Biology subjects

Gluenz, E.

Publications and source records attributed to Gluenz, E..

11 recordsLinked to original sources

bioRxiv2026

Vacuolar type H+ ATPase is involved in stress responses in Leishmania mexicana by regulating the lysosomal pH

Vacuolar H+ ATPases (v-ATPases) are conserved proton pumps that support diverse biological functions through acidification of cellular organelles. The protozoan parasite Leishmania requires its v-ATPase for survival in the sand fly vector and mammalian host, but genetic mutants remain viable in vitro. To gain further insight into this conditionally lethal phenotype, we first mapped organellar localization of the v-ATPase by co-localisation imaging of fluorescently tagged v-ATPase subunits and organelle markers. The v-ATPase signal was strongest in the flagellar pocket region, consistent with enrichment in the contractile vacuole complex (CVC). To define the conditions that require a functional v-ATPase, deletion mutants were exposed to different stresses (pH, temperature, osmolality, dense culture). All tested deviations from standard culture conditions affected the mutants' growth rate, viability or both. Despite differences in phenotype severity, all stressors triggered the formation of a large autolysosome, positive for the autophagy marker protein ATG8 and the lysosomal enzyme cysteine peptidase A, indicating an arrest at the final step of autophagy. Measurements with the pH sensor pHLuorin2 showed that the luminal pH of the lysosomes was 5.6 in unperturbed promastigotes and 7.1 in v-ATPase mutants. These data support a canonical function for the Leishmania v-ATPase in lysosome acidification and autophagy, which is essential for parasite differentiation, and identify the poorly characterized Leishmania CVC as another major site of v-ATPase concentration.

cell biology↗

bioRxiv2025

Axonemal dynein contributions to flagellar beat types and waveforms

Eukaryotic flagella, or motile cilia, are iconic molecular machines whose beating drives cell propulsion and fluid transport across diverse organisms. Beat type and waveform are tailored to function, differing between species and cell types, and individual flagella can switch between beat types. Aberrant beating causes ciliopathies and infertility in humans1 and prevents unicellular parasite transmission2. Eight distinct dynein motor protein complexes bind to axonemal doublet microtubules (DMTs) within flagella and drive beating, yet despite extensive structural analysis3-5, how this machinery achieves different beat types is unknown. Here, using the flagellate unicellular parasite Leishmania, we show a division of labour where specific dyneins drive specific beat types. Using cryo-EM, we determined the structure of the 96-nm repeat unit of the DMT and identified its dynein composition. We used CRISPR-Cas9 to systematically delete all 96-nm repeat proteins, comprehensively mapping necessity for swimming, and determined the contribution of each dynein to incidence and waveform of the preferred beat types. Outer dynein arms (ODAs) were required for symmetric tip-to-base beats, specific single-headed inner dynein arms (IDAs) were important for asymmetric base-to-tip beats (IDAd), and double-headed IDAf important for both. This systematic analysis indicates that the prevailing dogma that ODAs drive and IDAs shape the beat6-9 is either incomplete or not universal, and establishes new hypotheses for how different species, cell types and individual flagella achieve their necessary beat types.

biophysics↗

bioRxiv2025

Identification of transporters essential for survival of Leishmania promastigotes in the digestive tract of sand flies

