I-Propionic acid (PPA), i-antifungal agent kanye ne-common dietary additive, iboniswe ukuthi ibangela ukuthuthukiswa kwe-neurodevelopment okungavamile kumagundane okuhambisana nokungasebenzi kahle kwesisu, okungase kubangelwe yi-gut dysbiosis. Kuye kwaphakanyiswa ukuxhumana phakathi kokuvezwa kwe-PPA yokudla kanye ne-gut microbiota dysbiosis, kodwa akukaphenywanga ngokuqondile. Lapha, siphenye izinguquko ezihlobene ne-PPA ekwakhiweni kwe-gut microbiota okungase kuholele ku-dysbiosis. Ama-gut microbiomes amagundane adla ukudla okungalashwanga (n=9) kanye nokudla okucebile nge-PPA (n=13) kwahlelwa kusetshenziswa ukulandelana kwe-metagenomic okude ukuze kuhlolwe umehluko ekwakhiweni kwamagciwane kanye nezindlela ze-bacterial metabolic. I-Dietary PPA ihlotshaniswa nokwanda kobuningi be-taxa ebalulekile, kufaka phakathi izinhlobo eziningana ze-Bacteroides, i-Prevotella, ne-Ruminococcus, amalungu azo aseke ahileleka ekukhiqizweni kwe-PPA. Ama-microbiomes amagundane atholakale nge-PPA nawo ayenezindlela eziningi ezihlobene ne-lipid metabolism kanye ne-steroid hormone biosynthesis. Imiphumela yethu ikhombisa ukuthi i-PPA ingashintsha i-gut microbiota kanye nezindlela zayo ze-metabolic ezihambisanayo. Lezi zinguquko ezibonwe ziqokomisa ukuthi izilondolozi ezibhekwa njengeziphephile ukusetshenziswa zingathinta ukwakheka kwe-gut microbiota, futhi, impilo yabantu. Phakathi kwazo, i-P, i-G noma i-S ikhethwa kuye ngezinga lokuhlukaniswa elihlaziywayo. Ukuze kuncishiswe umthelela wokuhlukaniswa okungamanga, kwamukelwa umkhawulo omncane wenani elihlobene le-1e-4 (1/10,000 reads). Ngaphambi kokuhlaziywa kwezibalo, inani elihlobene elibikwe yi-Bracken (fraction_total_reads) laguqulwa kusetshenziswa ukuguqulwa kwe-log-ratio ephakathi (CLR) (Aitchison, 1982). Indlela ye-CLR yakhethwa ukuguqulwa kwedatha ngoba ayiguquki ngokwesilinganiso futhi yanele kumasethi edatha angaphelele (Gloor et al., 2017). Ukuguqulwa kwe-CLR kusebenzisa i-logarithm yemvelo. Idatha yokubala ebikwe yi-Bracken yalungiswa kusetshenziswa ukubonakaliswa kwe-log okuhlobene (RLE) (Anders noHuber, 2010). Izibalo zakhiqizwa kusetshenziswa inhlanganisela ye-matplotlib v. 3.7.1, seaborn v. 3.7.2 kanye nama-logarithms alandelanayo (Gloor et al., 2017). 0.12.2 kanye ne-stantanotations v. 0.5.0 (Hunter, 2007; Waskom, 2021; Charlier et al., 2022). Isilinganiso se-Bacillus/Bacteroidetes sibalwa ngesampula ngayinye kusetshenziswa ukubalwa kwamagciwane okujwayelekile. Amanani abikwe kumathebula azungezwe ezindaweni ezi-4 zamadesimali. Inkomba yokwehlukahlukana kwe-Simpson ibalwe kusetshenziswa iskripthi se-alpha_diversity.py esinikezwe kuphakheji ye-KrakenTools v. 1.2 (Lu et al., 2022). Umbiko we-Bracken unikezwe kuskripthi kanti inkomba ye-Simpson ethi “Si” inikezwe ipharamitha ye--an. Umehluko omkhulu ngobuningi uchazwe njengomehluko we-CLR omaphakathi ≥ 1 noma ≤ -1. Umehluko we-CLR omaphakathi we-±1 ukhombisa ukwanda okuphindwe ka-2.7 ngobuningi bohlobo lwesampula. Isibonakaliso (+/-) sibonisa ukuthi i-taxon iningi kakhulu kusampula ye-PPA kanye nesampula yokulawula, ngokulandelana. Ukubaluleka kutholakale kusetshenziswa isivivinyo sikaMann-Whitney U (Virtanen et al., 2020). Kwasetshenziswa ama-Statsmodels v. 0.14 (Benjamini and Hochberg, 1995; Seabold and Perktold, 2010), kanti inqubo kaBenjamini-Hochberg yasetshenziswa ekulungiseni ukuhlolwa okuningi. Inani le-p elilungisiwe ≤ 0.05 lasetshenziswa njengomkhawulo wokunquma ukubaluleka kwezibalo.
I-microbiome yomuntu ivame ukubizwa ngokuthi “isitho sokugcina somzimba” futhi idlala indima ebalulekile empilweni yomuntu (uBaquero noNombela, 2012). Ikakhulukazi, i-microbiome yamathumbu iyaziwa ngethonya layo ohlelweni lonke kanye nendima emisebenzini eminingi ebalulekile. Amabhaktheriya avamile atholakala emathunjini, ahlala ezindaweni eziningi zemvelo, esebenzisa izakhamzimba, futhi ancintisana namagciwane angaba khona (uJandhyala et al., 2015). Izingxenye ezahlukahlukene zamabhaktheriya ze-microbiota yamathumbu ziyakwazi ukukhiqiza izakhamzimba ezibalulekile njengamavithamini nokukhuthaza ukugaya ukudla (uRowland et al., 2018). Ama-metabolites amagciwane nawo aboniswe ukuthi athonya ukuthuthukiswa kwezicubu futhi athuthukise izindlela ze-metabolic kanye ne-immune (uHeijtz et al., 2011; uYu et al., 2022). Ukwakheka kwe-microbiome yamathumbu omuntu kuhluke kakhulu futhi kuncike ezicini zofuzo kanye nezemvelo njengokudla, ubulili, imithi, kanye nesimo sempilo (uKumbhare et al., 2019).
Ukudla komama kuyisici esibalulekile ekukhuleni kwengane kanye nosana olusanda kuzalwa kanye nomthombo obonakalayo wamakhemikhali angathonya intuthuko (Bazer et al., 2004; Innis, 2014). Enye yamakhemikhali anjalo athakazelisayo yi-propionic acid (PPA), umkhiqizo we-fatty acid ochungechungeni olufushane otholakala ekuvuthweni kwamagciwane kanye nesithasiselo sokudla (den Besten et al., 2013). I-PPA inezakhiwo zokulwa namagciwane kanye ne-antifungal ngakho-ke isetshenziswa njengesivikelo sokudla kanye nasezisetshenzisweni zezimboni ukuvimbela ukukhula kwesikhunta kanye namagciwane (Wemmenhove et al., 2016). I-PPA inemiphumela ehlukene ezicutshini ezahlukene. Esibindini, i-PPA inemiphumela yokulwa nokuvuvukala ngokuthinta ukubonakaliswa kwe-cytokine kuma-macrophages (Kawasoe et al., 2022). Lo mphumela wokulawula ubonwe nakwamanye amaseli omzimba, okuholela ekunciphiseni ukuvuvukala (Haase et al., 2021). Kodwa-ke, umphumela ophambene ubonwe ebuchosheni. Izifundo zangaphambilini zikhombisile ukuthi ukuchayeka kwi-PPA kubangela ukuziphatha okufana ne-autism kumagundane (El-Ansary et al., 2012). Ezinye izifundo zikhombisile ukuthi i-PPA ingadala i-gliosis futhi isebenze izindlela zokuvuvukala ebuchosheni (Abdelli et al., 2019). Ngenxa yokuthi i-PPA iyi-asidi ebuthakathaka, ingasabalala nge-epithelium yamathumbu iye egazini ngaleyo ndlela iwele izithiyo ezivimbelayo okuhlanganisa isithiyo segazi nobuchopho kanye ne-placenta (Stinson et al., 2019), okugqamisa ukubaluleka kwe-PPA njenge-metabolite elawulayo ekhiqizwa amabhaktheriya. Nakuba indima engaba khona ye-PPA njengesici esiyingozi se-autism isaphenywa okwamanje, imiphumela yayo kubantu abane-autism ingadlulela ngale kokudala ukuhlukahluka kwezinzwa.
