Bioinformatics: how it is being used to identify bacterial vaccine candidates.

Genomic sequencing has provided a tremendous amount of information that can be useful in vaccine target identification. The sheer volume of information available necessitates the use of new research disciplines and techniques. Using bioinformatics, researchers sift through available data to identify appropriate candidates for biological analysis. This review provides an overview of available bioinformatic techniques for vaccine candidate identification and a few examples of how these techniques are being applied to specific bacterial pathogens.

Characterization of the Acinetobacter baumannii growth phase-dependent and serum responsive transcriptomes.

Acinetobacter baumannii has emerged as a bacterial pathogen of considerable healthcare concern. Yet, little is known about the organism's basic biological processes and the regulatory networks that modulate expression of its virulence factors and antibiotic resistance. Using Affymetrix GeneChips , we comprehensively defined and compared the transcriptomes of two A. baumannii strains, ATCC 17978 and 98-37-09, during exponential and stationary phase growth in Luria-Bertani (LB) medium. Results revealed that in addition to expected growth phase-associated metabolic changes, several putative virulence factors were dramatically regulated in a growth phase-dependent manner. Because a common feature between the two most severe types of A. baumannii infection, pneumonia and septicemia, includes the organism's dissemination to visceral organs via the circulatory system, microarray studies were expanded to define the expression properties of A. baumannii during growth in human serum. Growth in serum significantly upregulated iron acquisition systems, genes associated with epithelial cell adherence and DNA uptake, as well as numerous putative drug efflux pumps. Antibiotic susceptibility testing verified that the organism exhibits increased antibiotic tolerance when cultured in human serum, as compared to LB medium. Collectively, these studies provide researchers with a comprehensive database of A. baumannii's expression properties in LB medium and serum and identify biological processes that may contribute to the organism's virulence and antibiotic resistance.

The early interferon response of nasal-associated lymphoid tissue to Streptococcus pyogenes infection.

Streptococcus pyogenes is a major causative agent of tonsillitis or pharyngitis in children. Streptococcus pyogenes can persist in tonsils, and one-third of children treated with antibiotics continue to shed streptococci and have recurrent infections. Mouse nasal-associated lymphoid tissue (NALT) is functionally analogous to human oropharyngeal lymphoid tissues, and serves as a model for characterization of the mucosal innate immune response to S. pyogenes. Wild-type S. pyogenes induces transcription of both type I and interferon-gamma (IFN-gamma)-responsive genes, proinflammatory genes and acute-phase response proteins 24 h after intranasal infection. Invasion of NALT and the induction of the interferon response were not dependent on expression of antiphagocytic M protein. Intranasal infection induces a substantial influx of neutrophils into NALT at 24 h, which declines by 48 h after infection. Infection of IFN-gamma(-/-) [IFN-gamma knock-out mouse (GKO)] C57BL/6 mice with wild-type S. pyogenes resulted in local dissemination of bacteria to draining lymph nodes (LN), but did not lead to systemic infection by 48 h after infection. Infected GKO mice had an increased influx of neutrophils into NALT compared with immunocompetent mice. Thus, IFN-gamma-induced responses are required to prevent local dissemination of streptococci to the draining LN.

Proteomic analysis and identification of Streptococcus pyogenes surface-associated proteins.

Streptococcus pyogenes is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of S. pyogenes. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in S. pyogenes. Several proteins were expressed in Escherichia coli, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzyme-linked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by S. pyogenes.

The pro-sequence domain of streptopain directs the folding of the mature enzyme.

The cysteine endopeptidase streptopain, an extracellular enzyme from pathogenic Streptococcus pyogenes, is synthesized as a precursor containing an NH2-terminal pro-sequence. The pro-sequence of streptopain was expressed in Escherichia coli and subjected to structural and functional investigation. Heat-induced denaturation of the pro-sequence studied using circular dichroism spectroscopy revealed that it forms a compact structure and represents an independently folded domain. The isolated pro-sequence exhibits high affinity towards mature streptopain and associates with its cognate enzyme by forming an equimolar complex. Refolding of denatured streptopain in the presence of pro-sequence in vitro facilitated recovery of active enzyme. Expression of the mature streptopain in E. coli either alone, or in trans with its pro-sequence as an independent polypeptide, led to the formation of insoluble protein aggregates or functionally active enzyme, respectively. These results demonstrate that the pro-sequence domain acts as an intramolecular chaperone that directs the correct folding of the mature streptopain.

Structure of the streptococcal cell wall C5a peptidase.

