Unveiling the genetic landscape and motility-associated functions of Type IV pili in the Bifidobacterium genus

Abstract

Background: Bifidobacteria are recognized as one of the most influential bacterial groups inhabiting the human gut, capable of modulating the host’s health. To understand how these bacteria interact with their hosts, it is essential to investigate their extracellular structures, such as pili. While the presence of sortase-dependent pili has already been investigated in the Bifidobacterium genus, limited information is available on Type IV pili (T4P), which have previously been identified in a few species as tight adherence (Tad) loci.

Methods: Here, we explored the T4P distribution across the currently 117 (sub)species representing all described taxa of the Bifidobacterium genus, revealing two distinct loci unevenly distributed within the genus through in silico genomic analyses supported by in vitro validation.

Results: Our analysis identified a conserved Type IVc pili (T4cP) structure across all bifidobacterial taxa, with minor predicted structural variations in members of the Bifidobacterium longum and Bifidobacterium boum phylogenetic groups. This T4cP structure, also known as Tad, exhibited an ancestral, non-retractile architecture typically associated with stable colonization and long-term persistence. In addition, a secondary Type IVa pili (T4aP) structure was detected in 13 bifidobacterial species. These species are associated with specific ecological niches, including primate, bovine, and porcine hosts, suggesting a link between this locus and host-associated adaptation.

Conclusion: Notably, twitching motility assays demonstrated that Bifidobacterium adolescentis strains harboring the T4aP locus exhibit motility in response to specific environmental signals, observed upon starch supplementation of the growth medium, thereby challenging the traditional view of bifidobacteria as a strictly non-motile bacterial genus.

Keywords

Bifidobacteria / genomics / phylogenetics / extracellular structures / T4P

References

[1] Hidalgo-Cantabrana C,Delgado S,Ruiz L,Ruas-Madiedo P,Sánchez B,Margolles A. Bifidobacteria and their health-promoting effects Microbiol Spectr. 2017 5 10.1128/microbiolspec.bad 0010 PMC11687494

[2] Lugli GA,Fontana F,Tarracchini C.et al. Exploring the biodiversity of Bifidobacterium asteroides among honey bee microbiomes Environ Microbiol. 2022 24 5666 79 PMC10092428

[3] Breselge S,Bellassi P,Barcenilla C,Álvarez-Ordóñez A,Morelli L,Cotter PD. Bifidobacterium fermentum sp. nov. and Bifidobacterium aquikefiricola sp. nov., isolated from water kefir Int J Syst Evol Microbiol. 2024 74 006549 PMC11500810

[4] Laureys D,Cnockaert M,De Vuyst L,Vandamme P. Bifidobacterium aquikefiri sp. nov., isolated from water kefir Int J Syst Evol Microbiol. 2016 66 1281 6

[5] Delcenserie V,Taminiau B,Gavini F.et al. Detection and characterization of Bifidobacterium crudilactis and B. mongoliense able to grow during the manufacturing process of French raw milk cheeses BMC Microbiol. 2013 13 239 PMC4231354

[6] Longhi G,Lugli GA,Tarracchini C.et al. From raw milk cheese to the gut: investigating the colonization strategies of Bifidobacterium mongoliense Appl Environ Microbiol. 2024 90 e0124424 PMC11409640

[7] Bondue P,Lebrun S,Taminiau B.et al. Effect of Bifidobacterium crudilactis and 3'-sialyllactose on the toddler microbiota using the SHIME® model Food Res Int. 2020 138 109755

[8] Milani C,Duranti S,Napoli S.et al. Colonization of the human gut by bovine bacteria present in Parmesan cheese Nat Commun. 2019 10 1286 PMC6426854

[9] Rohde M. The gram-positive bacterial cell wall Microbiol Spectr. 2019 7 10.1128/microbiolspec.gpp3 0044

[10] Wadhwa N,Berg HC. Bacterial motility: machinery and mechanisms Nat Rev Microbiol. 2022 20 161 73

[11] Limoli DH,Jones CJ,Wozniak DJ. Bacterial extracellular polysaccharides in biofilm formation and function Microbiol Spectr. 2015 3 10.1128/microbiolspec.MB 0011 4657554

