Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite

Xuye Yuan; Tatsuhiko Kadowaki

doi:10.1371/journal.pgen.1011195

. 2024 Mar 4;20(3):e1011195. doi: 10.1371/journal.pgen.1011195

Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite

Xuye Yuan ¹, Tatsuhiko Kadowaki ^1,^*

Editor: Ashley Soyong Byun²

PMCID: PMC10939215 PMID: 38437202

Abstract

The honey bee trypanosomatid parasite, Lotmaria passim, contains two genes that encode the flagellar calcium binding protein (FCaBP) through tandem duplication in its genome. FCaBPs localize in the flagellum and entire body membrane of L. passim through specific N-terminal sorting sequences. This finding suggests that this is an example of protein subcellular relocalization resulting from gene duplication, altering the intracellular localization of FCaBP. However, this phenomenon may not have occurred in Leishmania, as one or both of the duplicated genes have become pseudogenes. Multiple copies of the FCaBP gene are present in several Trypanosoma species and Leptomonas pyrrhocoris, indicating rapid evolution of this gene in trypanosomatid parasites. The N-terminal flagellar sorting sequence of L. passim FCaBP1 is in close proximity to the BBSome complex, while that of Trypanosoma brucei FCaBP does not direct GFP to the flagellum in L. passim. Deletion of the two FCaBP genes in L. passim affected growth and impaired flagellar morphogenesis and motility, but it did not impact host infection. Therefore, FCaBP represents a duplicated gene with a rapid evolutionary history that is essential for flagellar structure and function in a trypanosomatid parasite.

Author summary

Protein subcellular relocalization (PSR) was proposed as a mechanism for the functional divergence and retention of duplicate genes. We explored this hypothesis using flagellar calcium binding protein (FCaBP) genes duplicated in many trypanosomatid parasites. FCaBPs localize in the flagellum and entire body membrane of honey bee trypanosomatid parasite, Lotmaria passim through specific N-terminal sorting sequences. However, this is not common with all trypanosomatid parasites since one or both of the duplicated genes have become pseudogenes in Leishmania. N-terminal flagellar sorting sequence of FCaBP1 interacts with BBSome complex, and thus they must have co-evolved in each trypanosomatid species. FCaBPs are essential for the normal growth, flagellar morphogenesis, and motility but not host infection of L. passim. Our findings demonstrate that duplicated FCaBP genes have undergone PSR by changing amino acids in the N-terminal end with some but not all trypanosomatid parasites. In the parasites with multiple FCaBP genes, they play critical roles for the growth control as well as flagellar structure and function.

Introduction

Gene duplication is considered the main source of new genes [1–4]. However, to maintain duplicate genes in a genome, functional divergence is usually necessary, with a few exceptions. Functional divergence can occur through neofunctionalization, where one of the duplicates develops a new function while the other retains the original function of the ancestral gene [1]. Alternatively, subfunctionalization can take place, where the ancestral functions are divided between the duplicate genes. For example, their combined levels or patterns of activity could be equivalent to the original single gene [5–7]. These types of functional divergence can modify the function of the encoded protein or the gene’s expression pattern. Subcellular localization is also crucial for a protein’s function within a cell. Protein subcellular relocalization (PSR) has been proposed as a mechanism for the functional divergence and retention of duplicate genes [8,9]. PSR allows for the rapid subdivision of ancestral localization or acquisition of new localization following gene duplication. After neolocalization or sublocalization, the expression pattern and function of the genes can further diverge. Although this idea was questioned by comparing the frequency of PSR between singletons and duplicates [10], there are numerous examples of PSR for duplicated genes [11–13].

The flagellar calcium binding protein (FCaBP or Calflagin) was discovered as one of the abundant proteins in the flagellar membrane of Trypanosoma brucei and Trypanosoma cruzi. It contains four EF-hand calcium binding domains and is considered a conserved signaling protein among trypanosomatid parasites [14,15]. FCaBP is acylated with myristate and palmitate at the N-terminus, which is crucial for its localization in the flagellum and association with lipid raft microdomains [16–18]. Additionally, the N-terminal amino acid sequence of FCaBP is necessary for its targeting to the flagellum in T. cruzi [19]. Although the phenotypes of knocking down FCaBPs in T. brucei have been reported [20], the physiological functions of FCaBPs are not well understood. Proteins in the flagellum (and cilium) are initially synthesized in the cell body and then transported to the flagellum through intraflagellar transport (IFT). IFT requires IFT complexes (IFT-A and IFT-B) and motor proteins, including kinesin-2 for anterograde transport and IFT-dynein for retrograde transport, along the axonemal microtubules [21–23]. The IFT complexes act as carriers, and BBSome and tubby-like protein (TULP) function as adapters by binding to specific sets of flagellar membrane proteins [24–29]. For example, BBSome is necessary for the exit of a dually acylated phospholipase D from cilia in Chlamydomonas [26]. Therefore, FCaBP likely requires IFT complexes and either BBSome or TULP for its import and export to the flagellum.

Lotmaria passim is the most prevalent trypanosomatid parasite that infects honey bees worldwide [30–33]. It specifically colonizes the hindgut of honey bees, affecting the host’s physiology, and it may be associated with winter colony loss [34,35]. L. passim is a monoxenous parasite that only infects bees and can serve as a model to study the roles of flagellar formation and function for infecting the various hosts of trypanosomatid parasites.

In this study, we identified two FCaBP genes in the genomes of L. passim and closely related species, resulting from tandem duplication. By analyzing the gene in other trypanosomatid species’ genomes, the protein subcellular localizations, and N-terminal sorting sequences, we have uncovered their evolutionary history in relation to the flagellar protein transport machinery. Deletion of these genes in L. passim has also revealed novel physiological functions of FCaBPs. Therefore, FCaBP serves as an example to understand how duplicated genes have diversified during the evolution of trypanosomatid parasites.

Results

L. passim contains two FCaBPs with flagellar and entire body localizations

The genome of L. passim encodes two FCaBP genes (LpFCaBP1 and LpFCaBP2) that are separated by 1063 bp, suggesting tandem gene duplication. To determine their localizations, we expressed GFP-tagged versions of LpFCaBP1 and LpFCaBP2 (at C-terminus) in L. passim. LpFCaBP1-GFP was highly enriched in the flagellum, similar to FCaBPs in T. cruzi and T. brucei [16,17]. On the other hand, LpFCaBP2-GFP was present at the flagellum and the cell body membrane (Fig 1). We also observed that GFP alone is uniformly distributed in the entire body, while the paraflagellar rod protein 5 (LpPFRP5)-GFP fusion protein is specific to the flagellum (Fig 1). These findings indicate that the two duplicated FCaBP genes encode proteins enriched in the different part of parasite body with common localization in the flagellum.

Fig 1 — *Lotmaria passim* expressing LpFCaBP1-GFP, LpFCaBP2-GFP, LpPFRP5-GFP, or GFP were examined under visible (DIC) or fluorescence (Fluorescence) light. Merged images are also shown. Flagellum at the anterior end and cell body are indicated by arrows. The ratio of the mean fluorescence in the flagellum to that in the cell body (mean value ± SD) along with number of parasites analyzed (n) is presented on the right. Scale bar: 2 μm.

The N-terminal 16 amino acids of LpFCaBP function as either flagellum or cell body membrane sorting sequence

By aligning the amino acid sequences of LpFCaBP1 and LpFCaBP2, we found that they are almost identical except for their N-terminal ends (S1 Data). To determine the flagellar and entire body membrane sorting sequences, we swapped the N-terminal 16 or 28 amino acids between LpFCaBP1 and LpFCaBP2 in GFP fusion proteins. We also determined the ratio of mean fluorescence of flagellum to that of cell body using Image-J. There are no statistical differences between the ratios for LpFCaBP1-GFP (2.4 in average, Fig 1), LpFCaBP1N28/FCaBP2-GFP (2.3 in average, Fig 2), LpFCaBP1N16/FCaBP2-GFP (2.3 in average, Fig 2), and LpFCaBP1N16-GFP (2.1 in average, Fig 2). The ratio for LpFCaBP2-GFP (0.92 in average, Fig 1) is significantly higher than those of LpFCaBP2N28/FCaBP1-GFP (0.66 in average, P < 0.003, Fig 2), LpFCaBP2N16/FCaBP1-GFP (0.78 in average, P < 0.04, Fig 2), and LpFCaBP2N16-GFP (0.49 in average, P < 0.001, Fig 2). These results demonstrate that the N-terminal 16 amino acids of LpFCaBP1 and LpFCaBP2 are sufficient to direct GFP to the flagellum and cell body membrane, respectively (Fig 2). Thus, they represent the flagellar and cell body membrane sorting sequences. Since LpFCaBP2-GFP was present in the entire body (Fig 1), the EF-hand calcium binding domains (amino acids 38–189) contribute to the flagellar localization.

