Maria Scarselli

Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae

The 2,160,267 bp genome sequence of Streptococcus agalactiae, the leading cause of bacterial sepsis, pneumonia, and meningitis in neonates in the U.S. and Europe, is predicted to encode 2,175 genes. Genome comparisons among S. agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and the other completely sequenced genomes identified genes specific to the streptococci and to S. agalactiae. These in silico analyses, combined with comparative genome hybridization experiments between the sequenced serotype V strain 2603 V/R and 19 S. agalactiae strains from several serotypes using whole-genome microarrays, revealed the genetic heterogeneity among S. agalactiae strains, even of the same serotype, and provided insights into the evolution of virulence mechanisms.

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature. The number of surface-exposed proteins varied from strain to strain, most likely as a consequence of different capsule content. The surface-exposed proteins of the highly virulent M23_DSM2071 strain included 17 proteins, 15 in common with M1_SF370. When 14 of the 17 proteins were expressed in E. coli and tested in the mouse for their capacity to confer protection against a lethal dose of M23_DSM2071, one new protective antigen (Spy0416) was identified. This strategy overcomes the difficulties so far encountered in surface protein characterization and has great potential in vaccine discovery.

Genomic Approach for Analysis of Surface Proteins in Chlamydia pneumoniae

ABSTRACT Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.

Identification of protective and broadly conserved vaccine antigens from the genome of extraintestinal pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) are a common cause of disease in both mammals and birds. A vaccine to prevent such infections would be desirable given the increasing antibiotic resistance of these bacteria. We have determined the genome sequence of ExPEC IHE3034 (ST95) isolated from a case of neonatal meningitis and compared this to available genome sequences of other ExPEC strains and a few nonpathogenic E. coli. We found 19 genomic islands present in the genome of IHE3034, which are absent in the nonpathogenic E. coli isolates. By using subtractive reverse vaccinology we identified 230 antigens present in ExPEC but absent (or present with low similarity) in nonpathogenic strains. Nine antigens were protective in a mouse challenge model. Some of them were also present in other pathogenic non-ExPEC strains, suggesting that a broadly protective E. coli vaccine may be possible. The gene encoding the most protective antigen was detected in most of the E. coli isolates, highly conserved in sequence and found to be exported by a type II secretion system which seems to be nonfunctional in nonpathogenic strains.

Neisseria meningitidis GNA2132, a heparin-binding protein that induces protective immunity in humans

GNA2132 is a Neisseria meningitidis antigen of unknown function, discovered by reverse vaccinology, which has been shown to induce bactericidal antibodies in animal models. Here we show that this antigen induces protective immunity in humans and it is recognized by sera of patients after meningococcal disease. The protein binds heparin in vitro through an Arg-rich region and this property correlates with increased survival of the unencapsulated bacterium in human serum. Furthermore, two proteases, the meningococcal NalP and human lactoferrin, cleave the protein upstream and downstream from the Arg-rich region, respectively. We conclude that GNA2132 is an important protective antigen of N. meningitidis and we propose to rename it, Neisserial Heparin Binding Antigen (NHBA).

Multiple Factors Modulate Biofilm Formation by the Anaerobic Pathogen Clostridium difficile

Bacteria within biofilms are protected from multiple stresses, including immune responses and antimicrobial agents. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Although biofilms have been well studied for several gut pathogens, little is known about biofilm formation by anaerobic gut species. The obligate anaerobe Clostridium difficile causes C. difficile infection (CDI), a major health care-associated problem primarily due to the high incidence of recurring infections. C. difficile colonizes the gut when the normal intestinal microflora is disrupted by antimicrobial agents; however, the factors or processes involved in gut colonization during infection remain unclear. We demonstrate that clinical C. difficile strains, i.e., strain 630 and the hypervirulent strain R20291, form structured biofilms in vitro, with R20291 accumulating substantially more biofilm. Microscopic and biochemical analyses show multiple layers of bacteria encased in a biofilm matrix containing proteins, DNA, and polysaccharide. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella, and a putative quorum-sensing regulator, LuxS, are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in Spo0A, a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