Izimpawu zesisu ezifana nohudo kanye nokuqunjelwa zivame ezigulini ezinezinkinga ze-neurodevelopmental (Cao et al., 2021). Izifundo zangaphambilini zibonise ukuthi i-microbiome yeziguli ezine-autism spectrum disorders (ASD) ihlukile kweyabantu abaphilile, okuphakamisa ukuba khona kwe-gut microbiota dysbiosis (Finegold et al., 2010). Ngokufanayo, izici ze-microbiome zeziguli ezinezifo zamathumbu ezivuvukalayo, ukukhuluphala, isifo se-Alzheimer, njll. nazo zihlukile kwezabantu abaphilile (Turnbaugh et al., 2009; Vogt et al., 2017; Henke et al., 2019). Kodwa-ke, kuze kube manje, akukho buhlobo obubangela obukhona phakathi kwe-gut microbiome kanye nezifo noma izimpawu ze-neurological (Yap et al., 2021), yize izinhlobo eziningana zamagciwane kucatshangwa ukuthi zidlala indima kwezinye zalezi zimo zesifo. Isibonelo, i-Akkermansia, i-Bacteroides, i-Clostridium, i-Lactobacillus, i-Desulfovibrio kanye nezinye izinhlobo ziningi kakhulu kuma-microbiota eziguli ezine-autism (Tomova et al., 2015; Golubeva et al., 2017; Cristiano et al., 2018; Zurita et al., 2020). Okuphawulekayo ukuthi izinhlobo ezingamalungu ezinye zalezi zinhlobo zaziwa ukuthi zinezakhi zofuzo ezihlobene nokukhiqizwa kwe-PPA (Reichardt et al., 2014; Yun and Lee, 2016; Zhang et al., 2019; Baur and Dürre, 2023). Njengoba kunikezwe izakhiwo zokulwa namagciwane ze-PPA, ukwandisa ubuningi bayo kungaba yinzuzo ekukhuleni kwamagciwane akhiqiza i-PPA (Jacobson et al., 2018). Ngakho-ke, indawo ecebile nge-PFA ingaholela ezinguqukweni kuma-microbiota amathumbu, kufaka phakathi amagciwane amathumbu, okungaba yizinto ezingaholela ezimpawini zamathumbu.
Umbuzo oyinhloko ocwaningweni lwe-microbiome ukuthi ngabe umehluko ekwakhiweni kwamagciwane kuyimbangela noma uphawu lwezifo eziyisisekelo. Isinyathelo sokuqala sokucacisa ubudlelwano obuyinkimbinkimbi phakathi kokudla, i-gut microbiome, kanye nezifo zezinzwa ukuhlola imiphumela yokudla ekwakhiweni kwamagciwane. Ukuze sifeze lokhu, sisebenzise ukulandelana kwe-metagenomic okufundwa isikhathi eside ukuqhathanisa ama-gut microbiome enzalo yamagundane adla ukudla okucebile nge-PPA noma okuphelelwe yi-PPA. Inzalo yondliwa ukudla okufanayo nonina. Sacabanga ukuthi ukudla okucebile nge-PPA kuzoholela ekushintsheni ekwakhiweni kwamagciwane emathunjini kanye nezindlela zokusebenza kwamagciwane, ikakhulukazi lezo ezihlobene ne-metabolism ye-PPA kanye/noma ukukhiqizwa kwe-PPA.
Lolu cwaningo lusebenzise amagundane e-FVB/N-Tg(GFAP-GFP)14Mes/J transgenic (Jackson Laboratories) akhipha ngokweqile iphrotheni eluhlaza ekhanyayo (GFP) ngaphansi kokulawulwa komgqugquzeli we-GFAP othize we-glia ngokulandela iziqondiso zeKomiti Yokunakekelwa Kwezilwane Nokusetshenziswa kwe-University of Central Florida Institutional (UCF-IACUC) (Inombolo Yemvume Yokusetshenziswa Kwezilwane: PROTO202000002). Ngemva kokulunyulwa, amagundane ahlaliswa ngawodwana emakhejini anamagundane angu-1-5 obulili ngabunye ngekheji ngalinye. Amagundane anikwa ukudla okuhlanziwe okuhlanzekile (ukudla okujwayelekile okuvuliwe okushintshiwe, amafutha angu-16 kcal%) noma ukudla okungeziwe nge-sodium propionate (ukudla okujwayelekile okuvuliwe okushintshiwe, amafutha angu-16 kcal%, aqukethe i-sodium propionate engu-5,000 ppm). Inani le-sodium propionate elisetshenzisiwe lalilingana nesisindo sokudla esingu-5,000 mg PFA/kg. Lokhu kuhlushwa okuphezulu kakhulu kwe-PPA okuvunyelwe ukusetshenziswa njengesivikelo sokudla. Ukuze kulungiselelwe lolu cwaningo, amagundane angabazali anikwa ukudla kokubili amasonto ama-4 ngaphambi kokuzala futhi aqhubeka phakathi nokukhulelwa kwedamu. Amagundane azalwa [amagundane angu-22, izilawuli ezingu-9 (abesilisa abangu-6, abesifazane abangu-3) kanye nama-PPA angu-13 (abesilisa abangu-4, abesifazane abangu-9)] akhishwa ubisi lwebele abese eqhubeka nokudla okufanayo namadamu izinyanga ezi-5. Amagundane azalwa ahlatshwa eneminyaka engu-5 ubudala futhi okuqukethwe yindle yawo yamathumbu kwaqoqwa futhi ekuqaleni kwagcinwa emapayipini e-microcentrifuge angu-1.5 ml ku--20°C bese kudluliselwa efrijini elingu--80°C kuze kube yilapho i-DNA yomnikazi iphela futhi kukhishwe ama-microbial nucleic acid.
I-DNA yoMsingathi isusiwe ngokwephrothokholi eguquliwe (Charalampous et al., 2019). Kafushane, okuqukethwe yindle kudluliselwe ku-500 µl InhibitEX (Qiagen, Cat#/ID: 19593) futhi kwagcinwa kuqandisiwe. Kucutshungulwe ama-pellets endle angu-1-2 ngokukhipha ngakunye. Okuqukethwe yindle kwabe sekuhlanganiswa ngomshini kusetshenziswa i-pestle yepulasitiki ngaphakathi kwethubhu ukuze kwakhiwe i-slurry. Faka amasampula ku-10,000 RCF imizuzu emi-5 noma kuze kube yilapho amasampula esephethiwe, bese ufutha i-supernatant bese uphinda umise i-pellet ku-250 µl 1 × PBS. Engeza isixazululo se-saponin esingu-250 µl esingu-4.4% (TCI, inombolo yomkhiqizo S0019) kusampula njengesihlanzi ukuze kukhululeke ulwelwesi lwamaseli e-eukaryotic. Amasampula axutshwe kancane aze abe bushelelezi futhi afakwe ekushiseni kwegumbi imizuzu eyi-10. Okulandelayo, ukuze kuphazamiseke amangqamuzana e-eukaryotic, kwanezelwa amanzi angama-350 μl angena-nuclease kusampula, afakwa esikhumbeni imizuzwana engama-30, kwabe sekufakwa i-12 μl 5 M NaCl. Amasampula abe esefakwa ku-centrifuge ku-6000 RCF imizuzu emi-5. Fudumeza i-supernatant bese uphinda umise i-pellet ku-100 μl 1X PBS. Ukuze ususe i-DNA yomnikazi, engeza i-100 μl HL-SAN buffer (12.8568 g NaCl, 4 ml 1M MgCl2, 36 ml amanzi angena-nuclease) kanye ne-10 μl HL-SAN enzyme (ArticZymes P/N 70910-202). Amasampula axutshwe kahle ngokufaka amapayipi futhi afakwa ku-incubator ku-37 °C imizuzu engama-30 ngo-800 rpm ku-Eppendorf™ ThermoMixer C. Ngemva kokufuya, afakwa ku-centrifuge ku-6000 RCF imizuzu emi-3 futhi ahlanzwe kabili nge-800 µl kanye ne-1000 µl 1X PBS. Ekugcineni, phinda umise i-pellet ku-100 µl 1X PBS.
