In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence.

PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line, Δaslrim1, by embryonic genome editing using CRISPR-Cas9. Δaslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of Δaslrim1 mosquitoes and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when Δaslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1's regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males.

Whole genome sequencing of experimental hybrids supports meiosis-like sexual recombination in Leishmania.

Hybrid genotypes have been repeatedly described among natural isolates of Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L. major and L. infantum. We also generated and sequenced the first experimental hybrids in L. tropica. We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97-99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L. major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L. tropica, revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania. Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania, presenting clear opportunities for forward genetic analysis and positional cloning of important genes.

Divergent roles for Ly6C+CCR2+CX3CR1+ inflammatory monocytes during primary or secondary infection of the skin with the intra-phagosomal pathogen Leishmania major.

Inflammatory monocytes can be manipulated by environmental cues to perform multiple functions. To define the role of monocytes during primary or secondary infection with an intra-phagosomal pathogen we employed Leishmania major-red fluorescent protein (RFP) parasites and multi-color flow cytometry to define and enumerate infected and uninfected inflammatory cells in the skin. During primary infection, infected monocytes had altered maturation and were the initial mononuclear host cell for parasite replication. In contrast, at a distal site of secondary infection in mice with a healed but persistent primary infection, this same population rapidly produced inducible nitric oxide synthase (iNOS) in an IFN-γ dependent manner and was critical for parasite killing. Maturation to a dendritic cell-like phenotype was not required for monocyte iNOS-production, and enhanced monocyte recruitment correlated with IFN-γ dependent cxcl10 expression. In contrast, neutrophils appeared to be a safe haven for parasites in both primary and secondary sites. Thus, inflammatory monocytes play divergent roles during primary versus secondary infection with an intra-phagosomal pathogen.

The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major.

For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo.

Increased Transmissibility of Leishmania donovani From the Mammalian Host to Vector Sand Flies After Multiple Exposures to Sand Fly Bites.

Background: Patients with active visceral leishmaniasis are important reservoirs in the anthroponotic transmission cycle of Leishmania donovani. The role of the blood or skin as a source of infection to sand flies remains unclear, and the possible effect of multiple exposures to fly bites on transmissibility has not been addressed. Methods: L. donovani-infected hamsters underwent xenodiagnoses with Lutzomyia longipalpis on the same or different sites on the abdomen on 2 consecutive days or by artificial feeding on the skin or blood. Results: The transmission of L. donovani from sick hamsters to flies was surprisingly low (mean, 24% of fed flies). New flies fed on the same site acquired significantly more infections (mean, 61%; P < .0001). By artificial feeding, flies could acquire infection from blood and skin. However, only artificial feeding on blood produced infections that correlated with the natural feeding (R = 0.792; P < .0001). Infections acquired from blood increased dramatically for blood obtained after exposure to bites, as did the parasitemia level and the number of monocytes in the circulation. Conclusions: The bites of uninfected sand flies favor the transmissibility of L. donovani by infected hosts, owing to a systemic effect that exposure to bites has on the parasitemia. Patients with active visceral leishmaniasis are important reservoirs in the anthroponotic transmission cycle of Leishmania donovani. Using the hamster model of visceral disease, we demonstrate that prior exposure to bites of uninfected sand flies potentiates their ability to transmit infection to the vector.

Cross-species genetic exchange between visceral and cutaneous strains of Leishmania in the sand fly vector.

Genetic exchange between Leishmania major strains during their development in the sand fly vector has been experimentally shown. To investigate the possibility of genetic exchange between different Leishmania species, a cutaneous strain of L. major and a visceral strain of Leishmania infantum, each bearing a different drug-resistant marker, were used to coinfect Lutzomyia longipalpis sand flies. Eleven double-drug-resistant progeny clones, each the product of an independent mating event, were generated and submitted to genotype and phenotype analyses. The analysis of multiple allelic markers across the genome suggested that each progeny clone inherited at least one full set of chromosomes from each parent, with loss of heterozygosity at some loci, and uniparental retention of maxicircle kinetoplast DNA. Hybrids with DNA contents of approximately 2n, 3n, and 4n were observed. In vivo studies revealed clear differences in the ability of the hybrids to produce pathology in the skin or to disseminate to and grow in the viscera, suggesting polymorphisms and differential inheritance of the gene(s) controlling these traits. The studies, to our knowledge, represent the first experimental confirmation of cross-species mating in Leishmania, opening the way toward genetic linkage analysis of important traits and providing strong evidence that genetic exchange is responsible for the generation of the mixed-species genotypes observed in natural populations.