Leishmania amastigotes ingested by female phlebotomine sand flies are exposed to a harsh and dynamic environment that differs markedly from the intracellular niche in the mammalian host in temperature, pH and nutrient availability. Membrane transporter proteins, channels and pumps play a crucial role in maintaining cellular physiology under changing environments. A systematic loss-of-function screen of the L. mexicana transporter deletion mutants in macrophage and mouse infections previously identified transporter genes important for the amastigote stage. To test which transporters are important for the promastigote stage in the insect vector, we measured the fitness of gene deletion mutants in Lu. longipalpis sand flies. Pooled libraries of different complexities, consisting of 71 to 317 barcoded parasite lines allowed for an estimation of the bottleneck size in experimental infections, providing a foundation for similar experimental bar-seq studies. The fitness of each mutant parasite line was measured by tracking population composition over a course of 9 days in the sand flies and compared with the growth fitness of promastigotes over 7 days in laboratory cultures. There was a high correlation of fitness scores in vitro and in vivo, but 34 mutants showed a loss of fitness only in vivo, including deletion mutants of vacuolar H+ ATPase (V-ATPase) subunits. V-ATPase deletion mutants expressed low levels of the metacyclic-specific transcript sherp in vitro and failed to generate metacyclic promastigotes in sand flies, indicating that V-ATPase function is required for parasite differentiation and progression through the Leishmania life cycle. Author SummaryLeishmania parasites cause leishmaniases - a group of neglected tropical diseases that affect millions of people worldwide. These parasites must survive in two radically different environments: inside a mammalian host and within the gut of a blood-feeding sand fly. To thrive in the sand fly, Leishmania undergo extensive physiological changes and depend on transporter proteins to move nutrients and other molecules across their cell membranes. In this study, we focused on identifying which of these transporters are critical for the parasites survival inside the sand fly. We used a library of genetically engineered Leishmania promastigotes - the parasite form adapted to the insect vector - to assess the importance of more than 300 different transporter genes. We discovered that 34 of these transporters are essential for successful colonization of the sand fly. Among them, one key protein complex - the vacuolar H+ ATPase (V-ATPase) pump - was found to be crucial for parasite survival in the insect vector. Our findings deepen our understanding of how Leishmania adapts to life within the sand fly and highlight potential molecular targets for disrupting its transmission.

cell biology↗

bioRxiv2024

IFT and BBSome proteins are required for Leishmania mexicana pathogenicity, but flagellar motility is dispensable

Protists of the order Kinetoplastida possess a single multifunctional flagellum, which powers cellular displacement and mediates attachment to tissues of the arthropod vector. The kinetoplastid flagellar cytoskeleton consists of a nine-microtubule doublet axoneme; further structural elaborations, which can vary between species and life cycle stages, include the assembly of axonemal dynein complexes, a pair of singlet microtubules and the extra-axonemal paraflagellar rod. The intracellular amastigote forms of Leishmania spp. build a short, non-motile cilium whose function has remained enigmatic. Here we used a panel of 25 barcoded promastigote cell lines, including mutants lacking genes encoding flagellar assembly proteins, cytoskeletal proteins required for normal motility, or flagellar membrane proteins to examine how these defects impact on their virulence in macrophages and mice. Mutants lacking intraflagellar transport (IFT) protein 88 were severely attenuated indicating that assembly of a flagellum is necessary to allow for Leishmania survival in a mammalian host. A similarly severe loss of virulence was observed upon deletion of BBS2, a core component of the BBSome complex, which may act as a cargo adapter for IFT. By contrast, promastigotes that were unable to beat their flagella due to loss of PF16 could establish an infection and only showed a small reduction of parasite burden in vivo compared to the parental cell lines. These results confirm that flagellar motility is not necessary for mammalian infection but flagellum assembly and the integrity of the BBSome are essential for pathogenicity.

cell biology↗

bioRxiv2024

TransLeish: Identification of membrane transporters essential for survival of intracellular Leishmania parasites in a systematic gene deletion screen

For the protozoan parasite Leishmania, completion of its life cycle requires sequential adaptation of cellular physiology and nutrient scavenging mechanisms to the different environments of a sand fly alimentary tract and the acidic mammalian host cell phagolysosome. Transmembrane transporters are the gatekeepers of intracellular environments, controlling the flux of solutes and ions across membranes. To discover which transporters are vital for survival as intracellular amastigote forms, we carried out a systematic loss-of-function screen of the L. mexicana transportome. A total of 312 protein components of small molecule carriers, ion channels and pumps were identified and targeted in a CRISPR-Cas9 gene deletion screen in the promastigote form, yielding 188 viable null mutants. Forty transporter deletions caused significant loss of fitness in macrophage and mouse infections. A striking example is the Vacuolar H+ ATPase (V-ATPase), which, unexpectedly, was dispensable for promastigote growth in vitro but essential for survival of the disease-causing amastigotes.

cell biology↗

bioRxiv2023

Divergent Protein Kinase A contributes to the regulation of flagellar waveform of the motile flagellum of Leishmania mexicana

