Antifungal agent leh common dietary additive Propionic acid (PPA) hian mice-ah neurodevelopment danglam tak a thlen tih hmuhchhuah a ni a, hei hi gastrointestinal dysfunction nen a inzawm a, hei hi gut dysbiosis vang pawh a ni thei. Dietary PPA exposure leh gut microbiota dysbiosis inzawmna hi sawi a ni a, mahse direct-a zirchian a la ni lo. Hetah hian PPA nena inzawm gut microbiota composition inthlak danglamna, dysbiosis thlen thei tur kan zirchiang a. Mice-te gut microbiomes chu untreated diet (n=9) leh PPA-enriched diet (n=13) pek te chu long-range metagenomic sequencing hmangin sequence an siam a, microbial composition leh bacterial metabolic pathways danglamna tehna atan an hmang a ni. Dietary PPA hi taxa pawimawh tak tak tamna nen a inzawm a, chung zingah chuan Bacteroides, Prevotella, leh Ruminococcus chi hrang hrang te pawh an tel a, heng zinga member te hi a hmain PPA siamnaah an inrawlh tawh a ni. PPA-exposed mice-te microbiome-ah pawh lipid metabolism leh steroid hormone biosynthesis nena inzawm kawng tam zawk a awm bawk. Kan result hian PPA hian gut microbiota leh a kaihhnawih metabolic pathways te a tidanglam thei tih a tarlang. Heng inthlak danglamna hmuhchhuah te hian ei tur atana him anga classified preservatives te hian gut microbiota composition a nghawng thei tih a tilang a, chu chuan mihring hriselna a nghawng thei bawk. Chung zingah chuan classification level zirchian mek a zirin P, G emaw S emaw thlan a ni. False positive classification-in nghawng a neih tlem theih nan minimum relative abundance threshold 1e-4 (1/10,000 reads) hman a ni. Statistical analysis hma hian Bracken-a report relative abundances (fraction_total_reads) te chu centered log-ratio (CLR) transformation hmangin an thlak danglam a (Aitchison, 1982). CLR method hi data transformation atan thlan a ni a, a chhan chu scale-invariant a nih avangin non-sparse dataset tan a tawk a ni (Gloor et al., 2017). CLR transformation hian natural logarithm a hmang a. Bracken-a report count data chu relative log expression (RLE) hmangin normalized a ni (Anders and Huber, 2010). Figure hi matplotlib v. 3.7.1, seaborn v. 3.7.2 leh sequential logarithms te inzawmkhawm hmanga siam a ni (Gloor et al., 2017). 0.12.2 leh stantanotations v. 0.5.0 (Hunter, 2007; Waskom, 2021; Charlier leh a thawhpuiten, 2022) te a ni. Sample tin tan Bacillus/Bacteroidetes ratio chu normalized bacterial count hmangin chhut a ni. Table-a report value te chu decimal place 4-ah round a ni. Simpson diversity index hi KrakenTools v. 1.2 package-a alpha_diversity.py script pek hmangin chhut a ni (Lu et al., 2022). Bracken report chu script-ah dah a ni a, -an parameter atan Simpson index “Si” pek a ni bawk. Abundance-a danglamna langsar tak chu mean CLR differences ≥ 1 emaw ≤ -1 emaw tiin an sawi. Mean CLR difference ±1 chuan sample type pakhat abundance chu a let 2.7-in a pung tih a tarlang. Sign (+/-) hian PPA sample leh control sample-ah te taxon a tam zawk em tih a tarlang a ni. Significance chu Mann-Whitney U test hmangin an chhut a (Virtanen et al., 2020). Statsmodels v. 0.14 (Benjamini leh Hochberg, 1995; Seabold leh Perktold, 2010) hman a ni a, Benjamini-Hochberg procedure hmangin multiple testing atana siamthat a ni. Statistical significance hriat theihna tur threshold atan adjusted p-value ≤ 0.05 hman a ni.
Mihring microbiome hi “taksa peng hnuhnung ber” tia sawi a ni fo a, mihring hriselna atan hmun pawimawh tak a chang a ni (Baquero and Nombela, 2012). A bik takin, gut microbiome hi system-wide influence leh hna pawimawh tam takah a chanvo avangin hriat hlawh tak a ni. Commensal bacteria hi gut-ah a tam hle a, ecological niche tam tak an luah a, nutrients an hmang a, natna hrik awm thei te nen an inel bawk (Jandhyala et al., 2015). Gut microbiota-a bacterial component hrang hrangte hian vitamin ang chi nutrient pawimawh tak tak an siam chhuak thei a, chaw ei khamna a tichak thei bawk (Rowland et al., 2018). Bacterial metabolites hian tissue hmasawnna a nghawng a, metabolic leh immune pathways a tichak tih hmuhchhuah a ni bawk (Heijtz et al., 2011; Yu et al., 2022). Mihring gut microbiome composition hi a inang lo hle a, genetic leh environmental factors, ei leh in, mipa leh hmeichhia, damdawi, leh hriselna dinhmunah a innghat a ni (Kumbhare et al., 2019).
Nu ei leh in hi nausen leh nausen piang tharte thanlenna atana thil pawimawh tak a ni a, hmasawnna tidanglam thei compound awmna nia ngaih a ni (Bazer et al., 2004; Innis, 2014). Chutiang compound ngaihven hlawh tak pakhat chu propionic acid (PPA) a ni a, hei hi short-chain fatty acid by-product a ni a, bacteria fermentation atanga hmuh a ni a, food additive a ni bawk (den Besten et al., 2013). PPA hian antibacterial leh antifungal property a nei a, chuvangin ei tur humhalhtu atan leh industrial application-ah te hman a ni a, mold leh bacteria te tihpunna tur a ni (Wemmenhove et al., 2016). PPA hian tissue hrang hrangah nghawng hrang hrang a nei a. Liver-ah chuan PPA hian macrophages-a cytokine expression a nghawng avangin anti-inflammatory effect a nei a (Kawasoe et al., 2022). He regulatory effect hi immune cell dang ah pawh hmuh a ni tawh a, hei hian inflammation downregulation a thlen thin (Haase et al., 2021). Mahse, a kalh zawnga nghawng chu thluak-ah hmuh a ni. Tun hmaa zirchianna hrang hrangah PPA exposure hian mice-ah autism ang chi thiltih a thlen tih hmuhchhuah a ni tawh (El-Ansary et al., 2012). Zirna dangte chuan PPA hian gliosis a thlen thei a, thluaka pro-inflammatory pathways a tichak thei tih hmuhchhuah a ni bawk (Abdelli et al., 2019). PPA hi acid chak lo tak a nih avangin intestinal epithelium kaltlangin thisenah a darh thei a, chu chuan thisen-thluak inkharkhip bakah placenta te pawh huamin restrictive barrier a paltlang thei a ni (Stinson et al., 2019), hei hian PPA hi bacteria siam chhuah regulatory metabolite a nih a pawimawhzia a tilang chiang hle. Tunah hian autism vei theihna tur risk factor anga PPA chanvo awm thei chu zirchian mek ni mah se, autism vei mimalte chunga a nghawng dan chu neural differentiation inducing aiin a zau thei a ni.
