Stage- and gender-specific proteomic analysis of Brugia malayi excretory-secretory products

Abstract

Introduction: While we lack a complete understanding of the molecular mechanisms by which parasites establish and achieve protection from host immune responses, it is accepted that many of these processes are mediated by products, primarily proteins, released from the parasite. Parasitic nematodes occur in different life stages and anatomical compartments within the host. Little is known about the composition and variability of products released at different developmental stages and their contribution to parasite survival and progression of the infection.

Methodology/principal findings: To gain a deeper understanding on these aspects, we collected and analyzed through 1D-SDS PAGE and LC-MS/MS the Excretory-Secretory Products (ESP) of adult female, adult male and microfilariae of the filarial nematode Brugia malayi, one of the etiological agents of human lymphatic filariasis. This proteomic analysis led to the identification of 228 proteins. The list includes 76 proteins with unknown function as well as also proteins with potential immunoregulatory properties, such as protease inhibitors, cytokine homologues and carbohydrate-binding proteins. Larval and adult ESP differed in composition. Only 32 proteins were shared between all three stages/genders. Consistent with this observation, different gene ontology profiles were associated with the different ESP.

Conclusions/significance: A comparative analysis of the proteins released in vitro by different forms of a parasitic nematode dwelling in the same host is presented. The catalog of secreted proteins reflects different stage- and gender-specific related processes and different strategies of immune evasion, providing valuable insights on the contribution of each form of the parasite for establishing the host-parasite interaction.

Figure 1. SDS-PAGE of ESP from microfilariae (Mf), females (F) and males (M) B. malayi.

37.3 µg, 65.3 µg and 15.4 µg of protein from Mf, F and M, respectively, were separated by electrophoresis through a 2.4 cm gradient SDS gel (7.5–14%). Protein loaded is the amount recovered from 3 pooled sets of independent incubations. Following staining with Coomassie Brilliant blue G, the entire lanes were subjected to automated band excision to obtain 15 pieces per lane. Proteins from gel bands were digested with trypsin and analyzed by LC-MS/MS. Intensity of lane F was adjusted in order to allow better visualization of the staining.

Figure 2. Venn diagram showing the distribution of proteins identified in ESP from microfilariae, female and male B. malayi.

Figure 3. Distribution of Gene Ontology terms (level 2) for proteins identified in ESP from microfilariae, female and male B. malayi.

A. Molecular Function. B. Biological Process.

Figure 4. Distribution of the most abundant Gene Ontology terms (level 4) assigned for proteins identified in ESP from microfilariae, female and male worms of B. malayi.

A. Molecular Function. B. Biological Process.

Figure 5. Gene Ontology terms concentrated on individual ESP from microfilariae, female and male B. malayi compared to the total protein set.

A. Enriched molecular function terms. B. Enriched biological process terms.