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Group B Streptococcus (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.

There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Unlabelled: The pneumococcal Pilus-1 enhances attachment to epithelial cells in the respiratory tract and subsequent invasion. Pilus-1 expression is bi-stable and positively regulated by the RlrA transcriptional regulator. To delineate the role of pilus-1 in Experimental Otitis Media (EOM), we evaluated colonization and disease due to a Streptococcus pneumoniae (SP) wild type strain (Taiwan19F-14 wt) and its otherwise isogenic pilus-1 and pilus-2 deficient mutant (Taiwan19F-14 ΔPI-1/PI-2-) as well as potential for a chimeric protein (RrgB321) vaccine candidate for prevention of middle ear (ME) disease., Methods: Chinchillas were challenged intranasally with either Taiwan19F-14 wt or Taiwan19F-14PI-1/PI-2 deficient mutant. ME status was assessed and direct cultures performed. New cohorts of animals were immunized with RrgB321 or alum. Intranasal challenge with Taiwan19F-14 wt [erythromycin susceptible E(S)] was performed. Subsequently, a second cohort of animals was immunized and challenged with either Taiwan19F-14 wt or a Pilus-1 over-expressing mutant [Taiwan19F-14+pMU1328_Pc-rlrA mutant; E resistant (R)] strain. Pilus-1 expression was analyzed in SP isolated from nasopharynx (NP) and ME fluids by flow cytometry., Results: Culture positive EOM developed following challenge with either wild type SP (Taiwan19F-14) or its pilus-1 deficient mutant. Culture positive EOM developed following challenge with wild type in both RrgB321 immunized and control animals. Pilus-1 expression in ME fluids was significantly higher in controls compared to immunized chinchillas. In second cohort of immunized and control animals challenged with the over-expressing Pilus-1 mutant, delayed development of EOM in the immunized animals was observed. Pneumococci recovered from ME fluid of immunized animals were no longer E(R) signifying the loss of the pMU1328_Pc-rlrA plasmid., Conclusion: Pneumococcal pilus-1 was not essential for EOM. Regulation of Pilus-1 expression in ME fluids in the presence of anti RrgB321 antibody was essential for survival of S. pneumoniae. Pneumococci have evolved mechanisms of regulation of non-essential surface proteins to evade host defenses.

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis.

Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.

Studies of meningococcal evolution and genetic population structure, including the long-term stability of non-random associations between variants of surface proteins, are essential for vaccine development. We analyzed the sequence variability of factor H-binding protein (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and Neisseria adhesin A (NadA), three major antigens in the multicomponent meningococcal serogroup B vaccine 4CMenB. A panel of invasive isolates collected in the Netherlands over a period of 50 years was used. To our knowledge, this strain collection covers the longest time period of any collection available worldwide. Long-term persistence of several antigen sub/variants and of non-overlapping antigen sub/variant combinations was observed. Our data suggest that certain antigen sub/variants including those used in 4CMenB are conserved over time and promoted by selection.

Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.

Although the contribution of carbohydrate catabolism to bacterial colonization and infection is well recognized, the transcriptional changes during these processes are still unknown. In this study, we have performed comparative global gene expression analysis of GBS in sugar-free versus high glucose milieu. The analysis revealed a differential expression of genes involved in metabolism, transport and host-pathogen interaction. Many of them appeared to be among the genes previously reported to be controlled by the CovRS two-component system. Indeed, the transcription profile of a ΔcovRS strain grown in high-glucose conditions was profoundly affected. In particular, of the total genes described to be regulated by glucose, ∼27% were under CovRS control with a functional role in protein synthesis, transport, energy metabolism and regulation. Among the CovRS dependent genes, we found bibA, a recently characterized adhesin involved in bacterial serum resistance and here reported to be down-regulated by glucose. ChIP analysis revealed that in the presence of glucose, CovR binds bibA promoter in vivo, suggesting that CovR may act as a negative regulator or a repressor. We also demonstrated that, as for other target promoters, chemical phosphorylation of CovR in aspartic acid increases its affinity for the bibA promoter region. The data reported in this study contribute to the understanding of the molecular mechanisms modulating the adaptation of GBS to glucose.

