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Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro , may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets.

The virulence of Bacillus anthraci s, the causative agent of anthrax, stems from its antiphagocytic capsule, encoded by pXO2, and the tripartite toxins encoded by pXO1. The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play major roles in pathogenicity. We tested this assumption by a systematic study of mutants with combined deletions of the pag , lef , and cya genes, encoding protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively. The resulting seven mutants (single, double, and triple) were evaluated following subcutaneous (s.c.) and intranasal (i.n.) inoculation in rabbits and guinea pigs. In the rabbit model, virulence is completely dependent on the presence of PA. Any mutant bearing a pag deletion behaved like a pXO1-cured mutant, exhibiting complete loss of virulence with attenuation indices of over 2,500,000 or 1,250 in the s.c. or i.n. route of infection, respectively. In marked contrast, in guinea pigs, deletion of pag or even of all three toxin components resulted in relatively moderate attenuation, whereas the pXO1-cured bacteria showed complete attenuation. The results indicate that a pXO1-encoded factor(s), other than the toxins, has a major contribution to the virulence mechanism of B. anthracis in the guinea pig model. These unexpected toxin-dependent and toxin-independent manifestations of pathogenicity in different animal models emphasize the importance and need for a comprehensive evaluation of B. anthracis virulence in general and in particular for the design of relevant next-generation anthrax vaccines.

Bacillus anthracis secretes three major components, which assemble into two bipartite toxins: lethal toxin (LT), composed of lethal factor (LF) and protective antigen (PA) and edema toxin (ET), composed of edema factor (EF) and PA. EF is a potent calmodulin-dependent adenylate cyclase, which is internalized into the target cell following PA binding. Once inside the cell, EF elevates cAMP levels, interrupting intracellular signaling. Effects of ET were demonstrated on monocytes, neutrophils and T-cells. In an earlier work we demonstrated that a deletion of LF in a fully virulent strain had no effect in guinea pigs and a significant, but not major, effect in the rabbit model. These results suggested that EF might play an important role in the development of infection and mortality following exposure to B. anthracis spores. To evaluate the role of EF in B. anthracis pathogenicity we deleted the cya gene, which encodes the EF protein, in the fully virulent Vollum strain. The Δcya mutant was fully virulent in the guinea pig model as determined by LD(50) experiments. In the rabbit model, when infected subcutaneously, the absence of EF had no effect on the virulence of the mutant. However an increase of two orders of magnitude in the LD(50) was demonstrated when the rabbits were infected by intranasal instillation accompanied with partial mortality and increased mean time to death. These results argue that in the guinea pig model the presence of one of the toxins, ET or LT is sufficient for the development of the infection. In the rabbit model ET plays a role in respiratory infection, most probably mediating the early steps of host colonization.

The major virulence factor of Bacillus anthracis is the tripartite anthrax toxin, comprising the protective antigen (PA), lethal factor (LF) and edema factor (EF). The LF of B. anthracis is a metalloprotease that has been shown to play an important role in pathogenicity. Deletion of this gene (lef) in the Sterne strain was reported to dramatically reduce the pathogenicity of this strain in mice, and was reported to be as dramatic as the deletion of PA. We evaluated the effect on pathogenicity of the lef deletion in the fully virulent Vollum strain in guinea pigs and NZW rabbits by either subcutaneous injection or intranasal instillation. In guinea pigs, no major differences between the mutant strain and the wild type could be detected in the LD50 or mean time to death values. On the other hand, the lef deletion caused death of 50–70% of all rabbits infected with the mutant spores at doses equivalent or higher than the wild type LD50. The surviving rabbits, which were infected with spore doses higher than the wild type LD50, developed a protective immune response that conferred resistance to challenge with the wild type strain. These findings may indicate that the mutant lacking the LF is capable of host colonization which causes death in 50–70% of the animals and a protective immune response in the others. These results indicate that unlike the data obtained in mice, the LF mutation does not abolish B. anthracis pathogenicity.

Respiratory anthrax, in the absence of early antibiotic treatment, is a fatal disease. This study aimed to test the efficiency of antibiotic therapy in curing infected animals and those sick with anthrax. Postexposure prophylaxis (24 h postinfection [p.i.]) of guinea pigs infected intranasally with Bacillus anthracis Vollum spores with doxycycline, ofloxacin, imipenem, and gentamicin conferred protection. However, upon termination of treatment, the animals died from respiratory anthrax. Combined treatment with antibiotics and active vaccination with a protective antigen-based vaccine leads to full protection even after cessation of treatment. Delaying the initiation of antibiotic administration to over 24 h p.i. resulted in treatment of animals with anthrax exhibiting various degrees of bacteremia and toxemia. Treatment with doxycycline or ciprofloxacin cured sick guinea pigs and rabbits exhibiting bacteremia levels up to 10 5 CFU/ml. Addition of anti-protective antigen (PA) antibodies augmented the efficiency of protection, allowing the cure of guinea pigs and rabbits with 10- to 20-fold-higher bacteremia levels, up to 7 × 10 5 CFU/ml and 2 × 10 6 CFU/ml, respectively. Treatment with ciprofloxacin and a monoclonal anti-PA antibody rescued rabbits with bacteremia levels up to 4 × 10 6 CFU/ml. During antibiotic administration, all surviving animals developed a protective immune response against development of a fatal disease and subcutaneous challenge with Vollum spores. In conclusion, these results demonstrate that antibiotic treatment can prevent the development of fatal disease in respiratory-anthrax-infected animals and can cure animals after disease establishment. A therapeutic time window of 40 h to 48 h from infection to initiation of efficient antibiotic-mediated cure was observed.

