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1.
Proc Natl Acad Sci U S A ; 120(3): e2216237120, 2023 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-36626560

RESUMEN

Type 4 filaments (T4F)-of which type 4 pili (T4P) are the archetype-are a superfamily of nanomachines nearly ubiquitous in prokaryotes. T4F are polymers of one major pilin, which 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 bacterial 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 stabilizes the minor pilin PilC. We determined the structure of PilC, showing that it is a modular pilin with a lectin module binding a 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. This has general implications for T4F.


Asunto(s)
Proteínas Fimbrias , Streptococcus sanguis , Humanos , Proteínas Fimbrias/genética , Proteínas Fimbrias/química , Fimbrias Bacterianas/metabolismo
2.
Appl Environ Microbiol ; 90(9): e0088024, 2024 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-39140741

RESUMEN

The efficient natural transformation of Neisseria meningitidis allows the rapid construction of bacterial mutants in which the genes of interest are interrupted or replaced by antibiotic-resistance cassettes. However, this proved to be a double-edged sword, i.e., although facilitating the genetic characterization of this important human pathogen, it has limited the development of strategies for constructing markerless mutants without antibiotic-resistance markers. In addition, efficient tools for complementation or labeling are also lacking in N. meningitidis. In this study, we significantly expand the meningococcal genetic toolbox by developing new and efficient tools for the construction of markerless mutants (using a dual counterselection strategy), genetic complementation (using integrative vectors), and cell labeling (using a self-labeling protein tag). This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.IMPORTANCENeisseria meningitidis and Neisseria gonorrhoeae are two important human pathogens. Research focusing on these bacteria requires genetic engineering, which is facilitated by their natural ability to undergo transformation. However, the ease of mutant engineering has led the Neisseria community to neglect the development of more sophisticated tools for gene editing, particularly for N. meningitidis. In this study, we have significantly expanded the meningococcal genetic toolbox by developing novel and efficient tools for markerless mutant construction, genetic complementation, and cell tagging. This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.


Asunto(s)
Edición Génica , Neisseria meningitidis , Neisseria meningitidis/genética , Edición Génica/métodos , Prueba de Complementación Genética
3.
Proc Natl Acad Sci U S A ; 118(22)2021 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-34031252

RESUMEN

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 Streptococcus sanguinis displays an unusual bimodular three-dimensional structure with a bulky von Willebrand factor A-like (vWA) module "grafted" onto a small pilin module via a short loop. Structural modeling suggests that PilB is only compatible with a localization at the tip of T4P. By performing a detailed functional analysis, we found that 1) the vWA module contains a canonical metal ion-dependent adhesion site, preferentially binding Mg2+ and Mn2+, 2) abolishing metal binding has no impact on the structure of PilB or piliation, 3) metal binding is important for S. sanguinis T4P-mediated twitching motility and adhesion to eukaryotic cells, and 4) 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 a functional 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.


Asunto(s)
Proteínas Bacterianas/fisiología , Adhesión Celular , Proteínas Fimbrias/fisiología , Oxidorreductasas/fisiología , Streptococcus sanguis/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Células CHO , Cricetulus , Escherichia coli , Proteínas Fimbrias/química , Humanos , Oxidorreductasas/química , Conformación Proteica , Streptococcus sanguis/química
4.
Microbiology (Reading) ; 169(3)2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36947586

RESUMEN

Type 4 filaments (T4F) are a superfamily of filamentous nanomachines - virtually ubiquitous in prokaryotes and functionally versatile - of which type 4 pili (T4P) are the defining member. T4F are polymers of type 4 pilins, assembled by conserved multi-protein machineries. They have long been an important topic for research because they are key virulence factors in numerous bacterial pathogens. Our poor understanding of the molecular mechanisms of T4F assembly is a serious hindrance to the design of anti-T4F therapeutics. This review attempts to shed light on the fundamental mechanistic principles at play in T4F assembly by focusing on similarities rather than differences between several (mostly bacterial) T4F. This holistic approach, complemented by the revolutionary ability of artificial intelligence to predict protein structures, led to an intriguing mechanistic model of T4F assembly.


Asunto(s)
Inteligencia Artificial , Proteínas Fimbrias , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Bacterias/genética , Bacterias/metabolismo , Factores de Virulencia/metabolismo
5.
J Biol Chem ; 295(19): 6594-6604, 2020 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-32273343

RESUMEN

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 modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.


