Role for the flagellum attachment zone in Leishmania anterior cell tip morphogenesis

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum. These are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenesis of the anterior cell tip. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division

Lipophosphoglycan Polymorphisms Do Not Affect \u3cem\u3eLeishmania amazonensis\u3c/em\u3e Development in the Permissive Vectors \u3cem\u3eLutzomyia migonei\u3c/em\u3e and \u3cem\u3eLutzomyia longipalpis\u3c/em\u3e

Background: Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmaniapromastigotes. Its species-specific polymorphisms are found mainly in the sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Leishmania amazonensis is one of the most important species causing human cutaneous leishmaniasis in the New World. Here, we describe LPG intraspecific polymorphisms in two Le. amazonensis reference strains and their role during the development in three sand fly species. Results: Strains isolated from Lutzomyia flaviscutellata (PH8) and from a human patient (Josefa) displayed structural polymorphism in the LPG repeat units, possessing side chains with 1 and 2 β-glucose or 1 to 3 β-galactose, respectively. Both strains successfully infected permissive vectors Lutzomyia longipalpis and Lutzomyia migonei and could colonize their stomodeal valve and differentiate into metacyclic forms. Despite bearing terminal galactose residues on LPG, Josefa could not sustain infection in the restrictive vector Phlebotomus papatasi. Conclusions: LPG polymorphisms did not affect the ability of Le. amazonensis to develop late-stage infections in permissive vectors. However, the non-establishment of infection in Ph. papatasi by Josefa strain suggested other LPG-independent factors in this restrictive vector

Leishmania flagellum attachment zone is critical for flagellar pocket shape, development in the sand fly and pathogenicity in the host

Leishmania kinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical for Leishmania pathogenicity. The Leishmania FP has a bulbous region posterior to the FP collar, and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host-parasite interactions. Deletion of the FAZ protein FAZ5 clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: mutants were unable to develop late stage infections in sand flies and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity

Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases

Abstract Background Leishmania development in sand flies is confined to the alimentary tract and is closely connected with blood meal digestion. Previously, it has been published that activities of sand fly midgut proteases are harmful to Leishmania, especially to amastigote-promastigote transition forms. However, our experiments with various Leishmania-sand fly pairs gave quite opposite results. Methods We evaluated the effect of semi-digested midgut content on different life stages of Leishmania donovani and Leishmania major in vitro. Various morphological forms of parasites, including macrophage-derived amastigotes and transition forms, were incubated 2 h with midguts dissected at various intervals (6–72 h) post-blood meal or with commercially available proteinase, and their viability was determined using flow cytometry. In parallel, using amastigote-initiated experimental infections, we compared development of L. donovani in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to this parasite. Results In vitro, sand fly midgut homogenates affected L. major and L. donovani in a similar way; in all sand fly species, the most significant mortality effect was observed by the end of the blood meal digestion process. Surprisingly, the most susceptible Leishmania stages were promastigotes, while mortality of transforming parasites and amastigotes was significantly lower. Parasites were also susceptible to killing by rabbit blood in combination with proteinase, but resistant to proteinase itself. In vivo, L. donovani developed late-stage infections in both natural vectors; in P. argentipes the development was much faster than in P. orientalis. On the other hand, in refractory species P. papatasi and S. schwetzi, promastigotes survived activity of digestive enzymes but were lost during defecation. Conclusions We demonstrated that Leishmania transition forms are more resistant to the killing effect of semi-digested blood meal than 24 h-old promastigotes. Data suggest that Leishmania mortality is not caused directly by sand fly proteases, we assume that this mortality results from toxic products of blood meal digestion. Survival of L. donovani promastigotes in refractory sand flies until blood meal defecation, together with similar mortality of Leishmania parasites incubated in vitro with midgut homogenates of susceptible as well as refractory species, contradict the previously raised hypotheses about the role of midgut proteases in sand fly vector competence to Leishmania

Additional file 1: Figure S1. of Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases

Gating strategy. a Gating on single GFP+ cells on GFP-A/GFP-H dotplot. b Gating on Leishmania cells on FSC-A/SSC-A dotplot. c Analysis of DAPI fluorescence on histogram – enumeration of % DAPI+ (i.e. dead) Leishmania cells. Figure S2. Different Leishmania donovani stages  were incubated with midguts of P. argentipes dissected at 24 h post-blood meal. Analysis of dead Leishmania was performed using flow cytometry. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. Figure S3. Different Leishmania donovani stages were incubated with midguts of P. orientalis dissected at 24 h post-blood meal. Analysis of dead Leishmania was performed using flow cytometry. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. Figure S4. Different Leishmania donovani stages were incubated with midguts of P. papatasi dissected at 24 h post-blood meal. Analysis of dead Leishmania was performed using flow cytometry. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. Figure S5. Different Leishmania donovani stages were incubated with midguts of S. schwetzi dissected at 24 h post-blood meal. Analysis of dead Leishmania was performed using flow cytometry. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. Figure S6. Negative control; different Leishmania donovani stages were incubated with saline. Analysis of dead Leishmania was performed using flow cytometry. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. Figure S7. Positive control; parasites killed by 1% formaldehyde and permeabilised by 0.5% Triton X-100. Percentage of dead cells was assessed on histogram of DAPI fluorescence on gated single GFP-positive Leishmania. (PPTX 346 kb

The effect of initial infective dose on the development of <i>L</i>. <i>donovani</i> in <i>P</i>. <i>argentipes</i>.

<p>Females of <i>P</i>. <i>argentipes</i> were infected by feeding on a suspension of 2×10³, 2×10⁴ or 5×10⁵promastigotes/ml of blood and kept at 26°C. <b>8A</b>: Females were examined microscopically 2 and 6 days post infection. Intensities of infection were classified into three categories according to their intensity: light (< 100 parasites/gut), moderate (100 − 1000 parasites/gut), or heavy (> 1000 parasites/gut). Numbers above the bars indicate the number of dissected females. <b>8B:</b> The number of parasites was measured individually by Q-PCR in 20 females of each group on day 8 PBM.</p

Defecation times of <i>P. argentipes</i>, <i>P. orientalis</i>, <i>P. papatasi</i> and <i>S</i>. <i>schwetzi</i>.

<p>Females were fed either on anesthetized BALB/c mice (<b>2A</b>) or on heat-inactivated rabbit blood via a chick-skin membrane (<b>2B</b>), individually placed in small glass vials and checked twice a day for defecation.</p

Chymotrypsin activity in <i>P</i>. <i>argentipes</i>, <i>P</i>. <i>orientalis</i>, <i>P</i>. <i>papatasi</i> and <i>S</i>. <i>schwetzi</i> midguts.

<p>Chymotrypsin activity was measured at 12, 24, 36, 48, 72, 80 and 96 hours PBM in midgut homogenates (c = 0.5 gut/ml) of bloodfed females using fluorogenic substrate Suc-Ala-Ala-Pro-Phe-AMC (40 μM). Data from two independent experiments were pooled. <b>7A:</b> Females fed on anesthetized BALB/c mice. <b>7B:</b> Females fed through a chick-skin membrane on heat-inactivated rabbit blood.</p