Deep mutational scanning of the Neisseria meningitidis major pilin reveals the importance of pilus tip‐mediated adhesion

Abstract

Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are crucial for virulence. Indeed, these homopolymers of the major pilin PilE mediate interbacterial aggregation and adhesion to host cells. However, the mechanisms behind these functions remain unclear. Here, we simultaneously determined regions of PilE involved in pilus display, auto-aggregation, and adhesion by using deep mutational scanning and started mining this extensive functional map. For auto-aggregation, pili must reach a minimum length to allow pilus-pilus interactions through an electropositive cluster of residues centered around Lys140. For adhesion, results point to a key role for the tip of the pilus. Accordingly, purified pili interacting with host cells initially bind via their tip-located major pilin and then along their length. Overall, these results identify functional domains of PilE and support a direct role of the major pilin in TFP-dependent aggregation and adhesion.

Bacteria autoaggregation: how and why bacteria stick together

“…The enteropathogenic Escherichia coli bundle-forming pili are autoaggregation effectors that mediate localized adherence on epithelial cells, resulting in microcolonies, which are tightened by pilus retraction and stabilized by other adhesins such as the Escherichia common pilus (Figure 1) [7,8]. Other type IV fimbriae mediating autoaggregation include the many types of enteroaggregative E. coli aggregative adherence fimbriae [9][10][11][12] and Neisseria meningitidis type IV pili [13,14]. These retractable pili typically mediate host cell adherence by the binding of pilus tip proteins to specific receptors and autoaggregation by lateral, bundling interactions among the main structural subunits of different pili [13].…”

“…Other type IV fimbriae mediating autoaggregation include the many types of enteroaggregative E. coli aggregative adherence fimbriae [9][10][11][12] and Neisseria meningitidis type IV pili [13,14]. These retractable pili typically mediate host cell adherence by the binding of pilus tip proteins to specific receptors and autoaggregation by lateral, bundling interactions among the main structural subunits of different pili [13]. These dual function adhesins are important in the initiation, growth, maintenance and disassembly of auto-aggregates (microcolonies) within infection niches.…”

“…Adhesins may bind to heterologous receptors but autoaggregation is often achieved through self-association. The N. meningitidis PilE pilus structural subunit mediates autoaggregation by electrostatic means, requiring a C-terminal lysine residue [ 13 ]. In contrast, the heat-resistant agglutinin 1 (Hra1) and its allelic variant Hek [ 24 ] depend on specific interaction motifs, but their structural basis for self-association is presently unknown [ 25 ].…”

Variable disruption of epithelial monolayers by Neisseria meningitidis carriage isolates of the hypervirulent MenW cc11 and MenY cc23 lineages

“…The mechanistic basis for the differences between MenY:cc23 and MenW:cc11 carriage strains in their ability to disrupt epithelial monolayers is currently unclear. Retraction of the pilus facilitates intimate adhesion of meningococci to host cells, twitching motility and host cell remodelling, all of these phenotypes have the potential to contribute to tissue disruption [46][47][48]. Retraction of the meningococcal pilus is controlled by PilT but is also dependent on the PilC proteins, with the PilC1 protein being upregulated in a pilT mutant [49].…”

Tranception: protein fitness prediction with autoregressive transformers and inference-time retrieval

“…Wavenet Tranception w/o retrieval Tranception w/ retrieval A0A1J4YT16 9PROT (Davidi et al, 2020) 0.117 0.178 0.191 B1LPA6 ECOSM (Russ et al, 2020) 0.385 0.321 0.415 BLAT ECOLX (Gonzalez et al, 2019) 0.546 0.296 0.357 PTEN HUMAN (Mighell et al, 2018) 0.699 0.563 0.598 CAPSD AAV2S (Sinai et al, 2021) 0.457 0.549 0.586 HIS7 YEAST (Pokusaeva et al, 2019) 0.680 0.707 0.692 P53 HUMAN (Kotler et al, 2018) 0.001 0.395 0.401 Average 0.412 0.430 0.463 Deng et al, 2012;Weile et al, 2017;Klesmith et al, 2015;Doud & Bloom, 2016;Wu et al, 2014;Findlay et al, 2018;Stiffler et al, 2015;Faure et al, 2022;Jacquier et al, 2013;Kennouche et al, 2019;Sourisseau et al, 2019;Wrenbeck et al, 2017;Dandage et al, 2018;Seuma et al, 2021;Haddox et al, 2018;Doud et al, 2015;Mishra et al, 2016;Flynn et al, 2020;Amorosi et al, 2021;McLaughlin Jr et al, 2012;Nutschel et al, 2020;Kitzman et al, 2015;Kelsic et al, 2016;Lee et al, 2018;Mattenberger et al, 2021;Matreyek et al, 2018;Thompson et al, 2020;Romero et al, 2015;Qi et al, 2014;Roscoe et al, 2013;Bandaru et al, 2017;Young et al, 2021;…”