I-DNA yonke yebhaktheriya yahlukaniswa kusetshenziswa i-New England Biolabs Monarch Genomic DNA Purification Kit (New England Biolabs, Ipswich, MA, Cat# T3010L). Inqubo yokusebenza ejwayelekile enikezwe nekhithi ishintshiwe kancane. Faka amanzi angenawo ama-nuclease ku-60°C ngaphambi kokuhlinzwa ukuze kulungiswe okokugcina. Engeza i-10 µl Proteinase K kanye ne-3 µl RNase A kusampula ngayinye. Bese ufaka i-100 µl Cell Lysis Buffer bese uxuba ngobumnene. Amasampula abe esefakwa ku-Eppendorf™ ThermoMixer C ku-56°C kanye ne-1400 rpm okungenani ihora eli-1 kanye namahora afinyelela ku-3. Amasampula afakwe ku-centrifuge ku-12,000 RCF imizuzu emi-3 kanti i-supernatant evela kusampula ngayinye yadluliselwa kushubhu elihlukile le-1.5 mL microcentrifuge eliqukethe i-400 µL yesisombululo sokubopha. Amapayipi abe esevunguzwa nge-pulse imizuzwana emi-5-10 ngezikhawu zesekhondi eli-1. Dlulisa konke okuqukethwe uketshezi kwesampula ngayinye (cishe ama-600-700 µL) kukhatriji yesihlungi ebekwe kuthubhu lokuqoqwa lokugeleza. Amapayipi afakwa ku-centrifuge ku-1,000 RCF imizuzu emi-3 ukuvumela ukubopha kwe-DNA kokuqala bese efakwa ku-centrifuge ku-12,000 RCF umzuzu o-1 ukuze kususwe uketshezi olusele. Ikholomu yesampula yadluliselwa kuthubhu lokuqoqwa elisha bese igezwa kabili. Ekugezeni kokuqala, engeza ama-500 µL we-wash buffer kuthubhu ngalinye. Phendula ithubhu izikhathi ezi-3-5 bese ufaka ku-centrifuge ku-12,000 RCF umzuzu o-1. Lahla uketshezi kuthubhu lokuqoqwa bese ubeka i-filter cartridge emuva kuthubhu lokuqoqwa elifanayo. Ekugezeni kwesibili, engeza ama-500 µL we-wash buffer kusihlungi ngaphandle kokuguqula. Amasampula afakwa ku-centrifuge ku-12,000 RCF umzuzu o-1. Dlulisa isihlungi ku-1.5 mL LoBind® tube bese ufaka u-100 µL wamanzi afudumele angenayo i-nuclease. Izihlungi zifakwe ekushiseni kwegumbi umzuzu owodwa bese zifakwa ku-centrifuge ku-12,000 RCF umzuzu owodwa. I-DNA e-Eluted igcinwe ku--80°C.
Ukuhlushwa kwe-DNA kwalinganiswa kusetshenziswa i-Qubit™ 4.0 Fluorometer. I-DNA yalungiswa kusetshenziswa i-Qubit™ 1X dsDNA High Sensitivity Kit (Cat. No. Q33231) ngokwemiyalelo yomenzi. Ukusatshalaliswa kobude bezingcezu ze-DNA kwalinganiswa kusetshenziswa i-Aglient™ 4150 noma i-4200 TapeStation. I-DNA yalungiswa kusetshenziswa ama-Agilent™ Genomic DNA Reagents (Cat. No. 5067-5366) kanye ne-Genomic DNA ScreenTape (Cat. No. 5067-5365). Ukulungiswa komtapo wolwazi kwenziwa kusetshenziswa i-Oxford Nanopore Technologies™ (ONT) Rapid PCR Barcoding Kit (SQK-RPB004) ngokwemiyalelo yomenzi. I-DNA yahlelwa kusetshenziswa i-ONT GridION™ Mk1 sequencer ene-Min106D flow cell (R 9.4.1). Izilungiselelo zokulandelelana zaziyi: ukubizwa kwesisekelo okunembe kakhulu, inani elincane le-q elingu-9, ukusethwa kwebhakhodi, kanye nokunqunywa kwebhakhodi. Amasampula ahlelwe ngokulandelana amahora angama-72, ngemva kwalokho idatha yocingo oluyisisekelo yathunyelwa ukuze icutshungulwe futhi ihlaziywe kabanzi.
Ukucutshungulwa kwe-Bioinformatics kwenziwa kusetshenziswa izindlela ezichazwe ngaphambilini (Greenman et al., 2024). Amafayela e-FASTQ atholwe ngokulandelana ahlukaniswe ngama-directory esampula ngayinye. Ngaphambi kokuhlaziywa kwe-bioinformatics, idatha yacutshungulwa kusetshenziswa ipayipi elilandelayo: okokuqala, amafayela e-FASTQ amasampula ahlanganiswa abe ifayela elilodwa le-FASTQ. Ngemuva kwalokho, ama-ready amafushane kune-1000 bp ahlungwa kusetshenziswa i-Filtlong v. 0.2.1, kanti ipharamitha kuphela eshintshiwe ingu--min_length 1000 (Wick, 2024). Ngaphambi kokuhlunga okwengeziwe, ikhwalithi yokufunda yalawulwa kusetshenziswa i-NanoPlot v. 1.41.3 ngamapharamitha alandelayo: –fastq –plots dot –N50 -o
Ngokuhlukaniswa kwe-taxonomic, ama-reads nama-contig ahlanganisiwe ahlukaniswe kusetshenziswa i-Kraken2 v. 2.1.2 (Wood et al., 2019). Khiqiza imibiko namafayela okukhiphayo okufundwayo nama-assembly, ngokulandelana. Sebenzisa inketho ye-–use-names ukuhlaziya ama-reads nama-assembly. Izinketho ze-–gzip-compressed kanye ne-paired zichazwe ngezigaba zokufunda. Ubuningi obuhlobene be-taxa kuma-metagenomes bulinganiswe kusetshenziswa i-Bracken v. 2.8 (Lu et al., 2017). Siqale sakha isizindalwazi se-kmer esiqukethe izisekelo eziyi-1000 sisebenzisa i-bracken-build ngamapharamitha alandelayo: -d
Isichasiselo sezakhi zofuzo kanye nokulinganisa ubungako obuhlobene kwenziwe kusetshenziswa inguqulo eguquliwe yephrothokholi echazwe nguMaranga et al. (Maranga et al., 2023). Okokuqala, ama-contig afushane kune-500 bp asusiwe kuzo zonke izinhlangano kusetshenziswa i-SeqKit v. 2.5.1 (Shen et al., 2016). Izinhlangano ezikhethiwe zabe sezihlanganiswa zaba yi-pan-metagenome. Amafreyimu okufunda avulekile (ama-ORF) ahlonzwa kusetshenziswa i-Prodigal v. 1.0.1 (inguqulo efanayo ye-Prodigal v. 2.6.3) enamapharamitha alandelayo: -d
Izakhi zofuzo zaqala ukuhlanganiswa ngokwezihlonzi ze-Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog (KO) ezinikezwe yi-eggNOG ukuze kuqhathaniswe ubungako bendlela yezakhi zofuzo. Izakhi zofuzo ezingenayo i-knockouts noma izakhi zofuzo ezine-knockouts eziningi zasuswa ngaphambi kokuhlaziywa. Inani elimaphakathi le-KO ngayinye ngesampula ngayinye labe selibalwa futhi kwenziwa ukuhlaziywa kwezibalo. Izakhi zofuzo ze-metabolism ye-PPA zachazwa njenganoma yiliphi i-gene elabelwe umugqa we-ko00640 kukholomu ye-KEGG_Pathway, okubonisa indima ekusetshenzisweni kwe-propionate ngokusho kwe-KEGG. Izakhi zofuzo ezihlonzwe njengezihlobene nokukhiqizwa kwe-PPA zibhalwe kuThebula Elingeziwe 1 (Reichardt et al., 2014; Yang et al., 2017). Ukuhlolwa kwe-permutation kwenziwa ukuze kutholakale izakhi zofuzo ze-PPA metabolism kanye nokukhiqiza ezaziningi kakhulu ohlotsheni ngalunye lwesampula. Izinkulungwane ze-permutations zenziwa ku-gene ngayinye ehlaziyiwe. Inani le-p elingu-0.05 lasetshenziswa njenge-cutoff ukuze kunqunywe ukubaluleka kwezibalo. Izakhi zofuzo ezisebenzayo zabelwa izakhi zofuzo ngazinye ngaphakathi kweqembu ngokusekelwe ezichasisweni zezakhi zofuzo ezimele ngaphakathi kweqembu. I-Taxa ehlotshaniswa ne-metabolism ye-PPA kanye/noma ukukhiqizwa kwe-PPA ingabonakala ngokufanisa ama-contig ID kumafayela okukhipha e-Kraken2 nama-contig ID afanayo agcinwe ngesikhathi sokuchasiswa okusebenzayo kusetshenziswa i-eggNOG. Ukuhlolwa kokubaluleka kwenziwe kusetshenziswa ukuhlolwa kwe-Mann-Whitney U okuchazwe ngaphambilini. Ukulungiswa kokuhlolwa okuningi kwenziwe kusetshenziswa inqubo ye-Benjamini-Hochberg. Inani le-p elingu-≤ 0.05 lisetshenziswe njenge-cutoff ukunquma ukubaluleka kwezibalo.
