Bridging Bacteria and the Gut: Functional Aspects of Type IV Pili
2020; Elsevier BV; Volume: 28; Issue: 5 Linguagem: Inglês
10.1016/j.tim.2020.02.003
ISSN1878-4380
AutoresKate Ligthart, Clara Belzer, Willem M. de Vos, Hanne L. P. Tytgat,
Tópico(s)Clostridium difficile and Clostridium perfringens research
ResumoPili are key interaction molecules in the context of the gut microbiota.Type IV pili (T4P) are ubiquitous in bacteria and are of great functional importance.About 30%, and potentially even up to 45%, of microbiota members are thought to express T4P, illustrating their importance in the context of microbiota–host interactions.Functions are diverse and include (but are not limited to): adhesion, biofilm formation, motility, and molecule exchange [e.g., double stranded (ds)DNA and proteins].T4P are found in all monoderm and diderm bacteria and in members of the Archaea. Cell-surface-located proteinaceous appendages, such as flagella and fimbriae or pili, are ubiquitous in bacterial communities. Here, we focus on conserved type IV pili (T4P) produced by bacteria in the intestinal tract, one of the most densely populated human ecosystems. Computational analysis revealed that approximately 30% of known intestinal bacteria are predicted to produce T4P. To rationalize how T4P allow intestinal bacteria to interact with their environment, other microbiota members, and host cells, we review their established role in gut commensals and pathogens with respect to adherence, motility, and biofilm formation, as well as protein secretion and DNA uptake. This work indicates that T4P are widely spread among the known members of the intestinal microbiota and that their contribution to human health might be underestimated. Cell-surface-located proteinaceous appendages, such as flagella and fimbriae or pili, are ubiquitous in bacterial communities. Here, we focus on conserved type IV pili (T4P) produced by bacteria in the intestinal tract, one of the most densely populated human ecosystems. Computational analysis revealed that approximately 30% of known intestinal bacteria are predicted to produce T4P. To rationalize how T4P allow intestinal bacteria to interact with their environment, other microbiota members, and host cells, we review their established role in gut commensals and pathogens with respect to adherence, motility, and biofilm formation, as well as protein secretion and DNA uptake. This work indicates that T4P are widely spread among the known members of the intestinal microbiota and that their contribution to human health might be underestimated. Microbial interactions are essential for growth and survival. These include interactions of microbes with the abiotic environment as well as with other cells, including other microbes, viruses, and eukaryotic cells. In many cases these interactions are promoted by cell-surface-located structures that form protruding appendages, such as flagella and pili or fimbriae (see Glossary). Pili are proteinaceous hair-like appendages that offer bacteria a wide range of functional adaptations. Especially in the human gut, which is colonized by large bacterial communities, pili can be crucial for adhesion to host cells and molecules. The gut is an extremely versatile environment, characterized by constant turnover and flow as a result of food intake, mucin renewal, epithelial cell turnover, and peristalsis [1.Tytgat H.L.P. et al.Bowel biofilms: tipping points between a healthy and compromised gut?.Trends Microbiol. 2019; 27: 17-25Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. Previous deep metagenomic analyses of the gut microbiota have identified type I pili as a major factor enhancing survival and persistence of low-abundant species in the gut [2.Arumugam M. et al.Enterotypes of the human gut microbiome.Nature. 