Gastrointestinal symptoms, diarrhea leh constipation te hi neurodevelopmental disorder nei damlote zingah a awm fo thin (Cao et al., 2021). Tun hmaa zirchianna hrang hrangah chuan autism spectrum disorders (ASD) vei te microbiome hi mi hrisel te microbiome nen a danglam tih hmuhchhuah a ni a, hei hian gut microbiota dysbiosis a awm tih a tilang a ni (Finegold et al., 2010). Chutiang bawkin, inflammatory bowel diseases, obesity, Alzheimer’s disease, etc. vei te microbiome characteristic pawh hi mimal hrisel tak takte nen a danglam a ni (Turnbaugh et al., 2009; Vogt et al., 2017; Henke et al., 2019). Mahse, tun thleng hian gut microbiome leh neurological diseases emaw symptoms te hi causal relationship a la awm lo (Yap et al., 2021), mahse heng natna state thenkhatah hian bacteria chi hrang hrangin hmun an chang nia ngaih a ni. Entirnan, autism vei te microbiota-ah hian Akkermansia, Bacteroides, Clostridium, Lactobacillus, Desulfovibrio leh chi dangte hi an tam zawk a ni (Tomova et al., 2015; Golubeva et al., 2017; Cristiano et al., 2018; Zurita et al., 2020). Hriat tur pawimawh tak chu heng genera thenkhat member species te hian PPA siamna nena inzawm gene an nei tih hriat a ni (Reichardt et al., 2014; Yun and Lee, 2016; Zhang et al., 2019; Baur and Dürre, 2023). PPA hian antimicrobial property a neih avangin a tamna tihpun hi PPA siamtu bacteria te thanlenna atan a hlawkpui thei a ni (Jacobson et al., 2018). Chutiang chuan PFA tamna hmun chuan gut microbiota-ah inthlak danglamna a thlen thei a, chutah chuan gastrointestinal pathogens te pawh a tel a, chu chu gastrointestinal symptoms thlen theitu a ni thei bawk.
Microbiome research-a zawhna pawimawh tak chu microbial composition inang lo hi natna hnuaia awm chhan emaw, a lan chhuahna emaw a ni em tih hi a ni. Diet, gut microbiome leh neurological diseases inzawmna khirh tak chiang zawka hriat theihna tura hmalakna hmasa ber chu ei leh in hian microbial composition a nghawng dan tehna a ni. Chumi atan chuan PPA-rich emaw PPA-depleted diet ei mice thlahte gut microbiomes tehkhin nan long-read metagenomic sequencing kan hmang a. An thlahte chu an nute ei leh in ang chiah chiah an pe a. PPA tamna ei leh in hian gut microbial composition leh microbial functional pathways a tidanglam dawn tih kan hypothesis a, a bik takin PPA metabolism leh/ emaw PPA production nena inzawm te a tidanglam dawn a ni.
He zirchiannaah hian University of Central Florida Institutional Animal Care and Use Committee (UCF-IACUC) kaihhruaina angin (Animal Use Permit Number: PROTO202000002) glia-specific GFAP promoter control hnuaia green fluorescent protein (GFP) overexpress FVB/N-Tg(GFAP-GFP)14Mes/J transgenic mice (Jackson Laboratories) te hman a ni. Nau hnute an hnek hnuah mice chu pakhat zel cage-ah dahin, cage khatah mipa leh hmeichhia 1–5 mice awmin. Mice te chu ad libitum-ah purified control diet (modified open-label standard diet, 16 kcal% fat) emaw, sodium propionate-supplemented diet (modified open-label standard diet, 16 kcal% fat, 5,000 ppm sodium propionate awmna) emaw pek an ni. Sodium propionate hman zat chu 5,000 mg PFA/kg ei tur zawng zawng nen a inang a ni. Hei hi ei tur humhalhna atana hman phalna PPA concentration sang ber a ni. He zirchianna atana inbuatsaih nan hian nu leh pa mice te chu an inpawl hmain kar 4 chhung ei tur pahnih pek an ni a, dam naupai chhung zawngin an pe chhunzawm zel a ni. Offspring mice [mice 22, control 9 (mipa 6, hmeichhia 3) leh PPA 13 (mipa 4, hmeichhia 9)] te chu hnute hnek an hnek a, chutah chuan thla 5 chhung chu dam-te ei leh in ang chiah an ei chhunzawm leh a ni. Offspring mice te chu thla 5 an tlin chuan thah an ni a, an intestinal faecal contents te chu an la khawm a, a tirah chuan 1.5 ml microcentrifuge tube ah -20°C ah dah a ni a, chumi hnuah -80°C freezer ah host DNA a tlakchham thlengin microbial nucleic acids lakchhuah thlengin dah a ni.
