Markus Hilleringmann

A Second Pilus Type in Streptococcus pneumoniae Is Prevalent in Emerging Serotypes and Mediates Adhesion to Host Cells

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element containing genes typical of gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (consisting of pitA, sipA, pitB, srtG1, and srtG2) codes for a second functional pilus in pneumococcus. Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. Presence of PI-2 correlates with the genotype as defined by multilocus sequence typing and clonal complex (CC). The PI-2-positive CCs are associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging serotypes in both industrialized and developing countries. Interestingly, strains belonging to CC271 (where sequence type 271 is the predicted founder of the CC) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that play a role in the initial host cell contact to the respiratory tract and are potential antigens for inclusion in a new generation of pneumococcal vaccines.

Streptococcus pneumoniae pilus subunits protect mice against lethal challenge.

ABSTRACT Streptococcus pneumoniae is a major public health threat worldwide. The recent discovery that this pathogen possesses pili led us to investigate their protective abilities in a mouse model of intraperitoneal infection. Both active and passive immunization with recombinant pilus subunits afforded protection against lethal challenge with the S. pneumoniae serotype 4 strain TIGR4.

Pneumococcal Pili Are Composed of Protofilaments Exposing Adhesive Clusters of Rrg A

Pili have been identified on the cell surface of Streptococcus pneumoniae, a major cause of morbidity and mortality worldwide. In contrast to Gram-negative bacteria, little is known about the structure of native pili in Gram-positive species and their role in pathogenicity. Triple immunoelectron microscopy of the elongated structure showed that purified pili contained RrgB as the major compound, followed by clustered RrgA and individual RrgC molecules on the pilus surface. The arrangement of gold particles displayed a uniform distribution of anti-RrgB antibodies along the whole pilus, forming a backbone structure. Antibodies against RrgA were found along the filament as particulate aggregates of 2–3 units, often co-localised with single RrgC subunits. Structural analysis using cryo electron microscopy and data obtained from freeze drying/metal shadowing technique showed that pili are oligomeric appendages formed by at least two protofilaments arranged in a coiled-coil, compact superstructure of various diameters. Using extracellular matrix proteins in an enzyme-linked immunosorbent assay, ancillary RrgA was identified as the major adhesin of the pilus. Combining the structural and functional data, a model emerges where the pilus RrgB backbone serves as a carrier for surface located adhesive clusters of RrgA that facilitates the interaction with the host.

In Vitro Analysis of Protein-Operator Interactions of the NikR and Fur Metal-Responsive Regulators of Coregulated Genes in Helicobacter pylori

Two important metal-responsive regulators, NikR and Fur, are involved in nickel and iron homeostasis and controlling gene expression in Helicobacter pylori. To date, they have been implicated in the regulation of sets of overlapping genes. We have attempted here dissection of the molecular mechanisms involved in transcriptional regulation of the NikR and Fur proteins, and we investigated protein-promoter interactions of the regulators with known target genes. We show that H. pylori NikR is a tetrameric protein and, through DNase I footprinting analysis, we have identified operators for NikR to which it binds with different affinities in a metal-responsive way. Mapping of the NikR binding site upstream of the urease promoter established a direct role for NikR as a positive regulator of transcription and, through scanning mutagenesis of this binding site, we have determined two subsites that are important for the binding of the protein to its target sequence. Furthermore, by alignment of the operators for NikR, we have shown that the H. pylori protein recognizes a sequence that is distinct from its well-studied orthologue in Escherichia coli. Moreover, we show that NikR and Fur can bind independently at distinct operators and also compete for overlapping operators in some coregulated gene promoters, adding another dimension to the previous suggested link between iron and nickel regulation. Finally, the importance of an interconnection between metal-responsive gene networks for homeostasis is discussed.

Molecular architecture of Streptococcus pneumoniae TIGR4 pili

Although the pili of Gram‐positive bacteria are putative virulence factors, little is known about their structure. Here we describe the molecular architecture of pilus‐1 of Streptococcus pneumoniae, which is a major cause of morbidity and mortality worldwide. One major (RrgB) and two minor components (RrgA and RrgC) assemble into the pilus. Results from TEM and scanning transmission EM show that the native pili are approximately 6 nm wide, flexible filaments that can be over 1 μm long. They are formed by a single string of RrgB monomers and have a polarity defined by nose‐like protrusions. These protrusions correlate to the shape of monomeric RrgB–His, which like RrgA–His and RrgC–His has an elongated, multi‐domain structure. RrgA and RrgC are only present at the opposite ends of the pilus shaft, compatible with their putative roles as adhesin and anchor to the cell wall surface, respectively. Our structural analyses provide the first direct experimental evidence that the native S. pneumoniae pilus shaft is composed exclusively of covalently linked monomeric RrgB subunits oriented head‐to‐tail.

