1,021 research outputs found
In-vitro and in-vivo expression analysis of pneumococcal vaccine candidates: pilus-1 components
No full textStreptococcus pneumoniae (S. pneumoniae) is a Gram-positive commensal of the nasopharyngeal tract of both children and healthy adults. However, S. pneumoniae is also a leading cause of morbidity and mortality worldwide, being responsible for non-invasive and invasive diseases such as acute otitis media, pneumonia, sepsis and meningitis. Despite the unquestionable efficacy of the available pneumococcal glycoconjugate vaccines, the limited coverage, along with the observed phenomenon of serotype replacement, could reduce their long-term effectiveness. For these reasons, the development of a serotype-independent vaccine relying on the use of surface-exposed protein antigens represents a valid alternative. In this context, pneumococcal pilus-1 components, and in particular the pilus backbone RrgB, demonstrated significant efficacy in protecting mice from lethal challenge.
The S. 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. Since a combination of the three RrgB variants could broad the efficacy of a pilus-based vaccine, a fusion protein (RrgB321) containing the three RrgB variants in a head to tail organization was constructed. It was recently reported that RrgB321 elicites an antibody response against each of the variants and protectes mice against piliated pneumococcal strains of the three clades both by active and passive immunization, supporting the validity of this candidate as a potential antigen for the generation of a multi-component protein-based vaccine against S. pneumoniae.
The data reported in this work contribute to the characterization of pilus-1 expression regulation in in vitro and in vivo experiments providing evidence that pilus expression is biphasic and demonstrating that the pilus expression level does not impair the protection induced by RrgB321 immunization in mouse models of infection.
Analyzing the strains at the single-cell level, two phenotypically different sub-populations of bacteria (one that expresses the pilus, while the other does not) could be identified. 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. Moreover, evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria, was reported. Overall the in vitro data presented suggest that the observed biphasic pilus expression phenotype is an example of bistability in pneumococcus.
Additionally, in this study, the ability of RrgB321 antibodies to kill both H and L S. pneumoniae populations in the opsonophagocytosis assay, as well as the ability of RrgB321 to confer protection in vivo against both populations were analyzed.
The results obtained demonstrate that: i) the opsonophagocytic killing mediated in vitro by RrgB321 antisera is strictly dependent on the pilus expression ratio of the strain used; ii) during the opsonophagocytosis assay pilus-expressing pneumococci are selectively killed, and iii) no switch towards the pilus non-expressing phenotype can be observed. Furthermore, in sepsis and pneumonia models, mice immunized with RrgB321 are significantly protected against challenge with either the H or the L pilus-expressing population of strains representative of the three RrgB variants. This suggests that the pilus-1 expression is not down-regulated, and also that the expression of the pilus-1 could be up-regulated in vivo. In conclusion, these data provide evidence that RrgB321 is protective against PI-1 positive strains regardless of their pilus expression level, and support the rationale for the inclusion of this fusion protein into a multi-component protein-based pneumococcal vaccine
Crystal structure of the P pilus rod subunit PapA
No full textP pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE) mechanism, whereby the chaperone's G(1) beta-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte) of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it
Mass spectrometric analysis of pilus assembly, amyloid fibril formation, and membrane proteins in their native state
No full textStructural analysis of proteins and their complexes is crucial to understanding protein function. This thesis demonstrates the application of mass spectrometry (MS) to the study of pilus assembly on Gram-negative bacteria and amyloid formation in dialysisrelated amyloidosis. In addition, it also tackles the challenging area of membrane protein analysis by MS.
Pili are hair-like appendages located on the outer membrane of bacteria that are involved in the transmission of infection. A periplasmic chaperone and an outer membrane usher protein coordinate pilin subunit assembly. Electrospray ionisation (ESI)-MS was used to elucidate the mechanism of subunit assembly. Experiments revealed the specific amino acids on the N-terminal extension of the pilus subunits that are important in catalysing
the subunit assembly process. Further MS/MS analysis indicated differences in the stability of the chaperone-subunit-usher ternary complexes formed, providing new
insights into the role of the usher in orchestrating pilus biogenesis.