Host DNA chu modified protocol hmangin lakchhuah a ni (Charalampous et al., 2019). A tawi zawngin, fecal contents chu 500 μl InhibitEX (Qiagen, Cat#/ID: 19593) ah dahin frozen-ah dah a ni. Extraction khatah fecal pellets 1-2 aia tam process tur a ni. Chumi hnuah chuan fecal contents chu tube chhunga plastic pestle hmangin mechanically homogenized niin slurry siam a ni. Sample te chu 10,000 RCF ah min 5 emaw, sample te pellet a nih hma emaw centrifuge la, chutah chuan supernatant chu aspirate la, pellet chu 250 μl 1× PBS ah resuspend leh rawh. Sample-ah hian eukaryotic cell membrane tihzauh nan detergent atan 250 μl 4.4% saponin solution (TCI, product number S0019) dah la. Sample te chu a hmin thlengin zawi zawiin an pawlh a, room temperature-ah 10 min chhung an incubate a. A dawtah chuan eukaryotic cells tihbuai nan sample-ah nuclease tel lo tui 350 μl dahin, 30 s chhung an incubate a, chutah chuan 12 μl 5 M NaCl dah a ni. Chumi hnuah sample te chu 6000 RCF ah 5 min chhung centrifuged an ni. Supernatant chu aspirate la, pellet chu 100 μl 1X PBS-ah resuspend leh rawh. Host DNA lakchhuah nan HL-SAN buffer 100 μl (12.8568 g NaCl, 4 ml 1M MgCl2, 36 ml nuclease-free water) leh HL-SAN enzyme 10 μl (ArticZymes P/N 70910-202) dah tur a ni. Sample te chu pipetting hmangin uluk takin an pawlh a, EppendorfTM ThermoMixer C-ah 37 °C-ah 800 rpm-ah min 30 chhung an incubate a, an incubate hnuah 6000 RCF-ah min 3 chhung centrifuge-in 800 μl leh 1000 μl 1X PBS hmangin vawi hnih silfai a ni. A tawpah chuan pellet chu 100 μl 1X PBS-ah resuspend leh ang che.
Bacteria DNA zawng zawng chu New England Biolabs Monarch Genomic DNA Purification Kit (New England Biolabs, Ipswich, MA, Cat# T3010L) hmangin an la chhuak a. Kit nena pek chhuah standard operating procedure chu tlem a inthlak danglam a ni. Final elution atan operation hmain nuclease tel lo tui 60°C-ah incubate la, vawng reng rawh. Sample khatah Proteinase K 10 μl leh RNase A 3 μl dah tur a ni. Tichuan Cell Lysis Buffer 100 μl dah la, zawi zawiin mix rawh. Chumi hnuah sample te chu EppendorfTM ThermoMixer C-ah 56°C leh 1400 rpm-ah darkar 1 tal leh darkar 3 thleng an incubate a. Incubated sample te chu 12,000 RCF ah minute 3 chhung centrifuged a ni a, sample tin atanga supernatant chu 1.5 mL microcentrifuge tube hran, binding solution 400 μL awmnaah dah a ni. Chumi hnuah tube te chu second 1 inkar ah second 5–10 chhung pulse vortex an ni. Sample tina liquid awm zawng zawng (600–700 μL vel) chu flow-through collection tube-a dah filter cartridge-ah dah vek tur a ni. Tube te chu DNA binding hmasa ber neih theih nan RCF 1,000 ah minute 3 chhung centrifuge a ni a, chutah chuan 12,000 RCF ah minute 1 chhung centrifuged a ni a, residual liquid chu paih chhuah a ni. Sample column chu collection tube tharah dahin vawi hnih silfai a ni. Wash hmasa ber atan chuan tube tinah wash buffer 500 μL dah tur a ni. Tube chu vawi 3–5 invert la, chutah chuan 12,000 RCF-ah minute 1 chhung centrifuge rawh. Collection tube atanga liquid chu paih la, filter cartridge chu collection tube khatah dah leh rawh. Wash vawi hnihna atan chuan inverting lovin filter-ah wash buffer 500 μL dah la. Sample te chu 12,000 RCF ah minute 1 chhung centrifuged an ni. Filter chu 1.5 mL LoBind® tube-ah dah la, pre-warmed nuclease-free water 100 μL dah la. Filter te chu room temperature-ah minute 1 chhung an incubate a, chutah chuan 12,000 RCF-ah minute 1 chhung centrifuged an ni. Eluted DNA chu -80°C ah dah a ni.
DNA concentration chu QubitTM 4.0 Fluorometer hmangin an chhut a ni. DNA chu siamtute thupek angin QubitTM 1X dsDNA High Sensitivity Kit (Cat. No. Q33231) hmangin siam a ni. DNA fragment sei zawng insem dan chu AglientTM 4150 emaw 4200 TapeStation hmangin an teh a ni. DNA chu AgilentTM Genomic DNA Reagents (Cat. No. 5067-5366) leh Genomic DNA ScreenTape (Cat. No. 5067-5365) hmangin siam a ni. Library buatsaih hi a siamtute thupek angin Oxford Nanopore TechnologiesTM (ONT) Rapid PCR Barcoding Kit (SQK-RPB004) hmangin an ti a ni. DNA chu ONT GridIONTM Mk1 sequencer hmangin Min106D flow cell (R 9.4.1) hmangin sequence a ni. Sequencing settings chu: base calling dikna sang tak, q value tlem ber 9, barcode setup, leh barcode trim te a ni. Sample te chu darkar 72 chhung sequenced a ni a, chumi hnuah base call data chu process leh analysis dang neih belh turin submit a ni.
