A lipophosphoglycan-independent development of Leishmania in permissive sand flies

Leishmaniases are serious parasitic diseases the etiological organisms of which are transmitted by insect vectors, phlebotominae sand flies. Two sand fly species, Phlebotomus papatasi and P. sergenti, display remarkable specificity for Leishmania parasites they transmit in nature, but many others are broadly permissive to the development of different Leishmania species. Previous studies have suggested that in 'specific' vectors the successful parasite development is mediated by parasite surface glycoconjugates and sand fly lectins, however we show here that interactions involving 'permissive' sand flies utilize another molecules. We did find that the abundant surface glycoconjugate lipophosphoglycan, essential for attachment of Leishmania major in the specific vector P. papatasi, was not required for parasite adherence or survival in the permissive vectors P. arabicus and Lutzomyia longipalpis. Attachment in several permissive sand fly species instead correlated with the presence of midgut glycoproteins bearing terminal N-acetyl-galactosamine and with the occurrence of a lectin-like activity on Leishmania surface. This new binding modality has important implications for parasite transmission and evolution. It may contribute to the successful spreading of Leishmania due to their adaptation into new vectors, namely transmission of L. infantum by Lutzomyia longipalpis; this event led to the establishment of L. infantum/chagasi in Latin America.     

Leishmania-sand fly interactions controlling species-specific vector competence

Leishmaniasis is caused by a wide range of parasites that are transmitted by an even wider range of sand fly vectors. The phlebotomine vectors of Leishmaniasis are in some cases only permissive to the complete development of the species of Leishmania that they transmit in nature. The parasite–sand fly interactions that control this specificity are related to differences in the ability of the parasite to inhibit or to resist killing by proteolytic enzymes released into the mid-gut soon after blood feeding, and/or to maintain infection in the mid-gut during excretion of the digested blood meal. In each case, surface expressed or released phosphoglycan-containing molecules appear to promote parasite survival. The evidence that the surface lipophosphoglycan (LPG) mediates promastigote attachment to the mid-gut epithelium so as to prevent their loss during blood-meal excretion is especially strong based on the comparison of development in sand flies using LPG-deficient mutants. LPG displays interspecies polymorphisms in their phosphoglycan domains that in most cases can fully account for species-specific vector competence.     

Leishmania major Glycosylation Mutants Require Phosphoglycans (lpg2 ?) but Not Lipophosphoglycan (lpg1 ?) for Survival in Permissive Sand Fly Vectors

Background

Sand fly species able to support the survival of the protozoan parasite Leishmania have been classified as permissive or specific, based upon their ability to support a wide or limited range of strains and/or species. Studies of a limited number of fly/parasite species combinations have implicated parasite surface molecules in this process and here we provide further evidence in support of this proposal. We investigated the role of lipophosphoglycan (LPG) and other phosphoglycans (PGs) in sand fly survival, using Leishmania major mutants deficient in LPG (lpg1 ⁻), and the phosphoglycan (PG)-deficient mutant lpg2 ⁻. The sand fly species used were the permissive species Phlebotomus perniciosus and P. argentipes, and the specific vector P. duboscqi, a species resistant to L. infantum development.

Principal Findings

The lpg2 ⁻ mutants did not survive well in any of the three sand fly species, suggesting that phosphoglycans and/or other LPG2-dependent molecules are required for parasite development. In vitro, all three L. major lines were equally resistant to proteolytic activity of bovine trypsin, suggesting that sand fly-specific hydrolytic proteases or other factors are the reason for the early lpg2 ⁻ parasite killing. The lpg1 ⁻ mutants developed late-stage infections in two permissive species, P. perniciosus and P. argentipes, where their infection rates and intensities of infections were comparable to the wild type (WT) parasites. In contrast, in P. duboscqi the lpg1 ⁻ mutants developed significantly worse than the WT parasites.

Conclusions

In combination with previous studies, the data establish clearly that LPG is not required for Leishmania survival in permissive species P. perniciosus and P. argentipes but plays an important role in the specific vector P. duboscqi. With regard to PGs other than LPG, the data prove the importance of LPG2-related molecules for survival of L. major in the three sand fly species tested.     

Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(beta1,4)Man(alpha1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis.     

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

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.     

Phlebotomine sand flies and Leishmania parasites: friends or foes?

Leishmania parasites need phlebotomine sand flies to complete their life cycle and to propagate. This review looks at Leishmania-sand fly interactions as the parasites develop from amastigotes to infectious metacyclics, highlighting recent findings concerning the evolutionary adaptations that ensure survival of the parasites. Such adaptations include secretion of phosphoglycans, which protect the parasite from digestive enzymes; production of chitinases that degrade the stomodeal valve of the sand fly; secretion of a neuropeptide that arrests midgut and hindgut peristalsis; and attaching to the midgut to avoid expulsion.     

Flypaper for parasites

In this issue, Kamhawi et al. (2004) describe the identification of an insect galectin as the receptor for the stage-specific Leishmania adhesin lipophosphoglycan (LPG). This interaction is critical for parasite survival in the midgut of its sand fly vector. The results open new avenues for studies of insect immunity, transmission binding vaccines, and host-parasite coevolution.     

Increased transmission potential of Leishmania major/Leishmania infantum hybrids

Development of Leishmania infantum/Leishmania major hybrids was studied in two sand fly species. In Phlebotomus papatasi, which supported development of L. major but not L. infantum, the hybrids produced heavy late-stage infections with high numbers of metacyclic promastigotes. In the permissive vector Lutzomyia longipalpis, all Leishmania strains included in this study developed well. Hybrids were found to express L. major lipophosphoglycan, apparently enabling them to survive in P. papatasi midgut. The genetic exchange of the hybrids thus appeared to have enhanced their transmission potential and fitness. A potentially serious consequence is the future spread of the hybrids using this peridomestic and antropophilic vector.     

Intra-species and stage-specific polymorphisms in lipophosphoglycan structure control Leishmania donovani-sand fly interactions.

The Leishmania lipophosphoglycan conveys the ability for the parasites to avoid destruction in diverse host environments. During its life cycle within the sand fly vector, the parasite differentiates from a dividing procyclic promastigote stage that avoids expulsion from the midgut by attaching to the gut wall, to a nondividing metacyclic promastigote stage that is unable to attach to the midgut and migrates to the mouth parts for reinfection of a mammalian host. Lipophosphoglycan plays an integral role during this transition. Structurally, lipophosphoglycan is a multidomain glycoconjugate whose polymorphisms among species lie in the backbone Gal(beta 1,4)Man(alpha 1)-PO(4) repeating units and the oligosaccharide cap. We have characterized the lipophosphoglycan from an Indian L. donovani isolate. Unlike East African isolates, which express unsubstituted repeats and a galactose- and mannose-terminating cap, procyclic lipophosphoglycan from the Indian isolate consists of beta1,3-linked glucose residues that branch off the backbone repeats (n approximately 17) and also terminate the cap. Of biological significance, metacyclic lipophosphoglycan lacks the glucose residues while doubling the number of repeats. The importance of these developmental modifications in lipophosphoglycan structure was determined using binding experiments to Phlebotomus argentipes midguts. Procyclic promastigotes and procyclic LPG were able to bind to sand fly midguts in vitro whereas metacyclic parasites and LPG lost this capacity. These results demonstrate that the Leishmania adapts the synthesis of terminally exposed sugars of its LPG to manipulate parasite-sand fly interactions.     

Leishmania parasites (Kinetoplastida: Trypanosomatidae) reversibly inhibit visceral muscle contractions in hemimetabolous and holometabolous insects