Ion mobility spectrometry (IMS)-MS was next used to characterise oligomeric intermediates formed during beta-2 microglobulin (β2m) assembly into amyloid fibrils.
Analysis of the oligomers formed by a range of β2m point mutants that affect the kinetics of amyloid fibril formation highlighted the complexity of this fibril-forming
process. Further detailed characterisation of the β2m mutant H51A revealed subtle differences in the subunit exchange dynamics of the oligomers involved in fibril
formation.
Finally, this thesis shows a novel method for solubilising membrane proteins for MS analysis. Amphipathic polymers, termed amphipols, were used to fold and enhance the
stability of two bacterial outer membrane proteins, OmpT and PagP. The utility of amphipols to study the structural and functional properties of membrane proteins by
ESI-IMS-MS was then developed. Together these data show the power of ESI-IMS-MS in separating conformationally-distinct populations of amphipathic polymers from the amphipol-membrane complex whilst maintaining a ‘native-like’ membrane protein structure in the gas phase
The structure of the PapD-PapGII pilin complex reveals an open and flexible P5 pocket
No full textP pili are hairlike polymeric structures that mediate binding of uropathogenic Escherichia coli to the surface of the kidney via the PapG adhesin at their tips. PapG is composed of two domains: a lectin domain at the tip of the pilus followed by a pilin domain that comprises the initial polymerizing subunit of the 1,000-plus-subunit heteropolymeric pilus fiber. Prior to assembly, periplasmic pilin domains bind to a chaperone, PapD. PapD mediates donor strand complementation, in which a beta strand of PapD temporarily completes the pilin domain's fold, preventing premature, nonproductive interactions with other pilin subunits and facilitating subunit folding. Chaperone-subunit complexes are delivered to the outer membrane usher where donor strand exchange (DSE) replaces PapD's donated beta strand with an amino-terminal extension on the next incoming pilin subunit. This occurs via a zip-in-zip-out mechanism that initiates at a relatively accessible hydrophobic space termed the P5 pocket on the terminally incorporated pilus subunit. Here, we solve the structure of PapD in complex with the pilin domain of isoform II of PapG (PapGIIp). Our data revealed that PapGIIp adopts an immunoglobulin fold with a missing seventh strand, complemented in parallel by the G1 PapD strand, typical of pilin subunits. Comparisons with other chaperone-pilin complexes indicated that the interactive surfaces are highly conserved. Interestingly, the PapGIIp P5 pocket was in an open conformation, which, as molecular dynamics simulations revealed, switches between an open and a closed conformation due to the flexibility of the surrounding loops. Our study reveals the structural details of the DSE mechanism
Genetic and Epidemiological Characterization of Streptococcus pneumoniae disease determinants.
No full textThe Gram-positive pathogen Streptococcus pneumoniae is a major cause of community acquired pneumonia as well as of upper respiratory tract infections such as acute otitis media and sinusitis, and invasive diseases like meningitis, bacteremia, and endocarditis. However, S. pneumoniae is also a commensal of the upper respiratory tract, especially of young children, which represent the reservoir for pneumococcal transmission within the community.
A major virulence factor of Streptococcus pneumoniae is the polysaccharide capsule. The pneumococcal capsule displays an extremely high variability (it exists in fact in at least ninety different types, known as serotypes) and provides an effective barrier against host-cell mediated phagocytosis allowing bacterial persistence in the blood. In addition to the capsular determinant, many other secreted or surface exposed factors have been described to be of importance for virulence through in vivo animal model studies and in vitro experiments (i.e. choline-binding protein A (CbpA), the pneumococcal toxin pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pilus components; however, their direct contribution to and essentiality for disease development in humans have still to be determined.