Bioinformatics processing hi a hmaa kan sawi tawh angin an ti a (Greenman et al., 2024). Sequencing atanga FASTQ file hmuhte chu sample tin atan directory-ah then a ni. Bioinformatics analysis hma hian a hnuaia pipeline hmang hian data te hi process a ni a: a hmasa berah chuan sample te FASTQ file te chu FASTQ file pakhatah an dah khawm a. Tichuan, 1000 bp aia tawi reads chu Filtlong v. 0.2.1 hmangin filter a ni a, parameter thlak danglam awmchhun chu –min_length 1000 a ni (Wick, 2024). Filter leh hmain read quality chu NanoPlot v. 1.41.3 hmangin a hnuaia parameter te hmang hian control a ni: –fastq –plots dot –N50 -o
Taxonomic classification atan chuan reads leh assembled contigs te chu Kraken2 v. 2.1.2 hmangin classified a ni ( Wood et al., 2019 ). Read leh assembly atan report leh output file siam. Read leh assembly te thlirletna atan –use-names option hmang la. –gzip-compressed leh –paired option te hi read segment hrang hrang atan tarlan a ni. Metagenome-a taxa awm zat (relative abundance) chu Bracken v. 2.8 hmangin an chhut a ( Lu et al., 2017 ). Bracken-build hmangin base 1000 awmna kmer database kan siam hmasa a, a hnuaia parameter te hi kan siam a: -d
Gene annotation leh relative abundance estimation chu Maranga leh a thawhpuiten an sawi protocol modified version hmangin an ti a. (Maranga leh a thawhpuiten, 2023). Pakhatnaah chuan SeqKit v. 2.5.1 hmangin assembly zawng zawng atangin contigs 500 bp aia tawi te chu lakchhuah a ni (Shen et al., 2016). Chumi hnuah chuan assembly thlan bikte chu pan-metagenome-ah an dah khawm ta a ni. Open reading frame (ORF) te chu Prodigal v. 1.0.1 (Prodigal v. 2.6.3 parallel version) hmangin a hnuaia parameter te nen hriatchhuah a ni: -d
Genes te hi Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog (KO) identifiers hmanga eggNOG-in gene pathway abundances tehkhin nan a ruat angin group-ah an dah hmasa ber a ni. Knockout nei lo gene emaw, knockout tam tak nei gene emaw chu an zirchian hmain an paih chhuak a ni. Chumi hnuah sample khata KO pakhat zel abundance average chu chhut a ni a, statistical analysis tih a ni. PPA metabolism genes chu KEGG_Pathway column-a row ko00640-a dah gene eng pawh tihna a ni a, hei hian KEGG angin propionate metabolism-ah chanvo a nei tih a tilang a ni. PPA siam chhuahna nena inzawm nia hriat genes te chu Supplementary Table 1-ah tarlan a ni (Reichardt et al., 2014; Yang et al., 2017). Permutation test hmangin PPA metabolism leh production genes sample type tinah a tam zawk tih hmuhchhuah a ni. Gene an zirchian apiangah permutation sangkhat an nei a. Statistical significance hriat nan p-value 0.05 chu cutoff atan hman a ni. Functional annotations chu cluster chhunga gene hrang hrangte hnenah cluster chhunga representative genes annotations hmangin an dah a ni. PPA metabolism leh/ emaw PPA siamna nena inzawm taxa chu Kraken2 output file-a contig ID-te chu eggNOG hmanga functional annotation neih laiin contig ID ang chiah vawn reng nen matching hmangin hriat theih a ni. Significance testing hi a hmaa kan sawi tawh Mann-Whitney U test hmangin an ti a. Multiple testing atana siamthatna chu Benjamini-Hochberg procedure hmangin an ti a. Statistical significance hriat nan p-value ≤ 0.05 chu cutoff atan hman a ni.
Mice-te gut microbiome hrang hrangte chu Simpson diversity index hmangin an zirchiang a. Control leh PPA sample te hi genus leh species diversity lamah danglamna lian tham a awm lo (genus tan p-value: 0.18, species tan p-value: 0.16) (Figure 1). Chumi hnuah chuan principal component analysis (PCA) hmangin microbial composition an khaikhin a. Figure 2-ah hian sample hrang hrangte chu an phyla hrang hranga clustering a ni a, hei hian PPA leh control sample-te inkara microbiomes-a species composition-ah danglamna a awm tih a tilang a ni. He clustering hi genus level-ah a langsar lo hle a, hei hian PPA hian bacteria thenkhat a nghawng tih a tilang (Supplementary Fig. 1).
Figure 1. Mouse gut microbiome-a genera leh species composition hrang hrangte alpha diversity. PPA leh control sample-a genera (A) leh species (B) te Simpson diversity indices tarlanna box plot. Significance chu Mann-Whitney U test hmangin an chhut a, Benjamini-Hochberg procedure hmangin multiple correction an ti bawk. ns, p-value a langsar lo (p>0.05).
Figure 2. Mouse gut microbiome composition species level-a principal component analysis result. Principal component analysis plot-ah hian sample hrang hrangte chu an principal component hmasa pahniha an insem dan a tarlang a. Color hian sample type a tarlang a: PPA-exposed mice chu purple niin control mice chu yellow a ni. Principal component 1 leh 2 te hi x-axis leh y-axis-ah te plot a ni a, an explained variance ratio angin tarlan a ni.
RLE transformed count data hmangin control leh PPA mice-ah median Bacteroidetes/Bacilli ratio a tlahniam nasa hle tih hmuhchhuah a ni (control: 9.66, PPA: 3.02; p-value = 0.0011). He danglamna hi PPA mice-a Bacteroidetes tam zawk vang a ni a, control-te nena khaikhin chuan a danglamna a langsar lo hle (control mean CLR: 5.51, PPA mean CLR: 6.62; p-value = 0.054), Bacteroidetes abundance erawh a inang thung (control mean CLR: 7.76, PPA mean CLR: 7.60; p-value = 0.18 a ni).
Gut microbiome-a taxonomic member tam zia an zirchiannaah chuan PPA leh control sample-ah hian phylum 1 leh species 77-ah danglamna nasa tak a awm tih hmuhchhuah a ni (Supplementary Table 2). PPA sample-a chi 59 awm zat chu control sample aiin a tam zawk tih a chiang a, control sample-a chi 16 chauh awm zat erawh PPA sample aiin a tam zawk thung (Figure 3).
Figure 3. PPA leh control mice-te gut microbiome-a taxa tam dan inang lo. Volcano plot-ah hian PPA leh control sample-te zingah hian genera (A) emaw species (B) emaw tam dan inang lo a awm a ni. Gray dots hian taxa tamnaah danglamna lian tham a awm lo tih a tarlang. Dot rawng hrang hrangte hian abundance-ah danglamna nasa tak a nei tih a tarlang (p-value ≤ 0.05). Sample chi hrang hrang tamna inang lo ber taxa top 20 te chu red leh light blue (control leh PPA sample)-in an tarlang a ni. Yellow leh purple dots hi control emaw PPA sample-ah chuan control aiin a let 2.7 tal a tam zawk a ni. Black dots hian taxa tamna inang lo tak tak a entir a, mean CLR danglamna chu -1 leh 1 inkar a ni a, P value te chu Mann-Whitney U test hmangin chhut a ni a, Benjamini-Hochberg procedure hmangin multiple testing atan siamthat a ni. Bold mean CLR danglamna hian abundance-ah danglamna nasa tak a lantir a ni.
