Fig 1.
L. mexicana FAZ7B localizes at the cell body side of the FAZ and is differentially expressed in promastigote and amastigote forms.
Immunofluorescence assay were performed as indicated in Materials and Methods. (A) Extracted cytoskeleton of a L. mexicana promastigote expressing FAZ7A-mNG (green) and FAZ7B-3xHA (red). (B-E) Whole promastigote cells of L. mexicana expressing FAZ7B-3xHA (red) and the indicated fusion proteins of FAZ components (green). (F-G) Whole promastigote (F) and axenic amastigote (G) cells from L. mexicana expressing FAZ7B-3xHA (green) and the flagellar membrane marker SMP1-mCh (red). DNA was labelled with DAPI (blue). Scale bar: 5μm. (H) Schematic of the FAZ protein localisations and anterior cell tip organisation in a promastigote cell, providing a side-on view of the FAZ (as inferred from our data and adapted from Halliday et al., PLoS Pathog 16(10): e1008494). The flagellar pocket is divided into two regions (bulbous and neck) joining by the flagellar pocket collar region; the microtubule quartet (MTQ) starts at the basal bodies and wraps around the bulbous region of the flagellar pocket before terminating in the neck region. The different FAZ domains and the proteins analysed in the present study are shown. Not to scale. (I-J) Western blot of promastigotes (PRO) and axenic amastigotes (AMA) of the parental cell line expressing FAZ7A-3xHA (I) and FAZ7B-3xHA (J); 35 μg of total protein extract were loaded on a 12% SDS-PAGE, transferred and immuno-probed with anti-HA and anti-LACK (loading control) antibodies.
Fig 2.
FAZ7B is required for normal cell growth in promastigote and amastigote stages.
(A-B) Representative growth curves (log scale) of promastigotes (A) and axenic amastigotes (B) of the parental, FAZ7B KO and FAZ7B add-back cell lines over a 4-day course. Cell density was determined by counting at 24 h intervals and the mean ± SD of three independent experiments was plotted. *p < 0.05, **p < 0.01 (Student’s t-test). (C) Fluorescence micrographs of whole cells from FAZ7B add-back (upper panel) and mutant-complemented cell lines: FAZ7B KO + FAZ7B G199A_K200A-mRED (middle panel) and FAZ7B KO + FAZ7B C-term-mRED (lower panel) cell lines. Scale bar: 5μm. Growth curves of promastigotes (D) and axenic amastigotes (E) of the FAZ7B KO, FAZ7B add-back, FAZ7B KO+ FAZ7B G199A_K200A-mRED and FAZ7B KO+ FAZ7B C-term-mRED cell lines over a 4-day course. Cell density was determined by counting at 24 h intervals and mean ± SD of triplicate values was plotted.
Fig 3.
Deletion of FAZ7B impairs cell division in promastigotes and amastigotes.
(A and C) Quantification of nuclei (N) and kinetoplasts (K) cell phenotypes in DAPI-stained parental and FAZ7B KO cell lines: (A) Promastigotes, (C) Axenic amastigotes. Error bars represent SDs calculated from three independent experiments (n = 300). ** p < 0.01, * p < 0.05 (Student’s t-test). (B) Examples of dividing cells in the promastigote FAZ7B KO cell line, Hoechst-stained after 14hs of time-lapse imaging, showing the indicated categories of N/K patterns; multiF: multiflagellated cell; MultiN: a cell with several nuclei and kinetoplasts; cytoplasts can be seen, with (1N0K0F and 0N1K0F) or without (left panel, top) DNA, as well as in a dividing process (1N multiF cytoplast). See also S5, S6 and S7 Movies. Scale bar: 5μm.
Fig 4.
Loss of FAZ7B alters promastigote cell morphology.
(A) Immunofluorescence assay. Parental, FAZ7B KO and add-back representative cells labelled using anti-PFR2 antibody (green). Scale bar: 5μm. (B) Flagellum and (C) cell body length measurements of parental, FAZ7B KO and add-back cell lines. Cells were fixed with PFA at a density of 1x107 cells/mL. Boxes and error bars indicate the median, upper and lower quartiles and 95th percentiles. *** p < 0.001 (Student’s t-test). (D-E) Measurement of the distance between the SMP1 signal (D), kinetoplast (E) and the anterior end of the cell body in parental and FAZ7B KO cell lines. One hundred cells were counted per sample. Boxes and error bars indicate the median, upper and lower quartiles and 95th percentiles. *** p < 0.001; * p < 0.05 (Student’s t-test).
Fig 5.
Flagellar pocket structure and cell division are perturbed in the FAZ7B null mutant.