Fig 2 — The N-terminal 16 or 28 amino acids of LpFCaBP1 (in red) and LpFCaBP2 (in blue) were swapped as illustrated in the diagram. The positions of amino acids in the cores of LpFCaBP1 and 2 proteins are also indicated. GFPs fused with the N-terminal 16 amino acids of LpFCaBP1 and 2 are shown at the bottom. The ratio of the mean fluorescence in the flagellum to that in the cell body (mean value ± SD) along with number of parasites analyzed (n) is presented on the right. Scale bar: 2 μm.

FCaBP genes have rapidly evolved in trypanosomatid parasites

We examined FCaBP genes in various trypanosomatid parasites’ genomes. T. brucei has four FCaBP and the related genes as previously reported [20]. T. cruzi appears to contain the large number of FCaBP genes due to its repetitive genome [36]. Trypanosoma theileri genome has three FCaBP genes (S2 Data). These results suggest that FCaBP gene duplicated in the common ancestor of Trypanosoma and Leishmaniinae and has undergone further duplication in each Trypanosoma species.

In the genomes of various Leishmaniinae species, microsynteny around FCaBP genes is well conserved with the same orders and orientations of genes encoding three novel proteins, dynein light chain, and sucrose-phosphate synthase-like protein (Fig 3). In Leishmania infantum, Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani, two FCaBP genes are present but FCaBP2 appears to have become a pseudogene by generating a stop codon in 5’ end of the ORF (open reading frame). If this gene is translated, the truncated protein lacking the N-terminal sorting sequence is synthesized and is likely to be present in the cytosol rather than the membrane, as it lacks the N-terminal glycine and cysteine residues necessary for myristoylation and palmitoylation. Although the protein should be able to bind calcium, its interactions with other membrane associated proteins as a signaling factor would be lost. Thus, above four Leishmania species may contain only FCaBP1. Intriguingly, two FCaBP genes in Leishmania major have become pseudogenes by accumulating multiple internal stop codons in the ORFs (Fig 3). These characteristics are shared with three independently assembled genome sequences derived from different strains of L. major (Friedlin, SD75.1, and LV39c5), suggesting that this is not the result of genome assembly error.

Fig 3 — The microsynteny containing *FCaBP* genes is highly conserved in *Leishmania infantum*, *Leishmania braziliensis*, *Leishmania mexicana*, *Leishmania donovani*, *Leishmania major*, *Lotmaria passim*, *Crithidia fasciculata*, *Crithidia expoeki*, *Crithidia bombi*, *Leptomonas seymouri*, and *Leptomonas pyrrhocoris*. *FCaBP2* is a pseudogene (indicated by ×) in L. *infantum*, L. *braziliensis*, L. *mexicana*, and L. *donovani*, while both *FCaBP1* and 2 are pseudogenes in L. *major*. *FCaBP1* and 2 genes (red and blue arrows) are flanked by three novel genes (beige, light grey, and light green arrows), as well as dynein light chain (light blue arrow) and sucrose-phosphate synthase-like protein genes (dark grey arrow). The orientation of each gene is shown.

In species related to L. passim (34), Crithidia fasciculata, Crithidia expoeki, and Crithidia bombi have two FCaBP genes, while Leptomonas seymouri has only FCaBP1 gene (Fig 3). At downstream of LsFCaBP1, there is 173 bp genomic sequence capable of encoding a protein with low similarity to C-terminal 56 amino acid of FCaBP (e-value: 0.013) if translated. This may represent the remnant of LsFCaBP2 pseudogene. We primarily characterized L. seymouri assembled genome sequence derived from the strain ATCC 30220 Lsey_0068 (Accession number: LJSK01000068); however, FCaBP genomic sequence is the same in another independently assembled genome sequence derived from the different strain, BHU1095 (Accession number: ANAF02000031). Thus, the loss of LsFCaBP2 is unlikely to be caused by the error of genome assembly. Leptomonas pyrrhocoris contains FCaBP1 and two FCaBP2 genes based on the comparison of N-terminal sorting sequences (S1 Data). These results demonstrate that FCaBP gene has rapidly evolved in trypanosomatid parasites.

Flagellar localization of FCaBP depends on both the sorting sequence and trans-acting factors in L. passim

Fig 4A shows the N-terminal ends of aligned FCaBPs from T. cruzi, T. brucei, L. donovani, C. fasciculata, L. seymouri, and L. passim. Glycine and cysteine residues targeted for myristoylation and palmitoylation are well conserved; however, there are more variations in the sorting sequences of FCaBPs compared to the main sequences with EF-hand calcium binding domains. To elucidate the pivotal amino acids within the sorting sequence governing the membrane localization of LpFCaBP in the flagellum or entire body, we performed targeted mutagenesis on LpFCaBP1 and LpFCaBP2. Specifically, lysine at position 11 (K11) in LpFCaBP1 was substituted with isoleucine, and vice versa, generating LpFCaBP1N16(K11I)-GFP and LpFCaBP2N16(I11K)-GFP. Additionally, serines at positions 9 (S9) and 10 (S10) in LpFCaBP1 were substituted with threonine and glutamine, respectively, and vice versa, resulting in LpFCaBP1N16-TQ-GFP and LpFCaBP2N16-SS-GFP. Comparative analyses with wild-type LpFCaBP1N16-GFP (Fig 2) revealed a substantial increase in cell body fluorescence and a corresponding decrease in relative flagellar fluorescence with LpFCaBP1N16-TQ-GFP (average 1.2, P < 0.001), while no significant change was observed with LpFCaBP1N16(K11I)-GFP (Fig 4B). In comparison to wild-type LpFCaBP2N16-GFP (Fig 2), LpFCaBP2N16(I11K)-GFP exhibited an increase in relative flagellar fluorescence (average 0.79, P < 0.001), while no significant difference was noted with LpFCaBP2N16-SS-GFP (Fig 4B). These results indicate that K11 of LpFCaBP1 is not essential to direct LpFCaBP1N16-GFP to the flagellum but contributes to the flagellar localization of LpFCaBP2N16-GFP. Furthermore, S9 and S10, which are conserved among FCaBP1s across four trypanosomatid species (S1 Data), are crucial for the efficient localization of LpFCaBP1N16-GFP to the flagellum in L. passim. Remarkably, the fluorescence pattern of LpFCaBP1N16-TQ-GFP and LpFCaBP2N16-SS-GFP is uniform within the cell body, akin to GFP (Fig 1), in contrast to the membrane localization observed with LpFCaBP2N16-GFP (Fig 2) and other GFP fusion proteins (Fig 4B and 4C). Thus, it is likely that S9, S10, T9, and Q10 are required for efficient acylation at glycine (G2) and cysteine (C3) in the N-termini of FCaBP1 and FCaBP2. These lipid modifications appear to play a crucial role in determining FCaBP localization, either in the flagellum as previously reported [17,19] or in the entire body membrane. However, LpFCaBP1N16-TQ-GFP and LpFCaBP2N16-SS-GFP may fail to associate with the cell body membrane due to a mechanism other than defective acylation.

To test the conservation and plasticity of the flagellar and cell body membrane sorting sequences of FCaBPs, we expressed GFP fusion proteins containing N-terminal sorting sequences from various trypanosomatid parasites in L. passim. Because TcFCaBP and TbFCaBP (Calflagin, Tb24) are flagellar proteins, their N-terminal amino acids should function as flagellar sorting sequences in T. cruzi and T. brucei, respectively [17,19]. Indeed, the N-terminal 24 but not 12 amino acids of the TcFCaBP were sufficient to direct GFP to the flagellum of T. cruzi [19]. However, we found that GFP fused with the sorting sequence of TbFCaBP is localized in the cell body membrane of L. passim. GFP with the sorting sequence of either TcFCaBP or LdFCaBP is present in both cell body membrane and the proximal part of flagellum (Fig 4C). The sorting sequences of CfFCaBP1 and 2 directed GFP to flagellum (most) as well as cell body membrane and cell body membrane, respectively. GFP with the sorting sequence of LsFCaBP1 is primarily localized in flagellum (Fig 4C). These results may suggest that flagellar localization of FCaBP requires the recognition of N-terminal sorting sequence by IFT-associated proteins (trans-acting factors).