2011; 473: 174-180Crossref PubMed Scopus (3679) Google Scholar]. While microscopic observations were used for their initial identification, pili are presently classified based on their structure and mode of biogenesis. Here, we focus on type IV pili (T4P) since they are widely spread, functionally diverse, and share a similar genetic organization, protein composition (Box 1), and conserved biogenesis (Box 2) [3.Denise R. et al.Diversification of the type IV filament superfamily into machines for adhesion, protein secretion, DNA uptake, and motility.PLoS Biol. 2019; 17e3000390Crossref PubMed Scopus (33) Google Scholar]. T4P are involved in adherence, DNA uptake, motility, biofilm formation, and protein secretion (Figure 1, Key Figure). Like many other surface appendages, T4P can also be functionally exploited by bacteriophages that use these pili as receptors [4.Bradley D.E. Pitt T.L. Pilus-dependence of four Pseudomonas aeruginosa bacteriophages with non-contractile tails.J. Gen. Virol. 1974; 24: 1-15Crossref PubMed Scopus (55) Google Scholar]. T4P are typically 5–8 nanometers in diameter and can range up to several micrometers in length, exceeding the size of bacteria [5.Craig L. et al.Type IV pilus structure and bacterial pathogenicity.Nat. Rev. Microbiol. 2004; 2: 363-378Crossref PubMed Scopus (525) Google Scholar].Box 1T4P ArchitectureThe most striking structural feature of T4P is the pilus, which primarily consists of major pilin subunits. This major pilin subunit is called PilA in the T4P model organism P. aeruginosa, and TcpA and BfpA in resp. V. cholerae and E. coli. [52.Giltner C.L. et al.Type IV pilin proteins: versatile molecular modules.Microbiol. Mol. Biol. Rev. 2012; 76: 740-772Crossref PubMed Scopus (236) Google Scholar]. The major pilins are synthesized as prepilins and targeted to the membrane by an αN-terminal sequence motif, the class III signal peptide. This positively charged signal peptide ensures proper orientation of PilA in the inner membrane [53.Craig L. et al.Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions.Mol. Cell. 2006; 23: 651-662Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar]. Once in the inner membrane, the prepilin peptidase PilD cleaves off this leader sequence and methylates PilA (Box 2) [54.Strom M.S. et al.A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2404-2408Crossref PubMed Scopus (216) Google Scholar]. After this, PilA can be incorporated into the growing pilus structure [54.Strom M.S. et al.A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2404-2408Crossref PubMed Scopus (216) Google Scholar]. The major pilins of T4aP and T4bP are distinct, with the leader peptides of T4aP typically being less than ten amino acid residues in length and mature pilins ranging between 140 and 160 residues [9.Strom M.S. Lory S. Structure–function and biogenesis of the type IV pili.Annu. Rev. Microbiol. 1993; 47: 565-596Crossref PubMed Scopus (398) Google Scholar]. In T4bP, leader peptides are longer, namely 15–30 amino acids, and they precede a larger mature pilin of 180–200 residues [9.Strom M.S. Lory S. Structure–function and biogenesis of the type IV pili.Annu. Rev. Microbiol. 1993; 47: 565-596Crossref PubMed Scopus (398) Google Scholar]. The pilus is thus mainly built of PilA but can be further decorated with minor pilins. In bacteria expressing T4aP, a group of minor pilins, PilV, PilW, PilX, FimU, and PilE, are thought to form a priming complex for major pilin assembly (Box 2) [55.Nguyen Y. et al.Pseudomonas aeruginosa minor pilins prime type IVa pilus assembly and promote surface display of the PilY1 adhesin.J. Biol. Chem. 2015; 290: 601-611Crossref PubMed Scopus (56) Google Scholar]. Prepilin peptidase PilD is shown to process these minor pilins similarly to PilA, cleaving the leader sequence and methylating the pilin [56.Giltner C.L. et al.Pseudomonas aeruginosa minor pilins are incorporated into type IV pili.J. Mol. Biol. 2010; 398: 444-461Crossref PubMed Scopus (97) Google Scholar]. The minor pilins are thought to form a short stem that reduces the energy barrier for extension of the major pilin and can both be found at the tip and throughout the pilus [56.Giltner C.L. et al.Pseudomonas aeruginosa minor pilins are incorporated into type IV pili.J. Mol. Biol. 2010; 398: 444-461Crossref PubMed Scopus (97) Google Scholar]. Finally, T4P complexes can accommodate additional minor pilins and adhesins which confer extra specificity and functionality to the pilus – for example, ComP in Neisseria binds DNA [57.Cehovin A. et al.Specific DNA recognition mediated by a type IV pilin.