UNIVERSITAR DEGLI STUDI DI SASSARI SCUOLA DI DOTTORATO IN SCIENZE BIOMOLECOLARI E BIOTECNOLOGICHE (Intenational PhD School in Biomolecular and Biotechnological Sciences) Indirizzo: Microbiologia molecolare e clinica Molecular identification and evolution of protozoa belonging to the Parabasalia group and the genus Blastocystis Direttore della scuola: Prof. Masala Bruno Relatore: Prof. Pier Luigi Fiori Correlatore: Dott. Eric Viscogliosi Tesi di Dottorato : Dionigia Meloni XXIV CICLO Nome e cognome: Dionigia Meloni Titolo della tesi : Molecular identification and evolution of protozoa belonging to the Parabasalia group and the genus Blastocystis Tesi di dottorato in scienze Biomolecolari e biotecnologiche. Indirizzo: Microbiologia molecolare e clinica Universit degli studi di Sassari UNIVERSITAR DEGLI STUDI DI SASSARI SCUOLA DI DOTTORATO IN SCIENZE BIOMOLECOLARI E BIOTECNOLOGICHE (Intenational PhD School in Biomolecular and Biotechnological Sciences) Indirizzo: Microbiologia molecolare e clinica Molecular identification and evolution of protozoa belonging to the Parabasalia group and the genus Blastocystis Direttore della scuola: Prof. Masala Bruno Relatore: Prof. Pier Luigi Fiori Correlatore: Dott. Eric Viscogliosi Tesi di Dottorato : Dionigia Meloni XXIV CICLO Nome e cognome: Dionigia Meloni Titolo della tesi : Molecular identification and evolution of protozoa belonging to the Parabasalia group and the genus Blastocystis Tesi di dottorato in scienze Biomolecolari e biotecnologiche. Indirizzo: Microbiologia molecolare e clinica Universit degli studi di Sassari Abstract My thesis was conducted on the study of two groups of protozoa: the Parabasalia and Blastocystis . The first part of my work was focused on the identification, pathogenicity, and phylogeny of parabasalids. We showed that Pentatrichomonas hominis is a possible zoonotic species with a significant potential of transmission by the waterborne route and could be the aetiological agent of gastrointestinal troubles in children. We also confirmed the frequent presence of Trichomonas tenax and Trichomonas vaginalis outside their natural habitats in humans such as in the lungs and intestinal tract. In parallel, in complement to the SSU rRNA gene, we inferred the phylogenetic relationships among parabasalids on the basis of protein sequences (GAPDH, actin, EF1a). Our data indicated that increasing the number of taxa as well as the addition of new molecular markers greatly improved the robustness of the parabasalid tree and suggested that the ancestral parabasalid cell exhibited a morphologically simple structure. In the second part of my work, the first molecular epidemiological surveys of human Blastocystis isolates were conducted in Italy and Lebanon. The frequency of each Blastocystis subtype was determined in both populations. In addition, we first demonstrated mixed intra- and inter-subtype infections with Blastocystis in a same individual exhibiting a high potential risk of mixed infections with this parasite according to her lifestyle in rural area and long history of travelling. Nome e cognome: Dionigia Meloni Titolo della tesi : Molecular identification and evolution of protozoa belonging to the Parabasalia group and the genus Blastocystis Tesi di dottorato in scienze Biomolecolari e biotecnologiche. Indirizzo: Microbiologia molecolare e clinica Universit degli studi di Sassari Riassunto La mia tesi si basa sullo studio di due gruppi di protozoi: Parabasalia e Blastocystis. La prima parte del mio lavoro si focalizzata sull'identificazione, la patogenicit e filogenesi dei Parabsalia. Abbiamo dimostrato che Pentatrichomonas hominis una possibile specie zoonotica con un significativo potenziale di trasmissione attraverso l'acqua e che potrebbe essere l'agente eziologico dei problemi gastrointestinali nei bambini. Abbiamo anche confermato che, nellRuomo, Trichomonas tenax e Trichomonas vaginalis sono presenti con elevata frequenza al di fuori dei loro habitat naturali, come nei polmoni e nel tratto intestinale. Inoltre, a complemento del gene SSU rRNA, abbiamo dedotto le relazioni filogenetiche tra i parabasalidi sulla base di altre sequenze proteiche (GAPDH, actina, EF1a). I nostri dati mostrano che l'aumento del numero dei taxa cos come l'aggiunta di nuovi marcatori molecolari migliorano notevolmente la solidit dellR albero dei parabasalidi e ci suggeriscono che la cellula ancestrale dei Parabasalia mostrava una struttura morfologicamente semplice. Nella seconda parte del mio lavoro, sono stati condotti i primi studi epidemiologico- molecolari per individuare la presenza di Blastocystis in Italia e Libano. In entrambe le popolazioni stata determanata la frequenza di ciascun sottotipo di Blastocystis . Inoltre, per la prima volta, abbiamo dimostrato la presenza di infezioni miste intra-e inter-sottotipo di Blastocystis in un medesimo individuo che presenta un elevato rischio di infezioni da parte di