Gut microbial composition kan zirchian hnuah microbiome functional annotation kan nei a. Gene quality hniam tak tak filter chhuah hnuah sample zawng zawngah unique genes 378,355 hmuhchhuah a ni. Heng genes transformed abundance hi principal component analysis (PCA) atan hman a ni a, result-ah hian sample chi hrang hrangte chu an functional profile atanga thlirin clustering sang tak a awm tih a lang (Figure 4).
Figure 4. Mouse gut microbiome functional profile hmanga PCA result. PCA plot-ah hian sample hrang hrangte chu an principal component hmasa pahnihah an insem darh dan a tarlang a ni. Color hian sample type a tarlang a: PPA-exposed mice chu purple niin control mice chu yellow a ni. Principal component 1 leh 2 te hi x-axis leh y-axis-ah te plot a ni a, an explained variance ratio angin tarlan a ni.
A dawtah chuan sample chi hrang hranga KEGG knockout tam zia kan enfiah leh a. Unique knockout 3648 hmuhchhuah a ni a, chung zinga 196 chu control sample-ah a tam zawk tih a chiang a, 106 chu PPA sample-ah a tam zawk bawk (Figure 5). Control sample-ah gene 145 hmuhchhuah a ni a, PPA sample-ah gene 61 hmuhchhuah a ni a, abundance pawh a inang lo hle. Lipid leh aminosugar metabolism nena inzawm kawng hrang hrangte chu PPA sample-ah hian a hausa zawk tih a chiang hle (Supplementary Table 3). Nitrogen metabolism leh sulfur relay system nena inzawm kawngte chu control sample-ah hian a hausa zawk tih a chiang hle (Supplementary Table 3). PPA sample-ah hian aminosugar/nucleotide metabolism (ko:K21279) leh inositol phosphate metabolism (ko:K07291) nena inzawm gene tamna chu a tam zawk tih a chiang hle (Figure 5). Control sample-ah hian benzoate metabolism (ko:K22270), nitrogen metabolism (ko:K00368), leh glycolysis/gluconeogenesis (ko:K00131) nena inzawm genes a tam zawk tih a chiang hle ( Figure 5 ).
Fig. 5. PPA leh control mice-te gut microbiome-a KO awm zat inang lo. Volcano plot-ah hian functional group (KO) tam dan inang lo tak tak a lang a. Gray dots hian sample chi hrang hrang (p-value > 0.05) abundance danglamna lian tham nei lo KO a tarlang a ni. Dot rawng hrang hrangte hian abundance-ah danglamna nasa tak a nei tih a tarlang (p-value ≤ 0.05). Sample chi hrang hrang tamna inang lo ber KO 20 te chu red leh light blue-a tarlan a ni a, control leh PPA sample te nen a inmil a ni. Yellow leh purple dots hian control leh PPA sample-ah KO a tlem berah a let 2.7-in a tam zawk tih a tarlang. Black dots hian abundance hrang hrang nei KO a tarlang a, mean CLR danglamna chu -1 leh 1 inkar a ni a, P value te chu Mann-Whitney U test hmangin chhut a ni a, Benjamini-Hochberg procedure hmangin multiple comparisons atan siamrem a ni. NaN hian KO hi KEGG-a pathway pakhatah a tel lo tih a tarlang. Bold mean CLR difference value hian abundance-ah danglamna nasa tak a awm tih a tarlang. KO tarlan te hi eng kawng nge an nih tih chipchiar zawka hriat duh chuan Supplementary Table 3 en rawh.
Annotated gene zingah hian gene 1601 chuan sample chi hrang hrangah abundance hrang hrang (p ≤ 0.05) an nei a, gene tin hi a let 2.7 tal a tam zawk a ni. Heng zingah hian control sample-ah hian gene 4 a tam zawk a, PPA sample-ah hian gene 1597 a tam zawk bawk. PPA hian antimicrobial property a neih avangin sample chi hrang hrang inkara PPA metabolism leh production genes tam zia kan enfiah a. PPA metabolism nena inzawm gene 1332 zingah hian control sample-ah gene 27 a tam zawk tih a chiang a, PPA sample-ah hian gene 12 a tam zawk tih a chiang hle. PPA siamna nena inzawm gene 223 zingah hian PPA sample-ah hian gene 1 a tam zawk tih a chiang hle. Figure 6A hian PPA metabolism-a inrawlh genes tam zawk a tarlang belh a, control sample-ah chuan a tam zawk tih a chiang hle a, effect size lian tak tak a awm bawk a, Figure 6B-ah hian PPA sample-a hmuh chhuah tamna sang zawk nei genes pakhat zel a tarlang bawk.
Fig. 6. Mouse gut microbiome-a PPA nena inzawm genes tam dan inang lo. Volcano plot-ah hian PPA metabolism (A) leh PPA production (B) nena inzawm gene tam dan inang lo tak tak a awm a. Gray dots hian sample chi hrang hrang (p-value > 0.05) a tamna a danglamna lian tham lo gene a tarlang a ni. Dot rawng hrang hrangte hian abundance-ah danglamna nasa tak a nei tih a tarlang (p-value ≤ 0.05). Gene 20 tamna inang lo ber chu red leh light blue (control leh PPA sample)-ah te tarlan a ni. Yellow leh purple dot tamna hi control sample aiin a let 2.7 tal a tam zawk a, control leh PPA sample-ah hian a tam zawk a ni. Black dots hian genes tamna inang lo tak tak a entir a, mean CLR difference chu -1 leh 1 inkar a ni a, P value te chu Mann-Whitney U test hmangin chhut a ni a, Benjamini-Hochberg procedure hmangin multiple comparisons atan siamthat a ni. Genes hi non-redundant gene catalog-a representative genes te nen a inmil a ni. Gene hmingte hi KO gene tarlanna KEGG symbol atanga siam a ni. Bold mean CLR danglamna hian abundances inang lo tak a lantir a ni. Dash (-) hian KEGG database-ah hian gene atan hian symbol a awm lo tih a tarlang.
PPA metabolism leh/ emaw production nena inzawm gene nei taxa te chu contigs te taxonomic identity leh gene contig ID nena inmil tir a ni. Genus level-ah chuan genera 130-in PPA metabolism nena inzawm gene an nei tih hmuhchhuah a ni a, genera 61-in PPA siamna nena inzawm gene an nei tih hmuhchhuah a ni bawk (Supplementary Table 4). Mahse, genera pakhat mah hian abundance-ah danglamna lian tham a nei lo (p > 0.05).