Female sand flies can acquire protozoan parasites in the genus Leishmania when feeding on an infected vertebrate host. The parasites complete a complex growth cycle in the sand fly gut until they are transmitted by bite to another host. Recently, a myoinhibitory peptide was isolated from Leishmania major promastigotes. This peptide caused significant gut distension and reversible, dose-dependent inhibition of spontaneous hindgut contractions in the enzootic sand fly vector, Phlebotomus papatasi. The current study further characterizes myoinhibitory activity in L. major and other kinetoplastid parasites, using the P. papatasi hindgut and other insect organ preparations. Myoinhibitory activity was greatest in cultured promastigotes and in culture medium in late log-phase and early stationary-phase, coinciding with development of infective Leishmania morphotypes in the sand fly midgut. L. major promastigote lysates inhibited spontaneous contractions of visceral muscle preparations from hemimetabolous (Blattaria and Hemiptera) and holometabolous (Diptera) insects. Inhibition of visceral muscle contractions in three insect orders indicates a conserved mode of action. Myoinhibitory activity was detected also in Leishmania braziliensis braziliensis, a Sudanese strain of Leishmania donovani, and the kinetoplastid parasite Leptomonas seymouri. Protozoan-induced myoinhibition mimics the effect of insect myotropins. Inhibiting host gut contractions protects Leishmania parasites from being excreted after blood meal and peritrophic matrix digestion, allowing development and transmission of infective forms.     

Experimental transmission of Leishmania tropica to hyraxes (Procavia capensis) by the bite of Phlebotomus arabicus

The ability of the sand fly Phlebotomus (Adlerius) arabicus to transmit Leishmania tropica was studied experimentally using hyraxes (Procavia capensis), natural reservoir hosts of the parasite. Sand flies became infected with L. tropica after feeding on a lesion of needle-inoculated hyrax. Moreover, P. arabicus fed with L. tropica promastigotes transmitted the parasite to hyraxes by bite during a second bloodmeal. Although the animals remained asymptomatic after infective sand fly bite, they were PCR positive and infectious for naive sand flies. We have thus demonstrated cyclical transmission of L. tropica by P. arabicus in hyraxes. This confirms experimentally the vectorial competence of P. (Adlerius) arabicus, and demonstrates that asymptomatic reservoir hosts are infectious to appropriate vectors.     

Caspar-like gene depletion reduces Leishmania infection in sand fly host Lutzomyia longipalpis

Female phlebotomine sand flies Lutzomyia longipalpis naturally harbor populations of the medically important Leishmania infantum (syn. Leishmania chagasi) parasite in the gut, but the extent to which the parasite interacts with the immune system of the insect vector is unknown. To investigate the sand fly immune response and its interaction with the Leishmania parasite, we identified a homologue for caspar, a negative regulator of immune deficiency signaling pathway. We found that feeding antibiotics to adult female L. longipalpis resulted in an up-regulation of caspar expression relative to controls. caspar was differentially expressed when females were fed on gram-negative and gram-positive bacterial species. caspar expression was significantly down-regulated in females between 3 and 6 days after a blood feed containing Leishmania mexicana amastigotes. RNA interference was used to deplete caspar expression in female L. longipalpis, which were subsequently fed with Leishmania in a blood meal. Sand fly gut populations of both L. mexicana and L. infantum were significantly reduced in caspar-depleted females. The prevalence of L. infantum infection in the females fell from 85 to 45%. Our results provide the first insight into the operation of immune homeostasis in phlebotomine sand flies during the growth of bacterial and Leishmania populations in the digestive tract. We have demonstrated that the activation of the sand fly immune system, via depletion of a single gene, can lead to the abortion of Leishmania development and the disruption of transmission by the phlebotomine sand fly.     

Real-time PCR to assess the Leishmania load in Lutzomyia longipalpis sand flies: screening of target genes and assessment of quantitative methods

Visceral Leishmaniasis is an endemic disease in Brazil caused by Leishmania infantum chagasi and its main vector species is the sand fly Lutzomyia longipalpis. Epidemiological studies have used conventional PCR techniques to measure the rate of infection of sand flies collected in the field. However, real-time PCR can detect lower parasite burdens, reducing the number of false negatives and improving the quantification of Leishmania parasites in the sand fly. This study compared genes with various copy numbers to detect and quantify L. infantum chagasi in L. longipalpis specimens by real-time PCR. We mixed pools of 1, 10 and 30 male sand flies with various amounts of L. infantum chagasi, forming groups with 50, 500, 5000 and 50,000 Leishmania parasites. For the amplification of L. infantum chagasi DNA, primers targeting kDNA, polymerase α and the 18S ribosome subunit were employed. Parasites were measured by absolute and relative quantification. PCR detection using the amplification of kDNA exhibited the greatest sensitivity among the genes tested, showing the capacity to detect the DNA equivalent of 0.004 parasites. Additionally, the relative quantification using these primers was more accurate and precise. In general, the number of sand flies used for DNA extraction did not influence Leishmania quantification. However, for low-copy targets, such as the polymerase α gene, lower parasite numbers in the sample produced inaccurate quantifications. Thus, qPCR measurement of L. infantum chagasi in L. longipalpis was improved by targeting high copy-number genes; amplification of high copy-number targets increased the sensitivity, accuracy and precision of DNA-based parasite enumeration.     