The mating competence of geographically diverse Leishmania major strains in their natural and unnatural sand fly vectors.

Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4-6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed '2n' DNA contents, but with a significant number of '3n' and one '4n' offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable 'mating types' that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating.

Trypanosoma brucei eflornithine transporter AAT6 is a low-affinity low-selective transporter for neutral amino acids.

Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the up-take of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter.

A versatile proline/alanine transporter in the unicellular pathogen Leishmania donovani regulates amino acid homoeostasis and osmotic stress responses.

Unlike all other organisms, parasitic protozoa of the family Trypanosomatidae maintain a large cellular pool of proline that, together with the alanine pool, serve as alternative carbon sources as well as reservoirs of organic osmolytes. These reflect adaptation to their insect vectors whose haemolymphs are exceptionally rich in the two amino acids. In the present study we identify and characterize a new neutral amino acid transporter, LdAAP24, that translocates proline and alanine across the Leishmania donovani plasma membrane. This transporter fulfils multiple functions: it is the sole supplier for the intracellular pool of proline and contributes to the alanine pool; it is essential for cell volume regulation after osmotic stress; and it regulates the transport and homoeostasis of glutamate and arginine, none of which are its substrates. Notably, we provide evidence that proline and alanine exhibit different roles in the parasitic response to hypotonic shock; alanine affects swelling, whereas proline influences the rate of volume recovery. On the basis of our data we suggest that LdAAP24 plays a key role in parasite adaptation to its varying environments in host and vector, a phenomenon essential for successful parasitism.

Lysine transporters in human trypanosomatid pathogens.

In previous studies we characterized arginine transporter genes from Trypanosoma cruzi and Leishmania donovani, the etiological agents of chagas disease and kala azar, respectively, both fatal diseases in humans. Unlike arginine transporters in higher eukaryotes that transport also lysine, these parasite transporters translocate only arginine. This phenomenon prompted us to identify and characterize parasite lysine transporters. Here we demonstrate that LdAAP7 and TcAAP7 encode lysine-specific permeases in L. donovani and T. cruzi, respectively. These two lysine permeases are both members of the large amino acid/auxin permease family and share certain biochemical properties, such as specificity and Km. However, we evidence that LdAAP7 and TcAAP7 differ in their regulation and localization, such differences are likely a reflection of the dissimilar L. donovani and T. cruzi life cycles. Failed attempts to delete both alleles of LdAAP7 support the premise that this is an essential gene that encodes the only lysine permeases expressed in L. donovani promastigotes and T. cruzi epimastigotes, respectively.

Self-Hybridization in Leishmania major.

Genetic exchange between different Leishmania strains in the sand fly vector has been experimentally demonstrated and is supported by population genetic studies. In nature, opportunities for Leishmania interstrain mating are restricted to flies biting multiply infected hosts or through multiple bites of different hosts. In contrast, self-mating could occur in any infected sand fly. By crossing two recombinant lines derived from the same Leishmania major strain, each expressing a different drug-resistance marker, self-hybridization in L. major was confirmed in a natural sand fly vector, Phlebotomus duboscqi, and in frequencies comparable to interstrain crosses. We provide the first high resolution, whole-genome sequencing analysis of large numbers of selfing progeny, their parents, and parental subclones. Genetic exchange consistent with classical meiosis is supported by the biallelic inheritance of the rare homozygous single nucleotide polymorphisms (SNPs) that arose by mutation during the generation of the parental clones. In contrast, heterozygous SNPs largely failed to be transmitted in Mendelian ratios for reasons not understood. SNPs that were heterozygous in both parents, however, recombined to produce homozygous alleles in some hybrids. For trisomic chromosomes present in both parents, transmittal to the progeny was only altered by self-hybridization, involving a gain or loss of somy in frequencies predicted by a meiotic process. Whole-genome polyploidization was also observed in the selfing progeny. Thus, self-hybridization in Leishmania, with its potential to occur in any infected sand fly, may be an important source of karyotype variation, loss of heterozygosity, and functional diversity. IMPORTANCE Leishmania are parasitic protozoa that cause a wide spectrum of diseases collectively known as the leishmaniases. Sexual reproduction in Leishmania has been proposed as an important source of genetic diversity and has been formally demonstrated to occur inside the sand fly vector midgut. Nevertheless, in the wild, opportunities for genetic exchange between different Leishmania species or strains are restricted by the capacity of different Leishmania strains to colonize the same sand fly. In this work, we report the first high resolution, whole-genome sequence analysis of intraclonal genetic exchange as a type of self-mating in Leishmania. Our data reveal that self-hybridization can occur with comparable frequency as interstrain mating under experimental lab conditions, leading to important genomic alterations that can potentially take place within every naturally infected sand fly.

