Raphael Ber

Generation of Yersinia pestis Attenuated Strains by Signature-Tagged Mutagenesis in Search of Novel Vaccine Candidates

ABSTRACT In a search for novel attenuated vaccine candidates for use against Yersinia pestis, the causative agent of plague, a signature-tagged mutagenesis strategy was used and optimized for a subcutaneously infected mouse model. A library of tagged mutants of the virulent Y. pestis Kimberley53 strain was generated. Screening of 300 mutants through two consecutive cycles resulted in selection of 16 mutant strains that were undetectable in spleens 48 h postinfection. Each of these mutants was evaluated in vivo by assays for competition against the wild-type strain and for virulence following inoculation of 100 CFU (equivalent to 100 50% lethal doses [LD50] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10−5 to 10−7 to virulent mutants exhibiting a delay in the mean time to death or mutants indistinguishable from the wild type in the two assays. Characterization of the phenotypes and genotypes of the selected mutants led to identification of virulence-associated genes coding for factors involved in global bacterial physiology (e.g., purH, purK, dnaE, and greA) or for hypothetical polypeptides, as well as for the virulence regulator gene lcrF. One of the avirulent mutant strains (LD50, >107 CFU) was found to be disrupted in the pcm locus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcm mutant was superior to the EV76 live vaccine strain because it induced 10- to 100-fold-higher antibody titers to the protective V and F1 antigens and because it conferred efficacious protective immunity.

Interaction of Yersinia pestis with Macrophages: Limitations in YopJ-Dependent Apoptosis

ABSTRACT The enteropathogenic Yersinia strains are known to downregulate signaling pathways in macrophages by effectors of the type III secretion system, in which YopJ/YopP plays a crucial role. The adverse effects of Yersinia pestis, the causative agent of plague, were examined by infecting J774A.1 cells, RAW264.7 cells, and primary murine macrophages with the EV76 strain and with the fully virulent Kimberley53 strain. Y. pestis exerts YopJ-dependent suppression of tumor necrosis factor alpha secretion and phosphorylation of mitogen-activated protein kinases and thus resembles enteropathogenic Yersinia. However, Y. pestis is less able to activate caspases, to suppress NF-κB activation, and to induce apoptosis in macrophages than the high-virulence Y. enterocolitica WA O:8 strain. These differences appear to be related to lower efficiency of YopJ effector translocation by Y. pestis. The efficiencies of effector translocation and of apoptosis induction can be enhanced either by using a high bacterial load in a synchronized infection or by overexpressing exogenous YopJ in Y. pestis. Replacing YopJ with the homologous Y. enterocolitica effector YopP can further enhance these effects. Overexpression of YopP in a yopJ-deleted Y. pestis background leads to rapid and effective translocation into target cells, providing Y. pestis with the high cytotoxic potential of Y. enterocolitica WA O:8. We suggest that the relative inferiority of Y. pestis in triggering cell death in macrophages may be advantageous for its in vivo propagation in the early stages of infection.

Effective Protective Immunity to Yersinia pestis Infection Conferred by DNA Vaccine Coding for Derivatives of the F1 Capsular Antigen

ABSTRACT Three plasmids expressing derivatives of the Yersinia pestis capsular F1 antigen were evaluated for their potential as DNA vaccines. These included plasmids expressing the full-length F1, F1 devoid of its putative signal peptide (deF1), and F1 fused to the signal-bearing E3 polypeptide of Semliki Forest virus (E3/F1). Expression of these derivatives in transfected HEK293 cells revealed that deF1 is expressed in the cytosol, E3/F1 is targeted to the secretory cisternae, and the nonmodified F1 is rapidly eliminated from the cell. Intramuscular vaccination of mice with these plasmids revealed that the vector expressing deF1 was the most effective in eliciting anti-F1 antibodies. This response was not limited to specific mouse strains or to the mode of DNA administration, though gene gun-mediated vaccination was by far more effective than intramuscular needle injection. Vaccination of mice with deF1 DNA conferred protection against subcutaneous infection with the virulent Y. pestis Kimberley53 strain, even at challenge amounts as high as 4,000 50% lethal doses. Antibodies appear to play a major role in mediating this protection, as demonstrated by passive transfer of anti-deF1 DNA antiserum. Taken together, these observations indicate that a tailored genetic vaccine based on a bacterial protein can be used to confer protection against plague in mice without resorting to regimens involving the use of purified proteins.

Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis

ABSTRACT Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.

Enrichment of Yersinia pestis from Blood Cultures Enables Rapid Antimicrobial Susceptibility Determination by Flow Cytometry

Mortality from plague is high if not treated with the proper antibiotics within 18-24 hours after onset of symptoms. The process of antibiotic susceptibility determination of Yersinia pestis isolated from blood samples may extend from 4 to more than 7 days, since the in vitro growth is very slow. To accelerate this process, we developed an enrichment protocol as well as a non-standard yet reliable method for rapid antibiotic susceptibility analysis of Y. pestis from blood cultures using flow cytometry technology. This rapid method is applicable to blood cultures containing low levels of Y. pestis.

