Capsule Synthesis Research Articles

Identification of a new promoter for the response regulatorrcsBexpression inSalmonella entericaserovar Typhimurium

The RcsCDB (Rcs) phosphorelay system regulates capsule synthesis, flagella production and other cellular activities in several enteric bacteria. This system consists of three proteins: the sensor RcsC, the cognate response regulator RcsB and the histidine-containing phosphotransfer protein RcsD (YojN), which is hypothesized to act as an intermediary in the phosphotransfer from RcsC to RcsB. The rcsC gene is convergently transcribed toward rcsB, which follows rcsD in what appears to be a two-gene operon. Here, it is reported that the overproduction of the rcsB gene represses rcsD transcription, but has a weak effect on its own expression. We demonstrated that the differential rcsD and rcsB expression is due to the activity of two promoters to transcribe the rcsB gene: (1) P(rcsDB) located upstream of rcsD and (2) P(rcsB) located within the rcsD coding region. In addition, here it was demonstrated that in Salmonella typhimurium, P(rcsB) is important to activate the rcsB expression during the stationary growth phase.

Coordinate Regulation of Glycan Degradation and Polysaccharide Capsule Biosynthesis by a Prominent Human Gut Symbiont

Bacteria in the distal human gut have evolved diverse abilities to metabolize complex glycans, including the capacity to degrade these compounds as nutrients and to assemble their component sugars into new polymers such as extracellular capsules. The human gut bacterium Bacteroides thetaiotaomicron is well endowed with the ability to metabolize both host- and diet-derived glycans. Its genome contains 88 different polysaccharide utilization loci (PULs) for complex glycan catabolism and eight different gene clusters for capsular polysaccharide biosynthesis. Here, we investigate one of the prominent mechanisms by which this gut symbiont regulates many PULs involved in host mucin O-glycan degradation; namely, transcriptional regulation via the concerted interactions of cell-envelope-localized TonB-dependent transporters, extra-cytoplasmic function σ factors and anti-σ factors, which participate together in a regulatory pathway termed trans-envelope signaling. Unexpectedly, we found that several different trans-envelope signaling switches involved in PUL-mediated O-glycan degradation also modulate capsular polysaccharide synthesis. A novel regulatory pathway, which is dependent on expression of O-glycan-targeting outer membrane proteins, governs this coordinated regulation of glycan catabolism and capsule synthesis. This latter finding provides a new link in the dynamic interplay between complex glycan metabolism, microbial physiology, and host responses that occurs during colonization of the gut.

Fungi pathogenic to humans: molecular bases of virulence of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus.

The frequency of severe systemic fungal diseases has increased in the last few decades. The clinical use of antibacterial drugs, immunosuppressive agents after organ transplantation, cancer chemotherapy, and advances in surgery are associated with increasing risk of fungal infections. Opportunistic pathogens from the genera Candida and Aspergillus as well as pathogenic fungi from the genus Cryptococcus can invade human organism and may lead to mucosal and skin infections or to deep-seated mycoses of almost all inner organs, especially in immunocompromised patients. Nowadays, there are some effective antifungal agents, but, unfortunately, some of the pathogenic species show increasing resistance. The identification of fungal virulence factors and recognition of mechanisms of pathogenesis may lead to development of new efficient antifungal therapies. This review is focused on major virulence factors of the most common fungal pathogens of humans: Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. The adherence to host cells and tissues, secretion of hydrolytic enzymes, phenotypic switching and morphological dimorphism contribute to C. albicans virulence. The ability to grow at 37 degrees C, capsule synthesis and melanin formation are important virulence factors of C. neoformans. The putative virulence factors of A. fumigatus include production of pigments, adhesion molecules present on the cell surface and secretion of hydrolytic enzymes and toxins.