The structure of a cell surface enzyme from a gram-positive pathogen has been determined to 2-A resolution. Gram-positive pathogens have a thick cell wall to which proteins and carbohydrate are covalently attached. Streptococcal C5a peptidase (SCP), is a highly specific protease and adhesin/invasin. Structural analysis of a 949-residue fragment of the [D130A,S512A] mutant of SCP from group B Streptococcus (S. agalactiae, SCPB) revealed SCPB is composed of five distinct domains. The N-terminal subtilisin-like protease domain has a 134-residue protease-associated domain inserted into a loop between two beta-strands. This domain also contains one of two Arg-Gly-Asp (RGD) sequences found in SCPB. At the C terminus are three fibronectin type III (Fn) domains. The second RGD sequence is located between Fn1 and Fn2. Our analysis suggests that SCP binding to integrins by the RGD motifs may stabilize conformational changes required for substrate binding.

Immunization with C5a peptidase from either group A or B streptococci enhances clearance of group A streptococci from intranasally infected mice.

Group A streptococci (S. pyogenes) are responsible for pharyngitis, impetigo and several more serious diseases. Emergence of toxic shock, and necrotizing fasciitis, associated with this pathogen over the past 10 years, has generated interest in development of a vaccine, which would prevent infections and potential serious complications. The highly conserved C5a peptidase that is expressed on the surface of group A streptococcus and other streptococcal species, associated with human infections, is a prime vaccine candidate. Here, we report construction of an inactive form of the peptidase and test its potential to induce protection in mice from intranasal challenge with either serotype M1 and M49 strains of streptococci. Mice were immunized by subcutaneous administration of recombinant proteins, mixed with Alum and monophosphoryl lipid A (MPL) adjuvants. Control mice were vaccinated with tetanus toxoid in the same adjuvants. Preparations of SCPA protein were highly immunogenic in mice. Antibody directed against protein from either group A (SCPAw) or group B (SCPBw) streptococci neutralized activity associated with both enzymes. Streptococci were cleared from the oral-nasal mucosa of mice immunized with vaccine protein more rapidly than those immunized with tetanus toxoid. Moreover, immunization with either protein enhanced clearance of group A streptococci from the lung. These results suggest that parenteral vaccination with SCPBw protein will provide protection against infection by either group A or B streptococci.

Immunization with C5a peptidase or peptidase-type III polysaccharide conjugate vaccines enhances clearance of group B Streptococci from lungs of infected mice.

Group B streptococci (GBS) are among the most common causes of life-threatening neonatal infections. Vaccine development since the late 1970s has focused on the capsular polysaccharides, but a safe, effective product is still not available. Our quest for a vaccine turned to the streptococcal C5a peptidase (SCPB). This surface protein is antigenically conserved across most if not all serotypes. A murine model was used to assess the impact of SCPB on clearance of GBS from the lungs of intranasally infected animals. Mutational inactivation of SCPB resulted in more-rapid clearance of streptococci from the lung. Immunization with recombinant SCPB alone or SCPB conjugated to type III capsular polysaccharide produced serotype-independent protection, which was evidenced by more-rapid clearance of the serotype VI strain from the lungs. Immunization of mice with tetanus toxoid-type III polysaccharide conjugate did not produce protection, confirming that protection induced by SCPB conjugates was independent of type III polysaccharide antigen. Histological evaluation of lungs from infected mice revealed that pathology in animals immunized with SCPB or SCPB conjugates was significantly less than that in animals immunized with a tetanus toxoid-polysaccharide conjugate. These experiments suggest that inclusion of C5a peptidase in a vaccine will both add another level to and broaden the spectrum of the protection of a polysaccharide vaccine.

Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes.

A recombinant streptococcal C5a peptidase was expressed in Escherichia coli and its catalytic properties and thermal stability were subjected to examination. It was shown that the NH2-terminal region of C5a peptidase (Asn32-Asp79/Lys90) forms the pro-sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease streptopain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range of temperatures between 40 and 43 degrees C. The process of heat denaturation of C5a peptidase investigated by fluorescence and circular dichroism spectroscopy revealed that the protein undergoes biphasic unfolding transition with Tm of 50 and 70 degrees C suggesting melting of different parts of the molecule with different stability. Unfolding of the less stable structures was accompanied by the loss of proteolytic activity. Using synthetic peptides corresponding to the COOH-terminus of human complement C5a we demonstrated that in vitro peptidase catalyzes hydrolysis of two His67-Lys68 and Ala58-Ser59 peptide bonds. The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis of its derivative Ac-SQLRANISH-pNA that lacks residues at P2'-P7' positions, suggest the importance of C5a peptidase interactions with the P' side of the substrate.