The lethal anthrax disease is caused by spores of the gram-positive Bacillus anthracis, a member of the cereus group of bacilli. Although the disease is very rare in the Western world, development of anthrax countermeasures gains increasing attention due to the potential use of B. anthracis spores as a bio-terror weapon. Protective antigen (PA), the non-toxic subunit of the bacterial secreted exotoxin, fulfills the role of recognizing a specific receptor and mediating the entry of the toxin into the host target cells. PA elicits a protective immune response and represents the basis for all current anthrax vaccines. Anti-PA neutralizing antibodies are useful correlates for protection and for vaccine efficacy evaluation. Post exposure anti-toxemic and anti-bacteremic prophylactic treatment of anthrax requires prolonged antibiotic administration. Shorter efficient postexposure treatments may require active or passive immunization, in addition to antibiotics. Although anthrax is acknowledged as a toxinogenic disease, additional factors, other than the bacterial toxin, may be involved in the virulence of B. anthracis and may be needed for the long-lasting protection conferred by PA immunization. The search for such novel factors is the focus of several high throughput genomic and proteomic studies that are already leading to identification of novel targets for therapeutics, for vaccine candidates, as well as biomarkers for detection and diagnosis.

Mounting an effective immune response, while also protecting tissue integrity, is critical for host survival. We used a combined genomic and proteomic approach to investigate the role of extracellular matrix (ECM) proteolysis in achieving this balance in the lung during influenza virus infection. We identified the membrane-tethered matrix metalloprotease MT1-MMP as a prominent host-ECM-remodeling collagenase in influenza infection. Selective inhibition of MT1-MMP protected the tissue from infection-related structural and compositional tissue damage. MT1-MMP inhibition did not significantly alter the immune response or cytokine expression. The available flu therapeutic Oseltamivir did not prevent lung ECM damage and was less effective than anti-MT1-MMP in influenza virus Streptococcus pneumoniae coinfection paradigms. Combination therapy of Oseltamivir with anti-MT1-MMP showed a strong synergistic effect and resulted in complete recovery of infected mice. This study highlights the importance of tissue resilience in surviving infection and the potential of such host-pathogen therapy combinations for respiratory infections.

Respiratory anthrax is a fatal disease in the absence of early treatment with antibiotics. Rabbits are highly susceptible to infection with Bacillus anthracis spores by intranasal instillation, succumbing within 2 to 4 days postinfection. This study aims to test the efficiency of antibiotic therapy to treat systemic anthrax in this relevant animal model. Delaying the initiation of antibiotic administration to more than 24 h postinfection resulted in animals with systemic anthrax in various degrees of bacteremia and toxemia. As the onset of symptoms in humans was reported to start on days 1 to 7 postexposure, delaying the initiation of treatment by 24 to 48 h (time frame for mass distribution of antibiotics) may result in sick populations. We evaluated the efficacy of antibiotic administration as a function of bacteremia levels at the time of treatment initiation. Here we compare the efficacy of treatment with clarithromycin, amoxicillin-clavulanic acid (Augmentin), imipenem, vancomycin, rifampin, and linezolid to the previously reported efficacy of doxycycline and ciprofloxacin. We demonstrate that treatment with amoxicillin-clavulanic acid, imipenem, vancomycin, and linezolid were as effective as doxycycline and ciprofloxacin, curing rabbits exhibiting bacteremia levels of up to 10 5 CFU/ml. Clarithromycin and rifampin were shown to be effective only as a postexposure prophylactic treatment but failed to treat the systemic (bacteremic) phase of anthrax. Furthermore, we evaluate the contribution of combined treatment of clindamycin and ciprofloxacin, which demonstrated improvement in efficacy compared to ciprofloxacin alone.

The most aggressive form of anthrax results from inhalation of airborne spores of Bacillus anthracis and usually progresses unnoticed in the early stages because of unspecific symptoms. The only reliable marker of anthrax is development of bacteremia, which increases with disease progress. Rapid diagnosis of anthrax is imperative for efficient treatment and cure. Herein we demonstrate that the presence and level of a bacterial antigen, the protective antigen (PA), a component of B. anthracis toxins, in host sera can serve as a reliable marker of infection. This was tested in two animal models of inhalation anthrax, rabbits and guinea pigs infected by intranasal instillation of Vollum spores. In both models, we demonstrated qualitative and quantitative correlations between levels of bacteremia and PA concentrations in the sera of sick animals. The average time to death in infected animals was about 16 h after the appearance of bacteremia, leaving a small therapeutic window. As the time required for immunodetection of PA can be very short, the use of this marker will be beneficial for faster diagnosis and treatment of inhalation anthrax.

Correlates between immunological parameters and protection against Bacillus anthracis infection in animals vaccinated with protective antigen (PA)-based vaccines could provide surrogate markers to evaluate the putative protective efficiency of immunization in humans. In previous studies we demonstrated that neutralizing antibody levels serve as correlates for protection in guinea pigs (S. Reuveny et al., Infect. Immun. 69:2888-2893, 2001; H. Marcus et al., Infect. Immun. 72:3471-3477, 2004). In this study we evaluated similar correlates for protection by active and passive immunization of New Zealand White rabbits. Full immunization and partial immunization were achieved by single and multiple injections of standard and diluted doses of a PA-based vaccine. Passive immunization was carried out by injection of immune sera from rabbits vaccinated with PA-based vaccine prior to challenge with B. anthracis spores. Immunized rabbits were challenged by intranasal spore instillation with one of two virulent strains (strains Vollum and ATCC 6605). The immune competence was estimated by measuring the level of total anti-PA antibodies, the neutralizing antibody titers, and the conferred protective immunity. The results indicate that total anti-PA antibody titers greater than 1 × 10 5 conferred protection, whereas lower titers (between 10 4 and 10 5 ) provided partial protection but failed to predict protection. Neutralizing antibody titers between 500 and 800 provided partial protection, while titers higher than 1,000 conferred protection. In conclusion, this study emphasizes that regardless of the immunization regimen or the time of challenge, neutralizing antibody titers are better predictors of protection than total anti-PA titers.

We reported previously on the development of a Bacillus anthracis vaccine strain expressing high levels of recombinant protective antigen (rPA) [Cohen et al., Infec Immun 2000;68(8):4549-58]. To further explore the potential of the B. anthracis platform, we generated several attenuated strains expressing lethal toxin components PA and LF, which are biologically inactive, yet retain their antigenic properties. A single injection of 5 x 10(7) spores of one of these strains, carrying PA mutation at a site involved in effector translocation (residues 313-314) was shown to resemble wild type PA in inducing production of high levels of anti-PA neutralizing antibodies and producing effective protective immunity for 12 months. Long-term protection and persistence of functional antibody titers was observed after the gradual elimination of spores from guinea pig tissues 3 months after injection and in the measurable absence of bacteria in tissues. The mutant toxin components could, thus be an effective alternatives to their native counterparts when presented to the immune system in context of a live B. anthracis strain. These live vaccine prototypes may serve as a platform for future multi-component vaccines.