The second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) have been implicated in the regulation of flagellar motility in diverse eukaryotes, yet the specific contributions of PKA isoforms remain unclear. Moreover, the kinetoplastid PKA is insensitive to cAMP. Here we investigated subcellular localisations of PKA subunits in Leishmania and study the flagellar waveform parameters in PKA deletion mutants. Expansion microscopy localised LmxPKAR1 between the paraflagellar rod and the axoneme and LmxPKAC1, LmxPKAC2 depend on LmxPKAR1 for enrichment in the flagellum. LmxPKAC3 requires LmxPKAR3 for co-localisation at the cell cortex. Deletion of LmxPKAR3 resulted in increased flagellar LmxPKAC3 signal and increased swimming speed. Conversely, deletion of LmxPKAC3 resulted in reduced flagellar beat frequencies and swimming speed. The absence of LmxPKAC1 resulted in a lower incidence of high frequency tip-to-base waveforms, and consequently reduced swimming speed. LmxPKAC1 dissociated from cytoskeletons upon addition of guanosine, adenosine or inosine, consistent with a novel nucleoside-based PKA activation mechanism. These data suggest that this divergent PKA pathway regulates the motility of Leishmania through the modulation of flagellar waveforms.

cell biology↗

bioRxiv2022

ULK4 and Fused/STK36 interact to mediate assembly of a motile flagellum

Unc-51-like kinase (ULK) family serine-threonine protein kinase homologs have been linked to the function of motile cilia in diverse species. Mutations in Fused/STK36 and ULK4 in mice resulted in hydrocephalus and other phenotypes consistent with ciliary defects. How either protein contributes to the assembly and function of motile cilia is not well understood. Here we studied the phenotypes of ULK4 and Fused gene knockout (KO) mutants in the flagellated protist Leishmania mexicana. Both KO mutants exhibited a variety of structural defects of the flagellum cytoskeleton. Biochemical approaches indicate spatial proximity of these proteins and indicates a direct interaction between the N-terminus of LmxULK4 and LmxFused. Both proteins display a dispersed localisation throughout the cell body and flagellum, with enrichment near the flagellar base and tip. Fused/STK36 was previously shown to localise to mammalian motile cilia and we demonstrate here that ULK4 also localises to the motile cilia in mouse ependymal cells. Taken together these data suggest a model where the pseudokinase ULK4 is a positive regulator of the kinase Fused/STK36 in a pathway required for stable assembly of motile cilia. Summary StatementKnockout phenotypes in Leishmania, and confirmation of ULK4 ciliary localisation in mouse, show ULK4 and Fused/STK36 interact in a conserved pathway for stable assembly of motile cilia.

cell biology↗

bioRxiv2020

The single flagellum of Leishmania has a fixed polarisation of its asymmetric beat

Eukaryotic flagella undergo different beat types necessary for their function. The single flagellum on Leishmania parasites, for example, undergoes a symmetric tip-to-base beat for forward swimming and an asymmetric base-to-tip beat to rotate the cell. Asymmetric beats are most commonly associated with multi-ciliated tissues or organisms where the asymmetry has a constant polarisation. We asked whether this also holds for the single Leishmania flagellum. To do so, we used high frame rate dual colour fluorescence microscopy to visualise intracellular and intraflagellar structure in live swimming cells. This showed that the asymmetric Leishmania beat has a fixed polarisation. As in Chlamydomonas, this asymmetry arose from an asymmetric static curvature combined with a symmetric dynamic curvature. Some axoneme protein deletion mutants give flagella which retain static curvature, but lack dynamic curvature. We saw that these retain a fixed polarisation. Similarly, deletion mutants which disrupt vital asymmetric extra-axonemal and rootlet-like flagellum-associated structures also retain a fixed polarisation. This indicated that beat asymmetry does not originate from rootlet-like and extra-axonemal structures and is likely intrinsic to either the nine-fold rotational symmetry of the axoneme structure or due to differences between the outer doublet decorations.