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 3065-3070Crossref PubMed Scopus (91) Google Scholar], or PilY1, which aids in the adhesion of P. aeruginosa [55.Nguyen Y. et al.Pseudomonas aeruginosa minor pilins prime type IVa pilus assembly and promote surface display of the PilY1 adhesin.J. Biol. Chem. 2015; 290: 601-611Crossref PubMed Scopus (56) Google Scholar]. An overview of T4P biogenesis is given in Box 2.Box 2Biogenesis of T4PBiogenesis of T4P is strongly conserved, and bacteria expressing them harbor similar biogenesis genes, related to T2SS [44.Ayers M. et al.Architecture of the type II secretion and type IV pilus machineries.Future Microbiol. 2010; 5: 1203-1218Crossref PubMed Scopus (90) Google Scholar]. The first step in pilus assembly is the attachment of an ATPase, PilB (yellow, Figure I), to the cytoplasmic ring formed by PilM (blue) and platform protein PilC (green) [58.Chang Y.-W. et al.Architecture of the type IVa pilus machine.Science. 2016; 351aad2001Crossref PubMed Scopus (182) Google Scholar] (Figure I). The cytoplasmic ring, PilM, forms together with subunits PilN (light gray), PilO (dark gray), and PilP (orange), an inner membrane alignment complex called PilMNOP [58.Chang Y.-W. et al.Architecture of the type IVa pilus machine.Science. 2016; 351aad2001Crossref PubMed Scopus (182) Google Scholar] (Figure I). Attachment of PilB to PilM causes conformational changes in PilN and PilO, resulting in a cage-like ring in the inner membrane and periplasm [58.Chang Y.-W. et al.Architecture of the type IVa pilus machine.Science. 2016; 351aad2001Crossref PubMed Scopus (182) Google Scholar,59.McCallum M. et al.The dynamic structures of the type IV pilus.Microbiol. Spectr. 2019; 7: 1-12Crossref Scopus (14) Google Scholar]. This allows PilA (black) subunits to enter the complex, enabling pilus assembly [60.Leighton T.L. et al.Novel role for PilNO in type IV pilus retraction revealed by alignment subcomplex mutations.J. Bacteriol. 2015; 197: 2229-2238Crossref PubMed Scopus (21) Google Scholar]. The ATPase PilB is also attached to platform protein PilC, resulting in the incorporation of PilA subunits into the pilus, thus elongating the pilus [61.Takhar H.K. et al.The platform protein is essential for type IV pilus biogenesis.J. Biol. Chem. 2013; 288: 9721-9728Crossref PubMed Scopus (67) Google Scholar]. Minor pilins are synthesized in a similar fashion to PilA and are thought to form a priming complex for major pilin assembly (cf. Box 1). They are mostly found at the pilus tip, but also along the length of the pilus [56.Giltner C.L. et al.Pseudomonas aeruginosa minor pilins are incorporated into type IV pili.J. Mol. Biol. 2010; 398: 444-461Crossref PubMed Scopus (97) Google Scholar]. In Gram-negative bacteria, the inner membrane PilMNOP complex needs an outer membrane pore to facilitate the transfer of the pilus through the periplasm during (de-)polymerization [62.Tammam S. et al.PilMNOPQ from the Pseudomonas aeruginosa type IV pilus system form a transenvelope protein interaction network that interacts with PilA.J. Bacteriol. 2013; 195: 2126-2135Crossref PubMed Scopus (65) Google Scholar]. This outer membrane pore is formed by PilQ (red) which is linked to the inner alignment complex PilMNOP by PilP [58.Chang Y.-W. et al.Architecture of the type IVa pilus machine.Science. 2016; 351aad2001Crossref PubMed Scopus (182) Google Scholar] (Figure I). PilQ has two internal gates, a secretin gate and a periplasmic gate, which are closed in the absence of a pilus to prevent leakage of molecules [63.Gold V.A.M. et al.Structure of a type IV pilus machinery in the open and closed state.eLife. 2015; 4: 1-12Crossref Scopus (66) Google Scholar]. Obviously, PilQ is absent in Gram-positive bacteria where the pilus is directly sticking out through the peptidoglycan layer. Once fully assembled, the pilus can attach to a surface, thus inducing tension in the pilus. In T4aP, the retraction ATPase PilT (brown) is responsible for pilus retraction. The PilB ATPase can be released from the platform protein PilC, allowing PilT (brown) to interact with PilC [58.Chang Y.