[12] Danne C,Dramsi S. Pili of gram-positive bacteria: roles in host colonization Res Microbiol. 2012 163 645 58

[13] Verbunt J,Jocken J,Blaak E,Savelkoul P,Stassen F. Gut-bacteria derived membrane vesicles and host metabolic health: a narrative review Gut Microbes. 2024 16 2359515 PMC11141482

[14] Isidro-Coxca MI,Ortiz-Jiménez S,Puente JL. Type 1 fimbria and P pili: regulatory mechanisms of the prototypical members of the chaperone-usher fimbrial family Arch Microbiol. 2024 206 373 PMC11316696

[15] Costa TRD,Patkowski JB,Macé K,Christie PJ,Waksman G. Structural and functional diversity of type IV secretion systems Nat Rev Microbiol. 2024 22 170 85 PMC11290344

[16] Shoji M,Shibata S,Sueyoshi T,Naito M,Nakayama K. Biogenesis of type V pili Microbiol Immunol. 2020 64 643 56

[17] Milani C,Mangifesta M,Mancabelli L.et al. The sortase-dependent fimbriome of the genus bifidobacterium: extracellular structures with potential to modulate microbe-host dialogue Appl Environ Microbiol. 2017 83 e01295 17 5601332

[18] Turroni F,Serafini F,Foroni E.et al. Role of sortase-dependent pili of Bifidobacterium bifidum PRL2010 in modulating bacterium-host interactions Proc Natl Acad Sci U S A. 2013 110 11151 6 PMC3703987

[19] Foroni E,Serafini F,Amidani D.et al. Genetic analysis and morphological identification of pilus-like structures in members of the genus Bifidobacterium Microb Cell Fact. 2011 10 Suppl 1 Suppl S16 PMC3231923

[20] O'Connell Motherway M,Zomer A,Leahy SC.et al. Functional genome analysis of Bifidobacterium breve UCC2003 reveals type IVb tight adherence (Tad) pili as an essential and conserved host-colonization factor Proc Natl Acad Sci U S A. 2011 108 11217 22 PMC3131351

[21] O'Connell Motherway M,Houston A,O'Callaghan G.et al. A bifidobacterial pilus-associated protein promotes colonic epithelial proliferation Mol Microbiol. 2019 111 287 301

[22] Wang H,An J,Fan C,Zhai Z,Zhang H,Hao Y. Transcriptome analysis revealed growth phase-associated changes of a centenarian-originated probiotic Bifidobacterium animalis subsp. lactis A6 BMC Microbiol. 2022 22 61 PMC8876546

[23] Egan M,Bottacini F,O'Connell Motherway M.et al. Staying alive: growth and survival of Bifidobacterium animalis subsp. animalis under in vitro and in vivo conditions Appl Microbiol Biotechnol. 2018 102 10645 63

[24] Ellison CK,Whitfield GB,Brun YV. Type IV Pili: dynamic bacterial nanomachines FEMS Microbiol Rev. 2022 46 fuab053

[25] Gorgel M,Ulstrup JJ,Bøggild A.et al. High-resolution structure of a type IV pilin from the metal-reducing bacterium Shewanella oneidensis BMC Struct Biol. 2015 15 4 PMC4376143

[26] Clock SA,Planet PJ,Perez BA,Figurski DH. Outer membrane components of the Tad (tight adherence) secreton of Aggregatibacter actinomycetemcomitans J Bacteriol. 2008 190 980 90 2223556

[27] Sonani RR,Sanchez JC,Baumgardt JK.et al. Tad and toxin-coregulated pilus structures reveal unexpected diversity in bacterial type IV pili Proc Natl Acad Sci U S A. 2023 120 e2316668120 PMC2223556

[28] O'Leary NA,Wright MW,Brister JR.et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation Nucleic Acids Res. 2016 44 D733 45 PMC4702849

[29] Jain C,Rodriguez-R LM,Phillippy AM,Konstantinidis KT,Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries Nat Commun. 2018 9 5114 PMC6269478

[30] Chklovski A,Parks DH,Woodcroft BJ,Tyson GW. CheckM2: a rapid, scalable and accurate tool for assessing microbial genome quality using machine learning Nat Methods. 2023 20 1203 12 12949516

