Biology subjects

Pelicic, V.

Publications and source records attributed to Pelicic, V..

11 recordsLinked to original sources

bioRxiv2026

A short C-terminal plug motif in a conserved type 4 pilus component controls pilus-tip localisation and filament biogenesis

Type 4a pili (T4aP), the most widespread and functionally versatile subtype of the type 4 filaments (T4F) superfamily, are functionalised at their tip by the adhesin PilC/PilY1. How this unusually large non-pilin protein is stably displayed at the pilus tip, and why is it required for pilus biogenesis, remains unknown. Here, using a multidisciplinary approach in Neisseria meningitidis, we show that the last 12 residues of PilC, representing ~1% of the protein, are both necessary and sufficient for pilus-tip localisation and filament biogenesis. X-ray crystallography reveals that this short "plug" motif binds the PilK subunit (within a complex of four minor pilins that caps the pilus) through {beta}-strand augmentation, a mode of interaction not previously characterised in T4F. Binding assays with synthetic peptides establish the specificity and affinity of this interaction, while the addition of a plug peptide extracellularly to cultures of a{Delta} pilC mutant restores pilus biogenesis. We show, using different methods, that the plug motif markedly stabilises PilK, providing an explanation for the requirement of PilC in pilus biogenesis. Consequently, expression of a PilK protein carrying a fused plug in N. meningitidis bypasses the requirement for PilC in pilus biogenesis. Together, these findings define the molecular basis of PilC/PilY1 function and localisation, reconcile all previous observations, and establish a broadly applicable model for T4aP biogenesis.

microbiology↗

bioRxiv2026

Structure of an eDNA-scaffolded Scp-like protease filament from Streptococcus sanguinis

C5a peptidases are surface-associated serine proteases that enable pathogenic streptococci to evade innate immunity by degrading neutrophil chemoattractants. These enzymes are usually anchored to the bacterial cell wall via an LPXTG motif and have only been characterized as monomers. Here, we report the cryo-EM structure of an unexpected supramolecular assembly from Streptococcus sanguinis (S. sanguinis), in which a Streptococcal C5a peptidase-like (ScpH) oligomerizes into an extended filament scaffolded on extracellular DNA (eDNA). The 3.1 [A] resolution structure reveals that this protease forms interlocked dimers through reciprocal contacts between the Ig and protease-Fn domains, and that these dimers propagate into a double-rowed filament via three additional inter-protomer interfaces. The central eDNA strand is engaged by an electropositive cleft formed by the C-terminal FIVAR3 domain and the DNA-binding tail, which replace the LPXTG anchor found in characterized orthologs. AlphaFold 3 predictions indicate that both dimerization and eDNA binding are conserved across a subfamily of streptococcal species that are prominent oral colonizers and eDNA producers. The catalytic triad is structurally intact and a co-purified peptide occupies the active site, although proteolytic activity remains to be demonstrated. This structure reveals a previously unknown mechanism of biofilm functionalization in which a putative immune evasion protease is assembled directly onto eDNA, transforming a passive structural component of the biofilm matrix into a potential catalytic platform.

microbiology↗

bioRxiv2025

Unravelling the molecular mechanisms of DNA capture by the Com pilus in naturally transformable monoderm bacteria

Transformation is a mechanism of horizontal gene transfer widespread in bacteria. The first step in transformation - capture of exogenous DNA - is mediated by surface-exposed filaments belonging to the type 4 filament (T4F) superfamily. How these protein polymers, composed of major and minor pilin subunits, interact with DNA remains poorly understood. Here, we address this question for the Com pilus, a widespread T4F mediating DNA capture in competent monoderm species. Our functional analysis, performed in Streptococcus sanguinis, was guided by a complete structural model of the Com pilus. We show that the major pilin ComGC does not bind DNA. In contrast, a systematic mutational analysis of electropositive residues exposed at the filament surface in the four minor pilins (ComGD, ComGE, ComGF, ComGG) reveals that the interface between ComGD and ComGF is important for DNA capture. Sequential mutations in these two interacting subunits lead to complete abolition of transformation, without affecting piliation. We further demonstrate the physical interaction between ComGD and ComGF using disulfide crosslinking, upon mutagenesis of two strategically positioned residues into cysteines. A structural model of the Com pilus tip interacting with DNA recapitulates all these findings and highlights a novel mode of DNA-binding, conserved in hundreds of monoderm species. IMPORTANCEBacteria are capable of evolving and diversifying very rapidly by acquiring new genetic material via horizontal gene transfer (HGT). Transformation is a widespread mechanism of HGT in bacteria, which results from the capture of extracellular DNA by surface-exposed pili belonging to the superfamily of type 4 filament (T4F). How T4F - that are composed of major and minor pilins - interact with DNA remains poorly understood, especially in competent monoderm species that use a unique T4F for DNA capture known as Com pilus or T4dP. The significance of this work is in characterizing a novel mode of DNA-binding by showing that the interface between two minor pilins part of a tip-located complex of four pilins - found in many different T4F - have been functionalized in monoderms to capture DNA. This is an evolutionary strategy used by bacteria to promote the exceptional functional versatility of T4F.

