Targeting the Midgut Secreted PpChit1 Reduces Leishmania major Development in Its Natural Vector, the Sand Fly Phlebotomus papatasi

For a successful development within the midgut of the sand fly vector, Leishmania must overcome several barriers which are imposed by the vector. The ability to overcome these barriers has been associated with species specificity, and interference with the sand fly vector-parasite balance can change the outcome of the infection in the vector. Recently, our group has carried out a transcriptome assessment of the sand fly Phlebotomus papatasi midgut, uncovering many transcripts possibly associated with the barrier to Leishmania development. In order to validate the role of such genes, we have developed a dedicated RNA interference (RNAi) platform to assess whether RNAi targeting such genes can reduce Leishmania major development. PpChit1 is a midgut-specific chitinase presumably involved in the maturation/degradation of the peritrophic matrix in the gut of the sand fly after a blood meal. Our results show that knockdown of PpChit1 via RNAi led to a significant reduction of Le. major within the gut, supporting the potential use of PpChit1 as a target for transmission blocking strategies against sand fly-transmitted leishmaniasis

Effect of anti-PpChit1 on sand fly fitness and transmission of Leishmania infantum in American foxhounds by sand fly bite

Master of ScienceDepartment of EntomologyMarcelo Ramalho-OrtigaoSand flies (Diptera:Psychodidae) are vectors of parasites if the genus Leishmania, the causative agent of leishmaniasis, a neglected tropical disease in several countries around the world. Sand flies transmit Leishmania to suitable vertebrates during the blood meal and following a complex development parasites undergo within the fly. Many aspects of the Leishmania development within the sand fly vector are well known, however details about how sand fly molecules affect the parasite are still not yet known. Our group previously identified that RNAi knockdown of PpChit1, a midgut specific chitinase from the sand fly Phlebotomus papatasi, led to a significant decrease in the load of Le. major. In this study, we assessed potential fitness effects of antisera anti-PpChit1 on three laboratory-reared sand fly species (P. papatasi, Phlebotomus duboscqi, and Lutzomyia longipalpis). Our results suggest that feeding sand flies with anti-PpChit1 sera led to a one day delay in the onset of oviposition, and also suggested that anti-PpChit1-fed flies survived on average up to three days longer that control flies. Analyses of the peritrophic matrix (PM) indicated a significant increase in thickness 72 hours post anti-PpChit1 feeding compared to control sera. Altogether the results suggest that feeding sand flies with anti-PpChit1 likely affects the kinetics of sand PM, which in turn affects the flow of nutrients and certain aspects of sand fly fitness. In the course of this study, we also evaluated the ability of American Foxhounds naturally infected with Leishmania infantum to transmit these parasites via bites of phlebotomine sand flies to suitable vertebrates. Since 1999, an outbreak of canine visceral leishmaniasis (CVL) has been reported in the U.S especially among Foxhounds. The ability of sand flies to pick up and transmit this pathogen represents an important health risk for companion dogs and humans. Our results indicate that Foxhounds naturally infected with Le. infantum are highly infectious to sand flies and that the parasites are able to fully develop within these vectors and de successfully transmitted during blood feeding. Thus, the risk exists for these parasites to become endemic in North America where sand flies are also known to occur

Significance of bacteria in oviposition and larval development of the sand fly Lutzomyia longipalpis