The N-terminal sorting sequence of LpFCaBP1 is in close proximity to the BBSome complex but not TULP

Since the import and export of flagellar membrane proteins depend on either the BBSome complex or TULP [37], we first tested the interaction between LpFCaBP1N16-GFP and Flag-tagged LpBBS1 or LpTULP in the parasites expressing both proteins by immunoprecipitation. However, we were unable to detect binding with either protein. This would be due to the transient interaction between a flagellar membrane protein and BBSome or TULP as the adaptor [37]. We thus used an engineered biotin ligase, ultraID [38], instead of GFP. This fusion protein allowed us to identify proteins in the close proximity through biotinylation. In L. passim expressing both LpFCaBP1N16-UltraID and Flag-tagged LpBBS1 or LpTULP, the ultraID fusion protein was detected in the proximal part of the flagellum (Fig 5A). Surprisingly, LpFCaBP2N16-UltraID concentrated at the anterior end of cell body (Fig 5A) in contrast to the uniform cell body membrane localization of LpFCaBP2N16-GFP (Fig 2). Thus, ultraID tends to anchor at the anterior side of cell body and this may also inhibit the localization of LpFCaBP1N16-UltraID to the distal part of flagellum in L. passim (Fig 5A). We detected biotinylated Flag-LpBBS1 but not Flag-LpTULP in the parasites expressing LpFCaBP1N16- but not LpFCaBP2N16-UltraID (Fig 5B), suggesting that LpFCaBP1N16 is in close proximity to LpBBS1. The apparent size of Flag-LpTULP was larger than that of Flag-LpBBS1 and the expected size (59 kDa) (Fig 5B). LpTULP may have post-translational modification or migrate slowly through 10% SDS-PAGE. These findings suggest that the flagellar localization of LpFCaBP1 may be mediated by the BBSome complex.

Fig 5 — (A) Subcellular localization of ultraID with the N-terminal sorting sequence of LpFCaBP1 (LpFCaBP1N16-UltraID), LpFCaBP2 (LpFCaBP2N16-UltraID), Flag-tagged LpBBS1 (Flag-LpBBS1), and LpTULP (Flag-LpTULP) by immunofluorescence. Scale bar: 2 μm (B) Lysates of parasites expressing both either LpFCaBP1N16-UltraID or LpFCaBP2N16-UltraID and Flag-LpBBS1 or Flag-LpTULP were subjected to immunoprecipitation using Flag Fab-Trap Agarose (Input) or streptavidin agarose precipitation (Streptavidin) to capture biotinylated proteins. One-fourth of the total protein in the Streptavidin sample was analyzed for the Input sample. The precipitates were analyzed by western blot using an anti-Flag tag antibody.

LpFCaBPs are essential for flagellar morphogenesis and motility but not the host infection of L. passim

To determine the functions of LpFCaBPs, we deleted both LpFCaBP1 and LpFCaBP2 genes in L. passim by replacing them with hygromycin resistance gene by CRISPR-mediated homology-directed repair (Fig 6A). Although LpFCaBP1 and 2 primarily localize in the flagellum and entire body membrane, they could be still functionally redundant. As we previously reported, expressing Cas9 and gRNA does not modify the target gene in L. passim. Thus, the off-target effects are basically absent (39). By genomic PCR, we identified both heterozygous (+/-) and homozygous (-/-) deletion clones of LpFCaBPs (Fig 6B). To confirm the disruption of genes, we tested LpFCaBP1 and 2 mRNA expression in wild type, heterozygous and homozygous deletion parasites by RT-PCR using the gene-specific reverse primer. As shown in Fig 6C, both mRNAs are absent in the homozygous mutant (LpFCaBP1/2 -/-) parasites.

Fig 6 — (A) Schematic representation of wild-type (WT) and deleted (KO) alleles of *LpFCaBPs* generated by CRISPR/Cas9-induced homology-directed repair. 5’ and 3’ untranslated regions (UTRs), open reading frames (ORFs), and the internal spacer of *FCaBP1* and 2, hygromycin resistance gene *(Hph)*, are shown in blue, green, orange, yellow, purple, and red, respectively. The expected sizes of PCR products to detect WT and KO alleles (not to scale) are also shown. (B) Genomic DNAs of wild-type L. *passim* (+/+), heterozygous (+/-), and homozygous (-/-) mutants of *LpFCaBPs* were analyzed by PCR to detect *5’WT*, *5’KO*, *3’WT*, and *3’KO* alleles. Sizes of the DNA molecular weight markers are shown on the left. (C) Detection of *LpFCaBP1* and 2, as well as *LpGAPDH* mRNAs, in *LpFCaBPs* heterozygous (+/-) and homozygous (-/-) mutants together with wild-type L. *passim* (+/+) by RT-PCR. Sizes of the DNA molecular weight markers are shown on the left.

We also established LpFCaBP1/2 -/- parasites, wherein either LpFCaBP1 or LpFCaBP2 was introduced on a plasmid DNA vector containing a bleomycin resistance gene. Comparative phenotypic analyses were performed with wild-type and the parent mutant parasites at 29°C. LpFCaBP1/2 -/- parasites exhibited reduced growth rates during the logarithmic phase, reaching stationary phase at a density comparable to wild-type parasites. The growth defect in LpFCaBP1/2 -/- parasites was rescued by the introduction of either LpFCaBP1 or LpFCaBP2 (Fig 7A and 7B). In the early stationary phase (at 60 h after culture initiation), wild-type parasites displayed active movement with extended flagella. Measurement of flagella and cell body lengths, as well as individual parasite movements, revealed that LpFCaBP1/2 -/- parasites exhibited shorter flagella and cell bodies with reduced motility compared to wild-type parasites (Fig 7C–7F and S1 and S2 Videos). These findings indicate the essential role of LpFCaBP1 and LpFCaBP2 in flagellar morphogenesis and motility of L. passim. While either LpFCaBP1 or LpFCaBP2 rescued the observed phenotypes, they did not fully complement to wild-type levels, except for LpFCaBP1, which fully restored the short flagella of LpFCaBP1/2 -/- parasites (Fig 7C–7F and S1–S4 Videos). In the late stationary phase (at 96 h after culture initiation), wild-type parasites formed rosettes, characterized by clusters of cells with flagella directed towards the center. However, LpFCaBP1/2 -/- parasites remained as individual cells without clustering at the culture plate bottom (Fig 7G). Both LpFCaBP1 and LpFCaBP2 restored rosette formation in LpFCaBP1/2 -/- parasites, although the sizes of rosettes were generally smaller than those observed with wild-type parasites (Fig 7G). These results suggest that the molecular functions of LpFCaBP1 and LpFCaBP2 remain the same as calcium-binding proteins. However, in L. passim, both LpFCaBP1 concentrated in the flagellum and LpFCaBP2 present throughout the entire parasite are necessary. We then compared the infection of wild-type and LpFCaBP1/2 -/- parasites in honey bee hindgut and found that they infect the host at the comparable level (Fig 7H). Thus, less motile L. passim with short flagellum by the loss-of function of LpFCaBP genes is still capable of infecting honey bee hindgut effectively.

Fig 7 — Growth rates of wild-type (WT, circle), *LpFCaBPs* homozygous mutant (clone D1, *LpFCaBP1/2 -/-*, square), *LpFCaBPs* homozygous mutant expressing either *LpFCaBP1* (*LpFCaBP1/2 -/- LpFCaBP1*, triangle) or *LpFCaBP2* (*LpFCaBP1/2 -/- LpFCaBP2*, reverse triangle) L. *passim* in the modified FP-FB medium were monitored at 29°C for three days (biological replicates, n = 3). (B) Density of WT, *LpFCaBP1/2 -/-*, *LpFCaBP1/2 -/- LpFCaBP1*, and *LpFCaBP1/2 -/- LpFCaBP2* parasites compared at 36 and 48 hours after culture. *P < 0*.001 between WT and *LpFCaBP1/2 -/-* at 36 and 48 hours. Statistical comparisons were carried out using Welch’s t-test. (C) Morphology of WT, *LpFCaBP1/2 -/-*, *LpFCaBP1/2 -/- LpFCaBP1*, and *LpFCaBP1/2 -/- LpFCaBP2* parasites. Scale bar: 2 μm. (D) Flagellar length of individual WT (n = 23), *LpFCaBP1/2 -/-* (n = 34), *LpFCaBP1/2 -/- LpFCaBP1* (n = 24), and *LpFCaBP1/2 -/- LpFCaBP2* (n = 27) parasites. *P < 0*.001 between WT and *LpFCaBP1/2 -/-* or *LpFCaBP1/2 -/- LpFCaBP2*. Statistical comparison was carried out using the Steel test. (E) Cell body length of individual WT (n = 23), *LpFCaBP1/2 -/-* (n = 34), *LpFCaBP1/2 -/- LpFCaBP1* (n = 24), and *LpFCaBP1/2 -/- LpFCaBP2* (n = 27) parasites. *P < 0*.001 between WT and *LpFCaBP1/2 -/-* or *LpFCaBP1/2 -/- LpFCaBP1*. *P < 0*.002 between WT and *LpFCaBP1/2 -/- LpFCaBP2*. (F) Motility (total distance moved for 1 minute) of individual WT (n = 841), *LpFCaBP1/2 -/-* (n = 1777), *LpFCaBP1/2 -/- LpFCaBP1* (n = 1085), and *LpFCaBP1/2 -/- LpFCaBP2* (n = 901) parasites shown by a violin plot. Median, as well as the first and third quartiles, are indicated by solid and dashed lines, respectively. *P < 0*.001 between WT and *LpFCaBP1/2 -/-*, *LpFCaBP1/2 -/- LpFCaBP1*, or *LpFCaBP1/2 -/- LpFCaBP2*. Statistical comparison was carried out using the Steel test. (G) Images of WT, *LpFCaBP1/2 -/-*, *LpFCaBP1/2 -/- LpFCaBP1*, and *LpFCaBP1/2 -/- LpFCaBP2* parasites at 96 hours after culture. The clusters of parasites represent rosettes. Scale bar: 20 μm (H) The relative abundance of L. *passim* in individual honey bees (n = 24) at 14 days after the infection compared between WT and *LpFCaBP1/2 -/-* parasites. One sample infected by the WT parasite was set as 1, and the median with 95% CI is shown. The Brunner-Munzel test was used for statistical analysis. ns: not significant.