Supramolecular Organization of the Repetitive Backbone Unit of the Streptococcus Pneumoniae Pilus.

Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus. The crystal structure of the D2-D4 domains of RrgB was solved at 1.6 Å resolution. Rigid-body fitting of the X-ray coordinates into the electron density map enabled us to define the arrangement of the backbone subunits into the S. pneumoniae native pilus. The quantitative fitting provide evidence that the pneumococcal pilus consists uniquely of RrgB monomers assembled in a head-to-tail organization. The presence of short intra-subunit linker regions connecting neighbouring domains provides the molecular basis for the intrinsic pilus flexibility.

The Two Variants of the Streptococcus pneumoniae Pilus 1 RrgA Adhesin Retain the Same Function and Elicit Cross-Protection In Vivo

ABSTRACT Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus composed of the backbone subunit RrgB and two ancillary proteins, RrgA and RrgC. RrgA is the major determinant of in vitro adhesion associated with pilus 1, is protective in vivo in mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

Sortase A confers protection against Streptococcus pneumoniae in mice.

ABSTRACT Streptococcus pneumoniae sortase A (SrtA) is a transpeptidase that is highly conserved among pneumococcal strains, whose involvement in adhesion/colonization has been reported. We found that intraperitoneal immunization with recombinant SrtA conferred to mice protection against S. pneumoniae intraperitoneal challenge and that the passive transfer of immune serum before intraperitoneal challenge was also protective. Moreover, by using the intranasal challenge model, we observed a significant reduction of bacteremia when mice were intraperitoneally immunized with SrtA, while a moderate decrease of lung infection was achieved by intranasal immunization, even though no influence on nasopharynx colonization was seen. Taken together, our results suggest that SrtA is a good candidate for inclusion in a multicomponent, protein-based, pneumococcal vaccine.

A novel strategy to over-express and purify homologous proteins from Streptococcus pneumoniae.

Functional studies of Streptococcus pneumoniae virulence factors are facilitated by the development of complementation/mutagenesis systems. These methods usually result in poor expression yields; therefore, biochemical and structural/functional characterizations are mostly performed with proteins expressed and purified from heterologous systems (e.g. Escherichia coli). However, heterologous expression does not guarantee correct protein structure and function. In this work, we developed a method to over-express and purify homologous proteins from S. pneumoniae. The system relies on the combined use of the shuttle plasmid pMU1328 and a natural constitutive pneumococcal promoter, P(96). Efficient over-expression of secreted, membrane or surface anchored proteins, either wild type or mutant, was achieved. As proof of principle the S. pneumoniae pilus-1 backbone RrgB was successfully purified as a His-tag secreted protein (RrgB-His_SP) from pneumococcal culture supernatants. N-terminal sequencing and mass spectrometry analysis of RrgB-His_SP allowed the determination of the leader sequence cleavage site in pneumococcus, while proteolysis studies confirmed the stability of RrgB-His_SP to trypsin digestion. The data presented here support the use of this novel homologous expression method for all S. pneumoniae proteins for which extensive characterization studies are planned. Moreover, given the promiscuity of the pMU1328 replicon, this system could be used in diverse bacterial species.

Influenza Cell-Culture Vaccine Production

Influenza vaccination is currently the principal means of reducing or counteracting influenza mortality and morbidity burden in the community. Since the early development of monovalent killed-virus vaccine formulations in the 1940s, different principal strategies were followed by vaccine manufactures resulting in a variety of influenza vaccines (e.g., inactivated whole-virus vaccines, live attenuated vaccines, detergent or solvent “split” vaccines, subunit vaccines, and adjuvanted vaccines). Actually two main production processes, the classical egg-based technology and more recently cell-culture-based operations, can be distinguished. In addition different routes of immunization allow the generation of intramuscular-, intradermal-, and intranasal-influenza vaccines.