Optimization of Phase Transfer Catalysis for In Situ Coating of Resorcinol Formaldehyde Targets

To produce foam capsule fusion energy targets with smoother surfaces, we have combined two previously reported resorcinol formaldehyde (RF) aerogel synthesis techniques. Using this new hybrid process, we have successfully produced aerogel spheres that are coated in situ with a smooth submicron-thick skin. The surface roughness of these spheres is compared to the conventionally synthesized RF capsules. We also illustrate the microscopic surface morphology of the new and traditional techniques. We propose that this new approach to capsule synthesis be investigated further as a target candidate; both the enhanced smoothness and the altered surface morphology make for a more desirable coating substrate.

Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.

Functional Characterization of the σ B -Dependent yabJ - spoVG Operon in Staphylococcus aureus : Role in Methicillin and Glycopeptide Resistance

The alternative sigma factor sigma(B) of Staphylococcus aureus controls the expression of multiple genes, including virulence determinants and global regulators; promotes capsule production; and increases the resistance levels of methicillin-resistant S. aureus (MRSA) and glycopeptide-intermediate-resistant S. aureus (GISA) strains. We show here that deletion of the sigma(B)-controlled yabJ-spoVG operon, which codes for potential downstream regulators of sigma(B), abolished capsule synthesis and reduced resistance in MRSA and GISA to the same extent that sigma(B) inactivation did. Introduction of the yabJ-spoVG operon in trans restored the original phenotype. By genetic manipulations, we show that SpoVG but not YabJ is required for complementation. We therefore postulate that SpoVG is the major factor of the yabJ-spoVG operon required in S. aureus for capsule formation and antibiotic resistance.

Modulation of the Rcs-mediated signal transfer by conformational flexibility

The Rcs (regulator of capsule synthesis) signalling complex comprises the membrane-associated hybrid sensor kinases RcsC and RcsD, the transcriptional regulator RcsB and the two co-inducers RcsA and RcsF. Acting as a global regulatory network, the Rcs phosphorelay controls multiple cellular pathways including capsule synthesis, cell division, motility, biofilm formation and virulence mechanisms. Signal-dependent communication of the individual Rcs domains showing histidine kinase, phosphoreceiver, phosphoryl transfer and DNA-binding activities is characteristic and essential for the modulation of signal transfer. We have analysed the structures of core elements of the Rcs network including the RcsC-PR (phosphoreceiver domain of RcsC) and the RcsD-HPt (histidine phosphotransfer domain of RcsD), and we have started to characterize the dynamics and recognition mechanisms of the proteins. RcsC-PR represents a typical CheY-like alpha/beta/alpha sandwich fold and it shows a large conformational flexibility near the active-site residue Asp(875). NMR analysis revealed that RcsC-PR is able to adopt preferred conformations upon Mg(2+) co-ordination, BeF(3)(-) activation, phosphate binding and RcsD-HPt recognition. In contrast, the alpha-helical structure of RcsD-HPt is conformationally stable and contains a recognition area in close vicinity to the active-site His(842) residue. Our studies indicate the importance of protein dynamics and conformational exchange for the differential response to the variety of signals perceived by complex regulatory networks.

Induction of group A Streptococcus virulence by a human antimicrobial peptide

Group A streptococci (Streptococcus pyogenes or GAS) freshly isolated from individuals with streptococcal sore throat or invasive ("flesh-eating") infection often grow as mucoid colonies on primary culture but lose this colony appearance after laboratory passage. The mucoid phenotype is due to abundant production of the hyaluronic acid capsular polysaccharide, a key virulence determinant associated with severe GAS infections. These observations suggest that signal(s) from the human host trigger increased production of capsule and perhaps other virulence factors during infection. Here we show that subinhibitory concentrations of the human antimicrobial cathelicidin peptide LL-37 stimulate expression of the GAS capsule synthesis operon (hasABC). Up-regulation is mediated by the CsrRS 2-component regulatory system: it requires a functional CsrS sensor protein and can be antagonized by increased extracellular Mg(2+), the other identified environmental signal for CsrS. Up-regulation was also evident for other CsrRS-regulated virulence genes, including the IL-8 protease PrtS/ScpC and the integrin-like/IgG protease Mac/IdeS, findings that suggest a coordinated GAS virulence response elicited by this antimicrobial immune effector peptide. LL-37 signaling through CsrRS led to a marked increase in GAS resistance to opsonophagocytic killing by human leukocytes, an in vitro measure of enhanced GAS virulence, consistent with increased expression of the antiphagocytic capsular polysaccharide and Mac/IdeS. We propose that the human cathelicidin LL-37 has the paradoxical effect of stimulating CsrRS-regulated virulence gene expression, thereby enhancing GAS pathogenicity during infection. The ability of GAS to sense and respond to LL-37 may explain, at least in part, the unique susceptibility of the human species to streptococcal infection.