Species level-ah chuan bacteria chi 144-in PPA metabolism nena inzawm gene an nei tih hmuhchhuah a ni a, bacteria chi 68-in PPA siamna nena inzawm gene an nei tih hmuhchhuah a ni bawk (Supplementary Table 5). PPA metabolizer zingah hian bacteria pariat chuan sample chi hrang hrang inkarah a tamna a pung nasa hle a, an vai hian an nghawng danah danglamna nasa tak an nei vek a ni (Supplementary Table 6). PPA metabolizer hmuhchhuah zawng zawng, a tamna inang lo tak takte chu PPA sample-ah an tam zawk a ni. Species-level classification-ah chuan sample chi hrang hrangah danglamna lian tham nei lo genera aiawh an hmuchhuak a, chung zingah chuan Bacteroides leh Ruminococcus chi hrang hrang bakah Duncania dubois, Myxobacterium enterica, Monococcus pectinolyticus, leh Alcaligenes polymorpha te pawh an tel. PPA siamtu bacteria zingah hian bacteria pali chuan sample chi hrang hrangah an tam danah danglamna nasa tak an nei a ni. A tamna inang lo tak tak chi hrang hrangte chu Bacteroides novorossi, Duncania dubois, Myxobacterium enteritidis, leh Ruminococcus bovis te an ni.
He zirchiannaah hian PPA exposure hian mice gut microbiota a nghawng dan kan enfiah a. PPA hian bacteria-ah hian chhanna hrang hrang a thlen thei a, a chhan chu chi thenkhat siam a nih vang a ni a, chi dangin ei tur atana an hman vang emaw, antimicrobial effect a neih vang emaw a ni. Chuvangin, dietary supplementation hmanga gut environment-a a dah belh hian tolerance, susceptibility, leh nutrient source atana hman theihna a zirin nghawng hrang hrang a nei thei a ni. Bacteria chi hrang hrang sensitive tak takte chu paih chhuah a, PPA laka invenna nei zawk emaw, ei tur atana hmang thei emaw te chuan an thlak thei a, chu chuan gut microbiota composition a tidanglam thei a ni. Kan result-ah hian microbial composition-ah danglamna nasa tak a awm tih kan hmu a, mahse microbial diversity pumpuiah erawh nghawng a nei lo. A nghawng lian ber chu species level-ah hmuh a ni a, taxa 70 chuang chu PPA leh control sample-te zingah a tam danah a inang lo hle (Supplementary Table 2). PPA-exposed sample-te composition endik belh leh chuan microbial species-te chu unexposed sample-te nena khaikhin chuan heterogeneity a sang zawk tih hmuhchhuah a ni a, hei hian PPA hian bacteria growth characteristic a tichak thei a, PPA tamna hmuna bacteria population dam khawchhuak thei tur a tihtlem thei tih a tilang. Chutiang chuan PPA hian gut microbiota diversity nasa taka tihbuai ai chuan a thlangin inthlak danglamna a thlen thei a ni.
PPA ang chi food preservatives hian a hmain gut microbiome components tam zia a tidanglam thei tih hmuhchhuah a ni tawh a, overall diversity a nghawng lo (Nagpal et al., 2021). Hetah hian phylum Bacteroidetes (a hmaa Bacteroidetes tia hriat) chhunga Bacteroidetes chi hrang hrangte inkara danglamna langsar ber kan hmu a, chungte chu PPA-exposed mice-ah nasa takin an hausa a ni. Bacteroides chi tam zawk hi mucus degradation sang zawk nen a inzawm a, hei hian natna kai theihna a tipung thei a, inflammation a tipung thei bawk (Cornick et al., 2015; Desai et al., 2016; Penzol et al., 2019). Zirna pakhatah chuan Bacteroides fragilis hmanga enkawl mipa piang tharte chuan autism spectrum disorder (ASD) hriatrengna social behavior an nei tih hmuhchhuah a ni (Carmel et al., 2023), zirchianna dangah chuan Bacteroides species hian immune activity a tidanglam thei a, autoimmune inflammatory cardiomyopathy a thlen thei tih hmuhchhuah a ni bawk (Gil-Cruz et al., 2019). PPA vei mice-ah pawh Ruminococcus, Prevotella, leh Parabacteroides chi hrang hranga tel chi hrang hrangte pawh nasa takin an pung bawk (Coretti et al., 2018). Ruminococcus chi thenkhat chu proinflammatory cytokines siam chhuah hmangin Crohn natna ang chi natna nen an inzawm a (Henke et al., 2019), Prevotella chi hrang hrang Prevotella humani te erawh chu metabolic natna, hypertension leh insulin sensitivity te nen an inzawm thung (Pedersen et al., 2016; Li et al., 2017). A tawp berah chuan Bacteroidetes (a hmaa Firmicutes tia hriat) leh Bacteroidetes ratio chu PPA-exposed mice-ah chuan control mice aiin a hniam zawk tih kan hmuchhuak a, hei hi Bacteroidetes species total abundance a sang zawk vang a ni. He ratio hi a hmain intestinal homeostasis tilangtu pawimawh tak a ni tih hmuhchhuah a ni tawh a, he ratio-a buaina hi natna dinhmun hrang hrang nen a inzawm tih hmuhchhuah a ni (Turpin et al., 2016; Takezawa et al., 2021; An et al., 2023), inflammatory bowel diseases te pawh a tel (Stojanov et al., 2020). A pum puia kan han thlir chuan phylum Bacteroidetes chi hrang hrangte chu ei leh in lama PPA sang hian a nghawng nasa ber niin a lang. Hei hi PPA tolerance sang zawk emaw, PPA chu energy source atana hman theihna vang emaw a ni thei a, hei hi chi khat tal Hoylesella enocea tan pawh a dik tih hmuhchhuah a ni (Hitch et al., 2022). A dang pawhin, nu PPA exposure hian nausen thanlenna a tichak thei a, mouse fate gut chu Bacteroidetes colonization laka hlauhawm zawkah a siam thei bawk mahse, kan zirchianna design chuan chutiang tehfung chu a phal lo.