-W. et al.Architecture of the type IVa pilus machine.Science. 2016; 351aad2001Crossref PubMed Scopus (182) Google Scholar], after which PilA subunits are released back into the membrane (Figure I) [14.Wolfgang M. et al.PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae.Mol. Microbiol. 1998; 29: 321-330Crossref PubMed Scopus (256) Google Scholar]. In T4bP this process occurs independently of the PilT ATPase [17.Anantha R.P. et al.Role of BfpF, a member of the PilT family of putative nucleotide-binding proteins, in type IV pilus biogenesis and in interactions between enteropathogenic Escherichia coli and host cells.Infect. Immun. 1998; 66: 122-131Crossref PubMed Google Scholar,18.Ng D. et al.The Vibrio cholerae minor pilin TcpB initiates assembly and retraction of the toxin-coregulated pilus.PLoS Pathog. 2016; 12e1006109Crossref PubMed Scopus (30) Google Scholar].Figure 1Key Figure. The Various Functions of Type IV Pili (T4P) in Gut-Related Species.Show full captionT4P-carrying cells are indicated by a cartoon of a bacillus covered in T4P throughout; this, however, is only one of the potential manifestations of T4P – which can also be less abundant and may be polarly localized. T4P have established roles in microbiota–host interactions, in both commensal and pathogenic bacteria. One of these roles is motility: bacteria use T4P to move around by growing their pilus, attaching it to a surface, and finally pulling themselves forward by retraction of the pilus. The T4P major pilin PilA, sometimes with the help of minor (tip) pilins, is also essential for adherence to surfaces and other bacteria, which can ultimately lead to biofilm formation. T4P also play a role in the exchange of molecules between the bacterium and its environment: T4P can bind and subsequently take up double stranded (ds)DNA and secrete proteins. In the latter process, growth of the pilus, through the membrane, pushes out proteins in the manner of a piston. It will be exciting to see if dedicated T4P functional studies uncover further roles for T4P in general, and in the specific context of the gut. Abbreviation: IEC, intestinal epithelial cell.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The most striking structural feature of T4P is the pilus, which primarily consists of major pilin subunits. This major pilin subunit is called PilA in the T4P model organism P. aeruginosa, and TcpA and BfpA in resp. V. cholerae and E. coli. [52.Giltner C.L. et al.Type IV pilin proteins: versatile molecular modules.Microbiol. Mol. Biol. Rev. 2012; 76: 740-772Crossref PubMed Scopus (236) Google Scholar]. The major pilins are synthesized as prepilins and targeted to the membrane by an αN-terminal sequence motif, the class III signal peptide. This positively charged signal peptide ensures proper orientation of PilA in the inner membrane [53.Craig L. et al.Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions.Mol. Cell. 2006; 23: 651-662Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar]. Once in the inner membrane, the prepilin peptidase PilD cleaves off this leader sequence and methylates PilA (Box 2) [54.Strom M.S. et al.A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2404-2408Crossref PubMed Scopus (216) Google Scholar]. After this, PilA can be incorporated into the growing pilus structure [54.Strom M.S. et al.A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2404-2408Crossref PubMed Scopus (216) Google Scholar]. The major pilins of T4aP and T4bP are distinct, with the leader peptides of T4aP typically being less than ten amino acid residues in length and mature pilins ranging between 140 and 160 residues [9.Strom M.S. Lory S. Structure–function and biogenesis of the type IV pili.Annu. Rev. Microbiol. 1993; 47: 565-596Crossref PubMed Scopus (398) Google Scholar]. In T4bP, leader peptides are longer, namely 15–30 amino acids, and they precede a larger mature pilin of 180–200 residues [9.Strom M.S. Lory S. Structure–function and biogenesis of the type IV pili.Annu. Rev. Microbiol. 