Beyond the Big Three: Systematic Analysis of Virulence Factors in Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans has three major virulence factors: growth at 37 degrees C, capsule synthesis, and melanin formation. Recently in Cell, Liu et al. (2008) employed signature tagged mutagenesis to systematically identify virulence genes. Remarkably, mutations in many of these genes did not influence the major virulence traits.

Suppression of ΔbipAPhenotypes inEscherichia coliby Abolishment of Pseudouridylation at Specific Sites on the 23S rRNA

The BipA protein of Escherichia coli has intriguing similarities to the elongation factor subfamily of GTPases, including EF-Tu, EF-G, and LepA. In addition, phenotypes of a bipA deletion mutant suggest that BipA is involved in regulation of a variety of pathways. These two points have led to speculation that BipA may be a novel regulatory protein that affects efficient translation of target genes through direct interaction with the ribosome. We isolated and characterized suppressors of the cold-sensitive growth phenotype exhibited by DeltabipA strains and identified insertion mutations in rluC. The rluC gene encodes a pseudouridine synthase responsible for pseudouridine modification of 23S rRNA at three sites, all located near the peptidyl transferase center. Deletion of rluC not only suppressed cold sensitivity but also alleviated the decrease in capsule synthesis exhibited by bipA mutants, suggesting that the phenotypic effects of BipA are manifested through an effect on the ribosome. The suppressor effect is specific to rluC, as deletion of other rlu genes did not relieve cold sensitivity, and further, more than a single pseudouridine residue is involved, as alteration of single residues did not produce suppressors. These results are consistent with a role for BipA in either the structure or the function of the ribosome and imply that wild-type ribosomes are dependent on BipA for efficient expression of target mRNAs and that the lack of pseudouridylation at these three sites renders the ribosomes BipA independent.

The crystal structure of capsule synthesis protein CapD, a major virulence factor inB. anthracis

The crystal structure of capsule synthesis protein CapD, a major virulence factor in<i>B. anthracis</i>

Survival of Burkholderia pseudomallei in Water

BackgroundThe ability of Burkholderia pseudomallei to survive in water likely contributes to its environmental persistence in endemic regions. To determine the physiological adaptations which allow B. pseudomallei to survive in aqueous environments, we performed microarray analyses of B. pseudomallei cultures transferred from Luria broth (LB) to distilled water.FindingsIncreased expression of a gene encoding for a putative membrane protein (BPSL0721) was confirmed using a lux-based transcriptional reporter system, and maximal expression was noted at approximately 6 hrs after shifting cells from LB to water. A BPSL0721 deficient mutant of B. pseudomallei was able to survive in water for at least 90 days indicating that although involved, BPSL0721 was not essential for survival. BPSL2961, a gene encoding a putative phosphatidylglycerol phosphatase (PGP), was also induced when cells were shifted to water. This gene is likely involved in cell membrane biosynthesis. We were unable to construct a PGP mutant suggesting that the gene is not only involved in survival in water but is essential for cell viability. We also examined mutants of polyhydroxybutyrate synthase (phbC), lipopolysaccharide (LPS) oligosaccharide and capsule synthesis, and these mutations did not affect survival in water. LPS mutants lacking outer core were found to lose viability in water by 200 days indicating that an intact LPS core provides an outer membrane architecture which allows prolonged survival in water.ConclusionThe results from these studies suggest that B. pseudomallei survival in water is a complex process that requires an LPS molecule which contains an intact core region.

