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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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Co-incubation of L. mexicana with PSG enhances BALB/c macrophage infections in vitro under a range of activation states.(A–D) Kinetic of parasite growth in infected BMMΦ (5×104; MOI 5∶1) in the presence or absence of classical activators and vector-derived products. (A) Control L. mexicana infections of PBS (unstimulated, open circles), IFNγ and TNFα-treated (classically activated: CAMΦ, open squares) or IL-4-treated (alternatively activated: AAMΦ, open diamonds) macrophages. The role of L. mexicana PSG (0.25 µg) (B), Lu. longipalpis sand fly saliva (0.25 µg) (C) or a combination of PSG and saliva (0.25 µg of each) (D) (closed diamonds) was assessed for infection in CAMΦ. (E&F) 48 hour parasite burden of unstimulated BMMΦ, AAMΦ and CAMΦ infected in the presence or absence of 0.25 µg PSG macrophages was assessed by microscopy of infected cells (E), or by transformation assay of amastigotes released from macrophages (F). Growth was determined by direct counting of transformed promastigotes by haemocytometer in triplicate. (G–I) Effect of PSG (0.25 µg), saliva (0.25 µg), PSG and saliva (0.25 µg of each), deglycosylated (degly) PSG (0.25 µg) or saline on LPG2−/− (phosphoglycan-deficient) L. mexicana infections of unstimulated (G), AA (H) and CA (I) macrophages. Amastigote burden of infected macrophages determined by microscopy of at least 200 Giemsa-stained cells in triplicate or quadruplicate. Data representative of triplicate experiments. Unless they are linked with a bar all test groups are compared to their relevant saline control; ns, not significant P>0.05;*, P<0.05; **, P<0.005; ***, P<0.0005.
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ppat-1000555-g004: Co-incubation of L. mexicana with PSG enhances BALB/c macrophage infections in vitro under a range of activation states.(A–D) Kinetic of parasite growth in infected BMMΦ (5×104; MOI 5∶1) in the presence or absence of classical activators and vector-derived products. (A) Control L. mexicana infections of PBS (unstimulated, open circles), IFNγ and TNFα-treated (classically activated: CAMΦ, open squares) or IL-4-treated (alternatively activated: AAMΦ, open diamonds) macrophages. The role of L. mexicana PSG (0.25 µg) (B), Lu. longipalpis sand fly saliva (0.25 µg) (C) or a combination of PSG and saliva (0.25 µg of each) (D) (closed diamonds) was assessed for infection in CAMΦ. (E&F) 48 hour parasite burden of unstimulated BMMΦ, AAMΦ and CAMΦ infected in the presence or absence of 0.25 µg PSG macrophages was assessed by microscopy of infected cells (E), or by transformation assay of amastigotes released from macrophages (F). Growth was determined by direct counting of transformed promastigotes by haemocytometer in triplicate. (G–I) Effect of PSG (0.25 µg), saliva (0.25 µg), PSG and saliva (0.25 µg of each), deglycosylated (degly) PSG (0.25 µg) or saline on LPG2−/− (phosphoglycan-deficient) L. mexicana infections of unstimulated (G), AA (H) and CA (I) macrophages. Amastigote burden of infected macrophages determined by microscopy of at least 200 Giemsa-stained cells in triplicate or quadruplicate. Data representative of triplicate experiments. Unless they are linked with a bar all test groups are compared to their relevant saline control; ns, not significant P>0.05;*, P<0.05; **, P<0.005; ***, P<0.0005.

Mentions: Knowing that PSG enhanced L. mexicana infections in vivo and that it can recruit its ultimate host cells, macrophages, we next wanted to dissect the mechanism by which PSG exacerbates Leishmania infections in macrophages, by modelling natural infection in vitro (Fig. 4). Mature BMMΦ were infected and polarized into classically or alternatively activated macrophages in the presence or absence of PSG and saliva. As expected, classical activation resulted in significant control and alternative activation exacerbated L. mexicana growth from 24 hours post-infection onwards, as compared to parasite growth in unstimulated macrophages (Fig. 4A). Intra-macrophage growth was assessed microscopically by counting amastigotes (Fig. 4A–E, G–I) and parasite viability was determined by transformation and growth of amastigotes released from macrophages in vitro (Fig. 4F), or by fluorescent growth assay (Fig. S2). Interestingly, the presence of PSG significantly increased survival in classically activated macrophages during the first 48 hours of infection (Fig. 4B, P = 0.0002). Both the proportion of macrophages infected (Fig. S3) and their parasite burdens (Fig. 4B, E & I) were increased in the presence of PSG during this time (4–8 fold increase). By contrast, Lu. longipalpis sand fly saliva had minimal influence, with only a moderate increase in the proportion of macrophages infected by 24 hours post infection (Fig. S3). Interestingly, the combination of PSG and saliva resulted in an intermediate pattern of infection that overall resembled the kinetics of adding PSG alone, such that infections were exacerbated for both the 24 and 48 hour time points (Fig. 4D). By 72 hours of exposure to classical activators all macrophage infections were significantly reduced and no differences were exhibited between the groups. In separate experiments, 48 hour L. mexicana infections revealed a clear enhancing effect of PSG on the growth and viability of L. mexicana infection in either unstimulated, AAMΦ or CAMΦ in vitro (Fig. 4E&F).