Metagenomic content assessment-ah chuan PPA metabolism leh production nena inzawm genes tamnaah danglamna nasa tak a awm tih hmuhchhuah a ni a, PPA-exposed mice-te chuan PPA siamna atana mawhphurtu genes tam zawk an lantir a, non-PPA-exposed mice-te erawh chuan PAA metabolism-a mawhphurtu genes tam zawk an lantir thung (Figure 6). Heng results te hian PPA hian microbial composition a nghawng dan hi a hman vang chauh a ni lo mai thei tih a tilang a, chutiang a nih loh chuan PPA metabolism nena inzawm genes tam lutuk hian PPA-exposed mice-te gut microbiome-ah a tam zawk tih a lantir tur a ni. A sawifiahna pakhat chu PPA hian bacteria tamna chu bacteria-in nutrient atana a hman aiin a antimicrobial effects hmangin a mediate ber a ni. Tun hmaa zirchianna hrang hrangah chuan PPA hian Salmonella Typhimurium hi a dose a zirin a tipung thei tih hmuhchhuah a ni (Jacobson et al., 2018). PPA concentration sang zawka inhriat hian a antimicrobial property do thei bacteria a thlang thei a, a metabolize thei lo emaw, a siam thei lo emaw pawh a ni thei. Entirnan, Parabacteroides chi engemaw zatah chuan PPA sample-ah hian a tam zawk tih hmuhchhuah a ni a, mahse PPA metabolism emaw production emaw nena inzawm gene erawh hmuhchhuah a ni lo (Supplementary Tables 2, 4, leh 5). Chubakah, fermentation byproduct anga PPA siamchhuah hi bacteria hrang hrangah a darh zau hle bawk (Gonzalez-Garcia et al., 2017). Bacteria hrang hrang tam zawk hi control sample-a PPA metabolism nena inzawm gene tam zawk chhan a ni thei (Averina et al., 2020). Tin, gene 1332 zinga 27 (2.14%) chauh chu PPA metabolism nena inzawm chauh gene an nih thu an sawi bawk. PPA metabolism nena inzawm gene tam tak hi metabolic pathway dang ah pawh an inrawlh ve bawk. Hei hian PPA metabolism-a inrawlh gene tamna chu control sample-ah a tam zawk tih a tilang lehzual a; heng genes te hian PPA hmanna emaw byproduct anga siam emaw thlen lohna kawngah an thawk thei a ni. Hetiang a nih chuan PPA generation nena inzawm gene pakhat chauh hian sample chi hrang hrangah abundance-ah danglamna nasa tak a nei a ni. PPA metabolism nena inzawm genes nen a danglamna chu PPA siamna atana marker genes thlan a ni a, a chhan chu PPA siamna atana bacterial pathway-ah direct-a inrawlh an nih vang a ni. PPA-exposed mice-ah chuan species zawng zawng hian an tamna leh PPA siam chhuah theihna nasa takin an tipung tih hmuhchhuah a ni. Hei hian PPA-te hian PPA siamtute an thlang ang tih sawi lawk chu a thlawp a, chuvangin PPA siam chhuah theihna a sang dawn tih an sawi lawk a ni. Mahse, gene tamna hi gene expression nen a inzawm tlat lo; chuvangin, PPA metabolism nena inzawm genes tamna chu control sample-ah chuan sang zawk mah se, expression rate chu a danglam thei (Shi et al., 2014). PPA siamtu gene tamna leh PPA siam chhuahna inzawmna finfiah nan PPA siamna atana inrawlh genes expression chungchang zirchianna neih a ngai a ni.
PPA leh control metagenome-a functional annotation hmangin danglamna engemaw zat a awm tih hmuhchhuah a ni. PCA hmanga gene content an zirchiannaah chuan PPA leh control sample inkarah hian discrete clusters a awm tih hmuhchhuah a ni (Figure 5). Within-sample clustering-ah chuan control gene content chu a hrang zawk tih hmuhchhuah a ni a, PPA sample erawh chu a cluster khawm thung. Gene content atanga clustering chu species composition atanga clustering nen tehkhin theih a ni. Chutiang chuan pathway abundance inang lo chu an chhunga species leh strain bik tamna inthlak danglamna nen a inmil a ni. PPA sample-ah chuan a tamna sang zawkna kawng pahnih chu aminosugar/nucleotide sugar metabolism (ko:K21279) leh multiple lipid metabolism pathways (ko:K00647, ko:K03801; Supplementary Table 3) nen a inzawm a ni. ko:K21279 nena inzawm genes te hi PPA sample-a chi hrang hrang tam zawkna chi khat Bacteroides chi nen an inzawm tih hriat a ni. He enzyme hian capsular polysaccharides a express a, immune response a pumpelh thei a ni (Wang et al., 2008). Hei hian PPA-exposed mice-a Bacteroidetes a punna chhan a ni thei. Hei hian PPA microbiome-a fatty acid synthesis tihpun hmuhchhuah chu a tichak hle. Bacteria hian FASIIko:K00647 (fabB) pathway hmangin fatty acids an siam a, hei hian host metabolic pathways a nghawng thei a ni (Yao and Rock, 2015; Johnson et al., 2020), lipid metabolism inthlak danglamna hian neurodevelopment-ah chanvo a nei thei bawk (Yu et al., 2020). PPA sample-a abundance tihpunna kawng dang chu steroid hormone biosynthesis (ko:K12343) a ni. Gut microbiota-in hormone level a nghawng theihna leh hormone-in a nghawng theihna inkarah hian inverse relationship a awm tih finfiahna a pung zel a, chutiang chuan steroid level sang chuan downstream health consequence a nei thei a ni (Tetel et al., 2018).
