The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite’s 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 paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these 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.

Background: Rhamnose-containing glycans are involved in host-pathogen interactions. Results: UDP-rhamnose was identified in fungi; recombinant enzymes involved in its synthesis were characterized, and the genes involved are expressed in a tissue-specific manner. Conclusion: Fungi containing rhamnose likely utilize the UDP-rhamnose pathway. Significance: Understanding rhamnose pathways in fungi may provide new insight to fungus-host interaction.

The UDP-sugar interconverting enzymes involved in UDPGlcA metabolism are well described in eukaryotes but less is known in prokaryotes. Here we identify and characterize a gene (RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which encodes a dual function enzyme not previously described. One activity is to decarboxylate UDP-glucuronic acid to UDP-␤-Lthreo-pentopyranosyl-4؆-ulose in the presence of NAD ؉ . The second activity converts UDP-␤-L-threo-pentopyranosyl-4؆-ulose and NADH to UDP-xylose and NAD ؉ , albeit at a lower rate. Our data also suggest that following decarboxylation, there is stereospecific protonation at the C5 pro-R position. The identification of the R. solanacearum enzyme enables us to propose that the ancestral enzyme of UDP-xylose synthase and UDP-apiose/UDP-xylose synthase was diverged to two distinct enzymatic activities in early bacteria. This separation gave rise to the current UDP-xylose synthase in animal, fungus, and plant as well as to the plant Uaxs and bacterial ArnA and U4kpxs homologs.

23The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during 24 the parasite's life cycle from a long motile flagellum in promastigote forms in the sand fly to a 25 short immotile flagellum in amastigotes residing in mammalian phagocytes. This study 26 examined the protein composition and in vivo function of the promastigote flagellum. Protein 27 mass spectrometry and label free protein enrichment testing of isolated flagella and 28 deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms 29 2 (available via ProteomeXchange with identifier PXD011057). This information was used to 30 generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This 31 first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered 32 swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster 33 swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including 34 aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar 35 rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode 36 sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in 37 vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed 38 promastigotes and uncoordinated swimmers were severely diminished in the fly after 39 defecation of the bloodmeal. Subsequent examination of flies infected with a single mutant 40 lacking the central pair protein PF16 showed that these paralysed promastigotes did not 41 reach anterior regions of the fly alimentary tract. These data show that L. mexicana need 42 directional motility for successful colonisation of sand flies. 43 Author Summary 44Leishmania are protozoan parasites, transmitted between mammals by the bite of 45 phlebotomine sand flies. Promastigote forms in the sand fly have a long flagellum, which is 46 motile and used for anchoring the parasites to prevent clearance with the digested blood 47 meal remnants. To dissect flagellar functions and their importance in life cycle progression, 48we generated here a comprehensive list of >300 flagellar proteins and produced a CRISPR-49Cas9 gene knockout library of 100 mutant Leishmania. We studied their behaviour in vitro 50 before examining their fate in the sand fly Lutzomyia longipalpis. Measuring mutant 51 swimming speeds showed that about half behaved differently compared to the wild type: a 52 few swam faster, many slower and some were completely paralysed. We also found a group 53 of uncoordinated swimmers. To test whether flagellar motility is required for parasite 54 migration from the fly midgut to the foregut from where they reach the next host, we infected 55 sand flies with a mixed mutant population. Each mutant carried a unique tag and tracking 56 these tags up to nine days after infection showed that paralysed and uncoordinated 57 Leishmania were rapidly lost from flies. ...

Apiose is a branched monosaccharide that is present in the cell wall pectic polysaccharides rhamnogalacturonan II and apiogalacturonan and in numerous plant secondary metabolites. These apiose-containing glycans are synthesized using UDPapiose as the donor. UDP-apiose (UDP-Api) together with UDPxylose is formed from UDP-glucuronic acid (UDP-GlcA) by UDP-Api synthase (UAS). It was hypothesized that the ability to form Api distinguishes vascular plants from the avascular plants and green algae. UAS from several dicotyledonous plants has been characterized; however, it is not known if avascular plants or green algae produce this enzyme. Here we report the identification and functional characterization of UAS homologs from avascular plants (mosses, liverwort, and hornwort), from streptophyte green algae, and from a monocot (duckweed). The recombinant UAS homologs all form UDP-Api from UDP-glucuronic acid albeit in different amounts. Apiose was detected in aqueous methanolic extracts of these plants. Apiose was detected in duckweed cell walls but not in the walls of the avascular plants and algae. Overexpressing duckweed UAS in the moss Physcomitrella patens led to an increase in the amounts of aqueous methanol-acetonitrile-soluble apiose but did not result in discernible amounts of cell wall-associated apiose. Thus, bryophytes and algae likely lack the glycosyltransferase machinery required to synthesize apiose-containing cell wall glycans. Nevertheless, these plants may have the ability to form apiosylated secondary metabolites. Our data are the first to provide evidence that the ability to form apiose existed prior to the appearance of rhamnogalacturonan II and apiogalacturonan and provide new insights into the evolution of apiose-containing glycans.