Here we describe the characterization of a lipoprotein previously proposed as a potential Bacillus anthracis virulence determinant and vaccine candidate. This protein, designated MntA, is the solute-binding component of a manganese ion ATP-binding cassette transporter. Coupled proteomic-serological screen of a fully virulent wild-type B. anthracis Vollum strain, confirmed that MntA is expressed both in vitro and during infection. Expression of MntA is shown to be independent of the virulence plasmids pXO1 and pXO2. An mntA deletion, generated by allelic replacement, results in complete loss of MntA expression and its phenotypic analysis revealed: (i) impaired growth in rich media, alleviated by manganese supplementation; (ii) increased sensitivity to oxidative stress; and (iii) delayed release from cultured macrophages. The DeltamntA mutant expresses the anthrax-associated classical virulence factors, lethal toxin and capsule, in vitro as well as in vivo, and yet the mutation resulted in severe attenuation; a 10(4)-fold drop in LD(50) in a guinea pig model. MntA expressed in trans allowed to restore, almost completely, the virulence of the DeltamntA B. anthracis strain. We propose that MntA is a novel B. anthracis virulence determinant essential for the development of anthrax disease, and that B. anthracisDeltamntA strains have the potential to serve as platform for future live attenuated vaccines.

Classification and differentiation of Bacillus anthracis isolates by genetic markers play an important role in anthrax research. We used a PCR based method – Random Amplification of Polymorphic DNA (RAPD) – to identify genetic markers in B. anthracis strains. Twenty-five differential genetic markers were identified which divided the strains into five different groups. Three selected RAPD-markers were cloned and sequenced. The five RAPD-derived genotypes could be defined by integration of these three markers. This system offers a simple non-expensive method to classify B. anthracis strains in laboratories involved in the research of this bacterium.

The genes that encode the enterotoxigenic Escherichia coli (ETEC) CS4 fimbriae, csaA , - B , - C , - E , and - D ′, were isolated from strain E11881A. The csa operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa tip-associated protein (CsaE), a 27-kDa chaperone-like protein (CsaA), a 97-kDa usher-like protein (CsaC), and a deleted regulatory protein (CsaD′). The predicted amino acid sequences of the CS4 proteins are highly homologous to structural and assembly proteins of other ETEC fimbriae, including CS1 and CS2, and to CFA/I in particular. The csaA , - B , - C , - E operon was cloned on a stabilized plasmid downstream from an osomotically regulated ompC promoter. pGA2-CS4 directs production of CS4 fimbriae in both E . coli DH5α and Shigella flexneri 2a vaccine strain CVD 1204, as detected by Western blot analysis and bacterial agglutination with anti-CS4 immune sera. Electron-microscopic examination of Shigella expressing CS4 confirmed the presence of fimbriae on the bacterial surface. Guinea pigs immunized with CVD 1204(pGA2-CS4) showed serum and mucosal antibody responses to both the Shigella vector and the ETEC fimbria CS4. Among the seven most prevalent fimbrial antigens of human ETEC, CS4 is the last to be cloned and sequenced. These findings pave the way for CS4 to be included in multivalent ETEC vaccines, including an attenuated Shigella live-vector-based ETEC vaccine.

Shigella and enterotoxigenic Escherichia coli (ETEC) continue to be important causes of diarrheal disease in infants and young children in developing countries and are major etiologic agents of traveler's diarrhea. Since attenuated strains of Shigella have been developed as live oral vaccines against shigellosis, we have adapted these attenuated Shigella strains to serve as carriers of ETEC antigens, thereby constituting a hybrid vaccine. Since protective immunity against ETEC is largely directed against fimbrial antigens (of which there are multiple antigenic types), we have individually expressed four different ETEC fimbriae, including CFA/I, CS2, CS3, and CS4, using deltaguaBA attenuated Shigella vaccine strain CVD 1204 as a prototype live vector. Following mucosal (intranasal) immunization of guinea pigs, serum IgG and mucosal IgA responses were elicited against each fimbrial type. An additional strain was constructed expressing a detoxified version of the human ETEC variant of heat labile toxin (LThK63). Following mucosal immunization of guinea pigs with a mixed inoculum containing five Shigella strains each expressing a different ETEC antigen, immune responses were observed against each ETEC antigen plus the Shigella vector.

The efficiency of postexposure prophylaxis against Bacillus anthracis infection was tested in guinea pigs infected intranasally with either Vollum or strain ATCC 6605 spores (75 times the 50% lethal dose [LD 50 ] and 87 times LD 50, respectively). Starting 24 h postinfection, animals were treated three times per day for 14 days with ciprofloxacin, tetracycline, erythromycin, cefazolin, and trimethoprim-sulfamethoxazole (TMP-SMX). Administration of cefazolin and TMP-SMX failed to protect the animals, while ciprofloxacin, tetracycline, and erythromycin prevented death. Upon cessation of treatment all erythromycin-treated animals died; of the tetracycline-treated animals, two of eight infected with Vollum and one of nine infected with ATCC 6605 survived; and of the ciprofloxacin group injected with either 10 or 20 mg/kg of body weight, five of nine and five of five animals, respectively, survived. To test the added value of extending the treatment period, Vollum-infected (46 times the LD 50 ) animals were treated for 30 days with ciprofloxacin or tetracycline, resulting in protection of eight of nine and nine of nine animals, respectively. Once treatment was discontinued, only four of eight and five of nine animals, respectively, survived. Following rechallenge (intramuscularly) of the survivors with 30 times the LD 50 of Vollum spores, all ciprofloxacin-treated animals were protected while none of the tetracycline-treated animals survived. In an attempt to confer protective immunity lasting beyond the termination of antibiotic administration, Vollum-infected animals were immunized with a protective antigen (PA)-based vaccine concurrently with treatment with either ciprofloxacin or tetracycline. The combined treatment protected eight of eight and nine of nine animals. Following cessation of antibiotic administration seven of eight and eight of eight animals survived, of which six of seven and eight of eight resisted rechallenge. These results indicate that a combined treatment of antibiotics together with a PA-based vaccine could provide long-term protection to prevent reoccurrence of anthrax disease.