Streptococcus pneumoniae pilus islet-1 (PI-1)-encoded pilus enhances in vitro adhesion to the respiratory epithelium and may contribute to pneumococcal nasopharyngeal colonization and transmission. The pilus subunits are regarded as potential protein vaccine candidates. In the first part of this study, we sought to determine PI-1 prevalence in carried pneumococcal isolates and explore its relationship with transmissibility or carriage duration. We studied 896 pneumococcal isolates collected during a longitudinal carriage study that included monthly nasopharyngeal swabbing of 234 infants and their mothers between the ages of 1 and 24 months. These were cultured according to the WHO pneumococcal carriage detection protocol. PI-1 PCR and genotyping by multilocus sequence typing were performed on isolates chosen according to specific carriage and transmission definitions. Overall, 35.2% of the isolates were PI-1-positive, but PI-1 presence was restricted to ten of the 34 serotypes studied and was most frequently associated with serotypes 19F and 23F; 47.5% of transmitted and 43.3% of non-transmitted isolates were PI-1-positive (OR 1.2; 95% CI 0.8–1.7; p 0.4). The duration of first-ever infant pneumococcal carriage was significantly longer with PI-1-positive organisms, but this difference was not significant at the individual serotype level. In conclusion, PI-1 is commonly found in pneumococcal carriage isolates, but does not appear to be associated with pneumococcal transmissibility or carriage duration.
In the second part of this work, we focused on non-typeable Streptococcus pneumoniae (NTPn). NTPns are typically isolated from nasopharyngeal carriage or from conjunctivitis. Since the isolation of NTPn from invasive disease is rare, we characterized the genetic basis for non-typeability of two isolates obtained in Italy from two cases of bacteremic pneumonia. Multi Locus Sequence Typing (MLST) revealed that both NTPn belonged to ST191, which, according to the MLST database, is associated with serotype 7F. Sequencing of the capsular locus (cps) confirmed the presence of a 7F cps in both strains and revealed the existence of distinct single point mutations in the wchA gene (a glycosyltransferase), both leading to the translation of proteins truncated at the C-terminus. To verify if these mutations were responsible for non-typeability of the isolates, a functional 7F WchA was over-expressed in both NTPn. The two NTPn along with their WchA over-expressing derivatives were analyzed by Transmission Electron Microscopy and by high-resolution magic angle spinning NMR spectroscopy. Both NTPn were devoid of a polysaccharide capsule and WchA over-expression was sufficient to restore the assembly of a serotype 7F capsule on the surface of the two NTPn. In conclusion, we identified two new naturally-occurring point-mutations leading to the non typeability in pneumococcus and demonstrated that WchA is essential for the biosynthesis of the serotype 7F capsule
Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones
No full textPseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for,10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e. g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup [1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 A crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5
Clostridium perfringens produces an adhesive pilus required for the pathogenesis of necrotic enteritis in poultry
No full textClostridium perfringens type G strains cause necrotic enteritis (NE) in poultry, an economically important disease that is a major target of in-feed antibiotics. NE is a multifactorial disease, involving not only the critically important NetB toxin but also additional virulence and virulence-associated factors. We previously identified a C. perfringens chromosomal locus (VR-10B) associated with disease-causing strains that is predicted to encode a sortase-dependent pilus. In the current study, we sought to provide direct evidence for the production of a pilus by C. perfringens and establish its role in NE pathogenesis. Pilus structures in virulent C. perfringens strain CP1 were visualized by transmission electron microscopy (TEM) of immunogold-labeled cells. Filamentous structures were observed extending from the cell surface in wild-type CP1 but not from isogenic pilin-null mutant strains. In addition, immunoblotting of cell surface proteins demonstrated that CP1, but not the null mutant strains, produced a high molecular weight ladder-like pattern characteristic of a pilus polymer. Binding to collagen types I, II, and IV was significantly reduced (Tukey’s test, P < 0.01) in all three pilin mutants compared to CP1 and could be specifically blocked by CnaA and FimA antisera, indicating that these pilins participate in adherence. Furthermore, fimA and fimB null mutants were both severely attenuated in their ability to cause disease in an in vivo chicken NE challenge model. Together, these results provide the first direct evidence for the production of a sortase-dependent pilus by C. perfringens and confirm its critical role in NE pathogenesis and collagen binding.
IMPORTANCE In necrotic enteritis (NE), an intestinal disease of chickens, Clostridium perfringens cells adhere tightly to damaged intestinal tissue, but the factors involved are not known. We previously discovered a cluster of C. perfringens genes predicted to encode a pilus, a hair-like bacterial surface structure commonly involved in adherence. In the current study, we have directly imaged this pilus using transmission electron microscopy (TEM). We also show that inactivation of the pilus genes stops pilus production, significantly reducing the bacterium's ability to bind collagen and cause disease. Importantly, this is the first direct evidence for the production of a sortase-dependent pilus by C. perfringens, revealing a promising new target for developing therapeutics to combat this economically important disease.Agriculture and Agri-Food Canada and Canadian Poultry Research Counci
Model of a Pneumococcal Pilus.
