In vitro infectivity and differential gene expression of Leishmania infantum metacyclic promastigotes: negative selection with peanut agglutinin in culture versus isolation from the stomodeal valve of Phlebotomus perniciosus

Abstract

BackgroundLeishmania infantum is the protozoan parasite responsible for zoonotic visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been reported in this area. The life cycle of the parasite is digenetic. The promastigote stage develops within the gut of phlebotomine sand flies, whereas amastigotes survive and multiply within phagolysosomes of mammalian host phagocytes. The major vector of L. infantum in Spain is Phlebotomus perniciosus. The axenic culture model of promastigotes is generally used because it is able to mimic the conditions of the natural environment (i.e. the sand fly vector gut). However, infectivity decreases with culture passages and infection of laboratory animals is frequently required. Enrichment of the stationary phase population in highly infective metacyclic promastigotes is achieved by negative selection with peanut agglutinin (PNA), which is possible only in certain Leishmania species such as L. major and L. infantum. In this study, in vitro infectivity and differential gene expression of cultured PNA-negative promastigotes (Pro-PNA−) and metacyclic promastigotes isolated from the sand fly anterior thoracic midgut (Pro-Pper) have been compared.ResultsIn vitro infectivity is about 30 % higher in terms of rate of infected cells and number of amastigotes per infected cell in Pro-Pper than in Pro-PNA−. This finding is in agreement with up-regulation of a leishmanolysin gene (gp63) and genes involved in biosynthesis of glycosylinositolphospholipids (GIPL), lipophosphoglycan (LPG) and proteophosphoglycan (PPG) in Pro-Pper. In addition, differences between Pro-Pper and Pro-PNA− in genes involved in important cellular processes (e.g. signaling and regulation of gene expression) have been found.ConclusionsPro-Pper are significantly more infective than peanut lectin non-agglutinating ones. Therefore, negative selection with PNA is an appropriate method for isolating metacyclic promastigotes in stationary phase of axenic culture but it does not allow reaching the in vitro infectivity levels of Pro-Pper. Indeed, GIPL, LPG and PPG biosynthetic genes together with a gp63 gene are up-regulated in Pro-Pper and interestingly, the correlation coefficient between both transcriptomes in terms of transcript abundance is R2 = 0.68. This means that the correlation is sufficiently high to consider that both samples are physiologically comparable (i.e. the experiment was correctly designed and performed) and sufficiently low to conclude that important differences in transcript abundance have been found. Therefore, the implications of axenic culture should be evaluated case-by-case in each experimental design even when the stationary phase population in culture is enriched in metacyclic promastigotes by negative selection with PNA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2672-8) contains supplementary material, which is available to authorized users.

RNA-seq analysis reveals differences in transcript abundance between cultured and sand fly-derived Leishmania infantum promastigotes

“…even after 2 weeks of sand fly infection by means of bloodfeeding from dogs [5], which is comparable to the percentage of metacyclics obtained from culture [3,4]. Caution advised for interpretation of all these studies, we postulate that: i) up-regulation of HASP, GP63 and autophagy genes in nectomonads and metacyclics of L. major as well as in pooled L. infantum promastigote samples confirm their contribution to the metacyclogenesis process; ii) only certain amastin genes are up-regulated in metacyclics for preparation in advance for life in the parasitophorous vacuole of the mammalian host phagocyte as described [25,26]; iii) the SHERP gene cluster is up-regulated only in L. major metacyclics, which is in agreement with the fact that SHERP is the only well validated marker of metacyclics exclusively in L. major [27]; iv) upregulation of genes involved in metacyclogenesis in L. infantum promastigote pools derived from the gut of P. perniciosus with respect to cultured ones (Table 1) is due to more efficacious metacyclogenesis, yielding metacyclic promastigotes showing exacerbated infectivity [2,3] presumably due to the influence of the sand fly gut microenvironment. Three genes in tandem array that encode a nucleoside transporter are among the mostly highly up-regulated genes in sfPro.…”

“…is coincident with the results found in the pooled L. infantum samples from sand-fly and culture (Table 1), whereas amastins and SHERP are only increased in metacyclic L. major promastigotes but not in the L. infantum pools. In a previous comparison of L. infantum metacyclics from culture (peanut lectin non-agglutinating) and the stomodeal valve of P. perniciosus [3], some genes of the amastin superfamily were also found as up-regulated in metacyclics from P.…”

“…Consequently, passage through laboratory animals is required to maintain virulence (reviewed in [1]). While promastigote culture is designed to mimic the conditions of the microenvironment of the vector host, previous transcriptome analyses and in vitro infection experiments revealed important differences between promastigotes from the stomodeal valve of the sand fly and from cultures in stationary phase [2,3]. In fact, peanut lectin non-agglutinating (PNA -) promastigotes obtained from L. infantum promastigote cultures in stationary phase are 30% less infective than sand flyderived promastigotes, and the whole populations in stationary phase 50% less infective.…”

“…In fact, peanut lectin non-agglutinating (PNA -) promastigotes obtained from L. infantum promastigote cultures in stationary phase are 30% less infective than sand flyderived promastigotes, and the whole populations in stationary phase 50% less infective. The proportion of L. infantum metacyclic promastigotes in culture [3,4] and within the P. perniciosus gut [5] is up to 10% depending on the elapsed time of development. Metacyclic promastigotes are found at the stomodeal valve, which is located at the front of the thoracic midgut.…”

“…Suspensions of promastigotes from 10 different infected sand flies were pooled in 100 µl PBS and immediately harvested by centrifugation at 2,000g for 10 min. Infections were mature because each thoracic midgut was fully populated with promastigotes up to the stomodeal valve, as shown in our previous experiments [2,3,8]. This included daily follow-up of infections at the light microscope after random sampling.…”