Asunto(s)
Proteínas Fimbrias/química , Fimbrias Bacterianas/química , Pliegue de Proteína , Streptococcus pneumoniae/química , Streptococcus sanguis/química , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Streptococcus pneumoniae/genética , Streptococcus sanguis/genética
6.
J Biol Chem ; 294(17): 6796-6808, 2019 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-30837269

RESUMEN

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 heteropolymeric 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.


Asunto(s)
Fimbrias Bacterianas/metabolismo , Streptococcus/metabolismo , Biopolímeros/metabolismo , Proteínas Fimbrias/química , Proteínas Fimbrias/metabolismo , Metilación , Conformación Proteica
7.
Mol Microbiol ; 112(6): 1674-1683, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31556183

RESUMEN

In the diverse world of bacterial pili, type IV pili (Tfp) are unique for two reasons: their multifunctionality and ubiquity. This latter feature offers an extraordinary possibility, that is, to perform comparative studies in evolutionarily distant species in order to improve our fragmentary understanding of Tfp biology. Regrettably, such potential has remained largely untapped, because, for 20 years, Tfp have only been characterised in diderm bacteria. However, recent studies of Tfp in monoderms have started closing the gap, revealing many interesting commonalities and a few significant differences, extending the frontiers of knowledge of Tfp biology. Here, I review the current state of the art of the Tfp field in monoderm bacteria and discuss resulting implications for our general understanding of the assembly and function of these widespread filamentous nanomachines.


Asunto(s)
Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Bacterias , Proteínas Bacterianas , Clostridiales , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Bacterias Grampositivas/genética , Bacterias Grampositivas/metabolismo , Proteínas de la Membrana/metabolismo , Streptococcus , Streptococcus sanguis
8.
Proc Natl Acad Sci U S A ; 114(25): E4978-E4986, 2017 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-28588140

RESUMEN

Type IV pili (Tfp), which are key virulence factors in many bacterial pathogens, define a large group of multipurpose filamentous nanomachines widespread in Bacteria and Archaea. Tfp biogenesis is a complex multistep process, which relies on macromolecular assemblies composed of 15 conserved proteins in model gram-negative species. To improve our limited understanding of the molecular mechanisms of filament assembly, we have used a synthetic biology approach to reconstitute, in a nonnative heterologous host, a minimal machinery capable of building Tfp. Here we show that eight synthetic genes are sufficient to promote filament assembly and that the corresponding proteins form a macromolecular complex at the cytoplasmic membrane, which we have purified and characterized biochemically. Our results contribute to a better mechanistic understanding of the assembly of remarkable dynamic filaments nearly ubiquitous in prokaryotes.


Asunto(s)
Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Periplasma/metabolismo , Archaea/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Células Procariotas/metabolismo , Factores de Virulencia/metabolismo
9.
Nature ; 502(7470): 237-40, 2013 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-24067614

RESUMEN

Neisseria meningitidis has several strategies to evade complement-mediated killing, and these contribute to its ability to cause septicaemic disease and meningitis. However, the meningococcus is primarily an obligate commensal of the human nasopharynx, and it is unclear why the bacterium has evolved exquisite mechanisms to avoid host immunity. Here we demonstrate that mechanisms of meningococcal immune evasion and resistance against complement increase in response to an increase in ambient temperature. We have identified three independent RNA thermosensors located in the 5' untranslated regions of genes necessary for capsule biosynthesis, the expression of factor H binding protein, and sialylation of lipopolysaccharide, which are essential for meningococcal resistance against immune killing. Therefore increased temperature (which occurs during inflammation) acts as a 'danger signal' for the meningococcus, enhancing its defence against human immune killing. Infection with viral pathogens, such as influenza, leads to inflammation in the nasopharynx with an increased temperature and recruitment of immune effectors. Thermoregulation of immune defence could offer an adaptive advantage to the meningococcus during co-infection with other pathogens, and promote the emergence of virulence in an otherwise commensal bacterium.


Asunto(s)
Evasión Inmune/fisiología , Infecciones Meningocócicas/inmunología , Neisseria meningitidis/fisiología , Temperatura , Regiones no Traducidas 5'/genética , Cápsulas Bacterianas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Evasión Inmune/genética , Lipopolisacáridos/metabolismo , Infecciones Meningocócicas/microbiología , Neisseria meningitidis/genética , ARN Bacteriano/química , ARN Bacteriano/genética , Sensación Térmica/genética
10.
Nucleic Acids Res ; 45(6): e40, 2017 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-27903891

RESUMEN

Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable.