Ukuhlukahluka kwe-microbiome yamathumbu yamagundane kuhlolwe kusetshenziswa inkomba yokwehlukahlukana kweSimpson. Akukho mehluko obalulekile owabonwa phakathi kwamasampula okulawula kanye ne-PPA ngokuya ngokuhlukahlukana kohlobo kanye nohlobo (inani le-p lohlobo: 0.18, inani le-p lohlobo: 0.16) (Isithombe 1). Ukwakheka kwama-microbial kwabe sekuqhathaniswa kusetshenziswa ukuhlaziywa kwengxenye eyinhloko (i-PCA). Isithombe 2 sibonisa ukuhlanganiswa kwamasampula nge-phyla yawo, okubonisa ukuthi kwakukhona umehluko ekwakhiweni kohlobo lwama-microbiome phakathi kwamasampula e-PPA kanye nokulawula. Lokhu kuhlanganiswa kwakungacaci kahle ezingeni lohlobo, okuphakamisa ukuthi i-PPA ithinta amagciwane athile (Isithombe Esingeziwe 1).
Isithombe 1. Ukuhlukahluka kwe-Alpha kohlobo kanye nokwakheka kwezinhlobo ze-microbiome yesisu segundane. Ama-plot ebhokisi abonisa izinkomba zokuhlukahluka kwe-Simpson zohlobo (A) kanye nezinhlobo (B) kumasampula e-PPA kanye nokulawula. Ukubaluleka kunqunywe kusetshenziswa ukuhlolwa kwe-Mann-Whitney U, futhi ukulungiswa okuningi kwenziwa kusetshenziswa inqubo ye-Benjamini-Hochberg. ns, inani le-p lalingekho elibalulekile (p>0.05).
Isithombe 2. Imiphumela yokuhlaziywa kwengxenye eyinhloko yokwakheka kwe-microbiome yamathumbu egundane ezingeni lezinhlobo. Isakhiwo sokuhlaziywa kwengxenye eyinhloko sibonisa ukusatshalaliswa kwamasampula kuzo zonke izingxenye zawo ezimbili zokuqala eziyinhloko. Imibala ikhombisa uhlobo lwesampula: Amagundane abonakala nge-PPA anombala onsomi kanti amagundane okulawula aphuzi. Izingxenye eziyinhloko 1 no-2 zidwetshwe ku-x-axis kanye ne-y-axis, ngokulandelana, futhi zivezwa njengesilinganiso sazo sokuhlukahluka esichaziwe.
Kusetshenziswa idatha yokubala eguquliwe ye-RLE, ukwehla okukhulu kwesilinganiso sama-Bacteroidetes/Bacilli esimaphakathi kwabonwa kumagundane okulawula kanye ne-PPA (ukulawula: 9.66, i-PPA: 3.02; inani le-p = 0.0011). Lo mehluko ubangelwe ukwanda kwama-Bacteroidetes kumagundane e-PPA uma kuqhathaniswa nezilawuli, yize umehluko wawungabalulekile (isilinganiso sokulawula i-CLR: 5.51, isilinganiso se-PPA i-CLR: 6.62; inani le-p = 0.054), kuyilapho ukugcwala kwama-Bacteroidetes kwakufana (isilinganiso sokulawula i-CLR: 7.76, i-PPA isho i-CLR: 7.60; inani le-p = 0.18).
Ukuhlaziywa kobuningi bamalungu e-taxonomic e-gut microbiome kwembule ukuthi uhlobo olulodwa lwe-phylum kanye nohlobo lwama-77 behluke kakhulu phakathi kwe-PPA kanye namasampula okulawula (Ithebula Elingeziwe 2). Ubuningi bezinhlobo ezingama-59 kumasampula e-PPA babuphezulu kakhulu kunalawo asemasampula okulawula, kanti ubuningi bezinhlobo eziyi-16 kuphela kumasampula okulawula babuphakeme kunalawo asemasampula e-PPA (Isithombe 3).
Isithombe 3. Ubuningi obuhlukile be-taxa ku-microbiome yamathumbu e-PPA kanye namagundane okulawula. Ama-volcano plots abonisa umehluko ngobuningi bezinhlobo (A) noma izinhlobo (B) phakathi kwe-PPA namasampula okulawula. Amachashazi ampunga awabonisi mehluko obalulekile ngobuningi be-taxa. Amachashazi anemibala abonisa umehluko obalulekile ngobuningi (inani le-p ≤ 0.05). Ama-taxa angu-20 aphezulu anomehluko omkhulu ngobuningi phakathi kwezinhlobo zamasampula aboniswa kubomvu nokuluhlaza okwesibhakabhaka okukhanyayo (amasampula okulawula kanye ne-PPA), ngokulandelana. Amachashazi aphuzi nansomi ayenobuningi okungenani izikhathi ezingu-2.7 kumasampula okulawula noma e-PPA kunakuma-control. Amachashazi amnyama amelela i-taxa enobuningi obuhluke kakhulu, ngomehluko ophakathi we-CLR phakathi kuka--1 no-1. Amanani e-P abalwa kusetshenziswa ukuhlolwa kwe-Mann-Whitney U futhi kwalungiswa ukuhlolwa okuningi kusetshenziswa inqubo ye-Benjamini-Hochberg. Umehluko ophakathi we-CLR ogqamile ubonisa umehluko obalulekile ngobuningi.
Ngemva kokuhlaziya ukwakheka kwamagciwane amathumbu, senze isichasiselo esisebenzayo se-microbiome. Ngemva kokuhlunga izakhi zofuzo ezisezingeni eliphansi, kwatholakala izakhi zofuzo ezihlukile ezingu-378,355 kuzo zonke izibonelo. Ubuningi balezi zakhi zofuzo obuguquliwe basetshenziselwa ukuhlaziywa kwezingxenye eziyinhloko (i-PCA), futhi imiphumela ibonise izinga eliphezulu lokuhlanganiswa kwezinhlobo zamasampula ngokusekelwe kumaphrofayili azo okusebenza (Isithombe 4).
