The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum. These are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenesis of the anterior cell tip. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division.

• MeSH
• buněčná membrána MeSH
• cytokineze MeSH
• cytoskelet metabolismus   MeSH
• flagella fyziologie   ultrastruktura   MeSH
• interakce hostitele a parazita * MeSH
• Leishmania růst a vývoj   ultrastruktura   MeSH
• leishmanióza parazitologie   MeSH
• morfogeneze * MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

Leishmania kinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical for Leishmania pathogenicity. The Leishmania FP has a bulbous region posterior to the FP collar and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host-parasite interactions. Deletion of the FAZ protein, FAZ5, clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: Mutants were unable to develop late-stage infections in sand flies, and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity.

• Klíčová slova
• Leishmania, flagellar pocket, morphogenesis, pathogenicity,
• MeSH
• buněčná membrána metabolismus   MeSH
• cilie genetika   fyziologie   ultrastruktura   MeSH
• delece genu MeSH
• endocytóza MeSH
• flagella genetika   fyziologie   ultrastruktura   MeSH
• interakce hostitele a parazita MeSH
• Leishmania genetika   patogenita   fyziologie   ultrastruktura   MeSH
• mezibuněčné spoje MeSH
• myši MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• virulence genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

The human parasite Trypanosoma brucei does not synthesize heme de novo and instead relies entirely on heme supplied by its vertebrate host or its insect vector, the tsetse fly. In the host bloodstream T. brucei scavenges heme via haptoglobin-hemoglobin (HpHb) receptor-mediated endocytosis occurring in the flagellar pocket. However, in the procyclic developmental stage, in which T. brucei is confined to the tsetse fly midgut, this receptor is apparently not expressed, suggesting that T. brucei takes up heme by a different, unknown route. To define this alternative route, we functionally characterized heme transporter TbHrg in the procyclic stage. RNAi-induced down-regulation of TbHrg in heme-limited culture conditions resulted in slower proliferation, decreased cellular heme, and marked changes in cellular morphology so that the cells resemble mesocyclic trypomastigotes. Nevertheless, the TbHrg KO developed normally in the tsetse flies at rates comparable with wild-type cells. T. brucei cells overexpressing TbHrg displayed up-regulation of the early procyclin GPEET and down-regulation of the late procyclin EP1, two proteins coating the T. brucei surface in the procyclic stage. Light microscopy of immunostained TbHrg indicated localization to the flagellar membrane, and scanning electron microscopy revealed more intense TbHrg accumulation toward the flagellar pocket. Based on these findings, we postulate that T. brucei senses heme levels via the flagellar TbHrg protein. Heme deprivation in the tsetse fly anterior midgut might represent an environmental stimulus involved in the transformation of this important human parasite, possibly through metabolic remodeling.

• Klíčová slova
• differentiation, flagellum, heme, import, parasite, procyclin, transporter, trypanosome,
• MeSH
• biologický transport MeSH
• down regulace MeSH
• flagella metabolismus   MeSH
• hem metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• membránové transportní proteiny metabolismus   MeSH
• mikroskopie elektronová rastrovací MeSH
• moucha tse-tse parazitologie   MeSH
• proliferace buněk MeSH
• protozoální proteiny metabolismus   MeSH
• receptory buněčného povrchu metabolismus   MeSH
• RNA interference MeSH
• sekvence aminokyselin MeSH
• stadia vývoje MeSH
• transgeny MeSH
• Trypanosoma brucei brucei metabolismus   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hem MeSH
• hemoglobin-haptoglobin receptor MeSH Prohlížeč
• membránové transportní proteiny MeSH
• protozoální proteiny MeSH
• receptory buněčného povrchu MeSH

The kinetoplast (k), the uniquely packaged mitochondrial DNA of trypanosomatid protists is formed by a catenated network of minicircles and maxicircles that divide and segregate once each cell cycle. Although many proteins involved in kDNA replication and segregation are now known, several key steps in the replication mechanism remain uncharacterized at the molecular level, one of which is the nabelschnur or umbilicus, a prominent structure which in the mammalian parasite Trypanosoma brucei connects the daughter kDNA networks prior to their segregation. Here we characterize an M17 family leucyl aminopeptidase metalloprotease, termed TbLAP1, which specifically localizes to the kDNA disk and the nabelschur and represents the first described protein found in this structure. We show that TbLAP1 is required for correct segregation of kDNA, with knockdown resulting in delayed cytokinesis and ectopic expression leading to kDNA loss and decreased cell proliferation. We propose that TbLAP1 is required for efficient kDNA division and specifically participates in the separation of daughter kDNA networks.

• MeSH
• buněčný cyklus fyziologie   MeSH
• kinetoplastová DNA genetika   MeSH
• leucylaminopeptidasa genetika   metabolismus   MeSH
• mitochondriální DNA genetika   MeSH
• mitochondrie metabolismus   ultrastruktura   MeSH
• protozoální DNA genetika   MeSH
• protozoální proteiny metabolismus   MeSH
• replikace DNA fyziologie   MeSH
• Trypanosoma brucei brucei genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kinetoplastová DNA MeSH
• leucylaminopeptidasa MeSH
• mitochondriální DNA MeSH
• protozoální DNA MeSH
• protozoální proteiny MeSH