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis.

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major.

Sand flies are the natural vectors for the Leishmania species that produce a spectrum of diseases in their mammalian hosts, including humans. Studies of sand fly/Leishmania interactions have been limited by the absence of genome editing techniques applicable to these insects. In this report, we adapted CRISPR (clustered regularly interspaced palindromic repeat)/Cas9 (CRISPR-associated protein 9) technology to the Phlebotomus papatasi sand fly, a natural vector for Leishmania major, targeting the sand fly immune deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in P. papatasi and were able to generate transmissible null mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. While the maintenance of a homozygous mutant stock was severely compromised, we were able to establish in an early generation their greater susceptibility to infection with L. major Flies carrying different heterozygous mutant alleles variably displayed a more permissive phenotype, presenting higher loads of parasites or greater numbers of infective-stage promastigotes. Together, our data show (i) the successful adaptation of the CRISPR/Cas9 technology to sand flies and (ii) the impact of the sand fly immune response on vector competence for Leishmania parasites.IMPORTANCE Sand flies are the natural vectors of Leishmania parasites. Studies of sand fly/Leishmania interactions have been limited by the lack of successful genomic manipulation of these insects. This paper shows the first example of successful targeted mutagenesis in sand flies via adaptation of the CRISPR/Cas9 editing technique. We generated transmissible null mutant alleles of relish, a gene known to be essential for the control of immune response in other insects. In addition to the expected higher level of susceptibility to bacteria, the mutant flies presented higher loads of parasites when infected with L. major, showing that the sand fly immune response impacts its vector competence for this pathogen.

The Transcriptome of Leishmania major Developmental Stages in Their Natural Sand Fly Vector.

The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress.IMPORTANCE The life cycle of Leishmania parasites in the sand fly vector includes their growth and development as morphologically distinct forms of extracellular promastigotes found within the different microenvironments of the gut. Based on RNA-Seq, we provide here the first high-resolution, transcriptomic analysis of Leishmania insect stages during their cyclical development in vivo, from tissue amastigotes ingested with the blood meal to infective, metacyclic promastigotes that initiate infection in the mammalian host. The most extensive genetic reprogramming occurred during the early transformation of amastigotes to rapidly dividing procyclic promastigotes in the blood-fed midgut, with major changes in the abundance of mRNAs for surface proteins and metabolism. The post-blood meal-adapted nectomonad stage was characterized by the downregulation of cell cycle-related genes and the upregulation of stress- and starvation-related genes. Finally, the transcriptome of metacyclic promastigotes shifted to a more amastigote-like profile, suggesting their preadaptation to the intracellular host environment.

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei.

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 µM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-ß-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 µM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

The Potential Use of Forensic DNA Methods Applied to Sand Fly Blood Meal Analysis to Identify the Infection Reservoirs of Anthroponotic Visceral Leishmaniasis.