Cryptococcal Xylosyltransferase 1 (Cxt1p) from Cryptococcus neoformans Plays a Direct Role in the Synthesis of Capsule Polysaccharides

The opportunistic yeast Cryptococcus neoformans causes serious disease in humans and expresses a prominent polysaccharide capsule that is required for its virulence. Little is known about how this capsule is synthesized. We previously identified a beta1,2-xylosyltransferase (Cxt1p) with in vitro enzymatic activity appropriate for involvement in capsule synthesis. Here, we investigate C. neoformans strains in which the corresponding gene has been deleted (cxt1Delta). Loss of CXT1 does not affect in vitro growth of the mutant cells or the general morphology of their capsules. However, NMR structural analysis of the two main capsule polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), showed that both were missing beta1,2-xylose residues. There was an approximately 30% reduction in the abundance of this residue in GXM in mutant compared with wild-type strains, and mutant GalXM was almost completely devoid of beta1,2-linked xylose. The GalXM from the mutant strain was also missing a beta1,3-linked xylose residue. Furthermore, deletion of CXT1 led to attenuation of cryptococcal growth in a mouse model of infection, suggesting that the affected xylose residues are important for normal host-pathogen interactions. Cxt1p is the first glycosyltransferase with a defined role in C. neoformans capsule biosynthesis, and cxt1Delta is the only strain identified to date with structural alterations of the capsule polysaccharide GalXM.

Biosynthesis of the Escherichia coli K1 group 2 polysialic acid capsule occurs within a protected cytoplasmic compartment.

Capsular polysaccharides are important virulence determinants in a wide range of invasive infectious diseases. Although capsule synthesis has been extensively investigated, understanding polysaccharide export from the cytoplasm to the external environment has been more difficult. Here we present the results of a novel protection assay indicating that synthesis and export of the Escherichia coli K1 group 2 capsular polysialic acid (K1 antigen) occur within a protected subcellular compartment designated the sialisome. In addition to the polymerase encoded by neuS, localization and complementation analyses indicated that the sialisome includes the accessory membrane protein NeuE. The requirement for NeuE was suppressed by overproducing NeuS, suggesting that NeuE functions by stabilizing the polymerase or facilitating its assembly in the sialisome. Although an interaction between NeuE and NeuS could not be demonstrated with a bacterial two-hybrid system that reconstitutes an intracellular cell-signalling pathway, interactions between NeuS and KpsC as well as other sialisome components were detected. The combined results provide direct evidence for specific protein–protein interactions in the synthesis and export of group 2 capsular polysaccharides under in vivo conditions. The approaches developed here will facilitate further dissection of the sialisome, suggesting similar methodology for understanding the biosynthesis of other group 2 capsules.

Safety of raw meat and shellfish in Vietnam: An analysis of Escherichia coli isolations for antibiotic resistance and virulence genes