No full text<p>Model showing T4 pneumococcal pilus composed of at least two RrgB protofilaments (green) arranged in a coiled-coil superstructure with surface located ancillary proteins (RrgA and RrgC) is based on cryo-EM, freeze drying/metal shadowing EM, IEM and biochemical data. (R) and (L) illustrate a possible right and left handed orientation of the thin pilus respectively. Outlines are not drawn to scale.</p
Neisseria meningitidis differentially controls host cell motility through PilC1 and PilC2 components of type IV pili
No full textNeisseria meningitidis is a strictly human pathogen that has two facets since asymptomatic carriage can unpredictably turn into fulminant forms of infection. Meningococcal pathogenesis relies on the ability of the bacteria to break host epithelial or endothelial cellular barriers. Highly restrictive, yet poorly understood, mechanisms allow meningococcal adhesion to cells of only human origin. Adhesion of encapsulated and virulent meningococci to human cells relies on the expression of bacterial type four pili (T4P) that trigger intense host cell signalling. Among the components of the meningococcal T4P, the concomitantly expressed PilC1 and PilC2 proteins regulate pili exposure at the bacterial surface, and until now, PilC1 was believed to be specifically responsible for T4P-mediated meningococcal adhesion to human cells. Contrary to previous reports, we show that, like PilC1, the meningococcal PilC2 component is capable of mediating adhesion to human ME180 epithelial cells, with cortical plaque formation and F-actin condensation. However, PilC1 and PilC2 promote different effects on infected cells. Cellular tracking analysis revealed that PilC1-expressing meningococci caused a severe reduction in the motility of infected cells, which was not the case when cells were infected with PilC2-expressing strains. The amount of both total and phosphorylated forms of EGFR was dramatically reduced in cells upon PilC1-mediated infection. In contrast, PilC2-mediated infection did not notably affect the EGFR pathway, and these specificities were shared among unrelated meningococcal strains. These results suggest that meningococci have evolved a highly discriminative tool for differential adhesion in specific microenvironments where different cell types are present. Moreover, the fine-tuning of cellular control through the combined action of two concomitantly expressed, but distinctly regulated, T4P-associated variants of the same molecule (i.e. PilC1 and PilC2) brings a new model to light for the analysis of the interplay between pathogenic bacteria and human host cells
Contribution of individual Ebp Pilus subunits of Enterococcus faecalis OG1RF to pilus biogenesis, biofilm formation and urinary tract infection.
No full textThe endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome of Enterococcus faecalis that has been shown to be important for biofilm formation, adherence to host fibrinogen, collagen and platelets, and in experimental endocarditis and urinary tract infection models. Here, we created single and double deletion mutants of the pilus subunits and sortases; next, by combining western blotting, immunoelectron microscopy, and using ebpR in trans to increase pilus production, we identified EbpA as the tip pilin and EbpB as anchor at the pilus base, the latter attached to cell wall by the housekeeping sortase, SrtA. We also confirmed EbpC and Bps as the major pilin and pilin-specific sortase, respectively, both required for pilus polymerization. Interestingly, pilus length was increased and the number of pili decreased by deleting ebpA, while control overexpression of ebpA in trans restored wild-type levels, suggesting a dual role for EbpA in both initiation and termination of pilus polymerization. We next investigated the contribution of each pilin subunit to biofilm formation and UTI. Significant reduction in biofilm formation was observed with deletion of ebpA or ebpC (P<0.001) while ebpB was found to be dispensable; a similar result was seen in kidney CFUs in experimental UTI (ΔebpA, ΔebpC, P≤0.0093; ΔebpB, non-significant, each vs. OG1RF). Hence, our data provide important structural and functional information about these ubiquitous E. faecalis pili and, based on their demonstrated importance in biofilm and infection, suggest EbpA and EbpC as potential targets for antibody-based therapeutic approaches
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