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Proteophosophoglycans regurgitated by Leishmania-infected sand flies target the L-arginine metabolism of host macrophages to promote parasite survival.

Rogers M, Kropf P, Choi BS, Dillon R, Podinovskaia M, Bates P, Müller I - PLoS Pathog. (2009)

Bottom Line: This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours.Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth.The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Imperial College of Science, Technology and Medicine, London, UK. matthew.rogers@imperial.ac.uk

ABSTRACT
All natural Leishmania infections start in the skin; however, little is known of the contribution made by the sand fly vector to the earliest events in mammalian infection, especially in inflamed skin that can rapidly kill invading parasites. During transmission sand flies regurgitate a proteophosphoglycan gel synthesized by the parasites inside the fly midgut, termed promastigote secretory gel (PSG). Regurgitated PSG can exacerbate cutaneous leishmaniasis. Here, we show that the amount of Leishmania mexicana PSG regurgitated by Lutzomyia longipalpis sand flies is proportional to the size of its original midgut infection and the number of parasites transmitted. Furthermore, PSG could exacerbate cutaneous L. mexicana infection for a wide range of doses (10-10,000 parasites) and enhance infection by as early as 48 hours in inflamed dermal air pouches. This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours. Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth. The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo. Thus, PSG is an essential component of the infectious sand fly bite for the early establishment of Leishmania in skin, which should be considered when designing and screening therapies against leishmaniasis.

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Transmitted L. mexicana dose in relation to amount of PSG regurgitated by sand flies.(A) Relationship between numbers of L. mexicana egested by Lu. longipalpis sand flies collected by capillary feeding and size of the sand fly midgut parasite load quantified by direct counting immediately after capillary feeding. (B) Dot-blots of L. mexicana PSG standards and capillary feeds from L. mexicana-infected sand flies probed with anti-PSG sera or normal rabbit serum (NRS). (C) Area under curve analysis of digitized PSG standards (inset) reveals high and low egestion of PSG. (D&E) Relationship between the total midgut parasite load and the amount of PSG egested. (F&G) Relationship between the transmitted dose of parasites and the amount of PSG egested. **, P<0.005.
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ppat-1000555-g001: Transmitted L. mexicana dose in relation to amount of PSG regurgitated by sand flies.(A) Relationship between numbers of L. mexicana egested by Lu. longipalpis sand flies collected by capillary feeding and size of the sand fly midgut parasite load quantified by direct counting immediately after capillary feeding. (B) Dot-blots of L. mexicana PSG standards and capillary feeds from L. mexicana-infected sand flies probed with anti-PSG sera or normal rabbit serum (NRS). (C) Area under curve analysis of digitized PSG standards (inset) reveals high and low egestion of PSG. (D&E) Relationship between the total midgut parasite load and the amount of PSG egested. (F&G) Relationship between the transmitted dose of parasites and the amount of PSG egested. **, P<0.005.

Mentions: Recently it has been shown that the dose of L. major parasites delivered by a sand fly relies to an extent on the size of the original sand fly infection [4]. PSG accumulates during Leishmania metacyclogenesis to obstruct the sand fly midgut. This blockage can greatly influence the feeding ability of the fly and limit the amount of blood intake [5],[30], forcing it to regurgitate PSG and parasites during transmission. Therefore, in accordance with these observations we hypothesised that the quantity of PSG egested correlates with the size of the sand fly infection. In order to test this we used the experimental combination of L. mexicana with Lu. longipalpis, a parasite-vector combination that is (i) fully permissive for parasite development, (ii) readily transmitted to mice by bite and (iii) generates large infections and large amounts of PSG plug [5]. To standardise the feeding process we capillary fed saline to day 8 infected sand flies for 10 minutes, and only those flies for which active feeding (cibarial pumping) was observed were included in the analysis. Following feeding the number of egested parasites was counted and each fly was dissected to determine the number of promastigotes remaining in the midgut. Using this method we observed that the number of L. mexicana egested positively correlated with the size of the fly infection (Fig. 1A: r2 = 0.28, P = 0.005). We have previously ascertained that the capillary feeding technique underestimates the dose of transmitted parasites by a factor of 10 [3]. Bearing this in mind these flies exhibited a roughly bimodal distribution such that two groups of flies with high (Average±S.E.: 1.47×105±1.5×104) and low (5.2×104±1.48×104) Leishmania infections were observed to transmit correspondingly large (195±240) and small (53±79) doses of parasites. By analysing the regurgitate of each fly by semi-quantitative dot-blot we could group these flies into those that regurgitated high (0.03±0.06 µg) and low (0.0004±0.00016 µg) amounts of PSG (Fig. 1B&C). This revealed that sand flies egesting high amounts of PSG had higher parasite infections (Fig. 1D: P = 0.003; Fig. 1E: r2 = 0.18, P = 0.0314), and importantly, these flies tended to transmit more parasites (Fig. 1F: P = 0.08; Fig. 1G: r2 = 0.28, P = 0.006). Previously we have shown that the quantity of PSG in a sand fly midgut is dictated by the number of leptomonad promastigotes in the infection [2],[5]. Therefore, collectively these results verify our hypothesis and show that the size of the PSG blockage determines the number of parasite transmitted and the proportion of co-regurgitated gel.