Transmission electron micrographs of promastigotes and axenic amastigotes of the FAZ7B KO cell line. Promastigotes (A-B): (A) Abnormal cytoplasmic inclusions are present in the lumen of the FP (white arrows). (B) Rare cells showed abnormal kinetoplasts and/or abnormal basal bodies: here, a mutant cell showing four kDNA networks (of which three are contigous) (white arrow) and four basal bodies asymmetrically distributed (white asterisks). The flagellum structure appears normal (ax, black arrows). Amastigotes (C-K): Abnormal cytoplasmic inclusions were frequently observed in the lumen of the FP (C-G). Depending on the cutting plane, this material could be seen as protrusions of the FP wall inside the lumen of the pocket (D, G; white arrows). (G) An amastigote cell showing two nuclei and six transversal sections of flagella in a single FP and two flagella in another one. (H) Longitudinal section of a dividing amastigote, with two nuclei and only one kinetoplast and one FP visible on this cutting plane. On the left side, a portion of cytoplasm apparently devoid of nucleus but containing lipid bodies and organites shows incomplete separation from the ‘donor’ cell, with discontinuous plasma membrane and cortical microtubules (white arrows), forming a cytoplast (cp, black arrow). (I-K) A transversal section of a dividing FAZ7B-KO amastigote showing multiple FPs each containing one or two flagella. At least three partially separated cell bodies can be distinguished. The cortical microtubule network is in place but the plasma membrane shows incomplete separation (J, white arrows). The microtubule quartet is clearly seen close to most flagella (J, black arrowheads). The flagellum structure in transversal sections appears normal. n: nucleus; k: kinetoplast; ax: Axoneme; cp: cytoplast. Scale bars: 0.5 μm.
Fig 6.
The molecular organization of the flagellar pocket collar is altered in the FAZ7B null mutant.
Immunofluorescence labelling of promastigotes from parental and FAZ7B KO cell lines expressing (A) FAZ7B-3xHA (red) and mNG-BILBO1(green), and (B) FAZ7B-3xHA (red) and FPC4-mNG (green) Scale bar: 5μm. See also S11 Fig. (C-D) Western blot of promastigotes from the parental and FAZ7B KO cell lines expressing 3xHA-Bilbo (C) and FPC4-3xHA (D), using anti-HA. 5x106 cells were loaded on a 12% SDS-PAGE, transferred and immuno-probed with anti-HA and anti-LACK (loading control) antibodies.
Fig 7.
Deletion of FAZ7B impairs motility, proliferation and development of Leishmania in the sandfly.
(A) Directionality (velocity/speed) and (B) swimming speed measurements of parental, FAZ7A KO and FAZ7B KO promastigotes were calculated from swimming tracks obtained from video microscopy using the plugin Trackmate. Boxes and error bars indicate the mean, upper and lower quartiles and 95th percentiles. *** p < 0.001 (Student’s t-test). (C) Infection rate and intensity of infection in Lutzomyia longipalpis females infected with parental, FAZ7B KO and FAZ7B add-back cells. After 2 days and 8 days post-blood meal (PBM), sandflies were dissected (numbers indicated above the column) and the parasite load in each fly was measured as weak (1–100 parasites), moderate (101–1000 parasites), or heavy (1000+ parasites). The differences between the sandfly infections caused by parental and KO cell lines on day 8 PBM were highly significant (Chi-squared test, X2 value: 22.237, p value: 7.02.E-7). (D) Localization of Leishmania parasites within infected sandflies after 2 days and 8 days PBM. ES: endoperitrophic space, AM: abdominal midgut, TM: thoracic midgut, SV: stomodeal valve. Differences were observed among infections caused by the three cell lines but were not found statistically significant.
Fig 8.
Loss of FAZ7B reduces pathogenicity in the mouse.
(A-B) Axenic amastigotes of parental, FAZ7B KO and FAZ7B add-back cells were used to infect differentiated THP-1 monocytes. Percentage of infected macrophages (A) and parasitic index (B) were determined at 72h post infection by Giemsa staining as described in Experimental procedures. The parasitic index (PI) was defined as the percentage of infected macrophages x number of intracellular parasites/macrophage. ** p < 0.01, * p < 0.05. (C) Measurement of mean footpad lesion size during a ten-week infection time course with parental, FAZ7B KO and FAZ7B add-back stationary-phase promastigotes. Error bars represent SDs. The p value was calculated using two-tailed unpaired Student’s t-test comparing each cell line at week 10. *** p < 0.001, * p < 0.05. (D) Relative amount of Leishmania DNA compared to mouse DNA by qPCR at 10 weeks post-infection by parental, FAZ7B KO and FAZ7B add-back cells. ** p < 0.01, * p < 0.05 (Student’s t-test).