Infection parameters in the sand fly vector that predict transmission of Leishmania major

To identify parameters of Leishmania infection within a population of infected sand flies that reliably predict subsequent transmission to the mammalian host, we sampled groups of infected flies and compared infection intensity and degree of metacyclogenesis with the frequency of transmission. The percentage of parasites within the midgut that were metacyclic promastigotes had the highest correlation with the frequency of transmission. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic "cutoff" value that predicted transmission competence. Sand fly infections initiated with variable doses of parasites resulted in correspondingly altered percentages of metacyclic promastigotes, resulting in altered transmission frequency and disease severity. Lastly, alteration of sand fly oviposition status and environmental conditions at the time of transmission also influenced transmission frequency. These observations have implications for transmission of Leishmania by the sand fly vector in both the laboratory and in nature, including how the number of organisms acquired by the sand fly from an infection reservoir may influence the clinical outcome of infection following transmission by bite.     

Breeding sites of Phlebotomus sergenti, the sand fly vector of cutaneous leishmaniasis in the Judean Desert

Phlebotomine sand flies transmit Leishmania, phlebo-viruses and Bartonella to humans. A prominent gap in our knowledge of sand fly biology remains the ecology of their immature stages. Sand flies, unlike mosquitoes do not breed in water and only small numbers of larvae have been recovered from diverse habitats that provide stable temperatures, high humidity and decaying organic matter. We describe studies designed to identify and characterize sand fly breeding habitats in a Judean Desert focus of cutaneous leishmaniasis. To detect breeding habitats we constructed emergence traps comprising sand fly-proof netting covering defined areas or cave openings. Large size horizontal sticky traps within the confined spaces were used to trap the sand flies. Newly eclosed male sand flies were identified based on their un-rotated genitalia. Cumulative results show that Phlebotomus sergenti the vector of Leishmania tropica rests and breeds inside caves that are also home to rock hyraxes (the reservoir hosts of L. tropica) and several rodent species. Emerging sand flies were also trapped outside covered caves, probably arriving from other caves or from smaller, concealed cracks in the rocky ledges close by. Man-made support walls constructed with large boulders were also identified as breeding habitats for Ph. sergenti albeit less important than caves. Soil samples obtained from caves and burrows were rich in organic matter and salt content. In this study we developed and put into practice a generalized experimental scheme for identifying sand fly breeding habitats and for assessing the quantities of flies that emerge from them. An improved understanding of sand fly larval ecology should facilitate the implementation of effective control strategies of sand fly vectors of Leishmania.     

Reactive oxygen species-mediated immunity against Leishmania mexicana and Serratia marcescens in the sand phlebotomine fly Lutzomyia longipalpis

Phlebotomine sand flies are the vectors of medically important Leishmania. The Leishmania protozoa reside in the sand fly gut, but the nature of the immune response to the presence of Leishmania is unknown. Reactive oxygen species (ROS) are a major component of insect innate immune pathways regulating gut-microbe homeostasis. Here we show that the concentration of ROS increased in sand fly midguts after they fed on the insect pathogen Serratia marcescens but not after feeding on the Leishmania that uses the sand fly as a vector. Moreover, the Leishmania is sensitive to ROS either by oral administration of ROS to the infected fly or by silencing a gene that expresses a sand fly ROS-scavenging enzyme. Finally, the treatment of sand flies with an exogenous ROS scavenger (uric acid) altered the gut microbial homeostasis, led to an increased commensal gut microbiota, and reduced insect survival after oral infection with S. marcescens. Our study demonstrates a differential response of the sand fly ROS system to gut microbiota, an insect pathogen, and the Leishmania that utilize the sand fly as a vehicle for transmission between mammalian hosts.     

