Structure of the Full-Length Major Pilin from Streptococcus pneumoniae: Implications for Isopeptide Bond Formation in Gram-Positive Bacterial Pili

The surface of the pneumococcal cell is adorned with virulence factors including pili. The major pilin RrgB, which forms the pilus shaft on pathogenic Streptococcus pneumoniae, comprises four immunoglobulin (Ig)-like domains, each with a common CnaB topology. The three C-terminal domains are each stabilized by internal Lys-Asn isopeptide bonds, formed autocatalytically with the aid of an essential Glu residue. The structure and orientation of the crucial N-terminal domain, which provides the covalent linkage to the next pilin subunit in the shaft, however, remain incompletely characterised. We report the crystal structure of full length RrgB, solved by X-ray crystallography at 2.8 Å resolution. The N-terminal (D1) domain makes few contacts with the rest of the RrgB structure, and has higher B-factors. This may explain why D1 is readily lost by proteolysis, as are the N-terminal domains of many major pilins. D1 is also found to have a triad of Lys, Asn and Glu residues in the same topological positions as in the other domains, yet mass spectrometry and the crystal structure show that no internal isopeptide bond is formed. We show that this is because β-strand G of D1, which carries the Asn residue, diverges from β-strand A, carrying the Lys residue, such that these residues are too far apart for bond formation. Strand G also carries the YPKN motif that provides the essential Lys residue for the sortase-mediated intermolecular linkages along the pilus shaft. Interaction with the sortase and formation of the intermolecular linkage could result in a change in the orientation of this strand, explaining why isopeptide bond formation in the N-terminal domains of some major pilins appears to take place only upon assembly of the pili

The Streptococcus pneumoniae Pilus-1 Displays a Biphasic Expression Pattern

The Streptococcus pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. In vitro and in vivo assays have demonstrated that pilus-1 is involved in attachment to epithelial cells and virulence, as well as protection in mouse models of infection. Several reports suggest that pilus-1 expression is tightly regulated and involves the interplay of numerous genetic regulators, including the PI-1 positive regulator RlrA. In this report we provide evidence that pilus expression, when analyzed at the single-cell level in PI-1 positive strains, is biphasic. In fact, the strains present two phenotypically different sub-populations of bacteria, one that expresses the pilus, while the other does not. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Finally, we provide evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria. Overall, the data presented here suggest that the observed biphasic pilus expression phenotype could be an example of bistability in pneumococcus

Bacterial Pili exploit integrin machinery to promote immune activation and efficient blood-brain barrier penetration

Group B Streptococcus (GBS) is the leading cause of meningitis in newborn infants. Bacterial cell surface appendages, known as pili, have been recently described in streptococcal pathogens, including GBS. The pilus tip adhesin, PilA, contributes to GBS adherence to blood-brain barrier (BBB) endothelium; however, the host receptor and the contribution of PilA in central nervous system (CNS) disease pathogenesis are unknown. Here we show that PilA binds collagen, which promotes GBS interaction with the α2β1 integrin resulting in activation of host chemokine expression and neutrophil recruitment during infection. Mice infected with the PilA-deficient mutant exhibit delayed mortality, a decrease in neutrophil infiltration and bacterial CNS dissemination. We find that PilA-mediated virulence is dependent on neutrophil influx as neutrophil depletion results in a decrease in BBB permeability and GBS–BBB penetration. Our results suggest that the bacterial pilus, specifically the PilA adhesin, has a dual role in immune activation and bacterial entry into the CNS

Etudes structurale et fonctionnelle de protéines impliquées dans la virulence chez S. pneumoniae et P. aeruginosa