1993; 47: 565-596Crossref PubMed Scopus (398) Google Scholar]. The pilus is thus mainly built of PilA but can be further decorated with minor pilins. In bacteria expressing T4aP, a group of minor pilins, PilV, PilW, PilX, FimU, and PilE, are thought to form a priming complex for major pilin assembly (Box 2) [55.Nguyen Y. et al.Pseudomonas aeruginosa minor pilins prime type IVa pilus assembly and promote surface display of the PilY1 adhesin.J. Biol. Chem. 2015; 290: 601-611Crossref PubMed Scopus (56) Google Scholar]. Prepilin peptidase PilD is shown to process these minor pilins similarly to PilA, cleaving the leader sequence and methylating the pilin [56.Giltner C.L. et al.Pseudomonas aeruginosa minor pilins are incorporated into type IV pili.J. Mol. Biol. 2010; 398: 444-461Crossref PubMed Scopus (97) Google Scholar]. The minor pilins are thought to form a short stem that reduces the energy barrier for extension of the major pilin and can both be found at the tip and throughout the pilus [56.Giltner C.L. et al.Pseudomonas aeruginosa minor pilins are incorporated into type IV pili.J. Mol. Biol. 2010; 398: 444-461Crossref PubMed Scopus (97) Google Scholar]. Finally, T4P complexes can accommodate additional minor pilins and adhesins which confer extra specificity and functionality to the pilus – for example, ComP in Neisseria binds DNA [57.Cehovin A. et al.Specific DNA recognition mediated by a type IV pilin.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 3065-3070Crossref PubMed Scopus (91) Google Scholar], or PilY1, which aids in the adhesion of P. aeruginosa [55.Nguyen Y. et al.Pseudomonas aeruginosa minor pilins prime type IVa pilus assembly and promote surface display of the PilY1 adhesin.J. Biol. Chem. 2015; 290: 601-611Crossref PubMed Scopus (56) Google Scholar]. An overview of T4P biogenesis is given in Box 2. Biogenesis of T4P is strongly conserved, and bacteria expressing them harbor similar biogenesis genes, related to T2SS [44.Ayers M. et al.Architecture of the type II secretion and type IV pilus machineries.Future Microbiol. 2010; 5: 1203-1218Crossref PubMed Scopus (90) Google Scholar]. The first step in pilus assembly is the attachment of an ATPase, PilB (yellow, Figure I), to the cytoplasmic ring formed by PilM (blue) and platform protein PilC (green) [58.Chang Y.-W. et al.Architecture of the type IVa pilus machine.Science. 2016; 351aad2001Crossref PubMed Scopus (182) Google Scholar] (Figure I). The cytoplasmic ring, PilM, forms together with subunits PilN (light gray), PilO (dark gray), and PilP (orange), an inner membrane alignment complex called PilMNOP [58.Chang Y.-W. et al.Architecture of the type IVa pilus machine.Science. 2016; 351aad2001Crossref PubMed Scopus (182) Google Scholar] (Figure I). Attachment of PilB to PilM causes conformational changes in PilN and PilO, resulting in a cage-like ring in the inner membrane and periplasm [58.Chang Y.-W. et al.Architecture of the type IVa pilus machine.Science. 2016; 351aad2001Crossref PubMed Scopus (182) Google Scholar,59.McCallum M. et al.The dynamic structures of the type IV pilus.Microbiol. Spectr. 2019; 7: 1-12Crossref Scopus (14) Google Scholar]. This allows PilA (black) subunits to enter the complex, enabling pilus assembly [60.Leighton T.L. et al.Novel role for PilNO in type IV pilus retraction revealed by alignment subcomplex mutations.J. Bacteriol. 2015; 197: 2229-2238Crossref PubMed Scopus (21) Google Scholar]. The ATPase PilB is also attached to platform protein PilC, resulting in the incorporation of PilA subunits into the pilus, thus elongating the pilus [61.Takhar H.K. et al.The platform protein is essential for type IV pilus biogenesis.J. Biol. Chem. 2013; 288: 9721-9728Crossref PubMed Scopus (67) Google Scholar]. Minor pilins are synthesized in a similar fashion to PilA and are thought to form a priming complex for major pilin assembly (cf. Box 1). They are mostly found at the pilus tip, but also along the length of the pilus [56.Giltner C.L. et al.Pseudomonas aeruginosa minor pilins are incorporated into type IV pili.J. Mol. Biol. 2010; 398: 444-461Crossref PubMed Scopus (97) Google Scholar]. In Gram-negative bacteria, the inner membrane PilMNOP complex needs an outer membrane pore to facilitate the transfer of the pilus through the periplasm during (de-)polymerization [62.Tammam S. et al.PilMNOPQ from the Pseudomonas aeruginosa type IV pilus system form a transenvelope protein interaction network that interacts with PilA.J. Bacteriol. 