Isithombe 4. Imiphumela ye-PCA isebenzisa iphrofayili yokusebenza kwe-microbiome yesisu segundane. Isakhiwo se-PCA sibonisa ukusatshalaliswa kwamasampula kuzo zonke izingxenye zawo ezimbili zokuqala eziyinhloko. Imibala ikhombisa uhlobo lwesampula: Amagundane abonakala nge-PPA anombala onsomi kanti amagundane okulawula aphuzi. Izingxenye eziyinhloko 1 no-2 zidwetshwe ku-x-axis kanye ne-y-axis, ngokulandelana, futhi zivezwa njengesilinganiso sazo sokuhlukahluka esichaziwe.
Ngokulandelayo sihlole ubuningi be-KEGG knockouts ezinhlotsheni ezahlukene zamasampula. Kwatholakala inani lama-knockouts ahlukile angu-3648, lapho angu-196 ayeningi kakhulu kumasampula okulawula kanti angu-106 ayeningi kakhulu kumasampula e-PPA (Isithombe 5). Kwatholakala inani lama-genes angu-145 kumasampula okulawula kanye nama-genes angu-61 kumasampula e-PPA, anobuningi obuhlukene kakhulu. Izindlela ezihlobene ne-lipid kanye ne-aminosugar metabolism zacetshiswa kakhulu kumasampula e-PPA (Ithebula Elingeziwe 3). Izindlela ezihlobene ne-nitrogen metabolism kanye nezinhlelo zokudlulisa i-sulfur zacetshiswa kakhulu kumasampula okulawula (Ithebula Elingeziwe 3). Ubuningi bezakhi zofuzo ezihlobene ne-aminosugar/nucleotide metabolism (ko:K21279) kanye ne-inositol phosphate metabolism (ko:K07291) zaziphezulu kakhulu kumasampula e-PPA (Isithombe 5). Amasampula okulawula ayenezakhi zofuzo eziningi kakhulu ezihlobene nokusetshenziswa kwe-benzoate (ko:K22270), ukusetshenziswa kwe-nitrogen (ko:K00368), kanye ne-glycolysis/gluconeogenesis (ko:K00131) (Isithombe 5).
Isithombe 5. Ubuningi obuhlukile bama-KO ku-microbiome yamathumbu e-PPA kanye namagundane okulawula. Isakhiwo sentaba-mlilo sibonisa umehluko ebuningini bamaqembu asebenzayo (ama-KO). Amachashazi ampunga abonisa ama-KO anobuningi obungahlukile kakhulu phakathi kwezinhlobo zamasampula (inani le-p > 0.05). Amachashazi anombala abonisa umehluko obalulekile ebuningini (inani le-p ≤ 0.05). Ama-KO angu-20 anomehluko omkhulu ebuningini phakathi kwezinhlobo zamasampula aboniswa ngombala obomvu noluhlaza okwesibhakabhaka okhanyayo, okuhambisana namasampula okulawula kanye ne-PPA, ngokulandelana. Amachashazi aphuzi nansomi abonisa ama-KO okungenani ayenobuningi obuphindwe ka-2.7 ekulawuleni kanye namasampula e-PPA, ngokulandelana. Amachashazi amnyama abonisa ama-KO anobuningi obuhlukene kakhulu, ngomehluko we-CLR ophakathi kuka--1 no-1. Amanani e-P abalwa kusetshenziswa ukuhlolwa kwe-Mann-Whitney U futhi kwalungiswa ukuze kuqhathaniswe okuningi kusetshenziswa inqubo ye-Benjamini-Hochberg. I-NaN ikhombisa ukuthi i-KO akuyona eyendlela ku-KEGG. Amanani omehluko we-CLR ophakathi ogqamile abonisa umehluko obalulekile ebuningini. Ukuze uthole ulwazi oluningiliziwe ngezindlela ama-KO abhalwe phansi angena kuzo, bheka iThebula Elingeziwe 3.
Phakathi kwezakhi zofuzo ezichazwe ngenhla, izakhi zofuzo ezingu-1601 zazinobuningi obuhlukene kakhulu phakathi kwezinhlobo zamasampula (p ≤ 0.05), kanti isakhi sofuzo ngasinye sasinobuningi obuphindwe okungenani izikhathi ezingu-2.7. Kulezi zakhi zofuzo, izakhi zofuzo ezingu-4 zazinobuningi kakhulu kumasampula okulawula kanti izakhi zofuzo ezingu-1597 zazinobuningi kakhulu kumasampula e-PPA. Ngenxa yokuthi i-PPA inezakhiwo zokulwa namagciwane, sihlole ubuningi bezakhi zofuzo ze-PPA metabolism kanye nokukhiqiza phakathi kwezinhlobo zamasampula. Phakathi kwezakhi zofuzo ezihlobene ne-PPA metabolism ezingu-1332, izakhi zofuzo ezingu-27 zazinobuningi kakhulu kumasampula okulawula kanti izakhi zofuzo ezingu-12 zazinobuningi kakhulu kumasampula e-PPA. Phakathi kwezakhi zofuzo ezihlobene ne-PPA ezingama-223, isakhi sofuzo esisodwa sasinobuningi kakhulu kumasampula e-PPA. Isibalo 6A sibonisa futhi ubuningi obukhulu bezakhi zofuzo ezihilelekile ku-PPA metabolism, ngobuningi obukhulu kakhulu kumasampula okulawula kanye nobukhulu obukhulu bemiphumela, kuyilapho Isibalo 6B siqokomisa izakhi zofuzo ngazinye ezinobuningi obukhulu kakhulu obubonwe kumasampula e-PPA.
Isithombe 6. Ubuningi obuhlukile bezakhi zofuzo ezihlobene ne-PPA ku-microbiome yesisu segundane. Ama-volcano plots abonisa umehluko ebuningini bezakhi zofuzo ezihlobene ne-PPA metabolism (A) kanye nokukhiqizwa kwe-PPA (B). Amachashazi ampunga abonisa izakhi zofuzo ezinobuningi obungahlukile kakhulu phakathi kwezinhlobo zamasampula (inani le-p > 0.05). Amachashazi anemibala abonisa umehluko omkhulu ebuningini (inani le-p ≤ 0.05). Izakhi zofuzo ezingu-20 ezinomehluko omkhulu ebuningini ziboniswa kubomvu nokuluhlaza okwesibhakabhaka okukhanyayo (amasampula okulawula kanye ne-PPA), ngokulandelana. Ubuningi bamachashazi aphuzi nansomi babukhulu okungenani izikhathi ezingu-2.7 kumasampula okulawula kanye ne-PPA kunakumasampula okulawula. Amachashazi amnyama amelela izakhi zofuzo ezinobuningi obuhlukene kakhulu, ngomehluko we-CLR ophakathi kuka--1 no-1. Amanani e-P abalwa kusetshenziswa ukuhlolwa kwe-Mann-Whitney U futhi kwalungiswa ukuze kuqhathaniswe okuningi kusetshenziswa inqubo ye-Benjamini-Hochberg. Izakhi zofuzo zihambisana nezakhi zofuzo ezimele kukhathalogi yezakhi zofuzo ezingaphindi zisetshenziswe. Amagama ezakhi zofuzo aqukethe uphawu lwe-KEGG oluchaza i-KO gene. Umehluko we-CLR ophakathi ogqamile ubonisa ubuningi obuhlukene kakhulu. Ideshi (-) ikhombisa ukuthi akukho phawu lwe-gene kusizindalwazi se-KEGG.