Consequences of Delayed Ciprofloxacin and Doxycycline Treatment Regimens against Francisella tularensis Airway Infection

ABSTRACT This study examines the efficacy, bacterial load, and humoral response of extensively delayed ciprofloxacin or doxycycline treatments following airway exposure of mice to Francisella tularensis subsp. holarctica (strain LVS) or to the highly virulent F. tularensis subsp. tularensis (strain SchuS4). A delay in onset of both antibiotic treatments allowed the rescue of all LVS-infected animals. However, for animals infected with SchuS4, only ciprofloxacin was efficacious and prolongation of treatment rescued all animals.

Vaccination with plasmid DNA expressing the Yersinia pestis capsular protein F1 protects mice against plague.

The fraction 1 capsular protein (F1) is considered an important but not essential virulence factor unique to Y. pestis (Welkos et al., 1995). Immunization with the F1 protein has been shown to protect mice against subcutaneous challenge with wild type Y. pestis (Andrews et al., 1996) and a combined formulation containing F1 and V antigen confers protection against airborne infection (Williamson et al., 1997). The protein has been associated with eliciting protective immune response in humans as well. The observation that genetic immunization is able to elicit a protective immunity has fostered a new generation in vaccine development. The caf1 gene, which codes for the F1 protein, was previously used as DNA vaccine. In this study inbred mice were found to be non responsive, and outbred mice responded by a weak anamnestic response (Brandler et al., 1998). The advent in genetic vaccination and the accumulating information on factors modulating the extent of response to DNA vaccines led us to re-examine genetic vaccination based on F1 antigen. Here we compare three F1 DNA derivatives carrying different signals for cellular localization and demonstrate that one such genetic derivative, which presumably targets expression to the cytosol induces an effective antibody response and confers protection against high doses of infective Y. pestis.

A rapid real-time quantitative PCR assay to determine the minimal inhibitory extracellular concentration of antibiotics against an intracellular Francisella tularensis Live Vaccine Strain

Francisella tularensis is a highly virulent facultative intracellular bacterium. The lack of a safe and efficient vaccine makes antibiotics the preferred treatment. F. tularensis antibiotic susceptibility tests are based on the in vitro standard CLSI-approved microdilution method for determining the MIC. However, limited data are available regarding the minimal inhibitory extracellular concentration (MIEC) needed to eradicate intracellular bacteria. Here, we evaluated the MIEC values of various WHO-recommended antibiotics and compared the MIEC values to the established MICs. We describe a rapid 3-h quantitative PCR (qPCR) intracellular antibiogram assay, which yields comparable MIEC values to those obtained by the classical 72-h cfu assay. This rapid qPCR assay is highly advantageous in light of the slow growth rates of F. tularensis. Our results showed that the MIECs obtained for doxycycline, chloramphenicol and ciprofloxacin were indicative of intracellular activity. Gentamicin was not effective against intracellular bacteria for at least 32 h post treatment, raising the question of whether slow-penetrating gentamicin should be used for certain stages of the disease. We suggest that the qPCR intracellular antibiogram assay may be used to screen for potentially active antibiotics against intracellular F. tularensis as well as to detect strains with acquired resistance to recommended antibiotics.

A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes

Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC) values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test.

Isolation of Francisella tularensis and Yersinia pestis from Blood Cultures by Plasma Purification and Immunomagnetic Separation Accelerates Antibiotic Susceptibility Determination

The early symptoms of tularemia and plague, which are caused by Francisella tularensis and Yersinia pestis infection, respectively, are common to other illnesses, resulting in a low index of suspicion among clinicians. Moreover, because these diseases can be treated only with antibiotics, rapid isolation of the bacteria and antibiotic susceptibility testing (AST) are preferable. Blood cultures of patients may serve as a source for bacteria isolation. However, due to the slow growth rates of F. tularensis and Y. pestis on solid media, isolation by plating blood culture samples on proper agar plates may require several days. Thus, improving the isolation procedure prior to antibiotic susceptibility determination is a major clinically relevant need. In this study, we developed a rapid, selective procedure for the isolation of F. tularensis and Y. pestis from blood cultures. We examined drop-plating and plasma purification followed by immunomagnetic separation (IMS) as alternative isolation methods. We determined that replacing the classical isolation method with drop-plating is advantageous with respect to time at the expense of specificity. Hence, we also examined isolation by IMS. Sub-localization of F. tularensis within blood cultures of infected mice has revealed that the majority of the bacteria are located within the extracellular fraction, in the plasma. Y. pestis also resides within the plasma. Therefore, the plasma fraction was isolated from blood cultures and subjected to an IMS procedure using polyclonal anti-F. tularensis live vaccine strain (LVS) or anti-Y. pestis antibodies conjugated to 50-nm nano-beads. The time required to reach an inoculum of sufficient bacteria for AST was shortest when using the plasma and IMSs for both bacteria, saving up to 2 days of incubation for F. tularensis and 1 day for Y. pestis. Our isolation procedure provides a proof of concept for the clinical relevance of rapid isolation for AST from F. tularensis- and Y. pestis-infected patients.