Leishmania chitinase facilitates colonization of sand fly vectors and enhances transmission to mice

Abstract

Chitinases of trypanosomatid parasites have been proposed to fulfil various roles in their blood-feeding arthropod vectors but so far none have been directly tested using a molecular approach. We characterized the ability of Leishmania mexicana episomally transfected with LmexCht1 (the L. mexicana chitinase gene) to survive and grow within the permissive sand fly vector, Lutzomyia longipalpis. Compared with control plasmid transfectants, the overexpression of chitinase was found to increase the average number of parasites per sand fly and accelerate the escape of parasites from the peritrophic matrix-enclosed blood meal as revealed by earlier arrival at the stomodeal valve. Such flies also exhibited increased damage to the structure of the stomodeal valve, which may facilitate transmission by regurgitation. When exposed individually to BALB/c mice, those flies with chitinase-overexpressing parasites spent on average 2.4-2.5 times longer in contact with their host during feeding, compared with flies with control infections. Furthermore, the lesions that resulted from these single fly bite infections were both significantly larger and with higher final parasite burdens than controls. These data show that chitinase is a multifunctional virulence factor for L. mexicana which assists its survival in Lu. longipalpis. Specifically, this enzyme enables the parasites to colonize the anterior midgut of the sand fly more quickly, modify the sand fly stomodeal valve and affect its blood feeding, all of which combine to enhance transmission.

Fig. 1

Anatomy of female sand flies. A. Light micrograph (×500 magnification) of longitudinal section through the head and thorax of an uninfected female sand fly showing the position of the stomodeal valve (SV) in relation to the salivary glands (SG), thoracic midgut (TM) and foregut (FG). B. Schematic of longitudinal section through a female sand fly highlighting the position occupied by a 7-day-old infection (stippled area). Dashed box highlights tissues of sand fly represented in (C) and (D). C and D. Sagital section (C) and composite cross-section image of light micrographs (D) (C: ×500; D: ×1000 magnification) from a mature wild-type L. mexicana infection showing the distension of the valve during infection. (D) was taken at an oblique angle through the infected oesophagus (foregut), stomodeal valve and thoracic midgut. Arrows point to examples of Leishmania promastigotes attached to and in the lumen of the sand fly gut. The salivary glands can be seen lying below the stomodeal valve and thoracic midgut.

Fig. 2

Excess chitinase in the blood meal exacerbates early mortality of Leishmania to sand fly midgut trypsins. Comparison of early growth of wild-type L. mexicana in blood or plasma supplemented with or without 1 mg ml⁻¹ soybean trypsin inhibitor (TI) or 1 U ml⁻¹ S. griseus chitinase. Data from a typical infection; each bar representing the average of 10 flies per group ±1 standard error from the mean. Asterisks indicate values that are statistically significant (*P < 0.05, **P < 0.01) using an unpaired t-test.

Fig. 3

Leishmania chitinase assists in the infection of Lutzomyia longipalpis sand flies and colonization of the stomodeal valve. Flies were infected with plasmid control (pKSNEO; ◊) or chitinase-overexpressing (pKSNEO::LmexCht1-HA; ▪) Leishmania mexicana amastigotes. A. Kinetics of parasite growth in entire fly gut. B. The kinetics of parasite colonization and growth at the stomodeal valve region. Data from a typical infection; each point representing the average of 10 flies per group ±1 standard error from the mean. Asterisks indicate values from overexpressor infections that are statistically significant (*P < 0.05) from plasmid control infections using an unpaired t-test.

Fig. 4

Expression of Leishmania chitinase in sand flies causes pathology to the stomodeal valve. Electron micrographs of stomodeal valves from: (A) uninfected sand flies, (B) wild-type, (C) plasmid control and (D and E) chitinase-overexpressing L. mexicana- infected sand flies. The position of filamentous structures in the stomodeal valve connecting the cylindrical epithelial cells of the valve to the chitinous cuticle is shown by lines. cu, cuticle; fs, filametous structures; mi, mitochondria; mg, midgut; n, nucleus of epithelial cells of the stomodeal valve; Lp, Leishmania promastigote. The arrowheads indicate hemidesome-like plaques on parasite flagellum and the asterisks indicate chitin fragments.

Fig. 5

Leishmania chitinase overexpression enhances transmission from Lutzomyia longipalpis sand flies. Flies were infected with wild type (○), plasmid control (pKSNEO; ◊) or chitinase-overexpressing (pKSNEO::LmexCht1-HA; ▪) Leishmania mexicana amastigotes. Individual BALB/c mice were infected by single fly bites to the foot. A. Lesion development was monitored by measuring the thickness of the bitten foot compared with the unbitten foot. Each point represents the average lesion growth of at least five mice per group ±1 standard error from the mean. Asterisk and hash symbols indicate values from overexpressor infections that are statistically significant (*P < 0.05, #P < 0.05, ##P < 0.005) using an unpaired t-test from wild-type or plasmid control infections respectively. B. Final lesion parasite burdens from single fly bite infections of BALB/c mice. Bars represent the median value for each group and asterisk symbols indicate statistical significance between groups indicated (*P < 0.05, **P < 0.01) using the Mann–Whitney U-test.