I-Taxa enezakhi zofuzo ezihlobene ne-metabolism ye-PPA kanye/noma ukukhiqizwa kwayo yahlonzwa ngokufanisa ubunikazi be-taxonomic bezingxenye ezihambisanayo ne-contig ID yezakhi zofuzo. Ezingeni lezakhi zofuzo, izizukulwane ezingu-130 zatholakala zinezakhi zofuzo ezihlobene ne-metabolism ye-PPA kanti izizukulwane ezingu-61 zatholakala zinezakhi zofuzo ezihlobene nokukhiqizwa kwe-PPA (Ithebula Elingeziwe 4). Kodwa-ke, akukho sizukulwane esabonisa umehluko omkhulu ngobuningi (p > 0.05).
Ezingeni lezinhlobo, izinhlobo zamabhaktheriya eziyi-144 zitholakale zinezakhi zofuzo ezihlobene ne-metabolism ye-PPA kanti izinhlobo zamabhaktheriya ezingama-68 zitholakale zinezakhi zofuzo ezihlobene nokukhiqizwa kwe-PPA (Ithebula Elingeziwe 5). Phakathi kwe-metabilizers ye-PPA, amabhaktheriya ayisishiyagalombili abonise ukwanda okukhulu kobuningi phakathi kwezinhlobo zamasampula, futhi wonke abonise izinguquko ezibalulekile ekusebenzeni (Ithebula Elingeziwe 6). Zonke i-metabilizers ye-PPA ezikhonjwe ezinomehluko omkhulu ngobuningi zaziningi kakhulu kumasampula e-PPA. Ukuhlukaniswa kwezinga lezinhlobo kwembule abamele izizukulwane ezazingahlukanga kakhulu phakathi kwezinhlobo zamasampula, kufaka phakathi izinhlobo eziningana ze-Bacteroides ne-Ruminococcus, kanye ne-Duncania dubois, i-Myxobacterium enterica, i-Monococcus pectinolyticus, kanye ne-Alcaligenes polymorpha. Phakathi kwamabhaktheriya akhiqiza i-PPA, amabhaktheriya amane abonise umehluko omkhulu ngobuningi phakathi kwezinhlobo zamasampula. Izinhlobo ezinomehluko omkhulu ngobuningi zazihlanganisa i-Bacteroides novorossi, i-Duncania dubois, i-Myxobacterium enteritidis, kanye ne-Ruminococcus bovis.
Kulolu cwaningo, sihlole imiphumela yokuchayeka kwe-PPA kuma-microbiota amathumbu amagundane. I-PPA ingaveza izimpendulo ezahlukene kuma-bacteria ngoba ikhiqizwa izinhlobo ezithile, isetshenziswa njengomthombo wokudla ezinye izinhlobo, noma inemiphumela yokulwa namagciwane. Ngakho-ke, ukungezwa kwayo endaweni yamathumbu ngokungezwa kokudla kungaba nemiphumela ehlukile kuye ngokubekezelelana, ukuzwela, kanye nekhono lokuyisebenzisa njengomthombo wezakhamzimba. Izinhlobo zama-bacteria ezibucayi zingasuswa futhi zithathelwe indawo yilezo ezimelana kakhulu ne-PPA noma ezikwazi ukuyisebenzisa njengomthombo wokudla, okuholela ezinguqukweni ekwakhiweni kwe-microbiota yamathumbu. Imiphumela yethu yembule umehluko omkhulu ekwakhiweni kwama-microbiota kodwa akukho mthelela ekwahlukeni kwama-microbiota jikelele. Imiphumela emikhulu yabonwa ezingeni lezinhlobo, ngama-taxa angaphezu kuka-70 ahluke kakhulu ngobuningi phakathi kwama-PPA namasampula okulawula (Ithebula Elingeziwe 2). Ukuhlolwa okwengeziwe kokwakheka kwamasampula avulwe yi-PPA kwembule ukungafani okukhulu kwezinhlobo zama-microbial uma kuqhathaniswa namasampula angavulwanga, okuphakamisa ukuthi i-PPA ingathuthukisa izici zokukhula kwama-bacteria futhi inciphise inani lama-bacteria elingasinda ezindaweni ezicebile nge-PPA. Ngakho-ke, i-PPA ingabangela izinguquko ngokukhetha kunokuba ibangele ukuphazamiseka okubanzi kokuhlukahluka kwama-microbiota amathumbu.
Izithako zokudla ezifana ne-PPA ziboniswe ngaphambilini ukuthi zishintsha ubuningi bezingxenye ze-microbiome yamathumbu ngaphandle kokuthinta ukuhlukahluka okuphelele (Nagpal et al., 2021). Lapha, sibone umehluko ophawulekayo phakathi kwezinhlobo ze-Bacteroidetes ngaphakathi kwe-phylum Bacteroidetes (ezaziwa ngokuthi ama-Bacteroidetes), ezazicetshiswe kakhulu kumagundane atholakale kwi-PPA. Ukwanda kobuningi bezinhlobo ze-Bacteroides kuhlotshaniswa nokwanda kokonakala kwe-mucus, okungase kwandise ingozi yokutheleleka futhi kukhuthaze ukuvuvukala (Cornick et al., 2015; Desai et al., 2016; Penzol et al., 2019). Ucwaningo olulodwa luthole ukuthi amagundane amaduna asanda kuzalwa aphathwe nge-Bacteroides fragilis abonise ukuziphatha komphakathi okufana ne-autism spectrum disorder (ASD) (Carmel et al., 2023), kanti ezinye izifundo zibonise ukuthi izinhlobo ze-Bacteroides zingashintsha umsebenzi wokuzivikela komzimba futhi ziholele ekushayweni yi-cardiomyopathy yokuvuvukala komzimba (Gil-Cruz et al., 2019). Izinhlobo zesizukulwane iRuminococcus, iPrevotella, kanye neParabacteroides nazo zanda kakhulu emagundwini atholakale ku-PPA (Coretti et al., 2018). Ezinye izinhlobo zeRuminococcus zihlotshaniswa nezifo ezifana nesifo sikaCrohn ngokukhiqizwa kwama-cytokines abangela ukuvuvukala (Henke et al., 2019), kanti izinhlobo zePrevotella ezifana nePrevotella humani zihlotshaniswa nezifo ze-metabolic ezifana nomfutho wegazi ophakeme kanye nokuzwela kwe-insulin (Pedersen et al., 2016; Li et al., 2017). Ekugcineni, sithole ukuthi isilinganiso samaBacteroidetes (ngaphambili ayaziwa ngokuthi amaFirmicutes) kumaBacteroidetes sasiphansi kakhulu emagundwini atholakale ku-PPA kunasemagundwini alawulayo ngenxa yenani elikhulu lezinhlobo zamaBacteroidetes. Lesi silinganiso siboniswe ngaphambilini njengesinkomba esibalulekile se-homeostasis yamathumbu, futhi ukuphazamiseka kulesi silinganiso kuye kwahlotshaniswa nezimo ezahlukene zezifo (Turpin et al., 2016; Takezawa et al., 2021; An et al., 2023), okuhlanganisa nezifo zamathumbu ezivuvukalayo (Stojanov et al., 2020). Ngokubambisana, izinhlobo ze-phylum Bacteroidetes zibonakala zithinteka kakhulu yi-PPA yokudla ephezulu. Lokhu kungase kubangelwe ukubekezelelana okuphezulu kwe-PPA noma ikhono lokusebenzisa i-PPA njengomthombo wamandla, okuye kwabonakala kuyiqiniso okungenani kohlobo olulodwa, uHoylesella enocea (Hitch et al., 2022). Ngaphandle kwalokho, ukuvezwa kwe-PPA kamama kungathuthukisa intuthuko yengane ngokwenza amathumbu ezingane zegundane abe sengozini enkulu yokuhlaselwa yi-Bacteroidetes; noma kunjalo, umklamo wethu wocwaningo awuzange uvumele ukuhlolwa okunjalo.