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)

Co-incubation of L. mexicana with PSG enhances BALB/c macrophage infections in vitro under a range of activation states.(A–D) Kinetic of parasite growth in infected BMMΦ (5×104; MOI 5∶1) in the presence or absence of classical activators and vector-derived products. (A) Control L. mexicana infections of PBS (unstimulated, open circles), IFNγ and TNFα-treated (classically activated: CAMΦ, open squares) or IL-4-treated (alternatively activated: AAMΦ, open diamonds) macrophages. The role of L. mexicana PSG (0.25 µg) (B), Lu. longipalpis sand fly saliva (0.25 µg) (C) or a combination of PSG and saliva (0.25 µg of each) (D) (closed diamonds) was assessed for infection in CAMΦ. (E&F) 48 hour parasite burden of unstimulated BMMΦ, AAMΦ and CAMΦ infected in the presence or absence of 0.25 µg PSG macrophages was assessed by microscopy of infected cells (E), or by transformation assay of amastigotes released from macrophages (F). Growth was determined by direct counting of transformed promastigotes by haemocytometer in triplicate. (G–I) Effect of PSG (0.25 µg), saliva (0.25 µg), PSG and saliva (0.25 µg of each), deglycosylated (degly) PSG (0.25 µg) or saline on LPG2−/− (phosphoglycan-deficient) L. mexicana infections of unstimulated (G), AA (H) and CA (I) macrophages. Amastigote burden of infected macrophages determined by microscopy of at least 200 Giemsa-stained cells in triplicate or quadruplicate. Data representative of triplicate experiments. Unless they are linked with a bar all test groups are compared to their relevant saline control; ns, not significant P>0.05;*, P<0.05; **, P<0.005; ***, P<0.0005.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2722086&req=5

ppat-1000555-g004: Co-incubation of L. mexicana with PSG enhances BALB/c macrophage infections in vitro under a range of activation states.(A–D) Kinetic of parasite growth in infected BMMΦ (5×104; MOI 5∶1) in the presence or absence of classical activators and vector-derived products. (A) Control L. mexicana infections of PBS (unstimulated, open circles), IFNγ and TNFα-treated (classically activated: CAMΦ, open squares) or IL-4-treated (alternatively activated: AAMΦ, open diamonds) macrophages. The role of L. mexicana PSG (0.25 µg) (B), Lu. longipalpis sand fly saliva (0.25 µg) (C) or a combination of PSG and saliva (0.25 µg of each) (D) (closed diamonds) was assessed for infection in CAMΦ. (E&F) 48 hour parasite burden of unstimulated BMMΦ, AAMΦ and CAMΦ infected in the presence or absence of 0.25 µg PSG macrophages was assessed by microscopy of infected cells (E), or by transformation assay of amastigotes released from macrophages (F). Growth was determined by direct counting of transformed promastigotes by haemocytometer in triplicate. (G–I) Effect of PSG (0.25 µg), saliva (0.25 µg), PSG and saliva (0.25 µg of each), deglycosylated (degly) PSG (0.25 µg) or saline on LPG2−/− (phosphoglycan-deficient) L. mexicana infections of unstimulated (G), AA (H) and CA (I) macrophages. Amastigote burden of infected macrophages determined by microscopy of at least 200 Giemsa-stained cells in triplicate or quadruplicate. Data representative of triplicate experiments. Unless they are linked with a bar all test groups are compared to their relevant saline control; ns, not significant P>0.05;*, P<0.05; **, P<0.005; ***, P<0.0005.
Mentions: Knowing that PSG enhanced L. mexicana infections in vivo and that it can recruit its ultimate host cells, macrophages, we next wanted to dissect the mechanism by which PSG exacerbates Leishmania infections in macrophages, by modelling natural infection in vitro (Fig. 4). Mature BMMΦ were infected and polarized into classically or alternatively activated macrophages in the presence or absence of PSG and saliva. As expected, classical activation resulted in significant control and alternative activation exacerbated L. mexicana growth from 24 hours post-infection onwards, as compared to parasite growth in unstimulated macrophages (Fig. 4A). Intra-macrophage growth was assessed microscopically by counting amastigotes (Fig. 4A–E, G–I) and parasite viability was determined by transformation and growth of amastigotes released from macrophages in vitro (Fig. 4F), or by fluorescent growth assay (Fig. S2). Interestingly, the presence of PSG significantly increased survival in classically activated macrophages during the first 48 hours of infection (Fig. 4B, P = 0.0002). Both the proportion of macrophages infected (Fig. S3) and their parasite burdens (Fig. 4B, E & I) were increased in the presence of PSG during this time (4–8 fold increase). By contrast, Lu. longipalpis sand fly saliva had minimal influence, with only a moderate increase in the proportion of macrophages infected by 24 hours post infection (Fig. S3). Interestingly, the combination of PSG and saliva resulted in an intermediate pattern of infection that overall resembled the kinetics of adding PSG alone, such that infections were exacerbated for both the 24 and 48 hour time points (Fig. 4D). By 72 hours of exposure to classical activators all macrophage infections were significantly reduced and no differences were exhibited between the groups. In separate experiments, 48 hour L. mexicana infections revealed a clear enhancing effect of PSG on the growth and viability of L. mexicana infection in either unstimulated, AAMΦ or CAMΦ in vitro (Fig. 4E&F).

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