Biotypizácia kvasiniek skupiny Cryptococcus laurentii hmotnostnou spektrometriou
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Vysoké učení technické v Brně. Fakulta chemická
Abstract
Cryptococcus laurentii je vo všeobecnosti považovaný za saprofitický druh, avšak už bolo hlásených niekoľko prípadov ochorení ľudí i zvierat spôsobených práve týmito kvasinkami. Jedná sa o heterogénnu skupinu kapsulárnych mikroorganizmov s nejednoznačne vymedze–nými taxonomickými hranicami. Na základe metód molekulárnej biológie bola skupina Cryptococcus laurentii rozdelená na dve fylogenetické skupiny, ktoré sa navzájom líšia. Je však pravdepodobné, že taxonómia tejto skupiny nie je definitívna a s pribúdajúcimi poznatkami sa bude meniť. Kvôli nejednoznačnému taxonomickému statusu kvasiniek Cryptococcus laurentii a ich možnej patogenite sme sa rozhodli biotypizovať kmene skupiny Cryptococcus laurentii pomocou MALDI – TOF hmotnostnej spektrometrie, ktorá je považovaná za rýchlu, spoľahlivú a dokonca i relatívne lacnú metódu, schopnú identifikovať mikroorganizmy i na úrovni variety. Na vybraných kmeňoch rodu Cryptococcus (Cr. neoformans, Cr. laurentii, Cr. magnus) zo Zbierky kultúr kvasiniek (CCY) bol optimalizovaný experimentálny postup MS analýzy pre tento rod. Zvolenou metódou bolo identifikovaných tridsaťtri kmeňov, pôvodne zaradených ako Cryptococcus laurentii. Na základe nameraných hmotnostných spektier boli tieto kmene prerozdelené do šiestich odlišných skupín. Z každej zo skupín bol vybraný aspoň jeden kmeň, u ktorého bola uskutočnená sekvenčná analýza D1/D2 domén LSU rRNA génu. Pomocou molekulárno biologickej analýzy bol zvolený kmeň identifikovaný a stal sa reprezentujúcim kmeňom danej skupiny. Skupinu I tvorí typový Cryptococcus laurentii CCY 17-3-2, ktorý vykazoval odlišné spektrum ribozomálnych proteínov ako ostatné biotypizované kmene. Dokonca i s kmeňmi identifikovanými ako Cryptococcus laurentii bola podobnosť spektier nízka, čo by mohlo byť zapríčinené odlišnou varietou. Skupinu II reprezentuje Cryptococcus laurentii CCY 17-3-17. Okrem neho bolo do skupiny II priradených ďalších 13 kmeňov. Skupinu III reprezentuje Cryptococcus flavescens CCY 17-3-29. Do tejto skupiny bolo zaradených ďalších 12 kmeňov s takmer identickým hmotnostným spektrom. Do skupiny IV bol zaradený len jeden kmeň (CCY 17-3-13), ktorý bol pomocou sekvenácie genómu identifikovaný ako Cryptococcus carnescens. Rovnako jedného zástupcu (CCY 17-3-5) má i skupina V. Kmeň CCY 17-3-5 bol identifikovaný ako Cryptococcus flavus. Posledná skupina VI má troch členov a reprezentuje ju kmeň 17-3-35 identifikovaný ako Bulleromyces albus. Kým Cr. laurentii a Cr. flavescens patria do I. fylogenetickej skupiny a Cr. carnescens do II. fylogenetickej skupiny „skupiny Cr. laurentii,“ identifikácia Cr. flavus a Bulleromyces albus viedla k vylúčeniu uvedených kmeňov z tejto „skupiny.“
Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human and animal infection have been already reported. This species is reported to be heterogenous. The taxonomy of yeast Cryptococcus laurentii was always highly ambiguous. The application of molecular biology and bioinformatic methods led to dividing of searched strains to two distinct phylogenetic groups, some varieties were recognized as species and the locution „Cryptococcus laurentii group“ was introduced. The taxonomy of this group is likely not definitive and with advancing knowledge will change. Our aim was the identification of individual species within this group based on matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI – TOF MS), which has recently been described as a rapid, reliable, cost-effective and powerful tool for analyzing of microorganisms, even on variety level. Generally, the yeasts of genus Cryptococcus form highly resistant polysaccharide capsules and produce large amount of extracellular polysaccharides, therefore belong to so called „difficult“ cases for biotyping. The experimental protocol has been optimized for MS analysis of this genus on the selected strains of Cr. neoformans, Cr. laurentii and Cr. magnus from the Culture Collection of Yeasts (CCY).Thirty-three strains, originally classified as Cryptococcus laurentii has been identified by chosen method. These strains were distributed into six different groups according their spectra similarities. It was selected at least one strain of each group, which was classified based on the sequence analysis of the D1/D2 domains of the LSU rRNA gene. This strain (with known sequence) became representative for its group. Type strain of Cryptococcus laurentii (CCY 17-3-2) belongs to the group I. Its MS spectrum of ribosomal proteins differ from mass spectra of all other biotyped species, even with strains identified as Cryptococcus laurentii was the similarity of the spectra low, which could be caused by identification of two different varieties. The group II is represented by Cryptococcus laurentii CCY 17-3-17. Except this