Hundreds of immune cell types work in coordination to maintain tissue homeostasis. Upon infection, dramatic changes occur with the localization, migration, and proliferation of the immune cells to first alert the body of the danger, confine it to limit spreading, and finally extinguish the threat and bring the tissue back to homeostasis. Since current technologies can follow the dynamics of only a limited number of cell types, we have yet to grasp the full complexity of global in vivo cell dynamics in normal developmental processes and disease. Here, we devise a computational method, digital cell quantification (DCQ), which combines genome‐wide gene expression data with an immune cell compendium to infer in vivo changes in the quantities of 213 immune cell subpopulations. DCQ was applied to study global immune cell dynamics in mice lungs at ten time points during 7 days of flu infection. We find dramatic changes in quantities of 70 immune cell types, including various innate, adaptive, and progenitor immune cells. We focus on the previously unreported dynamics of four immune dendritic cell subtypes and suggest a specific role for CD103+ CD11b− DCs in early stages of disease and CD8+ pDC in late stages of flu infection. ![][1] A method is presented to infer the changes in the quantities of 213 immune cell types within a complex in vivo cell population. High‐resolution temporal analysis during flu infection reveals specific roles of dendritic cell subtypes in early and late disease phases. Mol Syst Biol. (2014) 10: 720 [1]: /embed/graphic-1.gif

Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied for the characterization of Bacillus anthracis spore biomarkers. B. anthracis spores were extracted under a simple procedure, followed by linear mode analysis, using sinapinic acid as the matrix. Several markers with a mass range of 4-7 kDa were detected in three B. anthracis strains: Vollum, Sterne and V770-NP1-R. Similar spectra were also obtained for spore extracts of two members of the B. cereus group: B. thuringiensis and B. cereus, but not for B. mycoides, B. subtilis or B. licheniformis, suggesting that these markers are specific to closely related members of the B. cereus group. When alpha-cyano-4-hydroxycinnamic acid was used as the matrix, at least four additional new markers within a mass range of 2-4 kDa could be detected in all B. anthracis spore extracts. These markers, corresponding to a molecular weight of 2528.3, 2792.4, 3077.4, and 3590.7 Da, have not been observed in extracts of the three closely related Bacillus species - B. cereus, B. thuringiensis and B. mycoides. These unique B. anthracis biomarkers, which were isotopically resolved and reproducibly detected in the highly accurate MALDI-TOFMS reflectron mode, may be useful as a basis for rapid and specific identification of B. anthracis strains.

Two bacterial enteric pathogens that have been identified by the World Health Organization as constituting important targets for the development of vaccines are enterotoxigenic Escherichia coli (ETEC) and Shigella (35, 38). In developing countries, ETEC is a major cause of diarrheal dehydration in infants (4), whereas Shigella is the main agent of bacillary dysentery in young children (35). Both pathogens contribute in a major way to the mortality burden attributable to enteric pathogens (4, 35). ETEC is also the most frequent etiologic agent associated with traveler's diarrhea (3, 14, 29, 51), whereas in many studies Shigella is often the second most incriminated pathogen (14, 29). Traveler's diarrhea caused by Shigella tends to be clinically more severe and debilitating than that caused by ETEC. Both ETEC and Shigella are deemed to be worthy targets for immunoprophylaxis of travelers from industrialized countries who visit developing regions of the world (45). Among the promising candidate vaccines against Shigella are parenteral O polysaccharide-carrier protein conjugates (7, 8), intranasally administered proteosomes consisting of outer membrane protein vesicles of group B Neisseria meningitidis to which Shigella lipopolysaccharide is noncovalently bound (47, 48), and attenuated strains of Shigella used as live oral vaccines (9, 34). Within the four Shigella species (also referred to as groups), 39 main serotypes and subtypes are recognized (15, 35), and epidemiologic and experimental observations indicate that immunity is group-specific and, in many instances, serotype-specific (21, 22). Consequently, initial success with prototype vaccines will have to be followed by the development of a final vaccine formulation that incorporates a strategy for conferring broad-spectrum protection against the epidemiologically most important Shigella serotypes (35, 53). In recent years, candidate human vaccines against ETEC have been prepared that are based on stimulating intestinal antibodies against the colonization factor fimbriae by which ETEC attaches to enterocytes and on stimulating antitoxin to neutralize heat-labile enterotoxin (LT) (1, 19, 41, 43, 61, 66, 67). Antigens to stimulate anticolonization immunity have included inactivated fimbriated ETEC whole bacteria (1, 16, 19, 60), purified ETEC fimbriae administered in native form (18, 43) or contained within polylactide-polyglycolide microspheres (67), and live oral vaccines consisting of either fimbriated nontoxigenic ETEC strains (36, 37) or of attenuated Shigella or Salmonella enterica serovars Typhi or Typhimurium live vectors expressing ETEC fimbriae and mutant LT or the LT B subunit (26, 31, 42, 54, 55). ETEC vaccines must also address the considerable antigenic heterogeneity among ETEC strains that cause human diarrheal disease (24, 39, 42). It is widely agreed that an ETEC vaccine should include colonization factor antigen I (CFA/I) and coli surface antigens 1 to 6 (CS1 to CS6) fimbrial antigens (42). The candidate ETEC vaccine that is furthest along in clinical trials consists of an oral formulation containing a mixture of inactivated, fimbriated ETEC strains that express CFA/I and CS1-6, coadministered in combination with the cholera toxin B subunit (CT-BS) (60, 61). CT and CT-BS elicit cross-reacting antibodies that can neutralize the LT variant found in ETEC strains in humans (LTh) (46, 65); CT-BS, by itself, has conferred short-term protection (for several months) against diarrhea caused by LT-producing ETEC (6, 56). We have embarked on a long-term project to develop a multivalent hybrid vaccine to prevent both Shigella dysentery and ETEC diarrhea caused by the epidemiologically most important serotypes and antigenic types (34, 39, 53). The approach consists of engineering five attenuated Shigella strains (representing five epidemiologically and immunologically critical serotypes), each expressing two separate ETEC fimbrial antigens and an antigen to elicit antibodies against LTh (31, 39, 55). Towards this goal, we have prepared improved Shigella vaccine candidates by introducing a deletion mutation in the guaBA operon (which encodes two enzymes involved in the synthesis of guanine nucleotides) in wild-type S. flexneri 2a, resulting in vaccine strain CVD 1204 as a basis of further derivatives (34, 54). This serotype is used as a model because of its epidemiologic importance, the presence in its chromosome of a pathogenicity island that includes Shigella enterotoxin 1 (20, 52), and extensive experience with this serotype in experimental challenge studies in volunteers (11–13, 32, 33). The effect of introducing additional attenuating mutations into CVD 1204, such as deletions in virG (also referred to as icsA, encoding a protein involved with intracellular and intercellular spread of Shigella), resulting in CVD 1205 (54), and in the genes encoding Shigella enterotoxins 1 and 2 (Δset1A, Δsen), resulting in CVD 1207, have been evaluated (34). We have previously reported cloning the genes necessary for the expression of CFA/I by CVD 1204 and the ability of that live vector to elicit both anti-S. flexneri 2a and anti-CFA/I antibodies (31). The research reported herein describes the expression of rigid CS2 fimbriae and flexible CS3 fibrillae by attenuated S. flexneri 2a strain CVD 1204; an estimation of the stability of the expression plasmids in CVD 1204; the suitability of the osmolarity-activated ompC promoter in promoting fimbrial expression; the ability of the live vector to elicit antibodies to each fimbrial antigen individually and to both fimbriae simultaneously, in addition to S. flexneri O antigen; and, finally, a demonstration that expression of ETEC fimbriae by the live vector does not diminish its ability to protect against virulent S. flexneri 2a in a challenge model.