Rational Design of a Meningococcal Antigen Inducing Broad Protective Immunity

A single chimeric protein induces protective immunity against all meningococcal B strains with implications for producing broadly protective vaccines. All for One and One for All The three musketeers were a formidable team, but imagine combining all of their skills and valor into just one musketeer. That is precisely the approach that Rappuoli and his colleagues have taken with their design of a vaccine against meningococcus B, the bacterial pathogen that causes meningitis. Although mining of the genome sequence of this pathogen has yielded excellent targets that could be used in a vaccine, many of these antigens show a high degree of variation that has stymied attempts to use them as vaccine immunogens. For example, factor H binding protein is essential for the survival of meningococcus B in the human host because it protects the pathogen from the onslaught of the human immune system’s complement pathway. Because it is essential for survival, factor H binding protein should be a valuable immunogen, but because it has at least 300 sequence variants, it is impractical to make one vaccine that contains all of these variants. Rappuoli and his colleagues have tackled this problem by dividing the 300 sequence variants of factor H binding protein into three major groups. Using detailed structural information about these three major variants, they engineered variant 1 to carry patches of amino acids from the surfaces of variants 2 and 3. They then introduced groups of point mutations into the amino acids of these transplanted patches to mimic the natural variation of variant 2 and 3 strains of meningococcus B. They then tested which of the 54 engineered single chimeric immunogens could elicit bactericidal antibodies against many different strains of meningococcus B. To do this, they injected the immunogens into mice and assayed mouse sera in vitro for bactericidal activity against multiple bacterial strains. One chimeric immunogen, called G1, was capable of inducing bactericidal antibodies that could kill all strains of meningococcus B, suggesting that it could be used to produce a broadly protective vaccine. This structure-based approach to vaccine design may be useful not only for meningococcus B but also for other pathogens like HIV that show a high degree of antigenic variation. The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.

The Region Comprising Amino Acids 100 to 255 of Neisseria meningitidis Lipoprotein GNA 1870 Elicits Bactericidal Antibodies

ABSTRACT GNA 1870 is a novel surface-exposed lipoprotein, identified by genome analysis of Neisseria meningitidis strain MC58, which induces bactericidal antibodies. Three sequence variants of the protein were shown to be sufficient to induce bactericidal antibodies against a panel of strains representative of the diversity of serogroup B meningococci. Here, we studied the antigenic and immunogenic properties of GNA 1870, which for convenience was divided into domains A, B, and C. The immune responses of mice immunized with each of the three variants were tested using overlapping peptides scanning the entire protein length and using recombinant fragments. We found that while most of the linear epitopes are located in the A domain, the bactericidal antibodies are directed against conformational epitopes located in the BC domain. This was also confirmed by the isolation of a bactericidal murine monoclonal antibody, which failed to recognize linear peptides on the A, B, and C domains separately but recognized a conformational epitope formed only by the combination of the B and C domains. Arginine in position 204 was identified as important for binding of the monoclonal antibody. The identification of the region containing bactericidal epitopes is an important step in the design of new vaccines against meningococci.

Ng-MIP, a surface-exposed lipoprotein of Neisseria gonorrhoeae, has a peptidyl-prolyl cis/trans isomerase (PPIase) activity and is involved in persistence in macrophages.

Macrophage infectivity potentiators (MIPs) are a family of surface‐exposed virulence factors of intracellular microorganisms such as Legionella, Chlamydia and Trypanosoma. These proteins display peptidyl‐prolyl cis/trans isomerase (PPIase) activity that is inhibited by immunosuppressants FK506 and rapamycin. Here we describe the identification and characterization in Neisseria gonorrhoeae of Ng‐MIP, a surface‐exposed lipoprotein with high homology to MIPs. The protein is an homodimer with rapamycin‐inhibited PPIase activity confirming that it is a functional member of the MIP family. A knock‐out strain, generated by deletion of the mip gene in N. gonorrhoeae F62 strain, was evaluated for its role in infection of mouse and human macrophages. We show that Ng‐MIP promotes the intracellular survival of N. gonorrhoeae in macrophages, highlighting a possible role of this protein in promoting the persistence of gonococcal infection.

Molecular mechanisms of complement evasion: learning from staphylococci and meningococci

The complement system is a crucial component of the innate immune response in humans. Recent studies in Staphylococcus aureus and Neisseria meningitidis have revealed how these bacteria escape complement-mediated killing. In addition, new structural data have provided detailed insights into the molecular mechanisms of host defence mediated by the complement system and how bacterial proteins interfere with this process. This information is fundamental to our understanding of bacterial pathogenesis and may facilitate the design of better vaccines.