Cette thèse est composée de deux parties : Le première partie rend compte de l'étude structurale de la protéine RrgA. RrgA est associée au pilus du pathogène Streptococcus pneumoniae et participe aux premières étapes de colonisation chez l'hôte en se liant à plusieurs composés de la Matrice Extra Cellulaire. Nous avons résolu la structure de cette protéine à 1.9 Å par cristallographie aux rayons-X. RrgA posse de une structure allongée formée de quatre domaines alignés d'origine eucaryote et procaryote. En effet, trois domaines ayant des similarités structurales avec les IgG et le domaine Cna-B semblent servir de piédestal pour orienter et présenter le domaine fonctionnel de type Intégrine. Nous avons confirmé la formation de deux ponts isopeptidiques stabilisateurs par spectrométrie de masse. De plus, le domaine intégrine possède deux insertions particulières dont la présence pourrait être impliquée dans la reconnaissance des divers substrats par RrgA. La deuxième partie de cette thèse est axée sur l'étude structurale du complexe ATPase et de ExsB, la pilotine présumée du système de sécrétion de type III chez Pseudomonas aeruginosa, bactérie opportuniste et jouant un rôle majeur dans l'infection des patients atteints de mucoviscidose. Pour la première fois, nous avons mis au point un protocole d'expression et de purification sous forme soluble de l'ATPase PscN en complexe avec une protéine partenaire, PscL. Des cristaux de ce complexe ont été obtenus au robot du PSB. Par ailleurs, nous avons confirmé l'expression de la lipoprotéine ExsB chez P. aeruginosa que nous avons localisée au sein de la membrane externe. De plus, nous avons résolu la structure de cette protéine qui présente un nouveau repliement et qui établie les bases structurales pour l'étude des pilotines pour tous les systèmes de sécrétion de type III de la famille Ysc.This manuscript is made up of two parts The first part describes the structural study of RrgA from Streptococcus pneumoniae. This protein is a pilus-associated adhesin that is able to bind to several components of the Extra Cellular Matrix and thus, participates in the first steps of host colonization. We solved the structure of RrgA to 1.9 Å by X-Ray crystallography. We showed that RrgA folds into an elongated 4-domain structure, and these domains display both eukaryotic and prokaryotic origins. Actually, three out of the four domains are reminiscent of IgG and Cna-B structures and act like stalks to orient and display the large Integrin-like domain. We confirmed the presence of two isopeptide bonds by mass spectrometry and hypothesised that the two inserted arms in the integrin domain could explain the wide variety of substrates RrgA can bind. The second part of this manuscript focuses on the structural studies of the ATPase complex as well as ExsB, the putative pilotin of the type III secretion system from Pseudomonas aeruginosa. This bacterium is a major threat in hospital-acquired infections and the main pathogen found in cystic-fibrosis suffering patients. For the first time we were able to express and purify the ATPase PscN in complex with its partner PscL. Crystallization trials led to a very promising condition that is being refined. Moreover, we confirmed expression of the lipoprotein ExsB in P. aeruginosa that we localised in the outer membrane. To have a better understanding of this protein, we also solved its high-resolution structure that displays a novel fold and our study paves the way for coming studies concerning pilotins.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

The association between viral load and concurrent human papillomavirus infection at the genital and anal sites of young women and the impact of vaccination

Concurrent genital-anal human papillomavirus (HPV) infections may impose an increased anal cancer risk in women with HPV-related genital lesions. High viral load may facilitate genital-anal HPV concurrence. Genital and anal HPV is reduced by a bivalent HPV16/18 vaccine, yet the effect on concurrent genital-anal HPV remains unclear. This study analyzed viral load in concurrent genital-anal HPV infections, relative to genital-only and anal-only HPV infections and the impact of vaccination in young women. We included 1074 women, who provided both genital and anal swabs. HPV detection and genotyping was performed using the SPF10-DEIA-LiPA25. HPV copy numbers were measured with type-specific qPCRs and corrected for cellular content to obtain the viral load. Concurrent genital-anal HPV often had significantly higher genital viral load (0.09-371 c/cell) than genital-only HPV (3.17E-04-15.9 c/cell, p < 0.0001 to p < 0.05). Moreover, nearly all concurrent genital-anal HPV types had higher genital copy numbers per PCR reaction (157-416E04 c/rxn) than anal copy numbers (0.90-884E01 c/rxn, p < 0.0001 to p < 0.001). Vaccinated women had significantly less infections with HPV16/18 vaccine-types (2.8% vs 13.7%, p < 0.0001) and HPV31/35/45 cross-protective types (7.4% vs 21.1%, p < 0.0001) than unvaccinated women. In conclusion, particularly high genital viral load is found in concurrent genital-anal HPV infections, which are effectively reduced by vaccination

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F-420-0 in Mycobacteria

F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance.IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420

Structure of the magnetosome-associated actin-like MamK filament at subnanometer resolution

Magnetotactic bacteria possess cellular compartments called magnetosomes that sense magnetic fields. Alignment of magnetosomes in the bacterial cell is necessary for their function, and this is achieved through anchoring of magnetosomes to filaments composed of the protein MamK. MamK is an actin homolog that polymerizes upon ATP binding. Here, we report the structure of the MamK filament at ∼6.5 Å, obtained by cryo‐Electron Microscopy. This structure confirms our previously reported double‐stranded, nonstaggered architecture, and reveals the molecular basis for filament formation. While MamK is closest in sequence to the bacterial actin MreB, the longitudinal contacts along each MamK strand most closely resemble those of eukaryotic actin. In contrast, the cross‐strand interface, with a surprisingly limited set of contacts, is novel among actin homologs and gives rise to the nonstaggered architecture