Ukuhlolwa kokuqukethwe kwe-metagenomic kwembule umehluko omkhulu ebuningini bezakhi zofuzo ezihlotshaniswa nokuguqulwa kwe-PPA kanye nokukhiqizwa kwayo, lapho amagundane abonakala ku-PPA ebonisa inqwaba yezakhi zofuzo ezibhekene nokukhiqizwa kwe-PPA, kanti amagundane angewona ama-PPA abonise inqwaba yezakhi zofuzo ezibhekene nokuguqulwa kwe-PAA (Isithombe 6). Le miphumela iphakamisa ukuthi umphumela we-PPA ekwakhiweni kwamagciwane kungenzeka kungabi ngenxa yokusetshenziswa kwawo kuphela, ngaphandle kwalokho inqwaba yezakhi zofuzo ezihlotshaniswa nokuguqulwa kwe-PPA kufanele ngabe ibonise inqwaba ephezulu ku-microbiome yamathumbu yamagundane abonakala ku-PPA. Enye incazelo ukuthi i-PPA ilawula inqwaba yamagciwane ikakhulukazi ngemiphumela yayo yokulwa namagciwane kunokusetshenziswa kwayo ngamagciwane njengezakhi zofuzo. Izifundo zangaphambilini zibonise ukuthi i-PPA ivimbela ukukhula kwe-Salmonella Typhimurium ngendlela encike kumthamo (Jacobson et al., 2018). Ukuchayeka ekugxilweni okuphezulu kwe-PPA kungakhetha amabhaktheriya amelana nezakhiwo zawo zokulwa namagciwane futhi angase angakwazi ngempela ukuwaxuba noma ukuwakhiqiza. Isibonelo, izinhlobo eziningana ze-Parabacteroides zibonise inani elikhulu kakhulu kumasampula e-PPA, kodwa azikho izakhi zofuzo ezihlobene nokuguqulwa kwe-PPA noma ukukhiqizwa kwazo ezitholakele (Amathebula Angeziwe 2, 4, no-5). Ngaphezu kwalokho, ukukhiqizwa kwe-PPA njengomkhiqizo olandelanayo wokubilisa kusakazwa kabanzi phakathi kwamabhaktheriya ahlukahlukene (Gonzalez-Garcia et al., 2017). Ukuhlukahluka okuphezulu kwamabhaktheriya kungaba yimbangela yobuningi obukhulu bezakhi zofuzo ezihlobene nokuguqulwa kwe-PPA kumasampula okulawula (Averina et al., 2020). Ngaphezu kwalokho, ama-27 kuphela (2.14%) ezakhi zofuzo ezingu-1332 abikezelwe ukuthi azakhi zofuzo ezihlotshaniswa kuphela nokuguqulwa kwe-PPA. Izakhi zofuzo eziningi ezihlotshaniswa nokuguqulwa kwe-PPA nazo zihilelekile kwezinye izindlela zokuguqulwa kwe-metabolic. Lokhu kubonisa futhi ukuthi ubuningi bezakhi zofuzo ezihilelekile kukuguqulwa kwe-PPA babuphezulu kumasampula okulawula; lawa ma-gene angasebenza ezindleleni ezingaholeli ekusetshenzisweni noma ekwakhekeni kwe-PPA njengomkhiqizo olandelanayo. Kulesi simo, i-gene eyodwa kuphela ehlotshaniswa nokukhiqizwa kwe-PPA ibonise umehluko omkhulu ngobuningi phakathi kwezinhlobo zamasampula. Ngokungafani nezakhi zofuzo ezihlotshaniswa nokuguqulwa kwe-PPA, izakhi zofuzo ezimakayo zokukhiqizwa kwe-PPA zakhethwa ngoba zihileleke ngqo endleleni yamabhaktheriya yokukhiqizwa kwe-PPA. Kumagundane avulwe yi-PPA, zonke izinhlobo zezilwane zitholakale zinenani elikhulu kanye namandla okukhiqiza i-PPA. Lokhu kusekela isibikezelo sokuthi ama-PPA angakhetha abakhiqizi be-PPA ngakho-ke abikezela ukuthi amandla okukhiqiza i-PPA azokwanda. Kodwa-ke, inani lezakhi zofuzo alihlobene ngempela nokubonakaliswa kwezakhi zofuzo; ngakho-ke, yize inani lezakhi zofuzo ezihlobene nokusetshenziswa kwe-PPA liphezulu kumasampula okulawula, izinga lokuvezwa lingase lihluke (Shi et al., 2014). Ukuqinisekisa ubudlelwano phakathi kokusabalala kwezakhi zofuzo ezikhiqiza i-PPA kanye nokukhiqizwa kwe-PPA, kudingeka izifundo zokubonakaliswa kwezakhi zofuzo ezihilelekile ekukhiqizweni kwe-PPA.
Isichasiselo esisebenzayo se-PPA kanye nama-metagenome okulawula siveze umehluko othile. Ukuhlaziywa kwe-PCA kokuqukethwe kwezakhi zofuzo kwembule amaqoqo ahlukene phakathi kwe-PPA namasampula okulawula (Isithombe 5). Ukuhlanganiswa kwesampula ngaphakathi kwembula ukuthi okuqukethwe kwezakhi zofuzo zokulawula kwakuhlukahlukene kakhulu, kuyilapho amasampula e-PPA eqoqwe ndawonye. Ukuhlanganiswa ngokuqukethwe kwezakhi zofuzo kwakufana nokuhlanganiswa ngokwakhiwa kwezinhlobo. Ngakho-ke, umehluko ebuningini bendlela uyahambisana nezinguquko ebuningini bezinhlobo ezithile kanye nezinhlobo ezingaphakathi kwazo. Kumasampula e-PPA, izindlela ezimbili ezinobuningi obukhulu kakhulu zazihlobene ne-aminosugar/nucleotide sugar metabolism (ko:K21279) kanye nezindlela eziningi ze-lipid metabolism (ko:K00647, ko:K03801; Ithebula Elingeziwe 3). Izakhi zofuzo ezihlotshaniswa ne-ko:K21279 zaziwa ukuthi zihlotshaniswa nohlobo lwe-Bacteroides, olunye lwezinhlobo ezinenani eliphakeme kakhulu lezinhlobo kumasampula e-PPA. Le enzyme ingagwema impendulo yomzimba ngokuveza ama-capsular polysaccharides (Wang et al., 2008). Lokhu kungase kubangele ukwanda kwama-Bacteroidetes abonwe kumagundane ahlungwe yi-PPA. Lokhu kuhambisana nokwanda kokuhlanganiswa kwe-fatty acid okubonwa ku-PPA microbiome. Amabhaktheriya asebenzisa indlela ye-FASIIko:K00647 (fabB) ukukhiqiza ama-fatty acid, okungase kuthonye izindlela ze-metabolic zomsingathi (Yao and Rock, 2015; Johnson et al., 2020), futhi izinguquko ku-lipid metabolism zingadlala indima ekuthuthukisweni kwe-neurodevelopment (Yu et al., 2020). Enye indlela ekhombisa ukwanda kobuningi kumasampula e-PPA kwakuyi-steroid hormone biosynthesis (ko:K12343). Kunobufakazi obukhulayo bokuthi kukhona ubudlelwano obuphambene phakathi kwekhono le-gut microbiota lokuthonya amazinga ama-hormone nokuthonywa ama-hormone, kangangokuthi amazinga aphezulu e-steroid angaba nemiphumela yezempilo engezansi (Tetel et al., 2018).