cell biology↗

bioRxiv2020

Bar-seq strategies for the LeishGEdit toolbox

The number of fully sequenced genomes increases steadily but the function of many genes remains unstudied. To accelerate dissection of gene function in Leishmania spp. and other kinetoplastids we developed previously a streamlined pipeline for CRISPR-Cas9 gene editing, which we termed LeishGEdit [1]. To facilitate high-throughput mutant screens we have adapted this pipeline by barcoding mutants with unique 17-nucleotide barcodes, allowing loss-of-function screens in mixed populations [2]. Here we present primer design and analysis tools that facilitate these bar-seq strategies. We have developed a standalone easy-to-use pipeline to design CRISPR primers suitable for the LeishGEdit toolbox for any given genome and have generated a list of 14,995 barcodes. Barcodes and oligos are now accessible through our website www.leishgedit.net allowing to pursue bar-seq experiments in all currently available TriTrypDB genomes (release 41). This will streamline CRISPR bar-seq assays in kinetoplastids, enabling pooled mutant screens across the community. HighlightsO_LIDeveloping tools for pooled bar-seq mutant screens across the kinetoplastid community C_LIO_LIDevelopment of a standalone script to design primers suitable for the LeishGEdit toolbox C_LIO_LIGeneration of 14,995 barcodes that can be used for bar-seq strategies in kinetoplastids C_LIO_LIBar-seq primers for all TriTrypDB genomes (release 41) can be obtained from www.leishgedit.net C_LI

cell biology↗

bioRxiv2019

LAX28 is required for assembly of the inner dynein arm l1 and tether/tether head complex in the Leishmania flagellum

Motile eukaryotic flagella beat through coordinated activity of dynein motor proteins yet the mechanisms of dynein coordination and regulation are incompletely understood. The inner dynein arm IDA f/I1 complex and the tether/tether head (T/TH) complex are thought to be key regulators but, unlike IDA f/I1, T/TH proteins show limited conservation across flagellates. Here we characterised T/TH-associated proteins in the protist Leishmania mexicana. Proteome analysis of{Delta} CFAP44 mutant axonemes showed that they lacked IDA f/I1 protein IC140 and a novel 28-kDA axonemal protein, LAX28. Sequence analysis identified similarities between LAX28 and the uncharacterised human sperm tail protein TEX47, sharing features with sensory BLUF-domain proteins. Leishmania lacking LAX28, CFAP44 or IC140 retained some motility albeit with reduced swimming speed and directionality and a propensity for flagellar curling. Expression of tagged proteins in different null mutant backgrounds showed that the axonemal localisation of LAX28 requires CFAP44 and IC140, and the axonemal localisations of CFAP44 and IC140 both depend on LAX28. These data demonstrate a role for LAX28 in motility and show mutual dependencies of IDA f/1I and T/TH-associated proteins for axonemal assembly in Leishmania.\n\nSummary StatementThe inner dynein arm f/l1 complex is required for coordinating flagellar motility. Here we show that LAX28 is needed for its function and localization in the flagellum of Leishmania mexicana.

cell biology↗

bioRxiv2018

Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections

The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasites life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single mutant lacking the central pair protein PF16 showed that these paralysed promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.\n\nAuthor SummaryLeishmania are protozoan parasites, transmitted between mammals by the bite of phlebotomine sand flies. Promastigote forms in the sand fly have a long flagellum, which is motile and used for anchoring the parasites to prevent clearance with the digested blood meal remnants. To dissect flagellar functions and their importance in life cycle progression, we generated here a comprehensive list of >300 flagellar proteins and produced a CRISPR-Cas9 gene knockout library of 100 mutant Leishmania. We studied their behaviour in vitro before examining their fate in the sand fly Lutzomyia longipalpis. Measuring mutant swimming speeds showed that about half behaved differently compared to the wild type: a few swam faster, many slower and some were completely paralysed. We also found a group of uncoordinated swimmers. To test whether flagellar motility is required for parasite migration from the fly midgut to the foregut from where they reach the next host, we infected sand flies with a mixed mutant population. Each mutant carried a unique tag and tracking these tags up to nine days after infection showed that paralysed and uncoordinated Leishmania were rapidly lost from flies. These data indicate that directional swimming is important for successful colonisation of sand flies.

cell biology↗