Discussion

Rapid evolution of FCaBP genes in trypanosomatid parasites

We characterized FCaBP genes in the genomes of trypanosomatid parasites and observed that each species, except L. seymouri, has more than two FCaBP genes in the same genomic contigs (Fig 3). This suggests that gene duplication occurred in the common ancestor of Trypanosoma and Leishmaniinae. While T. brucei, T. theileri, and L. pyrrhocoris have further duplicated FCaBP genes, it appears that four Leishmania species and L. seymouri have lost FCaBP2 by pseudogenization and deletion, respectively. When we expressed GFP fused with the sorting sequence of TbFCaBP in L. passim, we observed the cell body membrane localization. Meanwhile, GFPs fused with the sorting sequences of TcFCaBP, LdFCaBP, and CfFCaBP1 were present in both the flagellum and cell body membrane (Fig 4C). These findings indicate that the BBSome complex in L. passim may not recognize the flagellar sorting sequence of TbFCaBP, but partially recognizes those of TcFCaBP, LdFCaBP, and CfFCaBP1. Therefore, it appears that dually acylated FCaBP is naturally targeted to the plasma membrane of the entire body, and specific N-terminal sorting sequences and the BBSome complex are required for flagellar enrichment. Additionally, the inefficient localization of GFP at the distal part of the flagellum using the TcFCaBP and LdFCaBP sorting sequences suggests a functional or structural separation in the flagellum (See also Fig 5A). Meanwhile, GFP fusion proteins may also exist in the flagellar pocket and pocket neck membranes in these parasites. The lack of functional FCaBP in L. major demonstrates that it is not essential for completing the life cycle in sand fly and mammalian hosts, suggesting the presence of other proteins that complement the absence of FCaBP in L. major.

In trypanosomatid parasites with two FCaBP genes, mutations have accumulated in the 5’ end of the ORF, modifying the N-terminal amino acid sequences after gene duplication. As a result, FCaBP1 and FCaBP2 have different subcellular localizations at the flagellum and entire body membrane, respectively. Thus, this can be considered as one example of PSR to retain duplicated genes. However, this process may not have occurred in the five Leishmania species and L. seymouri. Without an outgroup paralogous gene or a species with a single FCaBP gene, it is difficult to conclude whether this represents neolocalization or sublocalization. However, sublocalization is unlikely due to the absence of FCaBP2 in the aforementioned species. Since neolocalization to the flagellum requires co-evolution with the BBSome complex, the chance of this occurring is quite low. Therefore, we like to propose that neolocalization to the entire body membrane has occurred, and FCaBP2 in the plasma membrane of the cell body must provide some advantages for C. fasciculata, L. passim, C. bombi, C. expoeki, and L. pyrrhocoris.

We showed that S9 and S10 conserved among FCaBP1s across four trypanosomatid species are not only important for the efficient flagellar localization but also myristoylation and palmitoylation at G2 and C3. Similarly, T9 and Q10 of FCaBP2 appear to be essential for the acylation at the same amino acid residues (Fig 4B). Thus, myristoylation and palmitoylation at G2 and C3 in FCaBPs are likely to be context dependent influenced by the surrounding amino acid residues.

Interaction between LpFCaBP1 and the BBSome complex in L. passim

The flagellar localization of LpFCaBP1 may depend on the interaction between the N-terminal sorting sequence and the BBSome complex, rather than TULP (Fig 5). These results are consistent with the requirement of the BBSome complex for the exit of dually acylated phospholipase D to the cilia in Chlamydomonas [40]. Although we did not detect interaction between LpFCaBP2N16-UltraID and Flag-LpBBS1, this could be due to the mislocalization of LpFCaBP2N16-UltraID at the anterior end of L. passim. It remains to be determined whether the entry/exit of LpFCaBP1 to the flagellum requires the BBSome and IFT complexes.

Roles of FCaBPs in flagellar morphogenesis and motility of L. passim

We found that LpFCaBPs are not essential for the viability of L. passim. However, the loss of LpFCaBPs altered the growth characteristics of L. passim in the culture medium and impaired flagellar morphogenesis and motility. The formation of rosettes, which requires the clustering of parasites through flagellar movement [41], was also defective in LpFCaBPs-deleted parasites (Fig 7). In contrast, knocking down TbFCaBPs (Calflagin Tb17, Tb24, and Tb44) in T. brucei did not affect parasite growth and motility [20]. The effects of FCaBP loss-of-function may differ between monoxenous parasites like L. passim (infecting only honey bees) and dixenous parasites like T. brucei (infecting both tsetse flies and mammals). It is also possible that the small amount of TbFCaBPs remaining after gene knockdown [20] is sufficient to support the normal growth and motility of T. brucei in the culture medium. We also found that neither FCaBP1 nor FCaBP2 alone can fully rescue the phenotypes of LpFCaBPs-deleted parasites (Fig 7). Assuming that the amount of ectopic LpFCaBP1 or LpFCaBP2 protein is comparable to that of the endogenous protein, L. passim relies on both proteins with flagellar and entire body localizations. This is consistent with a PSR hypothesis [8,12]. FCaBP likely functions as a calcium signaling protein, and it will be important to uncover how downstream effectors are involved in the growth and flagellar formation of L. passim.

Roles of the flagellum in the host infection of L. passim

We observed that LpFCaBPs-deleted parasites can infect honey bee as effectively as wild-type parasites in our assay (Fig 7H). Although the mutant parasites are less active, they can reach the honey bee hindgut through normal gut flow. L. passim appears to attach to the hindgut wall through the structurally modified flagellum [42], and the short flagella of the mutant parasites would be sufficient for attachment. In contrast, TbFCaBPs-depleted T. brucei showed attenuated parasitemia in infected mice [20], but the underlying mechanism and the infection in the tsetse fly gut were not clarified. The precise in vivo roles of FCaBP in infecting insects and mammalian hosts remain to be answered.

Materials and methods

Establishing L. passim to express the GFP fusion proteins

To express the GFP fusion proteins LpFCaBP1-GFP, LpFCaBP2-GFP, and LpPFRP5-GFP, we amplified the complete ORFs of LpFCaBP1, LpFCaBP2, and LpPFRP5 genes using PCR with KOD-FX DNA polymerase (TOYOBO), L. passim genomic DNA, and the primer pairs: LpFCaBP1-5 and LpFCaBP1-3, LpFCaBP2-5 and LpFCaBP2-3, and LpPFRP5-5 and LpPFRP5-3. The PCR products were digested with XbaI, purified from the gel, and then cloned into the XbaI site of pTrex-n-eGFP plasmid DNA [43]. To construct plasmids expressing LpFCaBP1 and LpFCaBP2 chimeric GFP fusion proteins, we amplified DNA fragments encoding the N-terminal 16 or 28 amino acids of LpFCaBP1 and LpFCaBP2 using 1444F primer and LpFCaBP1-N16-Rev, LpFCaBP1-N28-Rev, LpFCaBP2-N16-Rev, or LpFCaBP2-N28-Rev primer. Similarly, we amplified DNA fragments encoding amino acids 17–154 or 29–154 of LpFCaBP1 and LpFCaBP2 using LpFCaBP-487R primer and LpFCaBP1-N17-For, LpFCaBP1-N29-For, LpFCaBP2-N17-For, or LpFCaBP2-N29-For primer. To make the four chimeras, two DNA fragments for the corresponding N-terminal and internal amino acids of LpFCaBP1 and 2 or vice versa were ligated by fusion PCR using 1444F and LpFCaBP-487R primers. The resulting fusion PCR products and plasmids expressing LpFCaBP1-GFP or LpFCaBP2-GFP were digested with BamHI and ligated. Plasmid DNAs expressing GFPs with N-terminal sorting sequences of LpFCaBP1, LpFCaBP1K11I, LpFCaBP1TQ, LpFCaBP2, LpFCaBP2I11K, LpFCaBP2SS, TcFCaBP, TbFCaBP, LdFCaBP1, CfFCaBP1, CfFCaBP2, or LsFCaBP1 were constructed by cloning the corresponding complementary oligonucleotides into the XbaI site of pTrex-n-eGFP.

