Expression plasticity of Phlebotomus papatasi salivary gland genes in distinct ecotopes through the sand fly season

<p>Abstract</p> <p>Background</p> <p>Sand fly saliva can drive the outcome of <it>Leishmania </it>infection in animal models, and salivary components have been postulated as vaccine candidates against leishmaniasis. In the sand fly <it>Phlebotomus papatasi</it>, natural sugar-sources modulate the activity of proteins involved in meal digestion, and possibly influence vectorial capacity. However, only a handful of studies have assessed the variability of salivary components in sand flies, focusing on the effects of environmental factors in natural habitats. In order to better understand such interactions, we compared the expression profiles of nine <it>P. papatasi </it>salivary gland genes of specimens inhabiting different ecological habitats in Egypt and Jordan and throughout the sand fly season in each habitat.</p> <p>Results</p> <p>The majority of investigated genes were up-regulated in specimens from Swaymeh late in the season, when the availability of sugar sources is reduced due to water deprivation. On the other hand, these genes were not up-regulated in specimens collected from Aswan, an irrigated area less susceptible to drought effects.</p> <p>Conclusion</p> <p>Expression plasticity of genes involved with vectorial capacity in disease vectors may play an important epidemiological role in the establishment of diseases in natural habitats.</p

Profiling of human acquired immunity against the salivary proteins of Phlebotomus papatasi reveals clusters of differential immunoreactivity

Citation: Geraci, Nicholas S., Rami M. Mukbel, Michael T. Kemp, Mariha N. Wadsworth, Emil Lesho, Gwen M. Stayback, Matthew M. Champion, et al. 2014. “Profiling of Human Acquired Immunity Against the Salivary Proteins of Phlebotomus Papatasi Reveals Clusters of Differential Immunoreactivity.” The American Journal of Tropical Medicine and Hygiene 90 (5): 923–38. https://doi.org/10.4269/ajtmh.13-0130.Phlebotomus papatasi sand flies are among the primary vectors of Leishmania major parasites from Morocco to the Indian subcontinent and from southern Europe to central and eastern Africa. Antibody-based immunity to sand fly salivary gland proteins in human populations remains a complex contextual problem that is not yet fully understood. We profiled the immunoreactivities of plasma antibodies to sand fly salivary gland sonicates (SGSs) from 229 human blood donors residing in different regions of sand fly endemicity throughout Jordan and Egypt as well as 69 US military personnel, who were differentially exposed to P. papatasi bites and L. major infections in Iraq. Compared with plasma from control region donors, antibodies were significantly immunoreactive to five salivary proteins (12, 26, 30, 38, and 44 kDa) among Jordanian and Egyptian donors, with immunoglobulin G4 being the dominant anti-SGS isotype. US personnel were significantly immunoreactive to only two salivary proteins (38 and 14 kDa). Using k-means clustering, donors were segregated into four clusters distinguished by unique immunoreactivity profiles to varying combinations of the significantly immunogenic salivary proteins. SGS-induced cellular proliferation was diminished among donors residing in sand fly-endemic regions. These data provide a clearer picture of human immune responses to sand fly vector salivary constituents

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

Identification of point mutations previously identified in MIL-resistant <i>L</i>. <i>donovani</i> (T421N, L856P, W210*, M1) and <i>L</i>. <i>major</i> (G582D, M547del).

<p>Previously identified mutations were sequenced and are indicated with an asterisk (*) and highlighted in bold font, using <i>L</i>. <i>major</i> FVI wild-type as the reference strain. No mutations were detected in any of the resistant lines.</p><p>Identification of point mutations previously identified in MIL-resistant <i>L</i>. <i>donovani</i> (T421N, L856P, W210*, M1) and <i>L</i>. <i>major</i> (G582D, M547del).</p

Growth curves of <i>L</i>. <i>major</i> WT and MIL-resistant promastigotes growing in the presence of 30μM MIL or absence of MIL selection.

<p>Log-phase promastigotes cultures were counted daily until they reached stationary phase. Concentration was determined microscopically by counting in a Neubauer chamber. Results are the average of triplicate experiments ± SD.</p

Metacyclogenesis in WT and MIL-resistant <i>L</i>. <i>major</i>.

<p><i>L</i>. <i>major</i> promastigotes resistant to MIL exhibit increased metacyclogenesis as determined by qRT-PCR of SHERP expression relative to housekeeping gene GAPDH and normalized to WT expression levels (left). 5-day stationary parasites were subjected to peanut agglutination and Ficoll-400 gradients and percentage of metacyclics is shown (right). Results are the average of triplicate experiments ± SD. Statistical differences determined with a Student’s <i>t</i> test relative to control values (* <i>p</i><0.05)</p

Host cell infection assay.

<p>Early stages of macrophage invasion are similar between <i>L</i>. <i>major</i> WT and R40, as determined by infection of RAW264.7 murine macrophages. Metacyclic parasites were incubated in the presence of macrophages at a MOI of 10 metacyclic parasites per macrophage and cells were collected at 6h, 12h, 24h, and 48h. Samples were stained and infection was determined through light microscopy. <b>(A)</b> The percentage of infected macrophages, and <b>(B)</b> the number of parasites/100 cells were recorded. Results are the average of triplicate experiments ± SD. Statistical differences determined with a Students <i>t</i> test relative to control values (* <i>p</i><0.05; ** <i>p</i><0.01)</p

Susceptibility of MIL-resistant <i>L</i>. <i>major</i> FVI populations generated by step-wise selection and determined by EC₅₀ analysis.

<p>1×10⁶ Log-phase parasites were incubated in the presence of a range of drug concentrations for 48 hours at 27°C, and the surviving cells were quantified with Cell Titer Blue proliferation assay using a Typhoon FLA-9500 laser scanner. Populations of parasites were grown in increasing concentrations of MIL ranging from 10 μM (R10) to 40 μM (R40), showing increased resistance to MIL. Horizontal dashed line represents WT threshold for MIL resistance. “Rno” are resistant lines grown in the absence of MIL for at least 75 passages. Results are the average of triplicate experiments ± SD.</p