Background : Microbial ecology of phlebotomine sand flies is not well understood although bacteria likely play an important role in the sand fly biology and vector capacity for Leishmania parasites. In this study, we assessed the significance of the microbial community of rabbit feces in oviposition and larval development of Lutzomyia longipalpis as well as bacterial colonization of the gut of freshly emerged flies. Methods : Sterile (by autoclaving) and non-sterile (control) rabbit feces were used in the two-choice assay to determine their oviposition attractiveness to sand fly females. Bacteria were identified by amplification and sequencing of the 16S rRNA gene with universal eubacterial primers. Sterile, control (non-sterile), and sterilized and inoculated rabbit feces were used to assess the significance of bacteria in L. longipalpis development. Newly emerged adult flies were surface-sterilized and screened for the bacterial population size and diversity by the culturing approach. The digestive tract of L4 sterile and control larvae was incubated with Phalloidin to visualize muscle tissues and DAPI to visualize nuclei. Results : Two-choice behavioural assays revealed a great preference of L. longipalpis to lay eggs on rabbit feces with an active complex bacterial community (control) (85.8 % of eggs) in comparison to that of sterile (autoclaved) rabbit feces (14.2 %). Bioassays demonstrated that L. longipalpis larvae can develop in sterile rabbit feces although development time to adult stage was greatly extended (47 days) and survival of larvae was significantly lower (77.8 %) compared to that of larvae developing in the control rabbit feces (32 days and 91.7 %). Larval survival on sterilized rabbit feces inoculated with the individual bacterial isolates originating from this substrate varied greatly depending on a bacterial strain. Rhizobium radiobacter supported larval development to adult stage into the greatest extent (39 days, 88.0 %) in contrast to that of Bacillus spp. (76 days, 36.0 %). From the complex natural bacterial community of rabbit feces, R. radiobacter survived pupation and colonized the newly emerged females most successfully (82.6 % of all bacteria cultured); however, only 25 % of females were positive for bacteria in the digestive tract upon emergence. Immunohistochemistry did not reveal any obvious differences in anatomy of the digestive tract between control and axenic larvae. Conclusions : The bacterial community in the sand fly larval habitat affects oviposition and larval development although bacteria are not essential for successful development of L. longipalpis. Different bacteria contribute to larval development to various degrees and some, e.g. Rhizobium radiobacter, survive pupation and colonize the digestive tract of newly emerged females. With the establishment of the axenic rearing system, this study opens new venues to study the effect of bacteria on the gut epithelial immunity and vector competence of sand flies for Leishmania parasites with a goal to develop paratransgenic approaches for Leishmania control

Effect of mouse antisera targeting the Phlebotomus papatasi midgut chitinase PpChit1 on sandfly physiology and fitness

In sandflies, the absence of the peritrophic matrix (PM) affects the rate of blood digestion. Also, the kinetics of PM secretion varies according to species. We previously characterised PpChit1, a midgut-specific chitinase secreted in Phlebotomus papatasi (PPIS) that is involved in the maturation of the PM and showed that antibodies against PpChit1 reduce the chitinolytic activity in the midgut of several sandfly species. Here, sandflies were fed on red blood cells reconstituted with naïve or anti-PpChit1 sera and assessed for fitness parameters that included blood digestion, oviposition onset, number of eggs laid, egg bouts, average number of eggs per bout and survival. In PPIS, anti-PpChit1 led to a one-day delay in the onset of egg laying, with flies surviving three days longer compared to the control group. Anti-PpChit1 also had a negative effect on overall ability of flies to lay eggs, as several gravid females from all three species were unable to lay any eggs despite having lived longer than control flies. Whereas the longer survival might be associated with improved haeme scavenging ability by the PM, the inability of females to lay eggs is possibly linked to changes in PM permeability affecting nutrient absorption

Targeted Disruption of the Inhibitor of DNA Binding 4 (Id4) Gene Alters Photic Entrainment of the Circadian Clock

Inhibitor of DNA binding (Id) genes comprise a family of four helix–loop–helix (HLH) transcriptional inhibitors. Our earlier studies revealed a role for ID2 within the circadian system, contributing to input, output, and core clock function through its interaction with CLOCK and BMAL1. Here, we explore the contribution of ID4 to the circadian system using a targeted disruption of the Id4 gene. Attributes of the circadian clock were assessed by monitoring the locomotor activity of Id4−/− mice, and they revealed disturbances in its operation. Id4-mutant mice expressed a shorter circadian period length, attenuated phase shifts in responses to continuous and discrete photic cues, and an advanced phase angle of entrainment under a 12:12 light:dark cycle and under short and long photoperiods. To understand the basis for these properties, suprachiasmatic nucleus (SCN) and retinal structures were examined. Anatomical analysis reveals a smaller Id4−/− SCN in the width dimension, which is a finding consistent with its smaller brain. As a result of this feature, anterograde tracing in Id4−/− mice revealed retinal afferents innovate a disproportionally larger SCN area. The Id4−/− photic entrainment responses are unlikely to be due to an impaired function of the retinal pathways since Id4−/− retinal anatomy and function tested by pupillometry were similar to wild-type mice. Furthermore, these circadian characteristics are opposite to those exhibited by the Id2−/− mouse, suggesting an opposing influence of the ID4 protein within the circadian system; or, the absence of ID4 results in changes in the expression or activity of other members of the Id gene family. Expression analysis of the Id genes within the Id4−/− SCN revealed a time-of-day specific elevated Id1. It is plausible that the increased Id1 and/or absence of ID4 result in changes in interactions with bHLH canonical clock components or with targets upstream and/or downstream of the clock, thereby resulting in abnormal properties of the circadian clock and its entrainment

Fitness and phenotypic characterization of miltefosine-resistant Leishmania major