The etiological agent of anthrax disease in animals and humans is the spore-forming bacterium Bacillus anthracis. The major factors of virulence of B. anthracis are located on two plasmids, pXO1 and pXO2. pXO2 encodes a poly-d-glutamic acid capsule (19, 41), while pXO1 encodes two binary exotoxins, the lethal toxin (LT) and the edema toxin (ET) (43, 46, 61). These two toxins are composed of three different proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF) (for a review, see reference 36). PA is the common receptor binding domain of the toxins and can interact with the two different effector domains, EF and LF, to mediate their entry into target cells (14). EF is a calmodulin-dependent adenylate cyclase (37) responsible for the edema seen at the site of infection in experimental animals (17). The LF is a metalloprotease (34) recently shown to cleave the amino termini of the mitogen-activated protein kinase kinases 1 and 2, which results in their inactivation (13). It remains to be determined whether these are the main physiological substrates for the LT activity in vivo (5, 22). Two types of anthrax vaccines are licensed for use in humans: the spores of the toxigenic, nonencapsulated B. anthracis STI-1 strain (55) and the cell-free PA-based vaccines consisting of aluminum hydroxide-adsorbed supernatant material from cultures of the toxigenic, nonencapsulated B. anthracis strain V770-NPI-R (49) or alum-precipitated culture filtrate from the Sterne strain (6). The use of the live attenuated STI-1 occasionally results in general and local adverse responses, observed both after primary application and revaccination, and the frequency of responses increases with the number of vaccinations (58). Furthermore, it was reported that the STI-1 vaccine has a relatively low immunogenicity (reviewed by Stepanov et al. in reference 58). To increase the immunogenicity, a combined vaccine of live STI-1 supplemented with cell-free PA formulation was evaluated and proposed for veterinary use (1). While the cell-free PA-based vaccines appear to be safer, they require numerous boosters (8) and were shown to have reduced ability to protect laboratory animals against certain virulent strains of B. anthracis (39, 60). In addition, these vaccines contain variable amounts of PA, as well as undefined quantities of LF and EF, adsorbed to aluminum hydroxide (4, 21, 49, 59). It appears, therefore, that there is a need for a safe and more efficient vaccine which could generate stable and prolonged immunity in humans (59). These conclusions led to the evaluation of various adjuvants with purified PA (2, 16, 29, 59) and to the creation of two types of live vaccines: vaccines based on nonvirulent B. anthracis (pXO1+) mutated strains (31, 47) and vaccines expressing PA from a cloned pagA gene using heterologous hosts such as the vaccinia virus, Bacillus subtilis, Salmonella typhimurium (10, 27, 28, 30, 31, 64), or a nontoxinogenic strain of B. anthracis (4). These pioneering studies suggest that recombinant B. anthracis live vaccines may have potential as a future anthrax vaccine. We report here the construction of several recombinant, nonencapsulated, and nontoxinogenic B. anthracis spore-forming strains expressing different levels of PA. We demonstrate that one of these strains, containing the pagA gene under a potent heterologous constitutive promoter, can be safely used to provide efficacious long-lasting immunity in experimental animals following a single immunization dose.

Respiration-deficient (petite) mutations have been induced in various yeasts, which are categorized as petite-positive. Candida albicans was classified among the petite-negative yeasts. Since then, a few reports have appeared, describing the isolation of petite mutants in C. albicans. We report in the present study on the isolation of a petite mutant of C. albicans-SAR1. This mutant was isolated from a histidine auxotroph of C. albicans after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, thus our petite mutant carries a double mutation. SAR1 was characterized morphologically, biochemically and ultrastructurally. The results revealed differences from the wild type in respect to morphological, physiological and biochemical characteristics. Electron microscopy showed that the cells of the petite mutant contain only very few mitochondria that looked 'thread like' without any cristae. The significance of the mutation in the virulence of the mutant vs. that of the wild-type is being assessed.