[31] Lugli GA,Fontana F,Tarracchini C.et al. MEGAnnotator2: a pipeline for the assembly and annotation of microbial genomes Microbiome Res Rep. 2023 2 15 PMC10696586

[32] Hyatt D,Chen GL,Locascio PF,Land ML,Larimer FW,Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification BMC Bioinformatics. 2010 11 119 PMC2848648

[33] Buchfink B,Reuter K,Drost HG. Sensitive protein alignments at tree-of-life scale using DIAMOND Nat Methods. 2021 18 366 8 PMC8026399

[34] Fu L,Niu B,Zhu Z,Wu S,Li W. CD-HIT: accelerated for clustering the next-generation sequencing data Bioinformatics. 2012 28 3150 2 PMC3516142

[35] Jones P,Binns D,Chang HY.et al. InterProScan 5: genome-scale protein function classification Bioinformatics. 2014 30 1236 40 PMC3998142

[36] Mistry J,Chuguransky S,Williams L.et al. Pfam: The protein families database in 2021 Nucleic Acids Res. 2021 49 D412 9 PMC7779014

[37] Page AJ,Cummins CA,Hunt M.et al. Roary: rapid large-scale prokaryote Pan genome analysis Bioinformatics. 2015 31 3691 3 PMC4817141

[38] Katoh K,Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability Mol Biol Evol. 2013 30 772 80 PMC3603318

[39] Li KB. ClustalW-MPI: ClustalW analysis using distributed and parallel computing Bioinformatics. 2003 19 1585 6

[40] Letunic I,Bork P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool Nucleic Acids Res. 2024 52 W78 82 PMC11223838

[41] Duranti S,Turroni F,Lugli GA.et al. Genomic characterization and transcriptional studies of the starch-utilizing strain Bifidobacterium adolescentis 22L Appl Environ Microbiol. 2014 80 6080 90 4178675

[42] Turnbull L,Whitchurch CB. Motility assay: twitching motility Methods Mol Biol. 2014 1149 73 86

[43] O'Hara MT,Shimozono TM,Dye KJ,Harris D,Yang Z. Surface hydrophilicity promotes bacterial twitching motility mSphere. 2024 9 e0039024 PMC11423576

[44] Melville S,Craig L. Type IV pili in Gram-positive bacteria Microbiol Mol Biol Rev. 2013 77 323 41 3811610

[45] Whitfield GB,Brun YV. The type IVc pilus: just a Tad different Curr Opin Microbiol. 2024 79 102468

[46] Tassinari M,Rudzite M,Filloux A,Low HH. Assembly mechanism of a Tad secretion system secretin-pilotin complex Nat Commun. 2023 14 5643 PMC10499894

[47] Lugli GA,Alessandri G,Milani C.et al. Evolutionary development and co-phylogeny of primate-associated bifidobacteria Environ Microbiol. 2020 22 3375 93

[48] Lugli GA,Milani C,Turroni F.et al. Comparative genomic and phylogenomic analyses of the Bifidobacteriaceae family BMC Genomics. 2017 18 568 PMC5540593

[49] Mayrhofer S,Domig KJ,Amtmann E.et al. Antibiotic susceptibility of Bifidobacterium thermophilum and Bifidobacterium pseudolongum isolates from animal sources J Food Prot. 2007 70 119 24

[50] Gavini F,Delcenserie V,Kopeinig K.et al. Bifidobacterium species isolated from animal feces and from beef and pork meat J Food Prot. 2006 69 871 7

[51] Gavzy SJ,Kensiski A,Lee ZL,Mongodin EF,Ma B,Bromberg JS. Bifidobacterium mechanisms of immune modulation and tolerance Gut Microbes. 2023 15 2291164 PMC10730214

[52] Ligthart K,Belzer C,de Vos WM,Tytgat HLP. Bridging bacteria and the gut: functional aspects of type IV Pili Trends Microbiol. 2020 28 340 8

[53] Biswas I,Machen A,Mettlach J. In vitro motility assays for acinetobacter species Methods Mol Biol. 2019 1946 177 87