He zirchianna hi tihkhawtlai leh ngaihtuah tur awm lo a ni lo. Inthliarna pawimawh tak chu rannungte physiological assessment kan ti lo tih hi a ni. Chuvangin, microbiome-a inthlak danglamna hi natna eng emaw nen a inzawm em tih direct-a thutlukna siam theih a ni lo. Ngaihtuah tur dang chu he zirchiannaa mice te hi an nu te ei leh in ang chiah an ei a ni. Nakin lawka zirchianna chuan PPA tamna ei leh in atanga PPA tel lo ei leh in lama inthlak hian microbiome-a a nghawng dan a ti \ha em tih a hriat theih ang. Kan zirchianna tihkhawtlai pakhat chu mi dang tam tak ang bawkin sample size tlem hi a ni. Thutlukna dik tak siam theih ni mah se, sample size lian zawk chuan result thlirletnaah statistical power nasa zawk a pe ang. Gut microbiome inthlak danglamna leh natna eng pawh inzawmna chungchangah pawh thutlukna siamnaah kan fimkhur hle bawk (Yap et al., 2021). Kum, mipa leh hmeichhia, leh ei leh in te bakah thil buaithlak tak takte hian microorganisms composition nasa takin a nghawng thei a ni. Heng thilte hian gut microbiome leh natna khirh tak tak inzawmna chungchanga literature-a inmil lohna hmuhchhuah te chu a hrilhfiah thei a ni (Johnson et al., 2019; Lagod and Naser, 2023). Entirnan, ASD vei rannung leh mihringah hian Bacteroidetes chi member te hi an pung emaw, an tlahniam emaw tih hmuhchhuah a ni (Angelis et al., 2013; Kushak et al., 2017). Chutiang bawkin, inflammatory bowel disease nei damlote gut composition zirchiannaah pawh taxa inangah hian a pung leh a tlahniam tih hmuhchhuah a ni bawk (Walters et al., 2014; Forbes et al., 2018; Upadhyay et al., 2023). Gender bias-in nghawng a neih tihtlem nan, mipa leh hmeichhia inthlauhna chu ei leh in atanga lo chhuak a nih a rinawm ber theih nan mipa leh hmeichhia inthlauhna (equal representation) kan neih theih nan kan bei a. Functional annotation-a harsatna pakhat chu redundant gene sequences tihbo hi a ni. Kan gene clustering method hian 95% sequence identity leh 85% length similarity a mamawh a, chubakah false clustering tihbo nan 90% alignment coverage a mamawh bawk. Mahse, a then phei chuan annotation inang (eg, MUT) nei COG kan hmu a (Fig. 6). Heng orthologs te hi an danglam em, genera bik nena inzawm nge, hei hi gene clustering approach tihkhawtlai a nih leh nih loh hriat chian nan zirchianna dang neih belh a ngai a ni. Functional annotation tihkhawtlai dang chu classification dik lo awm thei hi a ni a; bacteria gene mmdA hi propionate siamna atana inrawlh enzyme hriat lar tak a ni a, mahse KEGG hian propionate metabolic pathway nen a inzawm lo. Chumi danglamna chu scpB leh mmcD orthologs te hi an inzawm tlat a ni. Designated knockout nei lo gene tam lutuk hian gene tamna tehnaah PPA nena inzawm genes hriat theih lohna a thlen thei a ni. Nakin lawka zirchiannaah chuan metatranscriptome analysis hmangin hlawkna a awm dawn a, hei hian gut microbiota hnathawh dan hriatthiamna thuk zawk a pe thei a, gene expression chu downstream effect awm thei nen a thlunzawm thei bawk. Neurodevelopmental disorder bik emaw, inflammatory bowel diseases emaw chungchanga zirchianna atan chuan, microbiome composition inthlak danglamna chu heng disorders te nena inzawm tir turin rannungte physiological leh behavioral assessment neih a ngai a ni. Gut microbiome hi germ-free mice-a transplant-na zirchianna dang neih belh pawh hi microbiome hi natna driver emaw characteristic emaw a nih leh nih loh hriat nan a tangkai hle ang.
A tawi zawngin, dietary PPA hian gut microbiota composition tidanglamtu hna a thawk tih kan hmuchhuak a. PPA hi FDA-in a pawmpui preservative a ni a, ei tur hrang hrangah a tam hle a, hun rei tak a awm chuan gut flora pangngai a tibuai thei a ni. Bacteria engemaw zat tamnaah inthlak danglamna kan hmu a, hei hian PPA hian gut microbiota composition a nghawng thei tih a tilang. Microbiota inthlak danglamna hian metabolic pathway thenkhat level a tidanglam thei a, chu chuan host hriselna nena inzawm physiological change a thlen thei a ni. Dietary PPA hian microbial composition a nghawng dan hian dysbiosis emaw natna dang emaw a thlen thei em tih hriat nan zirchianna neih belh a ngai a ni. He zirchianna hian nakin lawka PPA-in gut composition a nghawng danin mihring hriselna a nghawng theih dan tur zirchianna tur lungphum a phum a ni.
He zirchiannaa dataset tarlan te hi online repository-ah a awm a. Repository hming leh accession number chu hetiang hi a ni: https://www.ncbi.nlm.nih.gov/, PRJNA1092431.
He rannung zirchianna hi University of Central Florida Institutional Animal Care and Use Committee (UCF-IACUC) (Animal Use Permit Number: PROTO202000002) chuan a pawm a ni. He zirchianna hian tualchhung dan, dan leh hrai leh institutional requirement te a zawm a ni.
NG: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Thuziak (original draft), Thuziak (review & editing) tih te hi a ni. LA: Conceptualization, Data curation, Methodology, Resources, Thuziak (review & editing) te hi a ni. SH: Formal analysis, Software, Thuziak (review & editing) te a ni. SA: Investigation, Thuziak (review & editing) tih te. Chief Judge: Investigation, Thuziak (review & editing) te a ni. SN: Conceptualization, Project enkawl dan, Resources, Supervision, Thuziak (review & editing) te a ni. TA: Conceptualization, Project enkawl, Supervision, Thuziak (review & editing) te a ni.
He thuziak ziaktute hian he thuziak zirchianna, ziaktu nihna leh/ emaw tihchhuahna atan hian sum leh paia tanpuina an dawng lo tih an puang.
Hemi ziaktute hian zirchianna hi sumdawnna emaw sum leh pai lama inzawmna awm lo, inhnialna awm thei anga ngaih theih a nih loh thu an puang. hman theih a ni lo.
He thuziaka ngaihdan tarlan zawng zawng hi a ziaktute ngaihdan chauh a ni a, an institution, publisher, editor, emaw reviewer-te ngaihdan a ni hauh lo. He thuziak-a thil siam ennawn apiang, emaw, a siamtute sawi chhuah emaw chu a chhuahtuin a guarantee emaw, a endorsed emaw a ni lo.
He thuziak atana thil pawimawh dangte chu online-ah hmuh theih a ni: https://www.frontiersin.org/articles/10.3389/frmbi.2024.1451735/full#supplementary-material
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Post hun chhung: Apr-18-2025