Asunto(s)
Proteínas Fimbrias/genética , Edición Génica/métodos , Streptococcus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ingeniería Celular , Endopeptidasas/genética , Endopeptidasas/metabolismo , Proteínas Fimbrias/fisiología , Eliminación de Gen , Marcadores Genéticos , Histidina , Mutación Missense , Oligopéptidos , Ingeniería de Proteínas , Streptococcus/fisiología
11.
Mol Microbiol ; 99(2): 380-92, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26435398

RESUMEN

Type IV pili (Tfp), which have been studied extensively in a few Gram-negative species, are the paradigm of a group of widespread and functionally versatile nano-machines. Here, we performed the most detailed molecular characterisation of Tfp in a Gram-positive bacterium. We demonstrate that the naturally competent Streptococcus sanguinis produces retractable Tfp, which like their Gram-negative counterparts can generate hundreds of piconewton of tensile force and promote intense surface-associated motility. Tfp power 'train-like' directional motion parallel to the long axis of chains of cells, leading to spreading zones around bacteria grown on plates. However, S. sanguinis Tfp are not involved in DNA uptake, which is mediated by a related but distinct nano-machine, and are unusual because they are composed of two pilins in comparable amounts, rather than one as normally seen. Whole genome sequencing identified a locus encoding all the genes involved in Tfp biology in S. sanguinis. A systematic mutational analysis revealed that Tfp biogenesis in S. sanguinis relies on a more basic machinery (only 10 components) than in Gram-negative species and that a small subset of four proteins dispensable for pilus biogenesis are essential for motility. Intriguingly, one of the piliated mutants that does not exhibit spreading retains microscopic motility but moves sideways, which suggests that the corresponding protein controls motion directionality. Besides establishing S. sanguinis as a useful new model for studying Tfp biology, these findings have important implications for our understanding of these widespread filamentous nano-machines.


Asunto(s)
Proteínas Bacterianas/metabolismo , Fimbrias Bacterianas/metabolismo , Streptococcus/citología , Streptococcus/metabolismo , Proteínas Bacterianas/genética , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/genética , Streptococcus/genética
12.
PLoS Genet ; 9(12): e1004014, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24385921

RESUMEN

Natural transformation is the widespread biological process by which "competent" bacteria take up free DNA, incorporate it into their genomes, and become genetically altered or "transformed". To curb often deleterious transformation by foreign DNA, several competent species preferentially take up their own DNA that contains specific DUS (DNA uptake sequence) watermarks. Our recent finding that ComP is the long sought DUS receptor in Neisseria species paves the way for the functional analysis of the DUS-ComP interdependence which is reported here. By abolishing/modulating ComP levels in Neisseria meningitidis, we show that the enhancement of transformation seen in the presence of DUS is entirely dependent on ComP, which also controls transformation in the absence of DUS. While peripheral bases in the DUS were found to be less important, inner bases are essential since single base mutations led to dramatically impaired interaction with ComP and transformation. Strikingly, naturally occurring DUS variants in the genomes of human Neisseria commensals differing from DUS by only one or two bases were found to be similarly impaired for transformation of N. meningitidis. By showing that ComPsub from the N. subflava commensal specifically binds its cognate DUS variant and mediates DUS-enhanced transformation when expressed in a comP mutant of N. meningitidis, we confirm that a similar mechanism is used by all Neisseria species to promote transformation by their own, or closely related DNA. Together, these findings shed new light on the molecular events involved in the earliest step in natural transformation, and reveal an elegant mechanism for modulating horizontal gene transfer between competent species sharing the same niche.


Asunto(s)
Secuencia de Bases/genética , Proteínas de Unión al ADN/genética , Transferencia de Gen Horizontal/genética , Neisseria meningitidis/genética , Transformación Bacteriana/genética , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Humanos , Neisseria meningitidis/crecimiento & desarrollo
13.
Proc Natl Acad Sci U S A ; 110(8): 3065-70, 2013 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-23386723

RESUMEN

Natural transformation is a dominant force in bacterial evolution by promoting horizontal gene transfer. This process may have devastating consequences, such as the spread of antibiotic resistance or the emergence of highly virulent clones. However, uptake and recombination of foreign DNA are most often deleterious to competent species. Therefore, model naturally transformable gram-negative bacteria, including the human pathogen Neisseria meningitidis, have evolved means to preferentially take up homotypic DNA containing short and genus-specific sequence motifs. Despite decades of intense investigations, the DNA uptake sequence receptor in Neisseria species has remained elusive. We show here, using a multidisciplinary approach combining biochemistry, molecular genetics, and structural biology, that meningococcal type IV pili bind DNA through the minor pilin ComP via an electropositive stripe that is predicted to be exposed on the filaments surface and that ComP displays an exquisite binding preference for DNA uptake sequence. Our findings illuminate the earliest step in natural transformation, reveal an unconventional mechanism for DNA binding, and suggest that selective DNA uptake is more widespread than previously thought.


