An Insight into the Proteome of Crithidia fasciculata Choanomastigotes as a Comparative Approach to Axenic Growth, Peanut Lectin Agglutination and Differentiation of Leishmania spp. Promastigotes

Abstract

The life cycle of the trypanosomatid Crithidia fasciculata is monogenetic, as the unique hosts of these parasites are different species of culicids. The comparison of these non-pathogenic microorganisms evolutionary close to other species of trypanosomatids that develop digenetic life cycles and cause chronic severe sickness to millions of people worldwide is of outstanding interest. A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein. The comparison of the outcome with previous gene expression profiling studies developed in the related human pathogens of the genus Leishmania has revealed substantial differences between the motile stages of these closely related organisms in abundance of proteins involved in catabolism, redox homeostasis, intracellular signalling, and gene expression regulation. As L. major and L. infantum agglutinate with peanut lectin and non-agglutinating parasites are more infective, the agglutination properties were evaluated in C. fasciculata. The result is that choanomastigotes are able to agglutinate with peanut lectin and a non-agglutinating subpopulation can be also isolated. As a difference with L. infantum, the non-agglutinating subpopulation over-expresses the whole machinery for maintenance of redox homeostasis and the translation factors eIF5a, EF1α and EF2, what suggests a relationship between the lack of agglutination and a differentiation process.

Valosin-containing protein VCP/p97 is essential for the intracellular development of Leishmania and its survival under heat stress

“…Prior to infection, we evaluated the ability of the VCP mutant lines to develop into metacyclic forms. No significant differences in the percentage of metacyclics (20% in average) were observed between the WT, Li VCP (NEO/+) , Li VCP (NEO/+) VCP QQ , and the Li VCP (NEO/HYG) + VCP HA strains as determined by the peanut‐mediated agglutination assay (Alcolea et al, ; Figure S6). The percentage of infected THP1 MØ at 6 hr postinfection was evaluated at 35%–50% depending on the experiment for all strains tested (Figures S7 and S8a).…”

“…FIGURE S6 Percentage of metacyclic L. infantum forms collected from stationary promastigote cultures of different VCP mutants. To evaluate the percentage of metacyclic promastigotes in LiVCP(+/+), LiVCP (NEO/+), LiVCP (NEO/+) + VCPQQ and the facilitated dKO mutant 3 LiVCP (NEO/HYG) + VCPHA, a suspension of 108/ml stationary phase promastigote cells grown in SDM‐79 medium without serum was supplemented with 50 μg/ml peanut agglutinin lectin (PNA) (Vector Laboratories) and incubated at 260 C with agitation for 30 min as described by Alcolea, Alonso, Garcia‐Tabares, Toraño, and Larraga (). The cells were harvested at 700–800 rpm for 5 min to remove the agglutinated promastigotes (PNA+) and the metacyclic parasites (PNA‐) in the supernatant were recuperated by centrifugation at 3000 rpm during 10 min and resuspended for quantification.…”

eIF5a reduction via decreased Klf5 leads to cell senescence by mitochondrial fission in VSMCs

“…Using quantitative real-time PCR, we further validated the expression of mitochondrial dynamics-, redox homeostasis-and ATP biosynthesis-related genes and obtained the results similar to those seen in mRNA microarray analysis ( Fig 4D). Importantly, we found that eIF5a, a regulator of cell redox homeostasis (Alcolea et al, 2014), was also highly upregulated in Klf5-overexpressed VSMCs, as shown by RNA-seq analysis and qRT-PCR ( Fig 4C and 4D).…”

“…Because alterations of mitochondrial dynamics, which is controlled by fission and fusion, induce the mtROS generation (Shi et al, 2018), and eIF5a is localized not only to the nucleus but also to the mitochondria (Miyake et al, 2015) and might be involved in the regulation of redox homeostasis (Alcolea et al, 2014), we wanted to determine whether eIF5a downregulation induced by Klf5 deficiency affects mitochondrial dynamics. Thus, the interaction of eIF5a with mitochondrial dynamics-related proteins, such as Mfn1, Drp1, Fis1 and Mtfr1, was examined in VSMCs stimulated chronically by Ang II.…”

“…It is well known that the dynamin-like GTPases mitofusin 1 and 2 (Mfn1, Mfn2) regulate the mitochondrial fusion process, and dynamin-related protein 1 (Drp1) plays a central role in the regulation of mitochondrial fission (Zhao et al, 2011). Eukaryotic translation initiation factor 5a (eIF5a) is localized not only to the nucleus but also to the mitochondria (Miyake et al, 2015) and is involved in the regulation of redox homeostasis (Alcolea et al, 2014). However, it remains unclear whether eIF5a, together with mitochondrial dynamics-related proteins, participates in the regulation of mitochondrial dynamics.…”