microbiology↗

bioRxiv2024

A Toolbox for Neisseria meningitidis: Gene Editing, Complementation and Labelling

The efficient transformation of Neisseria meningitidis and Neisseria gonorrhoeae facilitates the rapid construction of bacterial mutants with insertion of antibiotic resistance cassettes. However, this strategy limits the construction of strains with multiple mutations. Recent advances in markerless strategies for Neisseria species have enabled the construction of mutants without antibiotic resistance markers. However, these innovative approaches have potential limitations related to the selection strategy or the possible occurrence of spontaneous mutations in the selection marker genes. In addition, complementation tools or labelling strategies for N. meningitidis are also lacking. In this study, we have introduced new tools for markerless mutation, genetic complementation and labelling in N. meningitidis, thus improving the research possibilities for understanding and tackling these pathogens.

microbiology↗

bioRxiv2023

Systematic functional analysis of the Com pilus in Streptococcus sanguinis: a minimalistic type 4 filament dedicated to DNA uptake in monoderm bacteria

Type 4 filaments (T4F) are a superfamily of functionally versatile nanomachines, ubiquitous in prokaryotes, which use similar multi-protein machineries to assemble and operate filamentous polymers of type 4 pilins. The best studied T4F use very complex machineries, which has posed challenges to understanding the mechanisms of both filament assembly and the roles they facilitate. Here, we report the systematic functional analysis of the Com pilus, a widespread T4F mediating DNA uptake during natural transformation in monoderm bacteria. Using Streptococcus sanguinis as a model, we show that Com pili are bona fide type 4 pili (T4P), which represent a new pilus sub-type. We show that with only eight components necessary for their assembly and functioning - all "core" poteins universally conserved across this superfamily - the Com pilus epitomises a minimalistic T4F. We demonstrate that core T4F components are sufficient for filament assembly. Intriguingly, akin to more elaborate T4F, the Com pilus contains four minor pilins forming a complex likely to be situated at the apex of the filaments. Our results have global implications for T4F and make Com pili a model for elucidating the fundamental processes underpinning filament assembly.

microbiology↗

bioRxiv2023

Structure of a heteropolymeric type 4 pilus from a monoderm bacterium

Type 4 pili (T4P) are important virulence factors, which belong to a superfamily of nanomachines ubiquitous in prokaryotes, called type 4 filaments (T4F). T4F are defined as helical polymers of type 4 pilins. Recent advances in cryo-electron microscopy (cryo-EM) led to structures of several T4F. This revealed that the long N-terminal -helix, the trademark of pilins, packs in the centre of the filaments to form a hydrophobic core, which in bacteria is accompanied by the melting (unfolding) of a portion of 1. Since all available bacterial T4F structures are from diderm species, we tested whether this architecture is conserved in phylogenetically distant species by determining the structure of the T4P of the monoderm Streptococcus sanguinis. Our 3.7 A resolution cryo-EM structure of this heteropolymeric T4P, and the resulting full atomic model including all minor pilins, highlight universal features of bacterial T4F and have widespread implications in understanding their biology.

microbiology↗

bioRxiv2022

Full structure/function analysis of all the pilin subunits in a type 4 pilus: a complex of minor pilins in Streptococcus sanguinis mediates binding to glycans