We collected actively growing L. passim (4 × 10⁷), washed twice with 5 mL PBS, and then resuspended in 0.4 mL of Cytomix buffer without EDTA (20 mM KCl, 0.15 mM CaCl₂, 10 mM K₂HPO₄, 25 mM HEPES and 5 mM MgCl₂, pH 7.6) [44,45]. The parasites were electroporated twice (1 min interval) with 10 μg of each plasmid DNA constructed above using a Gene Pulser X cell electroporator (Bio-Rad) and cuvette (2 mm gap). We set the voltage, capacitance, and resistance at 1.5 kV, 25 μF, and infinity, respectively. The electroporated parasites were cultured in 4 mL of modified FP-FB medium [46], and then G418 (200 μg/mL, Sigma-Aldrich) was added after 24 h to select the G418-resistant clones. We washed live L. passim expressing the GFP fusion protein three times with 1 mL PBS, and then placed them on poly-L-lysine-coated slide glass for imaging using a NIKON eclipse Ni-U fluorescence microscope. The exposure time remained constant at 200 msec. Using Image-J, we measured the mean fluorescence of GFP in both the entire flagellum and cell body of individual parasites, as well as the background. Then, we calculated the ratio between the fluorescence in the flagellum and the cell body after subtracting the background fluorescence. Statistical comparison was performed using the Steel test.

Analysis of microsynteny with FCaBP genes in trypanosomatid parasites

We used TBLASTN and LpFCaBP1 as a query to identify the genomic contigs containing FCaBP in trypanosomatid parasites. The order and orientation of genes in the microsynteny with FCaBP of five Leishmania species, L. seymouri, and L. pyrrhocoris were determined based on annotated gene lists. For L. passim, C. fasciculata, C. bombi, and C. expoeki, we performed the analysis using TBLASTN. The amino acid sequences of 11 FCaBPs (S1 Data) were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).

Assessing the interaction between the N-terminal sorting sequence of LpFCaBP1 or LpFCaBP2 and either LpBBS1 or LpTULP

We constructed plasmid DNA for expressing ultraID with the N-terminal sorting sequence of either LpFCaBP1 or LpFCaBP2. The DNA fragment encoding the sorting sequence was PCR amplified using 1444F and Ultra-Rev-LpFCaBP1 or 2 primers. We also PCR amplified the DNA fragment encoding ultraID using Ultra-For-LpFCaBP1 or 2 and Ultra-3-XhoI primers and used pSF3-ultraID [38] (ADDGENE: #172878) as a template. The two DNA fragments were ligated through fusion PCR followed by BamHI and XhoI digestion and cloned into the pTrex-n-eGFP plasmid at the same restriction enzyme sites. We first constructed plasmid DNA carrying triple Flag tags by inserting the annealed complementary oligonucleotides (3Flag-5 and 3Flag-3) into the XbaI and HindIII sites of tdTomato/pTREX-b [47] (ADDGENE: #68709). Then, we PCR amplified the entire ORFs of LpBBS1 and LpTULP (S2 Data) using LpBBS1-5-HindIII and LpBBS1-3-ClaI stop primers as well as LpTULP-5-HindIII and LpTULP-3-ClaI stop primers. After digesting with HindIII and ClaI, the PCR products were cloned in the same sites of the above plasmid DNA with Flag tags.

L. passim was electroporated as described above with 10 μg each of LpFCaBP1N16-UltraID or LpFCaBP2N16-UltraID and either Flag-LpBBS1 or Flag-LpTULP. The parasites expressing both proteins were selected using G418 and blasticidin (50 μg/mL, Macklin). We detected the expressed proteins by immunofluorescence. The parasites were washed and mounted on a poly-L-lysine-coated 8-well chamber slide as above, fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% TX-100 (PT), and blocked with PT containing 5% normal goat serum (PTG). The samples were incubated overnight at 4°C with rabbit anti-Myc (500-fold dilution for the ultraID fusion proteins) or rabbit anti-Flag epitope (500-fold dilution for the Flag-tagged proteins) polyclonal antibodies (Proteintech) in PTG. The samples were washed five times with PT (5 minutes for each wash) and then incubated with Alexa Fluor 488 (for the ultraID fusion proteins) or Alexa Fluor 555 (for the Flag-tagged proteins) anti-rabbit IgG antibody (ThermoFisher) for 2 hours at room temperature. After another round of washing, the samples were observed under a microscope.

We incubated the parasites (4 × 10⁸) in 20 mL culture medium with 50 μM biotin for 3 hours at 28°C, followed by washing with 5 mL PBS twice. The samples were then suspended in 2 mL lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA) containing a protease inhibitor cocktail (Beyotime) and sonicated three times (5 seconds each) at amplitude 10 using a Q700 sonicator (Qsonica). After centrifugation, the supernatants were collected, and 50 μL of streptavidin agarose (Yeasen) and 12.5 μL of DYKDDDDK (Flag) Fab-Trap Agarose (Chromotek) were added to the half volume of each sample, respectively. The beads were washed five times (5 minutes each) with washing buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% NP-40, 0.5 mM EDTA) and then suspended in 60 μL of SDS-PAGE sample buffer (2% SDS, 10% glycerol, 10% β-mercaptoethanol, 0.25% bromophenol blue, 50 mM Tris-HCl, pH 6.8). The samples were heated at 95°C for 5 minutes, and then 7.5 μL and 30 μL of each sample precipitated by Flag Fab beads and streptoavidin beads, respectively was applied to a 10% SDS-PAGE gel. The proteins were transferred to a nitrocellulose membrane (Pall Life Sciences) and the membrane was blocked with PBST (PBS with 0.1% Tween-20) containing 5% BSA at room temperature for 30 minutes. The membrane was incubated with a 1000-fold diluted anti-Flag epitope polyclonal antibody overnight at 4°C. After washing five times with PBST (5 minutes each), the membrane was incubated with a 10,000-fold diluted IRDye 680RD donkey anti-rabbit IgG (H+L) secondary antibody (LI-COR Biosciences) in PBST containing 5% skim milk at room temperature for 2 hours. Following another round of washing, the membrane was visualized using ChemiDoc MP (BioRad).

Deletion of LpFCaBP genes by CRISPR

To delete LpFCaBP1 and 2 genes, we designed the gRNA sequence using a custom gRNA design tool (http://grna.ctegd.uga.edu) [48]. Complementary oligonucleotides (0.1 nmole each) corresponding to the sgRNA sequences (LpFCaBP-For and LpFCaBP-Rev) were phosphorylated by T4 polynucleotide kinase (TAKARA), annealed, and cloned into BbsI-digested pSPneogRNAH vector [49]. We electroporated L. passim expressing Cas9 [39] with 10 μg of the constructed plasmid DNA and selected the transformants by blasticidin and G418 to establish parasites expressing both Cas9 and LpFCaBP gRNA.

For the construction of donor DNA for LpFCaBP genes, we performed fusion PCR of three DNA fragments: 5’UTR of LpFCaBP1 (574 bp, LpFCaBP1 5’UTR-F and LpFCaBP1 5’UTR-R), the ORF of the Hygromycin B phosphotransferase (Hph) gene derived from pCsV1300 [50] (1026 bp, LpFCaBP1 Hph-F and LpFCaBP2 Hph-R), and 3’UTR of FCaBP2 (590 bp, LpFCaBP2 3’UTR-F and LpFCaBP2 3’UTR-R). The fusion PCR products were cloned into the EcoRV site of pBluescript II SK(+) and the linearized plasmid DNA (10 μg) by HindIII was used for electroporation of L. passim expressing both Cas9 and LpFCaBP gRNA as described above.

After electroporation, L. passim resistant to blasticidin, G418, and hygromycin (150 μg/mL, Sigma-Aldrich) were selected, and single parasites were cloned by serial dilutions in a 96-well plate. The genotype of each clone was initially determined through the detection of 5’ wild-type (WT) and knock-out (KO) alleles for LpFCaBPs by PCR. After identifying heterozygous (+/-) and homozygous (-/-) KO clones, and their 5’WT (LpFCaBP1 5’UTR-Outer-F and LpFCaBP-152R), 5’KO (LpFCaBP1 5’UTR-Outer-F and Hyg-159R), 3’WT (LpFCaBP-538F and LpFCaBP2 3’UTR-Outer-R), and 3’KO (Hyg-846F and LpFCaBP2 3’UTR-Outer-R) alleles were confirmed by PCR using the specific primer sets.