Proteophosophoglycans regurgitated by Leishmania-infected sand flies target the L-arginine metabolism of host macrophages to promote parasite survival.

Rogers M, Kropf P, Choi BS, Dillon R, Podinovskaia M, Bates P, Müller I - PLoS Pathog. (2009)

Transmitted L. mexicana dose in relation to amount of PSG regurgitated by sand flies.(A) Relationship between numbers of L. mexicana egested by Lu. longipalpis sand flies collected by capillary feeding and size of the sand fly midgut parasite load quantified by direct counting immediately after capillary feeding. (B) Dot-blots of L. mexicana PSG standards and capillary feeds from L. mexicana-infected sand flies probed with anti-PSG sera or normal rabbit serum (NRS). (C) Area under curve analysis of digitized PSG standards (inset) reveals high and low egestion of PSG. (D&E) Relationship between the total midgut parasite load and the amount of PSG egested. (F&G) Relationship between the transmitted dose of parasites and the amount of PSG egested. **, P<0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2722086&req=5

ppat-1000555-g001: Transmitted L. mexicana dose in relation to amount of PSG regurgitated by sand flies.(A) Relationship between numbers of L. mexicana egested by Lu. longipalpis sand flies collected by capillary feeding and size of the sand fly midgut parasite load quantified by direct counting immediately after capillary feeding. (B) Dot-blots of L. mexicana PSG standards and capillary feeds from L. mexicana-infected sand flies probed with anti-PSG sera or normal rabbit serum (NRS). (C) Area under curve analysis of digitized PSG standards (inset) reveals high and low egestion of PSG. (D&E) Relationship between the total midgut parasite load and the amount of PSG egested. (F&G) Relationship between the transmitted dose of parasites and the amount of PSG egested. **, P<0.005.
Mentions: Recently it has been shown that the dose of L. major parasites delivered by a sand fly relies to an extent on the size of the original sand fly infection [4]. PSG accumulates during Leishmania metacyclogenesis to obstruct the sand fly midgut. This blockage can greatly influence the feeding ability of the fly and limit the amount of blood intake [5],[30], forcing it to regurgitate PSG and parasites during transmission. Therefore, in accordance with these observations we hypothesised that the quantity of PSG egested correlates with the size of the sand fly infection. In order to test this we used the experimental combination of L. mexicana with Lu. longipalpis, a parasite-vector combination that is (i) fully permissive for parasite development, (ii) readily transmitted to mice by bite and (iii) generates large infections and large amounts of PSG plug [5]. To standardise the feeding process we capillary fed saline to day 8 infected sand flies for 10 minutes, and only those flies for which active feeding (cibarial pumping) was observed were included in the analysis. Following feeding the number of egested parasites was counted and each fly was dissected to determine the number of promastigotes remaining in the midgut. Using this method we observed that the number of L. mexicana egested positively correlated with the size of the fly infection (Fig. 1A: r2 = 0.28, P = 0.005). We have previously ascertained that the capillary feeding technique underestimates the dose of transmitted parasites by a factor of 10 [3]. Bearing this in mind these flies exhibited a roughly bimodal distribution such that two groups of flies with high (Average±S.E.: 1.47×105±1.5×104) and low (5.2×104±1.48×104) Leishmania infections were observed to transmit correspondingly large (195±240) and small (53±79) doses of parasites. By analysing the regurgitate of each fly by semi-quantitative dot-blot we could group these flies into those that regurgitated high (0.03±0.06 µg) and low (0.0004±0.00016 µg) amounts of PSG (Fig. 1B&C). This revealed that sand flies egesting high amounts of PSG had higher parasite infections (Fig. 1D: P = 0.003; Fig. 1E: r2 = 0.18, P = 0.0314), and importantly, these flies tended to transmit more parasites (Fig. 1F: P = 0.08; Fig. 1G: r2 = 0.28, P = 0.006). Previously we have shown that the quantity of PSG in a sand fly midgut is dictated by the number of leptomonad promastigotes in the infection [2],[5]. Therefore, collectively these results verify our hypothesis and show that the size of the PSG blockage determines the number of parasite transmitted and the proportion of co-regurgitated gel.

Bottom Line: This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours.Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth.The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Imperial College of Science, Technology and Medicine, London, UK. matthew.rogers@imperial.ac.uk

ABSTRACT
All natural Leishmania infections start in the skin; however, little is known of the contribution made by the sand fly vector to the earliest events in mammalian infection, especially in inflamed skin that can rapidly kill invading parasites. During transmission sand flies regurgitate a proteophosphoglycan gel synthesized by the parasites inside the fly midgut, termed promastigote secretory gel (PSG). Regurgitated PSG can exacerbate cutaneous leishmaniasis. Here, we show that the amount of Leishmania mexicana PSG regurgitated by Lutzomyia longipalpis sand flies is proportional to the size of its original midgut infection and the number of parasites transmitted. Furthermore, PSG could exacerbate cutaneous L. mexicana infection for a wide range of doses (10-10,000 parasites) and enhance infection by as early as 48 hours in inflamed dermal air pouches. This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours. Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth. The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo. Thus, PSG is an essential component of the infectious sand fly bite for the early establishment of Leishmania in skin, which should be considered when designing and screening therapies against leishmaniasis.

Show MeSH
Related in: MedlinePlus