Extracellular release of virulence factor major surface protease via exosomes in Leishmania infantum promastigotes

Abstract

Background: The Leishmania spp. protozoa are introduced into humans through a sand fly blood meal, depositing the infectious metacyclic promastigote form of the parasite into human skin. Parasites enter a variety of host cells, although a majority are found in macrophages where they replicate intracellularly during chronic leishmaniasis. Symptomatic leishmaniasis causes considerable human morbidity in endemic regions. The Leishmania spp. evade host microbicidal mechanisms partially through virulence-associated proteins such as the major surface protease (MSP or GP63), to inactivate immune factors in the host environment. MSP is a metalloprotease encoded by a tandem array of genes belonging to three msp gene classes, whose mRNAs are differentially expressed in different life stages of the parasite. Like other cells, Leishmania spp. release small membrane-bound vesicles called exosomes into their environment. The purpose of this study was to detect MSP proteins in exosomal vesicles of Leishmania spp. protozoa.

Methods: Using mass spectrometry data we determined the profile of MSP class proteins released in L. infantum exosomes derived from promastigotes in their avirulent procyclic (logarithmic) stage and virulent stationary and metacyclic stages. MSP protein isoforms belonging to each of the three msp gene classes could be identified by unique peptides.

Results: Metacyclic promastigote exosomes contained the highest, and logarithmic exosomes had the lowest abundance of total MSP. Among the MSP classes, MSPC class had the greatest variety of isoforms, but was least abundant in all exosomes. Nonetheless, all MSP classes were present at higher levels in exosomes released from stationary or metacyclic promastigotes than logarithmic promastigotes.

Conclusions: The data suggest the efficiency of exosome release may be more important than the identity of MSP isoform in determining the MSP content of Leishmania spp. exosomes.

Keywords: Exosome; Leishmania; Major surface protease; Promastigotes; Virulence factors.

Fig. 1

Transmission electron microscopy of the secreted nanovesicles. The secreted nanovesicles from the stationary promastigotes of L. infantum were processed and examined by TEM. The size and morphology of the secreted nanovesicles from logarithmic and metacyclic promastigotes were similar (not shown). Scale-bar: 200 nm

Fig. 2

MSP in the secreted nanovesicles of L. infantum promastigotes. Seven μg of exosome protein were loaded in each lane. Lanes 1–3: nanovesicles; Lanes 4–6: total cell lysates; Lanes 1 and 4: metacyclic; Lanes 2 and 5: stationary; Lanes 3 and 6: log phase promastigotes. Molecular weight in KDa is shown on the left. The panel was probed with sheep polyclonal antiserum to MPS. One representative among three repeat blots is shown

Fig. 3

MSP proteins released in exosomes by various stages of promastigotes. The mean ± SE total spectral values per repeat exosome preparation from each life-cycle stage was calculated from sequences of each of the individual exosome samples (n = 3). Significant differences were calculated using one-way ANOVA with Tukey post-hoc test

Fig. 4

Quantification of individual MSP isoforms in exosomes from log, stationary or metacyclic promastigotes. The relative quantities were calculated according to the spectral values. Data shown as mean ± SD

Fig. 5

The mean ± SD spectral values corresponding to products of each of the three MSP classes were calculated in exosomes released from logarithmic, stationary or metacyclic promastigotes. MSP classes are MSPL (L), MSPS (S) and MSPC (C). Differences were observed between MSPs in logarithmic exosomes and exosomes from either metacyclic or stationary promastigotes (two-way ANOVA, Tukey post-hoc test)