RT-PCR

Total RNA was extracted from wild-type, LpFCaBPs heterozygous, and homozygous mutant parasites using TRIzol reagent (Sigma-Aldrich). Reverse transcription of 0.2 μg of total RNA was performed using ReverTra Ace (TOYOBO) and random primers, followed by PCR with KOD-FX DNA polymerase. To specifically detect LpFCaBP1 and 2 mRNAs, we designed reverse primers corresponding to the N-terminal sorting sequences (LpFCaBP1-36R and LpFCaBP2-36R). L. passim splice leader sequence (LpSL-F) was used as the forward primer. To detect LpGAPDH mRNA, we used LpGAPDH-F and LpGAPDH-R primers.

Culture, flagellar and cell body length measurement, and motility measurement of L. passim

To construct plasmid DNA expressing LpFCaBP1 or LpFCaBP2, we first PCR amplified the ORF using LpFCaBP1-5 and LpFCaBP1-3-XhoI or LpFCaBP2-5 and LpFCaBP2-3-XhoI primers. The plasmid DNA to express the corresponding GFP fusion protein was used as a template. We digested the resulting PCR products with XbaI and XhoI followed by subcloning into the same restriction enzyme sites of pTrex-n-eGFP plasmid DNA wherein neomycin resistance gene was replaced by bleomycin resistance gene. We electroporated LpFCaBPs homozygous mutant (LpFCaBP1/2 -/-) parasites with above plasmid DNA and the parasites expressing LpFCaBP1 or LpFCaBP2 were selected using hygromycin and zeocin (50 μg/mL, InvivoGen).

For culture, flagellar and cell body length measurement, and motility measurement, we inoculated wild-type, LpFCaBP1/2 -/-, and LpFCaBP1/2 -/- expressing either LpFCaBP1 or LpFCaBP2 parasites in the culture medium at 10⁵/mL at 29°C. The number of parasites was counted every 12 hours using a hemocytometer, and images of the cultured parasites were captured simultaneously for a week. We measured the length of both the flagellum and cell body of individual parasites at 60 hours after culture, utilizing phase images with Image-J. To record the movement of parasites, videos were created by taking images for 1 minute every second. The movement of parasites was tracked using TrackMate v7.10.2 [51] as a Fiji [52] plugin. The videos were imported to Fiji, converted to 8-bit grayscale, and the brightness and contrast were adjusted for better tracking. The Laplacian of Gaussian detector was used with an estimated object diameter of 30.0–36.0 pixels and a quality threshold of 0.2–0.5 for the detection of individual parasites. We used the Simple Linear Assignment Problem tracker to track the parasites by adjusting the linking maximum distance and gap-closing maximum distance to 35.0–200.0 pixels and gap-closing maximum frame gap to 1. We characterized above phenotypes with two independent clones of LpFCaBP1/2 -/- parasites (D1 and F6) and their phenotypes were the same. We thus used the clone D1 for further experiments.

Honey bee infection

To infect honey bees with L. passim, parasites collected during the logarithmic growth phase (5 × 10⁵/mL) were washed with PBS and suspended in sterile 10% sucrose/PBS at 5 × 10⁴/μL. Newly emerged honey bee workers were collected by placing the frames with late pupae in 33°C incubator and starved for 2–3 hours. Twenty individual honey bees were fed with 2 μL of the sucrose/PBS solution containing either wild-type or LpFCaBPs homozygous mutant (10⁵ parasites in total). The infected honey bees were maintained in metal cages at 33°C for 14 days and then frozen at -80°C. This experiment was repeated three times. We sampled eight honey bees from each of the above three experiments, and thus analyzed 24 honey bees in total infected with either wild-type or LpFCaBPs homozygous mutant. Genomic DNAs were extracted from the whole abdomens of individual bees using DNAzol reagent (Thermo Fisher). We quantified L. passim in the infected honey bee by qPCR using LpITS2-F and LpITS2-R primers which correspond to the part of internal transcript spacer region 2 (ITS2) in the ribosomal RNA gene. Honey bee AmHsTRPA was used as the internal reference using AmHsTRPA-F and AmHsTRPA-R primers [35]. The relative abundances of L. passim in the individual honey bees (24 each infected by wild-type or mutant L. passim) were calculated by the ΔC_t method and we set one sample infected by wild-type as 1. The statistical analysis was performed using the Brunner-Munzel test. All of the above primers are listed in S3 Data.

Supporting information

S1 Data. DNA and amino acid sequences of 18 FCaBPs as well as the aligned sequences.

(DOCX)

pgen.1011195.s001.docx^{(24.1KB, docx)}

S2 Data. DNA and amino acid sequences of three Trypanosoma theileri FCaBPs, LpPFRP5, LpBBS1, and LpTULP.

(DOCX)

pgen.1011195.s002.docx^{(18.8KB, docx)}

S3 Data. List of primers used in this study.

(DOCX)

pgen.1011195.s003.docx^{(23.5KB, docx)}

S4 Data. Ratios of mean GFP fluorescence of flagellum to that of cell body.

(XLSX)

pgen.1011195.s004.xlsx^{(21.4KB, xlsx)}

S5 Data. Numerical data shown in Fig 7.

(XLSX)

pgen.1011195.s005.xlsx^{(92.3KB, xlsx)}

S1 Video. Movement of wild-type L. passim.

(MP4)

Download video file^{(10.3MB, mp4)}

S2 Video. Movement of LpFCaBPs deleted mutant L. passim.

(MP4)

Download video file^{(31.1MB, mp4)}

S3 Video. Movement of LpFCaBPs deleted mutant L. passim expressing LpFCaBP1.

(MP4)

Download video file^{(15.1MB, mp4)}

S4 Video. Movement of LpFCaBPs deleted mutant L. passim expressing LpFCaBP2.

(MP4)

Download video file^{(13.7MB, mp4)}

Acknowledgments

We thank Yizhen Shao for his contribution to this study.

Data Availability

All data are available in the submitted Supplementary data files.

Funding Statement

This work was supported by Jinji Lake Double Hundred Talents Programme to TK. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Genet. doi: 10.1371/journal.pgen.1011195.r001

Decision Letter 0

Ashley Soyong Byun, Eva H Stukenbrock

12 Oct 2023

Dear Dr Kadowaki,

Thank you very much for submitting your Research Article entitled 'Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important and interesting problem, but raised some substantial concerns about the current manuscript. For example, while Reviewer 1 agreed that the data presented does support that LPFCaBP1 and 2 are differentially localized via PSR, there were concerns that the situation is more complex and that more functional data on the paralogs needs to be provided. Reviewer 2 acknowledged the high quality of the cell data but had concerns regarding the depth of the evolutionary analysis and discussion. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

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Yours sincerely,

Ashley Soyong Byun, PhD

Guest Editor

PLOS Genetics

Eva Stukenbrock

Section Editor

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Overview

This study seeks to characterises the location, localisation-determinants and function of two tandemly-duplicated genes in the trypanosomatid parasite of bees, Lotmaria passim, with the aim of understanding more about the processes driving/maintaining gene duplication and diversification. These proteins are highly similar, with the main differences occurring towards the N terminus, close to the myristate and palmitate addition signals. The authors express tagged proteins, and protein chimeras to demonstrate that the paralogs have different localisation distributions and that this is primarily conferred by the N terminal sequences. By expressing the N terminal peptide of the flagellar-localised paralog fused to an enzyme that performs promiscuous biotinylation followed by streptavidin purification of biotinylated proteins and western blot analysis, the authors present evidence that flagellar localisation of LpFCaBP1 is done via the N-terminal 16 residues interacting with interacting with BBS1. The authors perform KO experiments to show that although an effect is observed in vitro, KO parasites can still infect bees, suggesting a non-essential role in vivo.

Although the claim that the differential distributions of the two proteins is (predominantly) conferred by the N terminal sequences is solid, differential localisation of paralogs is not new and several of the descriptions and conclusions in the text do not tie with the data as shown in the Figures. Most importantly, however, the authors give little functional insights into these proteins, and the functional significance, if any, of their different localisation distributions is not investigated.

Main issues:

Line 126: “The N-terminal 16 amino acids of LpFCaBP1 and LpFCaBP2 were sufficient to direct GFP to the flagellum and cell body, respectively (Fig. 2)”.

Although this statement is justified by the experiment fusing the LmFCaBP1/2(1-16) to GFP, the other data appears to be more complex than the authors suggest in the text. For example, while the LpFCaBP1N28/FCaBP2-GFP does indeed appear to reproduce the LpFCaBP1-GFP full length protein, the LpFCaBP1N16/FcaBP2 does not, as this latter chimera appears to be missing from the cell-body membrane altogether. Would this not suggest that there is an additional signal in LpFCaBP1(N16-28) that facilitates some distribution to the cell-body membrane?