Trypanosomatid parasites of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease with several clinical manifestations. Leishmania major is the causative agent of cutaneous leishmaniasis (CL), which is largely characterized by ulcerative lesions appearing on the skin. Current treatments of leishmaniasis include pentavalent antimonials and amphotericin B, however, the toxic side effects of these drugs and difficulty with distribution makes these options less than ideal. Miltefosine (MIL) is the first oral treatment available for leishmaniasis. Originally developed for cancer chemotherapy, the mechanism of action of MIL in Leishmania spp. is largely unknown. While treatment with MIL has proven effective, higher tolerance to the drug has been observed, and resistance is easily developed in an in vitro environment. Utilizing stepwise selection we generated MIL-resistant cultures of L. major and characterized the fitness of MIL-resistant L. major. Resistant parasites proliferate at a comparable rate to the wild-type (WT) and exhibit similar apoptotic responses. As expected, MIL-resistant parasites demonstrate decreased susceptibility to MIL, which reduces after the drug is withdrawn from culture. Our data demonstrate metacyclogenesis is elevated in MIL-resistant L. major, albeit these parasites display attenuated in vitro and in vivo virulence and standard survival rates in the natural sandfly vector, indicating that development of experimental resistance to miltefosine does not lead to an increased competitive fitness in L. major.Fil: Turner, Kimbra G.. University of Notre Dame-Indiana; Estados UnidosFil: Vacchina, Paola. University of Notre Dame-Indiana; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Robles Murguia, Maricela. University of Notre Dame-Indiana; Estados UnidosFil: Wadsworth, Mariha. University of Notre Dame-Indiana; Estados UnidosFil: McDowell, Mary Ann. University of Notre Dame-Indiana; Estados UnidosFil: Morales, Miguel A.. University of Notre Dame-Indiana; Estados Unido

Modulation of peritrophin mRNA expression upon <i>Le. major</i> infection.

<p>qRT-PCR assays depicting differences in PpPer1 (<b>A</b>), PpPer2 (<b>B</b>), and PpPer3 (<b>C</b>) mRNA levels between non-infected and <i>Le. major</i> infected midguts dissected at 24 h, 48 h, and 72 h PBM. Each dot (symbol) represents the mRNA expression levels in a single midgut whereas horizontal bars indicate mean expression levels. The cDNA encoding the S3 protein of the 40S ribosomal subunit was used as the housekeeping control gene. The mean expression of non-infected midguts was used as a standard (100%) for comparisons to the percentage of mRNA expression of <i>Le. major</i> infected midguts for each time point. NI: Non-infected. INF: <i>Le. major</i> infected. NS: Not significant. *: Statistically significant, p<0.05.</p

Effect of dsRNA injection on <i>Le. major</i> infection level in <i>P. papatasi</i>.

<p>Parasite load was categorized according to the number of <i>Le. major</i> per midgut. (A) Percentage of sand flies injected with either dsCtr or dsChit1 exhibiting no infection (0 parasites), as well as light (1–1,000 parasites), moderate (1,000–10,000 parasites), or heavy (>10,000 parasites) infection at 48 h PBM. Differences are statistically significant (Chi-square, p = 0.01). (B) Percentage of sand flies injected with either dsCtr or dsChit1 exhibiting either no parasites or light infection (0–1000 parasites), or moderate infection (>1,000 parasites) at 120 h PBM. Differences are statistically significant (Fisher's exact test, p = 0.04). n: Number of flies dissected.</p

Effects of PpPer1 knockdown on <i>Le. major</i> infection.

<p>Knockdown of PpPer1 leads to greater <i>Le. major</i> load in the midgut of <i>P. papatasi</i>. At 48 h post-infection (<b>A</b>), dsPpPer1 (dsPer1) injection caused an increased (39%; p<0.05 Mann-Whitney U test) in <i>Le. major</i> load compared to dsGFP-injected <i>P. papatasi</i>. Results shown are from combined data of two independent experiments. (<b>B</b>) Although not statistically significant, <i>PpPer1</i> knockdown led to 22% increase in <i>Le. major</i> load in <i>P. papatasi</i> midguts at 96 h post-infection when compared to dsGFP-injected. Each dot (filled circle or square) represents number of <i>Le. major</i> in a single midgut whereas horizontal bars indicate mean parasite number. <i>P. papatasi</i> were infected with 5×10⁶<i>Le. major</i> amastigotes/ml in heat-inactivated mouse blood. N: Number of <i>P. papatasi</i> midguts dissected. NS: Not significant. *: Statistically significant, p<0.05.</p