Streptococcus pyogenes is a major human pathogen worldwide, responsible for both local and systemic infections. These bacteria express the subtilisin-like protease SpyCEP which cleaves human IL-8 and related chemokines. We show that localization of SpyCEP is growth-phase and strain dependent. Significant shedding was observed only in a strain naturally overexpressing SpyCEP, and shedding was not dependent on SpyCEP autoproteolytic activity. Surface-bound SpyCEP in two different strains was capable of cleaving IL-8. To investigate SpyCEP action in vivo, we adapted the mouse air pouch model of infection for parallel quantification of bacterial growth, host immune cell recruitment and chemokine levels in situ. In response to infection, the predominant cells recruited were neutrophils, monocytes and eosinophils. Concomitantly, the chemokines KC, LIX, and MIP-2 in situ were drastically increased in mice infected with the SpyCEP knockout strain, and growth of this mutant strain was reduced compared to the wild type. SpyCEP has been described as a potential vaccine candidate against S. pyogenes, and we showed that surface-associated SpyCEP was recognized by specific antibodies. In vitro, such antibodies also counteracted the inhibitory effects of SpyCEP on chemokine mediated PMN recruitment. Thus, α-SpyCEP antibodies may benefit the host both directly by enabling opsonophagocytosis, and indirectly, by neutralizing an important virulence factor. The animal model we employed shows promise for broad application in the study of bacterial pathogenesis.

Gram-positive bacteria assemble pili through class C sortase enzymes specialized in polymerizing pilin subunits into covalently linked, high-molecular-weight, elongated structures. Here we report the crystal structures of two class C sortases (SrtC1 and SrtC2) from Group B Streptococcus (GBS) Pilus Island 1. The structures show that both sortases are comprised of two domains: an 8-stranded β-barrel catalytic core conserved among all sortase family members and a flexible N-terminal region made of two α-helices followed by a loop, known as the lid, which acts as a pseudo-substrate. In vitro experiments performed with recombinant SrtC enzymes lacking the N-terminal portion demonstrate that this region of the enzyme is dispensable for catalysis but may have key roles in substrate specificity and regulation. Moreover, in vitro FRET-based assays show that the LPXTG motif common to many sortase substrates is not the sole determinant of sortase C specificity during pilin protein recognition.

The human cytomegalovirus (HCMV) protein RL13 has recently been described to be present in all primary isolates but rapidly mutated in culture adapted viruses. Although these data suggest a crucial role for this gene product in HCMV primary infection, no function has so far been assigned to this protein. Working with RL13 expressed in isolation in transfected human epithelial cells, we demonstrated that recombinant RL13 from the clinical HCMV isolates TR and Merlin have selective human immunoglobulin (Ig)-binding properties towards IgG1 and IgG2 subtypes. An additional Fc binding protein, RL12, was also identified as an IgG1 and IgG2 binding protein but not further characterized. The glycoprotein RL13 trafficked to the plasma membrane where it bound and internalized exogenous IgG or its constant fragment (Fcγ). Analysis of RL13 ectodomain mutants suggested that the RL13 Ig-like domain is responsible for the Fc binding activity. Ligand-dependent internalization relied on a YxxL endocytic motif located in the C-terminal tail of RL13. Additionally, we showed that the tyrosine residue could be replaced by phenylalanine but not by alanine, indicating that the internalization signal was independent from phosphorylation events. In sum, RL13 binds human IgG and may contribute to HCMV immune evasion in the infected host, but this function does not readily explain the instability of the RL13 gene during viral propagation in cultured cells.

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.