Summary. This study investigated the effect of the chitin synthetase inhibitors, the nikkomycins (NZ and NZ + NX), on Candida albicans adhesion to buccal epithelial cells (BECs) in vitro. The effect was expressed in reduced chitin synthetase activity and chitin content of fungal cells. In vitro adhesion assays to BECs of Candida exposed to NZ and NZ + NX revealed reduced adhesion values. Light, scanning and transmission electron microscopy (SEM, TEM) of NZ-treated and untreated microorganisms showed changed fungal morphology and reduced adherence of the treated yeasts. Scanning electron microscopy of NZ-treated C. albicans labelled with gold-conjugated wheatgerm agglutinin (WGA) revealed less labelling than in the untreated organisms. A close contact between the fungus and the epithelial cell at a site with intense WGA-gold labelling was noted in TEM experiments. The data point to the involvement of chitin in the adhesion of C. albicans to epithelial cells. Zusammenfassung. Es wurde die Wirkung des Chitinsynthetase-Hemmers Nikkomycin auf die Adharenz von Candida albicans an Epithelzellen der Mundschleimhaut in vitro untersucht. Der Einflus wurde uber die Reduktion der Chitinsynthetase-Aktivitat und des Chitingehalts der Pilzzellen erfast. Lichtmikroskopisch, raster- und transmissions-elektronenmikroskopisch Zeigten die Nikkomycin-behandelten Hefezellen morphologische Veranderungen und reduzierte Adharenz. Nikkomycin-behandelte C. albicans-Zellen, die mit Gold-konjugiertem Weizenkeimagglutinin (WGA) inkubiert waren, erwiesen sich weniger markiert als nicht mit Nikkomycin-behandelte Hefezellen. Transmissionselektronenmikroskopisch wurde eine intensive WGA-Gold-Markierung am Ort intensiven Kontaktes zwischen Epithelzelle und adharenter Pilzzelle beobachtet. Die Ergebnisse sprechen fur die Beteiligung von Chitin bei der Adharenz von C. albicans an Epithelzellen.

The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play a major role in pathogenicity. In the guinea pig (GP) model we have previously shown that deletion of all three toxin components results in a relatively moderate attenuation in virulence, indicating that B. anthracis possesses an additional toxin-independent virulence mechanism. To characterize this toxin-independent mechanism in anthrax disease, we developed a new rabbit model by intravenous injection (IV) of B. anthracis encapsulated vegetative cells, artificially creating bacteremia. Using this model we were able to demonstrate that also in rabbits, B. anthracis mutants lacking the toxins are capable of killing the host within 24 hours. This virulent trait depends on the activity of AtxA in the presence of pXO2, as, in the absence of the toxin genes, deletion of either component abolishes virulence. Furthermore, this IV virulence depends mainly on AtxA rather than the whole pXO1. A similar pattern was shown in the GP model using subcutaneous (SC) administration of spores of the mutant strains, demonstrating the generality of the phenomenon. The virulent strains showed higher bacteremia levels and more efficient tissue dissemination; however our interpretation is that tissue dissemination per se is not the main determinant of virulence whose exact nature requires further elucidation.

The lethal anthrax disease is caused by spores of the gram-positive Bacillus anthracis, a member of the cereus group of bacilli. Although the disease is very rare in the Western world, development of anthrax countermeasures gains increasing attention due to the potential use of B. anthracis spores as a bio-terror weapon. Protective antigen (PA), the non-toxic subunit of the bacterial secreted exotoxin, fulfills the role of recognizing a specific receptor and mediating the entry of the toxin into the host target cells. PA elicits a protective immune response and represents the basis for all current anthrax vaccines. Anti-PA neutralizing antibodies are useful correlates for protection and for vaccine efficacy evaluation. Post exposure anti-toxemic and anti-bacteremic prophylactic treatment of anthrax requires prolonged antibiotic administration. Shorter efficient postexposure treatments may require active or passive immunization, in addition to antibiotics. Although anthrax is acknowledged as a toxinogenic disease, additional factors, other than the bacterial toxin, may be involved in the virulence of B. anthracis and may be needed for the long-lasting protection conferred by PA immunization. The search for such novel factors is the focus of several high throughput genomic and proteomic studies that are already leading to identification of novel targets for therapeutics, for vaccine candidates, as well as biomarkers for detection and diagnosis.

Respiratory anthrax is a fatal disease in the absence of early therapy with antibiotics. Guinea pigs are highly sensitive to infection with Bacillus anthracis spores by intranasal spore instillation succumbing within 2 to 4 days post infection. Post exposure prophylaxis that initiated 24 h post infection with tetracycline, doxycycline, ciprofloxacin, ofloxacin, erythromycin, gentamicin or imipenem protected the animals, but after termination of treatment the animals developed a fatal anthrax disease. Combined treatment with antibiotics and active immunization with PA based vaccine conferred an efficient immune response that prevented the development of a fatal anthrax disease after termination of antibiotic administration. The CDC recommended in case of a major bioterrorist attack a mass distribution of antibiotics within 48 h. As the onset of symptoms in humans were reported to start on day 1–7 post exposure, this delay in initiation of treatment may result in treatment of sick populations. We evaluated the efficacy of late antibiotic administration starting from 30 h post infections and every 6 h thereafter, to cure respiratory anthrax sick animals. Administration of ciprofloxacin provided efficient protection up to 44 h post infection, at which time 60% of the sick animals survived. Administration of doxycycline cured up to 48 h post infection, when up to 75% of the sick animals survived. The antibiotic administration protected most animals with bacteremia level up to 105 CFU/ml, but failed to protect animals exhibiting higher levels of bacteremia. In conclusion these results demonstrate the efficacy of antibiotic treatment to cure animals even after the onset of the disease.