2013; 195: 2126-2135Crossref PubMed Scopus (65) Google Scholar]. This outer membrane pore is formed by PilQ (red) which is linked to the inner alignment complex PilMNOP by PilP [58.Chang Y.-W. et al.Architecture of the type IVa pilus machine.Science. 2016; 351aad2001Crossref PubMed Scopus (182) Google Scholar] (Figure I). PilQ has two internal gates, a secretin gate and a periplasmic gate, which are closed in the absence of a pilus to prevent leakage of molecules [63.Gold V.A.M. et al.Structure of a type IV pilus machinery in the open and closed state.eLife. 2015; 4: 1-12Crossref Scopus (66) Google Scholar]. Obviously, PilQ is absent in Gram-positive bacteria where the pilus is directly sticking out through the peptidoglycan layer. Once fully assembled, the pilus can attach to a surface, thus inducing tension in the pilus. In T4aP, the retraction ATPase PilT (brown) is responsible for pilus retraction. The PilB ATPase can be released from the platform protein PilC, allowing PilT (brown) to interact with PilC [58.Chang Y.-W. et al.Architecture of the type IVa pilus machine.Science. 2016; 351aad2001Crossref PubMed Scopus (182) Google Scholar], after which PilA subunits are released back into the membrane (Figure I) [14.Wolfgang M. et al.PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae.Mol. Microbiol. 1998; 29: 321-330Crossref PubMed Scopus (256) Google Scholar]. In T4bP this process occurs independently of the PilT ATPase [17.Anantha R.P. et al.Role of BfpF, a member of the PilT family of putative nucleotide-binding proteins, in type IV pilus biogenesis and in interactions between enteropathogenic Escherichia coli and host cells.Infect. Immun. 1998; 66: 122-131Crossref PubMed Google Scholar,18.Ng D. et al.The Vibrio cholerae minor pilin TcpB initiates assembly and retraction of the toxin-coregulated pilus.PLoS Pathog. 2016; 12e1006109Crossref PubMed Scopus (30) Google Scholar]. T4P-carrying cells are indicated by a cartoon of a bacillus covered in T4P throughout; this, however, is only one of the potential manifestations of T4P – which can also be less abundant and may be polarly localized. T4P have established roles in microbiota–host interactions, in both commensal and pathogenic bacteria. One of these roles is motility: bacteria use T4P to move around by growing their pilus, attaching it to a surface, and finally pulling themselves forward by retraction of the pilus. The T4P major pilin PilA, sometimes with the help of minor (tip) pilins, is also essential for adherence to surfaces and other bacteria, which can ultimately lead to biofilm formation. T4P also play a role in the exchange of molecules between the bacterium and its environment: T4P can bind and subsequently take up double stranded (ds)DNA and secrete proteins. In the latter process, growth of the pilus, through the membrane, pushes out proteins in the manner of a piston. It will be exciting to see if dedicated T4P functional studies uncover further roles for T4P in general, and in the specific context of the gut. Abbreviation: IEC, intestinal epithelial cell. T4P are widely distributed in Gram-negative and Gram-positive bacteria, such as Pseudomonas aeruginosa, Neisseria gonorrhoeae, Vibrio cholerae, and Clostridium sp., as well as in several Archaea [6.Hospenthal M.K. et al.A comprehensive guide to pilus biogenesis in Gram-negative bacteria.Nat. Rev. Microbiol. 2017; 15: 365-379Crossref PubMed Scopus (114) Google Scholar, 7.Danne C. Dramsi S. Pili of Gram-positive bacteria: roles in host colonization.Res. Microbiol. 2012; 163: 645-658Crossref PubMed Scopus (78) Google Scholar, 8.Pohlschroder M. Esquivel R.N. Archaeal type IV pili and their involvement in biofilm formation.Front. Microbiol. 