Type 4 filaments (T4F) - of which type 4 pili (T4P) are the archetype - are a superfamily of filamentous nanomachines nearly ubiquitous in prokaryotes. T4F are polymers of one major pilin that also contain minor pilins whose roles are often poorly understood. Here, we complete the structure/function analysis of the full set of T4P pilins in the opportunistic pathogen Streptococcus sanguinis. We determined the structure of the minor pilin PilA, which is unexpectedly similar to one of the subunits of a tip-located complex of four minor pilins, widely conserved in T4F. We found that PilA interacts and dramatically stabilises the minor pilin PilC. We determined the structure of PilC, showing that it is a modular pilin with a lectin module binding a specific subset of glycans prevalent in the human glycome, the host of S. sanguinis. Altogether, our findings support a model whereby the minor pilins in S. sanguinis T4P form a tip-located complex promoting adhesion to various host receptors. Our findings have general implications for a group of minor pilins widely conserved in T4F.

microbiology↗

bioRxiv2021

PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion

Type IV pili (T4P) are functionally versatile filamentous nanomachines, nearly ubiquitous in prokaryotes. They are predominantly polymers of one major pilin, but also contain minor pilins whose functions are often poorly defined, and likely to be diverse. Here, we show that the minor pilin PilB from the T4P of S. sanguinis displays an unusual bimodular 3D structure, with a bulky von Willebrand factor A-like (vWA) module "grafted" onto a small pilin module via a short unstructured loop. Structural modelling suggests that PilB is only compatible with a localisation at the tip of T4P. By performing a detailed functional analysis, we found that (i) the vWA module contains a canonical metal ion-dependent adhesion site (MIDAS), preferentially binding Mg2+ and Mn2+, (ii) abolishing metal-binding has no impact on the structure of PilB or piliation, (iii) metal-binding is important for S. sanguinis T4P-mediated twitching motility and adhesion to eukaryotic cells, and (iv) the vWA module shows an intrinsic binding ability to several host proteins. These findings reveal an elegant, yet simple, evolutionary tinkering strategy to increase T4P functional versatility, by grafting an adhesive module onto a pilin for presentation by the filaments. This strategy appears to have been extensively used by bacteria, in which modular pilins are widespread and exhibit an astonishing variety of architectures.

microbiology↗

bioRxiv2020

Construction of a complete set of Neisseria meningitidis defined mutants - the NeMeSys collection - and its use for the phenotypic profiling of the genome of an important human pathogen

One of the great challenges in biology is to determine the function of millions of genes of unknown function. Even in model bacterial species, there is a sizeable proportion of such genes, which has fundamental and practical consequences. Here, we constructed a complete collection of defined mutants in protein-coding genes - named NeMeSys 2.0 - in the human pathogen Neisseria meningitidis, consisting of individual mutants in 1,584 non-essential genes. This effort identified 391 essential genes - broadly conserved in other bacteria - leading to a full overview of the essential meningococcal genome, associated with just four underlying basic biological functions: 1) expression of genome information, 2) preservation of genome information, 3) cell membrane structure/function, and 4) cytosolic metabolism. Subsequently, we illustrated the utility of the NeMeSys 2.0 collection for determining gene function by identifying 1) a novel and conserved family of histidinol-phosphatase, 2) 20 genes, including three new ones, involved in the biology of type IV pili, a widespread virulence factor, and 3) several conditionally essential genes found in regions of genome plasticity encoding antitoxins and/or immunity proteins, which become dispensable when the gene encoding the cognate toxin is deleted. These findings have implications beyond the meningococcus. The NeMeSys 2.0 collection is an invaluable resource paving the way for a global phenotypic landscape in a major human bacterial pathogen.

microbiology↗

bioRxiv2020

The major subunit of widespread competence (pseudo)pili exhibits a novel and conserved type IV pilin fold

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modelling analyses suggest that ComGC unusual structure is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

microbiology↗

bioRxiv2018

In depth biochemical and structural analysis of a Gram-positive type IV pilus

Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis. In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis. We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins, i.e. a methylated N-terminus; (iii) are found in the same hetero-polymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin fold with a highly unusual flexible C-terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff than bona fide Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that S. sanguinis Tfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.

microbiology↗