Additionally, both of the other chimeras (LpFCaBP2N28/FCaBP1-GFP and LpFCaBP2N16/FCaBP1-GFP) are missing from the flagellar membrane (at least in the example given), in contrast to FCaBP2-GFP. Would this not suggest that there are sequences in FCaBP2(16-204) that confer at least some flagellar membrane localisation?

Line 152 – 170

the authors express the N terminal region of a series of calmodulin homologs from different trypanosomatid species and note different localisation distributions. In particular, they note that expressing the N24 residues of Tb/TcFCaBP localises to the cell body membrane, and not the flagellar membrane as has been shown for the full length protein in T. brucei/cruzi. From this they conclude that a the default localisation is the cell body membrane, and that a trans-acting factor causes localisation to the flagellar membrane. Beyond showing that the N terminal peptide clearly causes differences in localisation distribution (already known from Figure 2) I am unconvinced by this line of experiments and the conclusions that were drawn because of the biological irrelevance of drawing conclusions when expressing orthologs from diverse species and lack of controls (e.g. expression in native species, expression of full length protein). A more useful line of enquiry would have been to use these “natural mutation experiment” as a basis to perform directed mutagenesis to obtain mechanistic insight into the different sorting signals.

Figure 5 and line 171-186.

The authors show that BBS1 appears to be somewhat biotinylated by LpFCaBP1N16-UltraID. However, the authors do not show that the an interaction of LpFCaBP1N16 (or, indeed, LpFCaBP1N16) with the BBSome is necessary for its flagellar localisation, for example with BBS KO cells. We should also note that this experiment was only done with the N terminal peptide, which is less biologically realistic than the full length protein

Although using Flag-LpTULP adds an important control, the BBS1 band is not very convincing and this data would be strengthened using label-free using mass spectrometry to confirm BBS1 presence and relative abundance.

Note that biotinylation does not necessarily indicate “interaction” but could simply indicate proximity.

I would advise the authors to investigate any interaction of the BBSome with LpFCaBP2, because the BBSome has been implicated in removal of proteins from the flagellum

Figure 7 and line 187-214:

The authors investigate the functional characterises of KO parasites. Here I think the authors have missed a trick. Given the emphasis on understanding paralogs with different localisations, performing a addback with the different paralogs individually would seem to be a good way of getting at their potential different function.

Additionally, further analysis of the KO parasites seem warranted, for example performing TEM on flagellar cross sections to detect defects, and a microscopical analysis of the KO parasites from in vivo infections, as a PCR-based assay seems to be rather coarse and may have missed a phenotype.

Minor points:

Line 115: “These findings indicate that the two duplicated FCaBP genes encode proteins with distinct subcellular localizations”.

The localisations are actually the same (flagellar membrane and cell-body membrane), but the key factor is their relative enrichment on each plasma-membrane sub-domain. Suggest to re-phrase as “differentially enriched, but overlapping, distributions”, or something that conveys the same idea

The miscopy figures all need scale bars. In several instances the cells appear to be different magnifications – would recommend standardising the magnification within a Figure.

Unclear in some cases if it DOES localise a cell-body/flagellar membrane subdomain, but is difficult to discern because of the high levels of expression of the other subdomain (e.g. Figure 1 bottom two rows, but also perhaps in other places where there is strong expression to a particular subdomain). Can deal with this in a multiplicity of ways, but some quantification would be helpful

Figure 1:

FCaBP2-GFP needs a better example exhibiting a clear edge effect to show it is on the flagellar membrane

Provide clearer annotation for the unfamiliar reader to point out the flagellar membrane and cell body membrane

Figure 6

Can the authors clarify whether the screening primers are specific to each paralog?

Figure 7A and B:

Clarify on whether the growth curve data and statistics are based on technical or biological replicates. If technical replicates (i.e. the same experiment done in triplicate) then the very mild phenotype observed could be simply due to differences in the starting “health” of the cultures, and not reproducible.

Clarify how the length of the flagellum was calculated (e.g. measured from phase images?)

Reviewer #2: In the trypanosomatid parasites gene duplications are common and likely represent the evolution of proteins with novel functions. In this manuscript the authors investigate the duplication of a calcium binding protein in Lotmaria passim. They demonstrate that one protein localises to the flagellum and the other to the cell body and that one or the other are required for flagellum assembly and function. Overall, the cell biology data presented is of a high quality and generally supports the conclusions the authors make; however, the evolutionary analysis focussed on a restricted range of organisms, with limited discussion.

Abstract ln 30/31 – the authors do not present any data to show that the Tbrucei and Ldonovani FCaBPs do not interact with the BBSome. These leader sequences do not direct GFP to the Lpassim flagellum but that might not be due to the lack of BBSome interaction. Therefore, it is a stretch to conclude that this is due specific co-evolution between the Lotmaria BBSome and FCaBP. To really conclude this, they would need to demonstrate interaction of paired FCaBPs with BBSomes i.e. does Tbrucei FCaBP interact with Tbrucei BBSome in Tbrucei.

This point leads onto my concern with the genome/sequence analysis in this paper, which could be improved. The authors look at the conservation/synteny etc. in a range of organisms but these are all relatively closely related – are FCaBP found in more distantly related organisms such as Paratrypanosoma or Bodo saltans? If so, does that help refine the relationship between the different species?

Have the authors examined the phylogenetic relationships between these genes? Do they match the speciation of these organisms? Can this help to inform the analysis? The authors rely on the reference genomes for these organisms as presented but are they sure that the loss of FCaBP2 in Lseymouri is real and not an issue with genome assembly/annotation – is there a pseudogene downstream of FCaBP1 in this organism? In Lmexicana etc FCaBP2 is described as a pseudogene but the genome describes this as a normal protein-coding gene just without the N-terminus found in other species and the transcriptomic evidence suggests it is expressed. This protein may be perfectly functional and in fact might have a different localisation and represent a specific evolution event in these species. Where does this protein localise? In Lmajor both genes appear to pseudogenes but is this backed up by transcriptomic datasets? I would urge caution of taking what is presented in genomes at face value, especially if it looks different to what is seen in other organisms – it maybe true but might be an issue with the automated assembly of the genome.

Does the alignment of these leader sequences suggest what is important for flagellum retention/exclusion – the dipeptide SS – TQ could be potentially important? Is this a charge effect there are lots of lysines, which are supposed to be important for flagellum localisation in Tbrucei/Tcruzi but those that do not localise to the Lpassim flagellum have a number of asparagine/glutamine residues in the leader sequence – is there any pattern in the overall charge of these sequences and their localisation?

Minor comments

Need to be specific with descriptions of localisation – presumably the proteins localise to the flagellum membrane and cell body membrane, not the cell body.

The localisation of PFR5 shown here is not how a tagged PFR protein would normally look like. Is the PFR discontinuous in Lotmaria?

Can you include geneIDs in the figure legends so it is easier to find the genes they are examining.

What do the colours represent in figure 3?

Ln169/170 – the authors suggest that flagellum localisation requires sorting into the flagellum but this could equally be about retrieval of FCaBP2 from the flagellum – additional nuances should be discussed about what might define the localisation of a protein.

There was a limited information about the blots in figure 5. What was the loading of each lane, how many cell equivalents? What is the relationship between the input and streptavidin material? Without this information it is hard to judge how strong/likely this interaction is. In the discussion, there is a caveat about the weak signal detected here. Additional evidence to show this was a reciprocal interaction with FCaBP1 detected when BBSome1 was tagged with ultraID would increase confidence in this conclusion.

The functional analysis used a cell line in which both FCaBP genes had been deleted. How many clones were examined? Can you add this information to the legends etc? By deleting both genes, it is not possible to determine the relative contribution of each gene to the phenotype. Have the authors deleted each gene individually? Could they add back FCaBP1 into the deletion line and see if this restores the phenotype? Without this, it is not possible to determine if these proteins actually have separate functions as would be suggested by the PSR hypothesis.

Ln261/262 – Lmajor and Lpassim have evolved separately for millions of years and have very different life cycles etc, so I think the dispensability of FCaBP in Lpassim is likely unrelated to the ability of Lmajor to survive without these genes.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: No

PLoS Genet. 2024 Mar 4;20(3):e1011195. doi: 10.1371/journal.pgen.1011195.r002

Author response to Decision Letter 0

18 Jan 2024

Attachment

Submitted filename: Response to reviewers.docx

pgen.1011195.s010.docx^{(1.5MB, docx)}

PLoS Genet. doi: 10.1371/journal.pgen.1011195.r003

Decision Letter 1

Ashley Soyong Byun, Eva H Stukenbrock

14 Feb 2024

Dear Dr Kadowaki,

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers remarked that the manuscript was greatly improved through your addition of data and more thorough analyses but identified some minor concerns that we ask you address in a revised manuscript.

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

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We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Ashley Soyong Byun, PhD

Guest Editor

PLOS Genetics

Eva Stukenbrock

Section Editor

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have done a substantial amount of work and the manuscript is greatly improved.