This study was conducted to examine a current baseline profile of antimicrobial resistance and virulence of Escherichia coli isolated from foods commonly sold in the market place in Vietnam. E. coli were isolated from 180 samples of raw meat, poultry and shellfish and also isolated from 43 chicken faeces samples. Ninety-nine E. coli isolates recovered from all sources were selected for the investigation of their susceptibility to 15 antimicrobial agents by the disk diffusion method. Eighty-four percent of the isolates were resistant to one or more antibiotics, and multi-resistance, defined as resistance to at least 3 different classes of antibiotics, was detected in all sources. The rates of multi-resistance were up to 89.5% in chicken, 95% in chicken faeces and 75% in pork isolates. Resistance was most frequently observed to tetracycline (77.8%), sulfafurazole (60.6%), ampicillin (50.5%), amoxicillin (50.5%), trimethoprim (51.5%), chloramphenicol (43.4%), streptomycin (39.4%), nalidixic acid (34.3%) and gentamicin (24.2%). In addition, the isolates also displayed resistance to fluoroquinolones (ciprofloxacin 16.2%, norfloxacin 17.2%, and enrofloxacin 21.2%), with chicken isolates showing the highest rates of resistance to these antibiotics (52.6–63.2%). Thirty-eight multi-resistant isolates were selected for further the examination of antibiotic resistance genes and were also evaluated for virulence gene profiles by multiplex and uniplex polymerase chain reaction. The beta-lactam TEM gene and tetracycline resistance tetA, tetB genes were frequently detected in the tested isolates (84.2% and 89.5% respectively). Genes which are responsible for resistance to streptomycin ( aadA) (68.4%), chloramphenicol ( cmlA) (42.1%), sulfonamides ( sulI) (39.5%), trimethoprim ( dhfrV) (26.3%) and kanamycin ( aphA-1) (23.7%) were also widely distributed. Plasmid-mediated ampC genes were detected in E. coli isolates from chicken and pork. The isolates were tested for the presence of 58 virulence genes for adhesins, toxins, capsule synthesis, siderophores, invasins and others from different E. coli pathotypes. All of the tested isolates contained at least one virulence gene and there were 16 genes detected. Virulence genes detected were fimH (92.1%), bmaE (84.2%), TSPE4.C2 (42.1%), aidA AIDA-I ( orfB) (31.6%), east1 (26.3%), traT (23.7%), and others including fyuA, iutA, chuA, yjaA, iss, iroN E. coli , ibeA, aah ( orfA), iha and papG allele III (10.5–2.6%). Typical toxin genes produced by enterohemorrhagic and enterotoxigenic E. coli pathotypes (a heat-stable toxin (ST), heat-labile toxin (LT) and Shiga toxin stx1, stx2) were not detected in any of these 38 isolates. The study has revealed that E. coli in raw foods is a significant reservoir of resistance and virulence genes.

A compendium of antibiotic-induced transcription profiles reveals broad regulation of Pasteurella multocida virulence genes

The transcriptional responses of Pasteurella multocida to eight antibiotics with known mode of actions (MoAs) and one novel antibiotic compound with an unknown MoA were collected to create a compendium of transcriptional profiles for MoA studies. At minimal inhibitory concentration the three bactericidal compounds enrofloxacin, cefquinome and the novel compound had a minor impact on gene regulation with approximately 1% of the P. multocida genome affected, whilst the bacteriostatic compounds florfenicol, tilmicosin, rifampin, trimethoprim and brodimoprim regulated 20% of the genome. Novobiocin was special in that it regulated 40% of all P. multocida genes. Regulation of target genes was observed for novobiocin, rifampin, florfenicol and tilmicosin and signature genes were identified for most antibiotics. The transcriptional profile induced by the novel compound was unrelated to the compendium profiles suggesting a new MoA. The transcription of many P. multocida virulence factors, particularly genes involved in capsule synthesis and export, LPS synthesis, competence, adherence and iron transport were altered in the presence of antibiotics. Virulence gene transcription was mainly negatively affected, however the opposite effect was also observed in the case of rifampin where the up-regulation of the tad locus involved in tight adherence was seen. Novobiocin and trimethoprim caused a marked reduction in the transcription of capsule genes, which correlated with a concomitant reduction of the capsular layer on the surface of P. multocida. The broad negative impact on virulence gene transcription supports the notion that the therapeutic effect of some antibiotics could be a combination of growth and virulence inhibition.