BACKGROUND: In the Indian sub-continent, visceral leishmaniasis (VL), also known as kala azar, is a fatal form of leishmaniasis caused by the kinetoplastid parasite Leishmania donovani and transmitted by the sand fly Phlebotomus argentipes. VL is prevalent in northeast India where it is believed to have an exclusive anthroponotic transmission cycle. There are four distinct cohorts of L. donovani exposed individuals who can potentially serve as infection reservoirs: patients with active disease, cured VL cases, patients with post kala azar dermal leishmaniasis (PKDL), and asymptomatic individuals. The relative contribution of each group to sustaining the transmission cycle of VL is not known. METHODOLOGY/PRINCIPAL FINDINGS: To answer this critical epidemiological question, we have addressed the feasibility of an approach that would use forensic DNA methods to recover human DNA profiles from the blood meals of infected sand flies that would then be matched to reference DNA sampled from individuals living or working in the vicinity of the sand fly collections. We found that the ability to obtain readable human DNA fingerprints from sand flies depended entirely on the size of the blood meal and the kinetics of its digestion. Useable profiles were obtained from most flies within the first 24 hours post blood meal (PBM), with a sharp decline at 48 hours and no readable profiles at 72 hours. This early time frame necessitated development of a sensitive, nested-PCR method compatible with detecting L. donovani within a fresh, 24 hours blood meal in flies fed on infected hamsters. CONCLUSION/SIGNIFICANCE: Our findings establish the feasibility of the forensic DNA method to directly trace the human source of an infected blood meal, with constraints imposed by the requirement that the flies be recovered for analysis within 24 hours of their infective feed.

Size does matter: 18 amino acids at the N-terminal tip of an amino acid transporter in Leishmania determine substrate specificity.

Long N-terminal tails of amino acid transporters are known to act as sensors of the internal pool of amino acids and as positive regulators of substrate flux rate. In this study we establish that N-termini of amino acid transporters can also determine substrate specificity. We show that due to alternative trans splicing, the human pathogen Leishmania naturally expresses two variants of the proline/alanine transporter, one 18 amino acid shorter than the other. We demonstrate that the longer variant (LdAAP24) translocates both proline and alanine, whereas the shorter variant (∆18LdAAP24) translocates just proline. Remarkably, co-expressing the hydrophilic N-terminal peptide of the long variant with ∆18LdAAP24 was found to recover alanine transport. This restoration of alanine transport could be mediated by a truncated N-terminal tail, though truncations exceeding half of the tail length were no longer functional. Taken together, the data indicate that the first 18 amino acids of the negatively charged N-terminal LdAAP24 tail are required for alanine transport and may facilitate the electrostatic interactions of the entire negatively charged N-terminal tail with the positively charged internal loops in the transmembrane domain, as this mechanism has been shown to underlie regulation of substrate flux rate for other transporters.

Succession of bacterial communities during early plant development: transition from seed to root and effect of compost amendment.

Compost amendments to soils and potting mixes are routinely applied to improve soil fertility and plant growth and health. These amendments, which contain high levels of organic matter and microbial cells, can influence microbial communities associated with plants grown in such soils. The purpose of this study was to follow the bacterial community compositions of seed and subsequent root surfaces in the presence and absence of compost in the potting mix. The bacterial community compositions of potting mixes, seed, and root surfaces sampled at three stages of plant growth were analyzed via general and newly developed Bacteroidetes-specific, PCR-denaturing gradient gel electrophoresis methodologies. These analyses revealed that seed surfaces were colonized primarily by populations detected in the initial potting mixes, many of which were not detected in subsequent root analyses. The most persistent bacterial populations detected in this study belonged to the genus Chryseobacterium (Bacteroidetes) and the family Oxalobacteraceae (Betaproteobacteria). The patterns of colonization by populations within these taxa differed significantly and may reflect differences in the physiology of these organisms. Overall, analyses of bacterial community composition revealed a surprising prevalence and diversity of Bacteroidetes in all treatments.

Competing factors of compost concentration and proximity to root affect the distribution of streptomycetes.

Streptomycetes are important members of soil microbial communities and are particularly active in the degradation of recalcitrant macromolecules and have been implicated in biological control of plant disease. Using a streptomycetes-specific polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (PCR-DGGE) methodology coupled with band excision and sequence analysis, we examined the effect of grape marc compost amendment to soil on cucumber plant-associated streptomycetes community composition. We observed that both compost amendment and proximity to the root surface influenced the streptomycetes community composition. A strong root selection for a soil-derived Streptomycete, most closely related to Streptomyces thermotolerans, S. iakyrus, and S. thermocarboxydus, was independent of compost amendment rate. However, while the impact of compost amendment was mitigated with increasing proximity to the root, high levels of compost amendment resulted in the detection of compost-derived species on the root surface. Conversely, in rhizosphere and non-rhizosphere soils, the community composition of streptomycetes was affected strongly even by modest compost amendment. The application of a streptomycetes-specific PCR primer set combined with DGGE analysis provided a rapid means of examining the distribution and ecology of streptomycetes in soils and plant-associated environments.