The Leishmania major BBSome subunit BBS1 is essential for parasite virulence in the mammalian host

Abstract

Bardet-Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole-like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild-type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity.

Figure 1

BBS1 gene deletion in Leishmania major.A. Schematic diagram of the LmBBS1 locus and the plasmid constructs used for targeted deletion of the locus by replacement with hygromycin/puromycin resistance genes (HYG/PAC). Flanking sequences used to generate the targeting vectors are shown. Solid black bars represent fragments used as hybridization probes. H, HindIII site. E, EcoRV site.B. Southern blot analysis of wild-type L. major (WT), BBS1 null (−/−) and complemented (−/−+) parasite lines. Five micrograms of genomic DNA from each parasite line was digested with HindIII/EcoRV, size separated through 0.8% agarose, blotted and hybridized with DIG-labelled DNA probes (∼ 200 bp) as indicated. Corresponding DNA marker positions are shown (Kb).C. Growth of promastigotes from wild-type L. major (WT), BBS1 null (BBS1−/−) and complemented (BBS1−/−[+BBS1]) monitored over a 7-day time-course. Mean values are shown (n = 3) ± SD (error bars are not visible).D. Scanning electron micrographs of L. major wild-type and BBS1 null cell lines as above. Bar, 5 μm.E. Transmission electron micrographs of promastigotes from L. major parasite lines as above. FP, flagellar pocket. Fl, flagellum. K, kinetoplast. Bar, 2 μm.F. Procyclic promastigote osmotaxis assay. Parasite lines as above (day 3 post-inoculation) were tested for their ability to migrate into capillary tubes containing agarose and 100 mM sucrose compared with movement into control capillary tubes containing agarose alone (n = 6).Data shown are the combined results of six independent experiments.

Figure 2

Effect of BBS1 gene deletion on L. major differentiation and macrophage infection in vitro.A. Procyclic promastigotes of L. major wild-type (WT), BBS1 null (BBS1−/−) and complemented (BBS1−/−[+BBS1]) lines were used to inoculate cultures at an initial concentration of 5 × 10⁴ ml⁻¹ and samples were collected at 2, 5 and 7 days. Total-cell lysates (1 × 10⁶ cells per lane) were immunoblotted and probed with antibodies against L. major HASPB and SHERP, with NMT as a constitutively expressed control.B. Immunofluorescence analysis of L. major parasite lines as above, following growth in culture for 7 days to promote differentiation from procyclic to metacyclic promastigotes. Parasites were probed with rabbit anti-HASPB (green) and co-stained with DAPI (blue). Neg, negative control wild-type procyclic promastigote which shows no HASPB expression. Bar, 5 μm.C. L. major lines analysed in (A) were harvested at day 7 post-inoculation and stained with antibodies against α-tubulin and L. major PFR1. A number of cellular dimensions were measured (200 parasites per sample) and the life cycle stage assigned to each parasite based on the strict set of criteria used to define developmental stages within the sand fly vector (Walters, ; Cihakova and Volf, 1997). Data shown represent one of two independent experiments.D. Flagellum length measurements divided by body length are shown for L. major lines as described in (C).E. Mouse peritoneal macrophages were infected with metacyclic promastigotes from wild-type and BBS1 mutant lines. The number of parasites per macrophage (200 per sample) was determined at 48 and 72 h post-infection by immunofluorescence using antibodies against α-tubulin and L. major PFR1, excluding extracellular PFR1-positive metacyclic parasites from the analysis. The percentage of macrophages with 0, 1 or ≥ 2 parasites is shown for each group. These data represent one of three independent experiments.F. Immunofluorescence of mouse peritoneal macrophages following infection with L. major metacyclic promastigotes for 4 h or 72 h (as described in E). Cells were probed with anti-α-tubulin (red) and anti-PFR1 (green) and co-stained with DAPI (blue). Bar, 20 μm.G. Transmission electron micrographs of mouse peritoneal macrophages infected with metacyclic promastigotes from L. major parasite lines as above for 72 h. Ac, acidocalcisome. N, nucleus. FP, flagellar pocket. Fl, flagellum. K, kinetoplast. Bar, 2 μm.H. Scanning electron micrographs of L. major amastigotes isolated from human monocytic cell line THP1 infected with metacyclic promastigotes from parasite lines as above for 72 h. FP, flagellar pocket. Fl, flagellum. Bar, 1 μm.

Figure 3

Effect of BBS1 gene deletion on L. major host infectivity.A. BALB/c mice were infected with L. major wild-type (WT), BBS1 null (BBS1−/−) and complemented (BBS1−/−[+BBS1]) lines by subcutaneous injection of 5 × 10⁶ metacyclic promastigotes into the right hind footpad. Developing lesions were monitored over 60 days. Mean lesion thickness is shown (n = 5) ± SD. Data presented here represent one of two independent experiments.B. Parasite burden was measured in three footpads from each group of infected mice as in (A), by a limiting dilution assay following termination. Mean parasite burden per footpad is shown, combining data from two independent experiments (n = 6) ± SD. By this method, no parasites were detected in five of six mice infected with the L. major BBS1 null line.C. Immunofluorescence of lymph nodes draining the site of infection in BALB/c mice, 60 days post-infection with L. major parasite lines as above (two areas of the lymph node are shown for BBS1 complemented line). Tissue sections were probed with antibodies against L. major HASPB (yellow), the macrophage marker F4/80 (green) and B-cell marker B220 (pink). Bar, 200 μm.D. Lymph node section from a mouse infected with BBS1 complemented line for 60 days, probed with anti-HASPB (yellow) and F4/80 (green) and co-stained with DAPI (blue). Bar, 20 μm.E. Infected mouse lymph node sections probed with anti-HASPB (yellow) and co-stained with DAPI (blue). Bar, 2.5 μm.F. Infected mouse lymph node sections probed with anti-HASPB (red) and anti-LAMP1 (green) and co-stained with DAPI (blue). Bar, 5 μm.G. Infected and naïve mouse lymph node sections probed with anti-HASPB (yellow), anti-acetylated α-tubulin (pink) and anti-tyrosinated α-tubulin (green) and co-stained with DAPI (blue). Bar, 20 μm.