Revisions:

Line 343: “We also found that either FCaBP1 or FCaBP2 alone can not fully rescue the phenotypes of LpFCaBPs-deleted parasites (Fig. 7), suggesting that L. passim depends on both proteins with the flagellar and entire body localization”

The authors should acknowledge (or rule out if they have data to indicate otherwise, such as a western blot) that the inability to fully rescue the KO phenotype could be because the individual FCaBP1 or FCaBP2 addbacks do not express as highly as the combined FCaBP1/ FCaBP2 in the wild type.

The microscopy still needs scale bars

Reviewer #2: On the whole the authors have suitably addressed my concerns. They have provided a more thorough genomic analysis and have provided additional functional data by examining LpFCaBP1/2 add back parasites to define their relative roles. However, while the inclusion of the enrichment analysis has improved the manuscript and helps to interpret their findings they need to more clearly explain the results and provide more details about the methodology.

More detail about measurement of fluorescence needed – was there a background correction? Was all the cell body/flagellum measured or just a part of it? What microscope was used? Was a consistent exposure time used etc.?

Figure 1 - If the ratio of LpFCaBP2 between flagellum and cell body is 0.92±0.28 then it is essentially evenly distributed between the flagellum and cell body and at the moment the manuscript suggests that it is enriched in the cell body.

Figure 2 - For the LpFCaBP1N16/FCaBP2-GFP fusion how was the measurement of the flagellum fluorescence intensity performed was this the entire flagellum or just the proximal domain? On a related note this localisation pattern could represent an enrichment within the flagellar pocket and pocket neck membranes which constitute another membrane domain in these parasites, rather than the proximal domain of the flagellum.

Figure 2 - The authors need to describe the localisation pattern more clearly. The enrichment analysis shows no difference but the LpFCaBP1N16/FCaBP2-GFP shows an enrichment in the proximal flagellum region whereas the LpFCaBP1N28/FCaBP2-GFP is enriched along the entire flagellum as is the LpFCaBP1N16-GFP fusion and this is not pointed out. Moreover, as the LpFCaBP2N16-GFP gives a different enrichment ratio to LpFCaBP2-GFP the authors are correct in stating that the EF hands likely have an influence on protein localisation but this does not come across well in the text. At what point in the proteins does the EF hand domain start? This should be indicated somewhere in the manuscript.

Line 146-9 – the authors should indicate that this protein would likely be in the cytosol as they have no evidence to indicate its localisation.

Figure 4 - As the SS/TQ mutants appear to be cytosolic the key conclusion which the authors make is that this seems to affect fatty acid modification but because of that the enrichment ratio is not overly informative as it is unlikely that the differential localisation is occurring through the same mechanism.

Line 190 – the conclusion here needs to be modified as the results show that K11 is not necessary for flagellum localisation of LpFCaBP1N16. It appears therefore to contribute to LpFCaBP2N16 flagellum localisation but is not necessary for LpFCaBP1N16 flagellum localisation.

Figure 4C – the authors point out the proximal flagellum enrichment of the Tc/Ld FCaBP leader sequences and again this relates to my comment about the flagellar pocket membrane domain that could be added to the discussion. The authors comment on the inefficient localisation (lines 298-300) but they should acknowledge the present of another membrane domain here as well.

Line 274 – can the authors clarify this sentence, it is unclear what they mean by function is the same as a calcium binding protein.

Finally, the language of the paper needs to be improved throughout and the manuscript should be read by a native speaker before publication.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Reviewer #1: Yes

Reviewer #2: No: The numerical data for the graphs in figure 7 and enrichment analysis was not provided.

**********

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Reviewer #2: No

PLoS Genet. 2024 Mar 4;20(3):e1011195. doi: 10.1371/journal.pgen.1011195.r004

Author response to Decision Letter 1

20 Feb 2024

Attachment

Submitted filename: Response to Reviewer 2nd.docx

pgen.1011195.s011.docx^{(20.6KB, docx)}

PLoS Genet. doi: 10.1371/journal.pgen.1011195.r005

Decision Letter 2

Ashley Soyong Byun, Eva H Stukenbrock

23 Feb 2024

Dear Dr Kadowaki,

We are pleased to inform you that your manuscript entitled "Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Comments from the reviewers (if applicable):

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PLoS Genet. doi: 10.1371/journal.pgen.1011195.r006

Acceptance letter

Ashley Soyong Byun, Eva H Stukenbrock

26 Feb 2024

PGENETICS-D-23-00696R2

Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite

Dear Dr Kadowaki,

We are pleased to inform you that your manuscript entitled "Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Data. DNA and amino acid sequences of 18 FCaBPs as well as the aligned sequences.

(DOCX)

pgen.1011195.s001.docx^{(24.1KB, docx)}

S2 Data. DNA and amino acid sequences of three Trypanosoma theileri FCaBPs, LpPFRP5, LpBBS1, and LpTULP.

(DOCX)

pgen.1011195.s002.docx^{(18.8KB, docx)}

S3 Data. List of primers used in this study.

(DOCX)

pgen.1011195.s003.docx^{(23.5KB, docx)}

S4 Data. Ratios of mean GFP fluorescence of flagellum to that of cell body.

(XLSX)

pgen.1011195.s004.xlsx^{(21.4KB, xlsx)}

S5 Data. Numerical data shown in Fig 7.

(XLSX)

pgen.1011195.s005.xlsx^{(92.3KB, xlsx)}

S1 Video. Movement of wild-type L. passim.

(MP4)

Download video file^{(10.3MB, mp4)}

S2 Video. Movement of LpFCaBPs deleted mutant L. passim.

(MP4)

Download video file^{(31.1MB, mp4)}

S3 Video. Movement of LpFCaBPs deleted mutant L. passim expressing LpFCaBP1.

(MP4)

Download video file^{(15.1MB, mp4)}

S4 Video. Movement of LpFCaBPs deleted mutant L. passim expressing LpFCaBP2.

(MP4)

Download video file^{(13.7MB, mp4)}

Attachment

Submitted filename: Response to reviewers.docx

pgen.1011195.s010.docx^{(1.5MB, docx)}

Attachment

Submitted filename: Response to Reviewer 2nd.docx

pgen.1011195.s011.docx^{(20.6KB, docx)}

Data Availability Statement

All data are available in the submitted Supplementary data files.

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PERMALINK

Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite

Xuye Yuan

Tatsuhiko Kadowaki

Roles

Abstract

Author summary

Introduction

Results

L. passim contains two FCaBPs with flagellar and entire body localizations

Fig 1. Subcellular localization of LpFCaBP1- and 2-GFP fusion proteins.

The N-terminal 16 amino acids of LpFCaBP function as either flagellum or cell body membrane sorting sequence

Fig 2. Flagellum and cell body membrane sorting sequences of LpFCaBP1 and 2.

FCaBP genes have rapidly evolved in trypanosomatid parasites

Fig 3. Conserved microsynteny of FCaBP genes in Leishmaniinae species.

Flagellar localization of FCaBP depends on both the sorting sequence and trans-acting factors in L. passim

Fig 4. Subcellular localization of GFPs with N-terminal sorting sequences of FCaBPs from various trypanosomatid parasites.

The N-terminal sorting sequence of LpFCaBP1 is in close proximity to the BBSome complex but not TULP

Fig 5. Interaction of LpFCaBP1 N-terminal sorting sequence with LpBBS1.

LpFCaBPs are essential for flagellar morphogenesis and motility but not the host infection of L. passim

Fig 6. Deletion of LpFCaBP1 and 2 genes by CRISPR.

Fig 7. Phenotypes of LpFCaBPs deleted mutant under culture conditions and in honey bees.

Discussion

Rapid evolution of FCaBP genes in trypanosomatid parasites

Interaction between LpFCaBP1 and the BBSome complex in L. passim

Roles of FCaBPs in flagellar morphogenesis and motility of L. passim

Roles of the flagellum in the host infection of L. passim

Materials and methods

Establishing L. passim to express the GFP fusion proteins

Analysis of microsynteny with FCaBP genes in trypanosomatid parasites

Assessing the interaction between the N-terminal sorting sequence of LpFCaBP1 or LpFCaBP2 and either LpBBS1 or LpTULP

Deletion of LpFCaBP genes by CRISPR

RT-PCR

Culture, flagellar and cell body length measurement, and motility measurement of L. passim

Honey bee infection

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Ashley Soyong Byun

Eva H Stukenbrock

Roles

Author response to Decision Letter 0

Decision Letter 1

Ashley Soyong Byun

Eva H Stukenbrock

Roles

Author response to Decision Letter 1

Decision Letter 2

Ashley Soyong Byun

Eva H Stukenbrock

Roles

Acceptance letter

Ashley Soyong Byun

Eva H Stukenbrock

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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