstrated that MDL28170 affected many biological aspects in T. cruzi and Leishmania amazonensis life cycles (Branquinha et al 2013): these studies have pointed at ultrastructural alterations (Branquinha et al 2013; Marinho et al 2014), fluctuations of the expression levels of calpain-like proteins (CALPs) and distinct peptidases, reduction in the adhesion to the insect midgut and in the interaction with macrophages, impairment of differentiation and viability of infective forms (Branquinha et al 2013), besides the induction of apoptosis (Marinho et al 2014). Moreover, addition of distinct calpain inhibitors to the culture medium of P. serpens led to a dose-dependent reduction in the proliferation of the parasite, being MDL28170 the most potent inhibitor (Oliveira et al 2017).…”

Activity of Piperaceae extracts and fractions in the control of Phytomonas serpens

“…Phytomonas serpens is also attributed to the production of auxin, a class of phytohormones that ends up causing interference with the plant metabolism, thus affecting plant development (IENNE et al, 2014). Despite the damage caused to crops, P. serpens is not considered a phytopathogen parasite, since the promastigote form of the parasite remains restricted to the place of infection (oLIVEIRA et al, 2017).…”

The evolution of trypanosomatid taxonomy

“…Female phlebotomine sand flies are the natural vectors for Leishmania transmission, and roughly 70 known animal species serve as reservoirs for human pathogenic Leishmania species, including rodents [20], dogs [21] and other mammals [22]. Approximately 20 species of Leishmania act as the aetiological agents of human leishmaniasis.  Phytomonas Phthia picta , Nezara viridula , Oncopeltus fasciatus Phytomonas is a dixenous genus that includes several plant pathogens transmitted by phytophagous insects [140]. They predominately exist as promastigotes and less frequently as choanomastigotes [47].…”