strain, thirteen more strains belong to the group II. The group III represents Cryptococcus flavescens CCY 17-3-29. This group included 12 additional strains with almost identical mass spectra. Group IV included only one strain (CCY 17-3-13), which was identified as Cryptococcus carnescens based on gene sequence analysis. Similarly, one representative (CCY 17-3-5) has the group V. Strain CCY 17-3-5 was identified as Cryptococcus flavus. The last group VI of three members represents strain 17-3-35 identified as Bulleromyces albus. While Cr. laurentii and Cr. flavescens belong to phylogenetic group I and Cr. carnescens to the phylogenetic group II, four strains giving two types of different MS spectra and identified as Cr. flavus (1 strain) and Bulleromyces albus (3 strains) were excluded from „Cr. laurentii group.“
Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human and animal infection have been already reported. This species is reported to be heterogenous. The taxonomy of yeast Cryptococcus laurentii was always highly ambiguous. The application of molecular biology and bioinformatic methods led to dividing of searched strains to two distinct phylogenetic groups, some varieties were recognized as species and the locution „Cryptococcus laurentii group“ was introduced. The taxonomy of this group is likely not definitive and with advancing knowledge will change. Our aim was the identification of individual species within this group based on matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI – TOF MS), which has recently been described as a rapid, reliable, cost-effective and powerful tool for analyzing of microorganisms, even on variety level. Generally, the yeasts of genus Cryptococcus form highly resistant polysaccharide capsules and produce large amount of extracellular polysaccharides, therefore belong to so called „difficult“ cases for biotyping. The experimental protocol has been optimized for MS analysis of this genus on the selected strains of Cr. neoformans, Cr. laurentii and Cr. magnus from the Culture Collection of Yeasts (CCY).Thirty-three strains, originally classified as Cryptococcus laurentii has been identified by chosen method. These strains were distributed into six different groups according their spectra similarities. It was selected at least one strain of each group, which was classified based on the sequence analysis of the D1/D2 domains of the LSU rRNA gene. This strain (with known sequence) became representative for its group. Type strain of Cryptococcus laurentii (CCY 17-3-2) belongs to the group I. Its MS spectrum of ribosomal proteins differ from mass spectra of all other biotyped species, even with strains identified as Cryptococcus laurentii was the similarity of the spectra low, which could be caused by identification of two different varieties. The group II is represented by Cryptococcus laurentii CCY 17-3-17. Except this strain, thirteen more strains belong to the group II. The group III represents Cryptococcus flavescens CCY 17-3-29. This group included 12 additional strains with almost identical mass spectra. Group IV included only one strain (CCY 17-3-13), which was identified as Cryptococcus carnescens based on gene sequence analysis. Similarly, one representative (CCY 17-3-5) has the group V. Strain CCY 17-3-5 was identified as Cryptococcus flavus. The last group VI of three members represents strain 17-3-35 identified as Bulleromyces albus. While Cr. laurentii and Cr. flavescens belong to phylogenetic group I and Cr. carnescens to the phylogenetic group II, four strains giving two types of different MS spectra and identified as Cr. flavus (1 strain) and Bulleromyces albus (3 strains) were excluded from „Cr. laurentii group.“
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Citation
JÄGER, J. Biotypizácia kvasiniek skupiny Cryptococcus laurentii hmotnostnou spektrometriou [online]. Brno: Vysoké učení technické v Brně. Fakulta chemická. 2014.
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Document version
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Language of document
sk
Study field
Potravinářská chemie a biotechnologie
Comittee
prof. Ing. Peter Šimko, DrSc. (předseda)
doc. Ing. Jiřina Omelková, CSc. (místopředseda)
prof. RNDr. Ivana Márová, CSc. (člen)
doc. RNDr. Alena Španová, CSc. (člen)
doc. Ing. Bohuslav Rittich, CSc. (člen)
Date of acceptance
2014-06-03
Defence
1. Diplomant seznámil členy komise s náplní a cílem diplomové práce.
2. Byly přečteny posudky na diplomovou práci.
3. Diplomant akceptoval všechny připomínky oponenta a na všechny otázky odpověděl v plné šíři.
Diskuse:
prof. Šimko: Jak funguje databáze MO ? Liší se totéž medium od různých dodavatelů ?
prof. Márová: Jak by se identifikoval neznámý vzorek ? Co za látky je konkrétně ve spektrech ?
Result of defence
práce byla úspěšně obhájena
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Standardní licenční smlouva - přístup k plnému textu bez omezení