Effect of sand fly saliva on Leishmania uptake by murine macrophages

Leishmania promastigotes are introduced into the skin by blood-sucking phlebotomine sand flies. In the vertebrate host, promastigotes invade macrophages, transform into amastigotes and multiply intracellularly. Sand fly saliva was shown to enhance the development of cutaneous leishmaniasis lesions by inhibiting some immune functions of the host macrophages. This study demonstrates that sand fly saliva promotes parasite survival and proliferation. First, macrophages gravitated towards increasing concentrations of sand fly saliva in vitro. Secondly, saliva increased the percentage of macrophages that became infected with Leishmania promastigotes and exacerbated the parasite load in these cells. Thus, during natural transmission, saliva probably reduces the exposure of promastigotes to the immune system by attracting macrophages to the parasite inoculation site and by accelerating the entry of promastigotes into macrophages. Saliva may also enhance lesion development by shortening the generation time of dividing intracellular amastigotes.     

First Evidence of Intraclonal Genetic Exchange in Trypanosomatids Using Two Leishmania infantum Fluorescent Transgenic Clones

Background

The mode of reproduction in Leishmania spp has been argued to be essentially clonal. However, recent data (genetic analysis of populations and co-infections in sand flies) have proposed the existence of a non-obligate sexual cycle in the extracellular stage of the parasite within the sand fly vector. In this article we propose the existence of intraclonal genetic exchange in the natural vector of Leishmania infantum.

Methodology/Principal findings

We have developed transgenic L. infantum lines expressing drug resistance markers linked to green and red fluorescent reporters. We hypothesized whether those cells with identical genotype can recognize each other and mate. Both types of markers were successfully exchanged within the sand fly midgut of the natural vector Phlebotomus perniciosus when individuals from these species were fed with a mixture of parental clones. Using the yellow phenotype and drug resistance markers, we provide evidence for genetic exchange in L. infantum. The hybrid progeny appeared to be triploid based on DNA content analysis. The hybrid clone analyzed was stable throughout the complete parasite life cycle. The progress of infections by the hybrid clone in BALB/c mice caused a reduction in parasite loads in both spleen and liver, and provided weight values similar to those obtained with uninfected mice. Spleen arginase activity was also significantly reduced relative to parental strains.

Conclusions/Significance

A L. infantum hybrid lineage was obtained from intraclonal genetic exchange within the midgut of the natural vector, suggesting the ability of this parasite to recognize the same genotype and mate. The yellow hybrid progeny is stable throughout the whole parasite life cycle but with a slower virulence, which correlates well with the lower arginase activity detected both in vitro and in vivo infections.     

Leishmania tropica: intraspecific polymorphisms in lipophosphoglycan correlate with transmission by different Phlebotomus species

Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes which has been shown to be critical for parasite-sand fly vector interactions. To provide additional evidence for its importance in transmission, the LPGs from three Leishmania tropica strains that differ in their capability to infect sand flies, were biochemically characterized. One of these strains, ISER/IL/98/LRC-L747, was isolated from a Phlebotomus sergenti female collected in the Judean Desert close to Jerusalem. The other strains originated from a different focus in the Galilee region of northern Israel. One was isolated from a patient (MHOM/IL/02/Ofri-LRC-L863) and the other from a naturally infected Phlebotomus arabicus female (IARA/IL/00/Amnunfly1-LRC-L810). The LPG structures of the isolates from the Galilee (L863 and L810) were similar to each other, but differed in the LPG repeat units from the Judean Desert isolate (L747). The terminal sugar in the side chains of the repeat units of LPG purified from L863 and L810 was beta-galactose and was not capped with glucose, unlike L747 and a previously characterized L. tropica strain from Iraq (L36). Since L810 was isolated from P. arabicus and L747 from P. sergenti, variations in the structure of their LPGs may explain their capacity to infect different sand fly species.