Asunto(s)
ADN Bacteriano/metabolismo , Proteínas Fimbrias/metabolismo , Neisseria meningitidis/genética , Western Blotting , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Proteínas Fimbrias/aislamiento & purificación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica
14.
mBio ; 15(1): e0266723, 2024 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-38095871

RESUMEN

IMPORTANCE: Type 4 filaments (T4F) are nanomachines ubiquitous in prokaryotes, centered on filamentous polymers of type 4 pilins. T4F are exceptionally versatile and widespread virulence factors in bacterial pathogens. The mechanisms of filament assembly and the many functions they facilitate remain poorly understood because of the complexity of T4F machineries. This hinders the development of anti-T4F drugs. The significance of our research lies in characterizing the simplest known T4F-the Com pilus that mediates DNA uptake in competent monoderm bacteria-and showing that four protein components universally conserved in T4F are sufficient for filament assembly. The Com pilus becomes a model for elucidating the mechanisms of T4F assembly.


Asunto(s)
Fimbrias Bacterianas , Streptococcus sanguis , Streptococcus sanguis/genética , Streptococcus sanguis/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Bacterias/genética , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , ADN/metabolismo
15.
Mol Microbiol ; 84(5): 857-73, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22486968

RESUMEN

The functionally versatile type IV pili (Tfp) are one of the most widespread virulence factors in bacteria. However, despite generating much research interest for decades, the molecular mechanisms underpinning the various aspects of Tfp biology remain poorly understood, mainly because of the complexity of the system. In the human pathogen Neisseria meningitidis for example, 23 proteins are dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. One of the important gaps in our knowledge concerns the topology of this multiprotein machinery. Here we have used a bacterial two-hybrid system to identify and quantify the interactions between 11 Pil proteins from N. meningitidis. We identified 20 different binary interactions, many of which are novel. This represents the most complex interaction network between Pil proteins reported to date and indicates, among other things, that PilE, PilM, PilN and PilO, which are involved in pilus assembly, indeed interact. We focused our efforts on this subset of proteins and used a battery of assays to determine the membrane topology of PilN and PilO, map the interaction domains between PilE, PilM, PilN and PilO, and show that a widely conserved N-terminal motif in PilN is essential for both PilM-PilN interactions and pilus assembly. Finally, we show that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Taken together, these findings have numerous implications for understanding Tfp biology and provide a useful blueprint for future studies.


Asunto(s)
Sistemas de Secreción Bacterianos , Fimbrias Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Neisseria meningitidis/fisiología , Sustancias Macromoleculares/metabolismo , Neisseria meningitidis/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Técnicas del Sistema de Dos Híbridos
16.
Mol Microbiol ; 83(5): 1064-1079, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22296581

RESUMEN

Although oxidative stress is a key aspect of innate immunity, little is known about how host-restricted pathogens successfully repair DNA damage. Base excision repair is responsible for correcting nucleobases damaged by oxidative stress, and is essential for bloodstream infection caused by the human pathogen, Neisseria meningitidis. We have characterized meningococcal base excision repair enzymes involved in the recognition and removal of damaged nucleobases, and incision of the DNA backbone. We demonstrate that the bi-functional glycosylase/lyases Nth and MutM share several overlapping activities and functional redundancy. However, MutM and other members of the GO system, which deal with 8-oxoG, a common lesion of oxidative damage, are not required for survival of N. meningitidis under oxidative stress. Instead, the mismatch repair pathway provides back-up for the GO system, while the lyase activity of Nth can substitute for the meningococcal AP endonuclease, NApe. Our genetic and biochemical evidence shows that DNA repair is achieved through a robust network of enzymes that provides a flexible system of DNA repair. This network is likely to reflect successful adaptation to the human nasopharynx, and might provide a paradigm for DNA repair in other prokaryotes.


Asunto(s)
Daño del ADN , Reparación del ADN , ADN-Formamidopirimidina Glicosilasa/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina)/metabolismo , Neisseria meningitidis/genética , Estrés Oxidativo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , ADN-Formamidopirimidina Glicosilasa/genética , Desoxirribonucleasa (Dímero de Pirimidina)/genética , Neisseria meningitidis/enzimología
17.
Nat Commun ; 14(1): 7143, 2023 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-37932265

RESUMEN

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, revealing that the long N-terminal α-helix (α1) - the trademark of pilins - packs in the centre of the filaments to form a hydrophobic core. In diderm bacteria - all available bacterial T4F structures are from diderm species - a portion of α1 is melted (unfolded). Here we report that this architecture is conserved in phylogenetically distant monoderm species by determining the structure of Streptococcus sanguinis T4P. Our 3.7 Å resolution cryo-EM structure of S. sanguinis heteropolymeric T4P and the resulting full atomic model including all minor pilins highlight universal features of bacterial T4F and have widespread implications in understanding T4F biology.