Hepatitis C Virus E1E2 heterodimers are components of the viral spike. Although there is a general agreement on the necessity of the co-expression of both E1 and E2 on a single coding unit for their productive folding and assembly, in a previous study using an in vitro system we obtained strong indications that E1 can achieve folding in absence of E2. Here, we have studied the folding pathway of unescorted E1 from stably expressing CHO cells, compared to the folding observed in presence of the E2 protein. A DTT-resistant conformation is achieved by E1 in both situations, consistent with the presence of an E2-independent oxidative pathway. However, while the E1E2 heterodimer is stable inside cells, E1 expressed alone is degraded within a few hours. On the other hand, the oxidation and stability of individually expressed E2 subunits is dependent on E1 co-expression. These data are consistent with E1 and E2 assisting each other for correct folding via different mechanisms: E2 assists E1 by stabilizing a semi-native conformation meanwhile E1 drives E2 towards a productive folding pathway.

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtA(ΔN40)) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtA(ΔN40) does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a gram-positive human pathogen responsible for a diverse variety of diseases, including pharyngitis, skin infections, invasive necrotizing fasciitis and autoimmune sequelae. We have recently shown that GAS cell adhesion and biofilm formation is associated with the presence of pili on the surface of these bacteria. GAS pilus proteins are encoded in the FCT (Fibronectin-Collagen-T antigen) genomic region, of which nine different variants have been identified so far. In the present study we undertook a global analysis of GAS isolates representing the majority of FCT-variants to investigate the effect of environmental growth conditions on their capacity to form multicellular communities. For FCT-types 2, 3, 5 and 6 and a subset of FCT-4 strains, we observed that acidification resulting from fermentative sugar metabolism leads to an increased ability of the bacteria to form biofilm on abiotic surfaces and microcolonies on epithelial cells. The higher biofilm forming capacity at low environmental pH was directly associated with an enhanced expression of the genes encoding the pilus components and of their transcription regulators. The data indicate that environmental pH affects the expression of most pilus types and thereby the formation of multicellular cell-adhering communities that assist the initial steps of GAS infection.

Streptococcus agalactiae is the primary colonizer of the anogenital mucosa of up to 30% of healthy women and can infect newborns during delivery and cause severe sepsis and meningitis. Persistent colonization usually involves the formation of biofilm and increasing evidences indicate that in pathogenic streptococci biofilm formation is mediated by pili. Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands. Here we have investigated the capacity of these strains to form biofilms. We have found that most of the biofilm-formers carry pilus 2a, and using insertion and deletion mutants we have confirmed that pilus type 2a, but not pilus types 1 and 2b, confers biofilm-forming phenotype. We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype. Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders.

Background: The evolution of bacterial organelles involved in host-pathogen interactions is subject to intense and competing selective pressures due to the need to maintain function while escaping the host immune response. To characterize the interplay of these forces in an important pathogen, we sequenced the rlrA islet, a chromosomal region encoding for a pilus-like structure involved in adherence to lung epithelial cells in vitro and in colonization in a murine model of infection, in 44 clinical isolates of Streptococcus pneumoniae., Results: We found that the rrgA and rrgB genes, encoding the main structural components of the pilus, are under the action of positive selection. In contrast, the rrgC gene, coding for a component present in low quantities in the assembled pilus, and the srtB, srtC and srtD genes, coding for three sortase enzymes essential for pilus assembly but probably not directly exposed to the host immune system, show no evidence of positive selection. We found several events of homologous recombination in the region containing these genes, identifying 4 major recombination hotspots. An analysis of the most recent recombination events shows a high level of mosaicism of the region coding for the rrgC, srtB, srtC and srtD genes., Conclusions: In the rlrA islet, the genes coding for proteins directly exposed to the host immune response are under the action of positive selection, and exist in distinct forms in the population of circulating strains. The genes coding for proteins not directly exposed on the surface of the bacterial cell are more conserved probably due to the homogenizing effect of recombination.