questo parassita, a causa del suo stile di vita, come il vivere in una zona rurale e il viaggiare frequantemente. Nome e cognome: Dionigia Meloni Titolo della tesi : Molecular identification and evolution of protozoa belonging to the Parabasalia group and the genus Blastocystis Tesi di dottorato in scienze Biomolecolari e biotecnologiche. Indirizzo: Microbiologia molecolare e clinica Universit degli studi di Sassari Summary Introduction GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG...1 I Introduction GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG2 Literature Review GGGGGGGGGGGGGGGGGGGGGGGGGGGGG..4 II Parabasalia: a complex assemblage of diverse species of protozoa GGGGGGGG.5 II-1 Diversity and sizeGGGGGGGGGGGGGGGGGGGGGGGGGGGG5 II-2 Cytoskeleton of ParabasaliaGGGGGGGGGGGGGGGGGGGG..GG.5 II-2-1 Microtubular structuresGGGGGGGGGGGGGGGGGGGGGGG...5 II-2-2 Microfibrillar structuresGGGGGGGGGGGGGGGGGGGGGGG..8 II-3 Division and hydrogenosomesGGGGGGGGGGGGGGGGGGGGG.11 II-4 Habitat, pathogenicity and treatmentGGGGGGGGGGGGGGGGGG...14 II-5 Unusual location of trichomonads and pulmonary trichomonosisGGGGGGG...18 II-6 Genome of Trichomonas vaginalis GGGGGGGGGGGGGGGGGGG..19 III Emergence of Parabasalia in eukaryogenesis GGGGGGGGGGGGGGGG20 IV Systematic and evolution of Parabasalia GGGGGGGGGGGGGGGGGG21 IV-1 Based on analysis of morphological charactersGGGGGGGGGGGGGGG..21 IV-2 Based on the analysis of SSU rRNA gene sequencesGGGGGGGGGGGGG.23 IV-3 Based on the analysis of protein sequencesGGGGGGGGGGGGGGGGG27 IV-4 The quest of the root of ParabasaliaGGGGGGGGGGGGGGGGGGGG29 V Blastocystis sp.: an enigmatic Stramenopile GGGGGGGGGGGGGGGGG.29 V-1 Classification, morphology and life cycleGGGGGGGG.........................................29 V-2 IdentificationGGGGGGGGGGGGGGGGGGGGGGGGGGGGG.30 V-3 Genetic diversity...............................................................................................................32 V-4 EpidemiologyGGGGGGGGGGGGGGGGGGGGGGGGGGGGG32 V-5 Predominance of ST3 in the human population and zoonotic potential and transmission of this parasiteGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG..35 V-6 Clinical aspects of blastocystosisGGGGGGGGGGGGGGGGGGGGG..36 V-7 Development of animal models of blastocystosis.............................................................37 V-8 In vitro studies and identification of potential virulence factors.......................................37 V-9 Treatment of blastocystosis...............................................................................................38 V-10 Genome sequenceGGGGGGGGGGGGGGGGGGGGGGGGGGG39 Results GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG..41 Part 1: Identification and Pathogenicity of trichomonadsGGGGGGGGGGGGG...42 I-1 IntroductionGGGGGGGGGGGGGGGGGGGGGGGGGGGGG43 I-2 ResultsGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG....43 I-3 Conclusions GGGGGGGGGGGGGGGGGGGGGGGGGGGG...44 Part 2: Evolution and systematics of parabasalidsGGGGGGGGGGGGGGGG..49 II-1 IntroductionGGGGGGGGGGGGGGGGGGGGGGGGGGGG..50 Nome e cognome: Dionigia Meloni Titolo della tesi : Molecular identification and evolution of protozoa belonging to the Parabasalia group and the genus Blastocystis Tesi di dottorato in scienze Biomolecolari e biotecnologiche. Indirizzo: Microbiologia molecolare e clinica Universit degli studi di Sassari II-2 ResultsGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG..50 II-3 Conclusions GGGGGGGGGGGGGGGGGGGGGGGGGGGG.50 Part 3: Molecular epidemiology of Blastocystis sp. in Italy and LebanonGGGGGGG.76 III-1 IntroductionGGGGGGGGGGGGGGGGGGGGGGGGGGGG77 III-2 ResultsGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG77 III-3 Conclusions GGGGGGGGGGGGGGGGGGGGGGGGGGG...78 Part 4: Multiple coinfections with Blastocystis sp. in a French patientGGGGGGGG..80 IV-1 IntroductionGGGGGGGGGGGGGGGGGGGGGGGGGGG.....81 IV-2 ResultsGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG.81 IV-3 Conclusions GGGGGGGGGGGGGGGGGGGGGGGGGGGG81 General conclusions and perspectives ....................................................................................93 I- Pentatrichomonas hominis : pathogenicity and transmission................................................94 II- Trichomonads colonize different unusual sitesGGGGGGGGGGGGGGGG...95 III- Phylogenies of parabasalids based on the concatenation of sequences of different markersGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG98 IV- Molecular epidemiology of Blastocystis sp. in Italy and Lebanon...................................99 V- Mixed infections with Blastocystis sp. isolates..................................................................101 References ..............................................................................................................................103 Appendix ................................................................................................................................119