Asunto(s)
Proteínas Fimbrias , Fimbrias Bacterianas , Proteínas Fimbrias/química , Microscopía por Crioelectrón/métodos , Fimbrias Bacterianas/química , Bacterias , Polímeros
18.
Infect Immun ; 79(8): 3028-35, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21646452

RESUMEN

Type IV pili (Tfp) are widespread filamentous bacterial organelles that mediate multiple functions and play a key role in pathogenesis in several important human pathogens, including Neisseria meningitidis. Tfp biology remains poorly understood at a molecular level because the roles of the numerous proteins that are involved remain mostly obscure. Guided by the high-resolution crystal structure we recently reported for N. meningitidis PilW, a widely conserved protein essential for Tfp biogenesis, we have performed a structure/function analysis by targeting a series of key residues through site-directed mutagenesis and analyzing the corresponding variants using an array of phenotypic assays. Here we show that PilW's involvement in the functionality of Tfp can be genetically uncoupled from its concurrent role in the assembly/stabilization of the secretin channels through which Tfp emerge on the bacterial surface. These findings suggest that PilW is a multifunctional protein.


Asunto(s)
Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/fisiología , Neisseria meningitidis/fisiología , Análisis Mutacional de ADN , Proteínas Fimbrias/química , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo
19.
Infect Immun ; 78(7): 3053-63, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20439474

RESUMEN

Type IV pili (Tfp), which mediate multiple phenotypes ranging from adhesion to motility, are one of the most widespread virulence factors in bacteria. However, the molecular mechanisms of Tfp biogenesis and associated functions remain poorly understood. One of the underlying reasons is that the roles played by the numerous genes involved in Tfp biology are unclear because corresponding mutants have been studied on a case-by-case basis, in different species, and using different assays, often generating heterogeneous results. Therefore, we have recently started a systematic functional analysis of the genes involved in Tfp biology in a well-characterized clinical isolate of the human pathogen Neisseria meningitidis. After previously studying 16 genes involved in Tfp biogenesis, here we report the characterization of 7 genes that are dispensable for piliation and potentially involved in Tfp biology. Using a battery of assays, we assessed piliation and each of the Tfp-linked functions in single mutants, double mutants in which filament retraction is abolished by a concurrent mutation in pilT, and strains overexpressing the corresponding proteins. This showed that each of the seven genes actually fine-tunes a Tfp-linked function(s), which brings us one step closer to a global view of Tfp biology in the meningococcus.


Asunto(s)
Fimbrias Bacterianas/genética , Genes Bacterianos/genética , Neisseria meningitidis/genética , Adhesión Bacteriana/genética , Adhesión Bacteriana/fisiología , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Proteínas Fimbrias/genética , Proteínas Fimbrias/aislamiento & purificación , Fimbrias Bacterianas/fisiología , Genes Bacterianos/fisiología , Humanos , Immunoblotting , Mutación/genética , Neisseria meningitidis/fisiología , Transformación Bacteriana/genética
20.
Nat Commun ; 11(1): 5541, 2020 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-33139723

RESUMEN

The bacterium Neisseria meningitidis causes life-threatening meningitis and sepsis. Here, we construct a complete collection of defined mutants in protein-coding genes of this organism, identifying all genes that are essential under laboratory conditions. The collection, named NeMeSys 2.0, consists of individual mutants in 1584 non-essential genes. We identify 391 essential genes, which are associated with basic functions such as expression and preservation of genome information, cell membrane structure and function, and metabolism. We use this collection to shed light on the functions of diverse genes, including a gene encoding a member of a previously unrecognised class of histidinol-phosphatases; a set of 20 genes required for type IV pili function; and several conditionally essential genes encoding antitoxins and/or immunity proteins. We expect that NeMeSys 2.0 will facilitate the phenotypic profiling of a major human bacterial pathogen.


Asunto(s)
Genes Bacterianos/genética , Genes Esenciales/genética , Mutación , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Fenotipo , Proteínas Bacterianas/metabolismo , Biología Computacional , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Humanos , Neisseria meningitidis/patogenicidad
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