Do malaria ookinete surface proteins P25 and P28 mediate parasite entry into mosquito midgut epithelial cells?

Abstract

Background: P25 and P28 are related ookinete surface proteins highly conserved throughout the Plasmodium genus that are under consideration as candidates for inclusion in transmission-blocking vaccines. Previous research using transgenic rodent malaria parasites lacking P25 and P28 has demonstrated that these proteins have multiple partially redundant functions during parasite infection of the mosquito vector, including an undefined role in ookinete traversal of the mosquito midgut epithelium, and it has been suggested that, unlike wild-type parasites, Dko P25/P28 parasites migrate across the midgut epithelium via an intercellular, rather than intracellular, route.

Presentation of the hypothesis: This paper presents an alternative interpretation for the previous observations of Dko P25/P28 parasites, based upon a recently published model of the route of ookinete invasion across the midgut epithelium. This model claims ookinete invasion is intracellular, with entry occurring through the lateral apical plasma membrane of midgut epithelial cells, and is associated with significant invagination of the midgut epithelium localised at the site of parasite penetration. Following this model, it is hypothesized that: (1) a sub-population of Dko P25/P28 ookinetes invaginate, but do not penetrate, the apical surface of the midgut epithelium and thus remain within the midgut lumen; and (2) another sub-population of Dko P25/P28 parasites successfully enters and migrates across the midgut epithelium via an intracellular route similar to wild-type parasites and subsequently develops into oocysts.

Testing the hypothesis: These hypotheses are tested by showing how they can account for previously published observations and incorporate them into a coherent and consistent explanatory framework. Based upon these hypotheses, several quantitative predictions are made, which can be experimentally tested, about the relationship between the densities of invading Dko P25/P28 ookinetes in different regions of the midgut epithelium and the number of oocyst stage parasites to which these mutant ookinetes give rise.

Implications of the hypothesis: The recently published model of ookinete invasion implies that Dko P25/P28 parasites are greatly, although not completely, impaired in their ability to enter the midgut epithelium. Therefore, P25 and/or P28 have a novel, previously unrecognized, function in mediating ookinete entry into midgut epithelial cells, suggesting that one mode of action of transmission-blocking antibodies to these ookinete surface proteins is to inhibit this function.

Figure 1

A general model of ookinete entry into the mosquito midgut epithelium. (a) Ookinetes (shown in green) enter the apical surface of the midgut epithelium, through the microvillar brush border (MV), where the lateral membranes (LM) of adjacent epithelial cells (EC) converge [47,51,52]. (b-c) Ookinete movement into the midgut epithelium causes substantial localized invagination of the latter (indicated by small blue arrows) [52,57]. (d) Ookinetes subsequently enter midgut epithelial cells through the lateral apical membrane immediately adjacent to the site where the intercellular junctions (IJ) begin [47,51,52]. (e) The ookinete proceeds intracellularly towards the basal membrane (BM) of the invaded midgut epithelial cell which exhibits morphological abnormalities including protrusion (indicated by large black arrow) into the midgut lumen (LUM) [44–48,52,54,55].

Figure 2

Dko P25/P28 P. berghei ookinete invasion of the midgut epithelium. The unified model of the route of ookinete invasion across the mosquito midgut epithelium (Figure 1) [51] implies that there are two sub-populations of Dko P25/28 parasites: (1) a major sub-population of Dko P25/28 ookinetes (shown in green) unable to penetrate midgut epithelial cells, which remain extracellular within the midgut lumen, embedded against the invaginated apical surface of the midgut epithelium (indicated by small blue arrow); and (2) a minor sub-population of Dko P25/28 ookinetes able to penetrate midgut epithelial cells, causing activation of mosquito immune responses and protrusion of invaded midgut cells, in a manner similar to wild-type parasites. Whether the latter parasites migrate through multiple adjacent midgut epithelial cells (as shown) is uncertain.