Classification and differentiation of Bacillus anthracis isolates by genetic markers play an important role in the study of anthrax epidemiology. We have applied a PCR based method – Random Amplification of Polymorphic DNA (RAPD) to identify twenty-five B. anthracis genetic markers. These markers allowed for classification of the studied strains into five different groups. Three selected RAPD markers were cloned and sequenced. Typical integration of the three markers allowed for specific definition of the five RAPD derived genotypes. To test the universal power of these markers to discriminate between diverse B. anthracis strains, the nucleotide sequence of each marker was searched against all available B. anthracis genome sequences (both finished and unfinished). The three markers system could differentiate between strains belonging to the genetic groups Aβ, A1a, A1b, A4 and B1 (as defined by Keim et al., 2000; Maho et al., 2006) and gave rise to a unique combination for group A3, but couldn’t distinguish between the sub groups A3a and A3b. In addition, this system could not distinguish between groups B2 and C (identical three marker combination). In an attempt to improve the resolution of this system we introduced a fourth marker. In silico analysis revealed that the resulting four markers system could now differentiate between group B2 and C, adding a second genotype to groups A1a, A3a and A4. This four marker system could potentially provide an accurate, simple, and inexpensive agarose-based system for classification B. anthracis strains in laboratories involved in research of this bacterium.

The involvement of TLR2 receptor in the innate response to infection with Bacillus anthracis was investigated. We studied the response to virulent or attenuated Vollum strains in either in vitro assays using macrophage cultures, or in an in vivo model comparing the sensitivity of Syrian hamster cells (expressing normal TLR2) to Chinese hamster cells (lacking functional TLR2) to infection by the various B. anthracis strains. Phagocytosis experiments with murine cell cultures or primary macrophages from both hamster strains, using virulent or attenuated Tox+Cap-, Tox-Cap+or Tox-Cap-spores indicated that the secretion of TNF-α was induced by all the bacterial spores and purified spore antigens. In contrast, capsular antigens induce secretion of TNF-α only by Syrian hamster macrophages indicating the involvement of a functional TLR2 in macrophage activation. Challenge experiments with both hamster strains by intranasal spore inoculation, indicated that, while both strains are equally sensitive to infection with the virulent strain, the Chinese hamster demonstrated a higher sensitivity to infection with the toxinogenic or encapsulated strains. In conclusion, our findings imply that TLR2 has an important role in the attempt of the innate immunity to control B. anthracis infection, although TNF-α secretion was found to be mediated by both TLR2-dependent and TLR2-independent pathways.

Bacillus anthracis proteins that possess antigenic properties and are able to evoke an immune response were identified by a reductive genomic-serologic screen of a set of in silico-preselected open reading frames (ORFs). The screen included in vitro expression of the selected ORFs by coupled transcription and translation of linear PCR-generated DNA fragments, followed by immunoprecipitation with antisera from B. anthracis- infected animals. Of the 197 selected ORFs, 161 were chromosomal and 36 were on plasmids pXO1 and pXO2, and 138 of the 197 ORFs had putative functional annotations (known ORFs) and 59 had no assigned functions (unknown ORFs). A total of 129 of the known ORFs (93%) could be expressed, whereas only 38 (64%) of the unknown ORFs were successfully expressed. All 167 expressed polypeptides were subjected to immunoprecipitation with the anti- B. anthracis antisera, which revealed 52 seroreactive immunogens, only 1 of which was encoded by an unknown ORF. The high percentage of seroreactive ORFs among the functionally annotated ORFs (37%; 51/129) attests to the predictive value of the bioinformatic strategy used for vaccine candidate selection. Furthermore, the experimental findings suggest that surface-anchored proteins and adhesins or transporters, such as cell wall hydrolases, proteins involved in iron acquisition, and amino acid and oligopeptide transporters, have great potential to be immunogenic. Most of the seroreactive ORFs that were tested as DNA vaccines indeed appeared to induce a humoral response in mice. We list more than 30 novel B. anthracis immunoreactive virulence-related proteins which could be useful in diagnosis, pathogenesis studies, and future anthrax vaccine development.

Here we describe the characterization of a lipoprotein previously proposed as a potential Bacillus anthracis virulence determinant and vaccine candidate. This protein, designated MntA, is the solute-binding component of a manganese ion ATP-binding cassette transporter. Coupled proteomic-serological screen of a fully virulent wild-type B. anthracis Vollum strain, confirmed that MntA is expressed both in vitro and during infection. Expression of MntA is shown to be independent of the virulence plasmids pXO1 and pXO2. An mntA deletion, generated by allelic replacement, results in complete loss of MntA expression and its phenotypic analysis revealed: (i) impaired growth in rich media, alleviated by manganese supplementation; (ii) increased sensitivity to oxidative stress; and (iii) delayed release from cultured macrophages. The DeltamntA mutant expresses the anthrax-associated classical virulence factors, lethal toxin and capsule, in vitro as well as in vivo, and yet the mutation resulted in severe attenuation; a 10(4)-fold drop in LD(50) in a guinea pig model. MntA expressed in trans allowed to restore, almost completely, the virulence of the DeltamntA B. anthracis strain. We propose that MntA is a novel B. anthracis virulence determinant essential for the development of anthrax disease, and that B. anthracisDeltamntA strains have the potential to serve as platform for future live attenuated vaccines.

An attenuated nontoxinogenic nonencapsulated Bacillus anthracis spore vaccine expressing high levels of recombinant mutant protective antigen (PA), which upon subcutaneous immunization provided protection against a lethal B. anthracis challenge, was found to have the potential to serve also as an oral vaccine. Guinea pigs immunized per os with the recombinant spore vaccine were primed to B. anthracis vegetative antigens as well as to PA, yet only a fraction of the animals (30% to 50%) mounted a humoral response to all of these antigens. Protective immunity provided by per os immunization correlated with a threshold level of PA neutralizing antibody titers and was long-lasting. Protection conferred by per os immunization was attained when the vaccine was administered in the sporogenic form, which, unlike the vegetative cells, survived passage through the gastrointestinal tract. A comparison of immunization of unirradiated spores with immunization of γ-irradiated spores demonstrated that germination and de novo synthesis of PA were prerequisites for mounting an immune protective response. Oral immunization of guinea pigs with attenuated B. anthracis spores resulted in a characteristic anti-PA immunoglobulin isotype profile (immunoglobulin [G1 IgG1] versus IgG2), as well as induction of specific anti-PA secretory IgA, indicating development of mucosal immunity.