Lolu cwaningo alunazo imikhawulo kanye nokucatshangelwa. Umehluko obalulekile ukuthi asizange senze ukuhlolwa kwempilo yezilwane. Ngakho-ke, akunakwenzeka ukuphetha ngokuqondile ukuthi izinguquko ku-microbiome zihlotshaniswa nanoma yisiphi isifo. Okunye okucatshangelwayo ukuthi amagundane kulolu cwaningo anikwa ukudla okufanayo nonina. Izifundo zesikhathi esizayo zinganquma ukuthi ukushintsha ekudleni okucebile nge-PPA kuya ekudleni okungena-PPA kuthuthukisa yini imiphumela yako ku-microbiome. Omunye umkhawulo wocwaningo lwethu, njengabanye abaningi, usayizi wesampula olinganiselwe. Nakuba iziphetho ezivumelekile zingatholakala, usayizi wesampula omkhulu unganikeza amandla amakhulu ezibalo lapho kuhlaziywa imiphumela. Siphinde siqaphele ngokufinyelela iziphetho mayelana nokuhlangana phakathi kwezinguquko ku-microbiome yamathumbu nanoma yisiphi isifo (Yap et al., 2021). Izici ezididayo ezifaka phakathi ubudala, ubulili, kanye nokudla kungathinta kakhulu ukwakheka kwama-microorganism. Lezi zici zingachaza ukungahambisani okubonwe ezincwadini mayelana nokuhlangana kwe-microbiome yamathumbu nezifo eziyinkimbinkimbi (Johnson et al., 2019; Lagod noNaser, 2023). Isibonelo, amalungu ohlobo lwe-Bacteroidetes aboniswe ukuthi ayanda noma ancipha ezilwaneni nakubantu abane-ASD (Angelis et al., 2013; Kushak et al., 2017). Ngokufanayo, izifundo zokwakheka kwamathumbu ezigulini ezinezifo zamathumbu ezivuvukalayo zithole kokubili ukwanda nokuncipha kwesilinganiso esifanayo (Walters et al., 2014; Forbes et al., 2018; Upadhyay et al., 2023). Ukuze sinciphise umthelela wokucwaswa ngokobulili, sizame ukuqinisekisa ukumelwa okulinganayo kobulili ukuze umehluko cishe uqhutshwe ukudla. Inselele eyodwa yokuchazwa okusebenzayo ukususwa kokulandelana kwezakhi zofuzo okungafuneki. Indlela yethu yokuqoqa izakhi zofuzo idinga ubunikazi bokulandelana okungu-95% kanye nokufana kobude obungu-85%, kanye nokumbozwa kokuqondanisa okungu-90% ukuze kuqedwe ukuqoqana okungamanga. Kodwa-ke, kwezinye izimo, sibone ama-COG anezincazelo ezifanayo (isb., MUT) (Isithombe 6). Kudingeka izifundo ezengeziwe ukuze kutholakale ukuthi la ma-ortholog ahlukile yini, ahlotshaniswa nezinhlobo ezithile, noma ukuthi lokhu kuwumkhawulo wendlela yokuhlanganisa izakhi zofuzo. Omunye umkhawulo wokuchazwa kokusebenza ukuhlukaniswa okungaba khona; i-gene yebhaktheriya i-mmdA iyi-enzyme eyaziwayo ehilelekile ekwakhiweni kwe-propionate, kodwa i-KEGG ayiyihlobanisi nendlela ye-metabolic ye-propionate. Ngokuphambene nalokho, ama-ortholog e-scpB kanye ne-mmcD ahlobene. Inani elikhulu lama-gene angenazo izigameko ezikhethiwe lingabangela ukungakwazi ukubona izakhi zofuzo ezihlobene ne-PPA lapho kuhlolwa ubuningi bezakhi zofuzo. Izifundo zesikhathi esizayo zizozuza ekuhlaziyweni kwe-metatranscriptome, okunganikeza ukuqonda okujulile kwezici zokusebenza kwe-gut microbiota futhi kuxhumanise ukubonakaliswa kwezakhi zofuzo nemiphumela engaba khona engezansi. Ezifundweni ezibandakanya ukuphazamiseka okuthile kwe-neurodevelopmental noma izifo zamathumbu ezivuvukalayo, ukuhlolwa komzimba nokuziphatha kwezilwane kuyadingeka ukuze kuxhunywe izinguquko ekubunjweni kwe-microbiome nalezi zinkinga. Izifundo ezengeziwe zokufakelwa i-gut microbiome kumagundane angenamagciwane nazo zingasiza ekunqumeni ukuthi i-microbiome iyimbangela noma isici sesifo.
Ngamafuphi, sibonise ukuthi i-PPA yokudla isebenza njengesici ekushintsheni ukwakheka kwe-microbiota yamathumbu. I-PPA iyisivikelo esivunyelwe yi-FDA esitholakala kabanzi ekudleni okuhlukahlukene, okuthi uma sichayeka isikhathi eside, singaholela ekuphazamisekeni kwezitshalo ezivamile zamathumbu. Sithole izinguquko ebuningini bamabhaktheriya amaningana, okuphakamisa ukuthi i-PPA ingathonya ukwakheka kwe-microbiota yamathumbu. Izinguquko ku-microbiota zingaholela ekushintsheni emazingeni ezindlela ezithile ze-metabolic, okungaholela ezinguqukweni zomzimba eziphathelene nempilo yomnikazi. Kudingeka izifundo ezengeziwe ukuze kutholakale ukuthi imiphumela ye-PPA yokudla ekwakhiweni kwamagciwane ingaholela yini ku-dysbiosis noma kwezinye izifo. Lolu cwaningo lubeka isisekelo sezifundo zesikhathi esizayo mayelana nokuthi imiphumela ye-PPA ekwakhiweni kwamathumbu ingathinta kanjani impilo yabantu.
Amasethi edatha avezwe kulolu cwaningo ayatholakala ezindaweni zokugcina eziku-inthanethi. Igama lendawo yokugcina kanye nenombolo yokungena yile: https://www.ncbi.nlm.nih.gov/, PRJNA1092431.
Lolu cwaningo lwezilwane luvunyiwe yiKomidi Lokunakekelwa Kwezilwane Nezikhungo lase-University of Central Florida (UCF-IACUC) (Inombolo Yemvume Yokusetshenziswa Kwezilwane: PROTO202000002). Lolu cwaningo luhambisana nemithetho yendawo, imithethonqubo, kanye nezimfuneko zezikhungo.
NG: Ukuqonda, Ukuhlelwa kwedatha, Ukuhlaziywa okusemthethweni, Uphenyo, Indlela, Isofthiwe, Ukubona ngeso lengqondo, Ukubhala (uhlaka lokuqala), Ukubhala (ukubuyekeza nokuhlela). LA: Ukuqonda, Ukuhlelwa kwedatha, Indlela, Izinsiza, Ukubhala (ukubuyekeza nokuhlela). SH: Ukuhlaziywa okusemthethweni, Isofthiwe, Ukubhala (ukubuyekeza nokuhlela). SA: Uphenyo, Ukubhala (ukubuyekeza nokuhlela). IJaji Eliyinhloko: Uphenyo, Ukubhala (ukubuyekeza nokuhlela). SN: Ukuqonda, Ukuphathwa kwephrojekthi, Izinsiza, Ukuqapha, Ukubhala (ukubuyekeza nokuhlela). TA: Ukuqonda, Ukuphathwa kwephrojekthi, Ukuqapha, Ukubhala (ukubuyekeza nokuhlela).
Ababhali bamemezele ukuthi abatholanga kusekelwa ngezimali ngocwaningo, ubunikazi bombhali, kanye/noma ukushicilelwa kwalesi sihloko.
Ababhali bathi ucwaningo lwenziwe ngaphandle kobudlelwano bezentengiselwano noma bezezimali obungabhekwa njengokungqubuzana kwezintshisekelo okungaba khona.
Yonke imibono evezwe kulesi sihloko ingeyababhali kuphela futhi ayibonisi ngempela imibono yezikhungo zabo, abashicileli, abahleli, noma ababuyekezi. Noma yimiphi imikhiqizo ehlolwe kulesi sihloko, noma yiziphi izimangalo ezenziwe abakhiqizi bayo, ayiqinisekiswanga noma ayivunyelwe ngumshicileli.
Izinto ezengeziwe zalesi sihloko zingatholakala ku-inthanethi: https://www.frontiersin.org/articles/10.3389/frmbi.2024.1451735/full#supplementary-material
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Isikhathi sokuthunyelwe: Ephreli-18-2025