2015; 6: 190Crossref PubMed Scopus (37) Google Scholar]. Bacterial T4P are further divided into subtypes, namely type IVa pili (T4aP), type IVb pili (T4bP), and Tad pili, which recently have been designated as type IVc pili (T4cP) [3.Denise R. et al.Diversification of the type IV filament superfamily into machines for adhesion, protein secretion, DNA uptake, and motility.PLoS Biol. 2019; 17e3000390Crossref PubMed Scopus (33) Google Scholar,9.Strom M.S. Lory S. Structure–function and biogenesis of the type IV pili.Annu. Rev. Microbiol. 1993; 47: 565-596Crossref PubMed Scopus (398) Google Scholar], which are all assembled using conserved mechanisms (see Figure I in Box 2). T4aP are characterized by the presence of a PilT retraction ATPase, resulting in retraction of the pilus which can generate large mechanical forces (nanoNewton range) [10.Biais N. et al.Cooperative retraction of bundled type IV pili enables nanonewton force generation.PLoS Biol. 2008; 6e87Crossref PubMed Scopus (103) Google Scholar]. This pilus retraction can result in motility (Box 2) [11.Berry J.-L. Pelicic V. Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives.FEMS Microbiol. Rev. 2015; 39: 134-154Crossref PubMed Scopus (124) Google Scholar]. However, V. cholerae T4aP have been shown to retract in the absence of a PilT retraction ATPase [12.Ellison C.K. et al.Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae.Nat. Microbiol. 2018; 3: 773-780Crossref PubMed Scopus (94) Google Scholar]. T4aP are widely spread and studied in detail in P. aeruginosa [13.Bradley D.E. A function of Pseudomonas aeruginosa PAO polar pili: twitching motility.Can. J. Microbiol. 1980; 26: 146-154Crossref PubMed Scopus (232) Google Scholar] and in N. gonorrhoeae [14.Wolfgang M. et al.PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae.Mol. Microbiol. 1998; 29: 321-330Crossref PubMed Scopus (256) Google Scholar]. T4bP are less prevalent and less uniform, and are produced by enteropathogenic Escherichia coli (EPEC) [15.Sohel I. et al.Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis.J. Bacteriol. 1996; 178: 2613-2628Crossref PubMed Google Scholar] and V. cholerae [toxin coregulated pili (TCP)] [16.Sun D. et al.Domains within the Vibrio cholerae toxin coregulated pilin subunit that mediate bacterial colonization.Gene. 1997; 192: 79-85Crossref PubMed Scopus (29) Google Scholar] amongst others. While most T4bP lack PilT, they can have another ATPase, for example, the BfpF ATPase of EPEC bundle-forming pili [17.Anantha R.P. et al.Role of BfpF, a member of the PilT family of putative nucleotide-binding proteins, in type IV pilus biogenesis and in interactions between enteropathogenic Escherichia coli and host cells.Infect. Immun. 1998; 66: 122-131Crossref PubMed Google Scholar]. They can also use a different mechanism entirely, as in V. cholerae where TCP retraction is thought to be triggered by the incorporation of a specific minor pilin leading to spontaneous disassembly and retraction [18.Ng D. et al.The Vibrio cholerae minor pilin TcpB initiates assembly and retraction of the toxin-coregulated pilus.PLoS Pathog. 2016; 12e1006109Crossref PubMed Scopus (30) Google Scholar]. Tight-adherence (Tad) pili have smaller pilin subunits than other T4P and are encoded at a single genetic locus. They used to be considered a subclass of T4bP, but recent phylogenetic studies showed that they form a distinct phylogenetic clade, namely T4cP [3.Denise R. et al.Diversification of the type IV filament superfamily into machines for adhesion, protein secretion, DNA uptake, and motility.PLoS Biol. 2019; 17e3000390Crossref PubMed Scopus (33) Google Scholar,19.Ellison C.K. et al.Obstruction of pilus retraction stimulates bacterial surface sensing.Science. 2017; 358: 535-538Crossref PubMed Scopus (105) Google Scholar]. T4cP retraction also takes place in the absence of PilT. For example, in Caulobacter crescentus, T4cP retraction was shown to be regulated by CpaF, a bifunctional pilus motor that is also responsible for extension [20.Ellison C.K. et al.A bifunctional ATPase drives tad pilus extension and retraction.Sci. Adv. 2019; 5eaay2591Crossref PubMed Scopus (11) Google Scholar]. Several omics studies and cultivation efforts have generated insights into species that are pres
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