The efficacy of passive immunization as a postexposure prophylactic measure for treatment of guinea pigs intranasally infected with Bacillus anthracis spores was evaluated. Antisera directed either against the lethal toxin components (PA or LF) or against a toxinogenic strain (Sterne) were used for this evaluation. All antisera exhibited high enzyme-linked immunosorbent assay titers against the corresponding antigens, high titers of neutralization of cytotoxicity activity in an in vitro mouse macrophages cell line (J774A.1), as well as in vivo neutralization of toxicity when administered either directly to Fisher rats prior to challenge with the lethal toxin or after incubation with the lethal toxin. In these tests, anti-LF antiserum exhibited the highest neutralization efficiency, followed by anti-Sterne and anti-PA. The time dependence and antibody dose necessary for conferring postexposure protection by the various antibodies of guinea pigs infected with 25 50% lethal doses of Vollum spores was examined. Rabbit anti-PA serum was found to be the most effective. Intraperitoneal injections of anti-PA serum given 24 h postinfection protected 90% of the infected animals, whereas anti-Sterne and anti-LF were less effective. These results further emphasizes the importance of anti-PA antibodies in conferring protection against B. anthracis infection and demonstrated the ability of such antibodies to be effectively applied as an efficient postexposure treatment against anthrax disease.

Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found ( r 2 = 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD 50 ) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.

Adhesion of Candida albicans to host cells is believed to represent a fungal virulence factor and a significant step in the development of candidiasis. As C. albicans strains may differ in their in vitro adhesion ability we initiated a study to investigate whether mutant strains differ in this respect from their parent wild-type. We assessed the in vitro adhesion of C. albicans CBS562 and two mutants obtained by mutagenesis with N′-nitrosoguanidine: a histidine auxotroph, SAG5, derived from CBS562, and a respiratory-deficient strain (a petite mutant), SAR1, derived from SAG5. The adhesion was tested in vitro using two target cell systems: (1) exfoliated human buccal epithelial cells (BEC); and (2) human keratinocyte tissue line cells (HaCaT cells). Adhesion to BEC was evaluated microscopically and that to HaCaT cells by a direct ELISA technique. The results indicated a 54% reduction in adhesion to BEC for SAG5 and 30% for SAR1 as compared to the wild-type, and a 25% reduction in adhesion to HaCaT cells for SAG5 and 20% for SAR1. To verify whether the prototrophy restores the adhesion ability, we complemented the his-negative auxotroph by transforming the strain with the HIS4 gene. Then we assayed the adhesion to BEC of the complemented his-negative mutant in comparison to that of the wild-type, the his-negative mutant (SAG5) and the plasmid-cured transformant. The adhesion values of the complemented his-negative strain were similar to those of the wild-type, whereas the values of the plasmid-cured strain were similar to those of SAG5.

Data from our previous studies suggested that the fungal cell wall component, chitin, is involved in the adhesion of Candida albicans to mucosal surfaces. In the present study, we investigated the effect of polyoxin D, an inhibitor of chitin synthase, on the interaction of the fungus with epithelial cells. The effect of polyoxin D on Candida was evaluated in in vitro assays for its capacity to adhere to buccal epithelial cells (BEC), and by fluorescent-microscopy photometry and flow cytometry using cells stained with cellufluor (CF), a fluorochrome with affinity for chitin. C. albicans grown with and without polyoxin D was stained with CF and examined in a fluorescent microscope equipped with a photometer. Measurements of fluorescence revealed a wide range of intensity among C. albicans cells and a decreased intensity in polyoxin D treated cultures. Flow cytometry analyses of yeasts revealed 2 peaks of fluorescence intensity, and pointed to differences between polyoxin D treated and non-treated microorganisms. C. albicans stained with CF were separated into 2 subpopulations by flow cytometry according to fluorescence intensity. In vitro adhesion of each subpopulation to BEC was similar. Polyoxin D treated fungi showed significantly reduced adherence to BEC, as evaluated by a radioactivity assay with radiolabelled yeasts and by microscopic readings. The reduction in adhesion was Polyoxin D concentration dependent. These observations support our previous findings suggesting involvement of chitin in the attachment process of C. albicans (CBS562) to epithelial cells.

The clinical importance of C. albicans requires the development of a genetic system in this organism for better understanding of its mechanism(s) of pathogenesis and to eventually develop improved therapies for candidal infection. Our previous studies focused on the attachment of the fungus to host mammalian tissues1–3. Those studies involved a specific C. albicans isolate-CBS562. We considered it important to develop a genetic system in this strain. Consequently, we describe herewith the isolation of an histidine auxotroph (his4), and isolation, characterization and chromosomal location of the homologous HIS4 gene.

Genetic studies were done with Candida albicans CBS 562. Various auxotrophs were isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. SAG5 (his4C), a stable histidine auxotroph defective in histidinol dehydrogenase activity, was characterized and chosen for further molecular studies. Therefore, the C. albicans HIS4 gene was isolated. The gene was obtained from a genomic library of the wild-type strain, which was constructed in plasmid YEp24. The HIS4 gene was isolated by transformation of a Saccharomyces cerevisiae strain that carried a his4 mutation. The isolated C. albicans HIS4 gene complemented S. cerevisiae his4A, his4B, his4C, and his4ABC mutant strains, which indicates that the clone contains the entire HIS4 gene. The gene was isolated on plasmid pSTC7, whose physical map was constructed with BamHI, SalI, and EcoRV restriction endonucleases, locating the HIS4 gene on a 14-kilobase-pair DNA fragment. Hybridization experiments with HIS4 and C. albicans genomic DNA showed correspondence between the restriction patterns of the gene with that of the chromosomal DNA, indicating that the gene originates from C. albicans and appears in a single copy. Chromosomes of C. albicans CBS562 and four other strains were resolved by orthogonal-field alteration gel electrophoresis. The electrokaryotyping results showed heterogeneity in chromosomal sizes. The electrokaryotyping of CBS 562 showed a resolution of six chromosomal bands, three of which seemed to be doublets. The C. albicans HIS4 gene was located on the largest resolvable chromosome in all of the strains.