Whole-genome comparison of disease and carriage strains provides insights into virulence evolution in Neisseria meningitidis

Neisseria meningitidis is a leading cause of infectious childhood mortality worldwide. Most research efforts have hitherto focused on disease isolates belonging to only a few hypervirulent clonal lineages. However, up to 10% of the healthy human population is temporarily colonized by genetically diverse strains mostly with little or no pathogenic potential. Currently, little is known about the biology of carriage strains and their evolutionary relationship with disease isolates. The expression of a polysaccharide capsule is the only trait that has been convincingly linked to the pathogenic potential of N. meningitidis. To gain insight into the evolution of virulence traits in this species, whole-genome sequences of three meningococcal carriage isolates were obtained. Gene content comparisons with the available genome sequences from three disease isolates indicate that there is no core pathogenome in N. meningitidis. A comparison of the chromosome structure suggests that a filamentous prophage has mediated large chromosomal rearrangements and the translocation of some candidate virulence genes. Interspecific comparison of the available Neisseria genome sequences and dot blot hybridizations further indicate that the insertion sequence IS1655 is restricted only to N. meningitidis; its low sequence diversity is an indicator of an evolutionarily recent population bottleneck. A genome-based phylogenetic reconstruction provides evidence that N. meningitidis has emerged as an unencapsulated human commensal from a common ancestor with Neisseria gonorrhoeae and Neisseria lactamica and consecutively acquired the genes responsible for capsule synthesis via horizontal gene transfer.

Carbohydrate mimetics as potential antiinfective treatments for Staphylococcus aureus

Antibiotic resistant strains of Staphylococcus aureus are an important cause of nosocomial infections. These strains are currently causing community‐acquired infections, resulting in an increased need for alternative therapies. S. aureus capsules appear to act as important virulence determinants, as bacteria with type 5 and 8 capsules cause 75–90% of nosocomial infections and anti‐capsular antibodies decrease mortality in infected mice. Our research involves construction of analogs of the aminosugars found in S. aureus capsules that we are testing for their ability to prevent capsule synthesis. To measure capsule production, bacteria were incubated with MV‐II‐065 or MV‐II‐063 (both n‐glucosyl 1,2,3‐triazoles with different functional groups), formalinized, trypsinized, and the bacteria used as an antigen in an ELISA. The presence of capsular carbohydrate was determined by incubation with anti‐capsular monoclonal antibodies, followed by a secondary incubation with peroxidase‐conjugate anti‐mouse Ig and a colorimetric substrate. Incubation type 5 S. Aureus with MV‐II‐065 decreased antibody binding, but the compound had no effect on Type 8 capsule synthesis. Studies with MV‐II‐063 (shown below) have indicated inhibition of capsule synthesis in Type 8 bacteria, while type 5 capsule synthesis remains to be tested.In addition to their possible therapeutic potential, the differential effects of these two compounds may be used to investigate the synthetic pathways for these capsules. Support: NIH R15 2003‐2006, YSU PACER.

The Rcs Phosphorelay Is a Cell Envelope Stress Response Activated by Peptidoglycan Stress and Contributes to Intrinsic Antibiotic Resistance

Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the beta-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to beta-lactam antibiotics.

Plasmid-based vaccination with candidate anthrax vaccine antigens induces durable type 1 and type 2 T-helper immune responses

The current anthrax vaccine imparts protective immunity by generating a humoral immune response against a single antigen, the PA exotoxin subunit. While this response neutralizes the two anthrax exotoxins and protects the recipient from toxin-related mortality, the recipient is not protected from spore germination, infection, and/or bacteremia. Moreover, protective immunity against PA must be generated via a lengthy injection schedule and maintained by a yearly booster. In an effort to improve upon the current vaccine formulation, we screened six of seven known virulence factors encoded by Bacillus anthracis epigenetic elements pXO1 and pXO2 as well as the major surface proteins EA1 and SAP. Screening was carried out in conjunction with a plasmid-based technology known for its ability to generate type 1 and type 2 T-helper responses. Long-term high level antibody titers were generated against the products of eag (EA1), sap (SAP), and the capA capsule synthesis subunit in vivo. Further analysis of PA- and EA1-vaccinated mice demonstrated antigen-specific type 1 helper responses including IFN-gamma secretion and lysis of EA1- or PA-loaded macrophages; further, an EA1 T-cell epitope was identified. The results demonstrate that anthrax antigens other than PA might be suitable for the generation of durable immune responses against anthrax.