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We have previously shown that the cell morphogenesis NlpD lipoprotein is essential for virulence of the plague bacteria, Yersinia pestis. To elucidate the role of NlpD in Y. pestis pathogenicity, we conducted a whole-genome comparative transcriptome analysis of the wild-type Y. pestis strain and an nlpD mutant under conditions mimicking early stages of infection. The analysis suggested that NlpD is involved in three phenomena: (i) Envelope stability/integrity evidenced by compensatory up-regulation of the Cpx and Psp membrane stress-response systems in the mutant; (ii) iron acquisition, supported by modulation of iron metabolism genes and by limited growth in iron-deprived medium; (iii) activity of the twin-arginine (Tat) system, which translocates folded proteins across the cytoplasmic membrane. Virulence studies of Y. pestis strains mutated in individual Tat components clearly indicated that the Tat system is central in Y. pestis pathogenicity and substantiated the assumption that NlpD essentiality in iron utilization involves the activity of the Tat system. This study reveals a new role for NlpD in Tat system activity and iron assimilation suggesting a modality by which this lipoprotein is involved in Y. pestis pathogenesis., Author summary We have previously shown that the NlpD lipoprotein, which is involved in the regulation of cell morphogenesis, is essential for virulence of the plague bacteria, Yersinia pestis. To uncover the role of NlpD in Y. pestis pathogenicity, we conducted a whole-genome comparative transcriptome analysis as well as phenotypic and virulence evaluation analyses of the nlpD and related mutants. The study reveals a new role for the Y. pestis NlpD lipoprotein in iron assimilation and Tat system activity.

BACKGROUND Plague is initiated by Yersinia pestis, a highly virulent bacterial pathogen. In late stages of the infection, bacteria proliferate extensively in the internal organs despite the massive infiltration of neutrophils. The ineffective inflammatory response associated with tissue damage may contribute to the low efficacy of antiplague therapies during late stages of the infection. In the present study, we address the possibility of improving therapeutic efficacy by combining corticosteroid administration with antibody therapy in the mouse model of bubonic plague. METHODS Mice were subcutaneously infected with a fully virulent Y. pestis strain and treated at progressive stages of the disease with anti-Y. pestis antibodies alone or in combination with the corticosteroid methylprednisolone. RESULTS The addition of methylprednisolone to antibody therapy correlated with improved mouse survival, a significant decrease in the amount of neutrophils and matrix metalloproteinase 9 in the tissues, and the mitigation of tissue damage. Interestingly, the combined treatment led to a decrease in the bacterial loads in infected organs. CONCLUSIONS Corticosteroids induce an unexpectedly effective antibacterial response apart from their antiinflammatory properties, thereby improving treatment efficacy.

Markers of the early stages of plague, a rapidly progressing deadly disease, are crucial for enabling the onset of an effective treatment. Here, we show that V-antigen protein (LcrV) is accumulated in the serum of Yersinia pestis-infected mice before bacterial colonization of the spleen and dissemination to blood, in a model of bubonic plague. LcrV accumulation is detected earlier than that of F1 capsular antigen, an established marker of disease. In a mouse model of pneumonic plague, LcrV can be determined in the bronchoalveolar lavage fluid somewhat later than F1, but before dissemination of Y. pestis to the blood. Thus, determination of soluble LcrV is suggested as a potential useful tool for monitoring disease progression in both bubonic and pneumonic plague. Moreover, it may be of particular advantage in cases of infections with F1 nonproducing strains.

The enteropathogenicYersiniastrains 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 ofYersinia 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. pestisexerts YopJ-dependent suppression of tumor necrosis factor alpha secretion and phosphorylation of mitogen-activated protein kinases and thus resembles enteropathogenicYersinia. However,Y. pestisis less able to activate caspases, to suppress NF-κB activation, and to induce apoptosis in macrophages than the high-virulenceY. enterocoliticaWA O:8 strain. These differences appear to be related to lower efficiency of YopJ effector translocation byY. 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 inY. pestis. Replacing YopJ with the homologousY. enterocoliticaeffector YopP can further enhance these effects. Overexpression of YopP in ayopJ-deletedY. pestisbackground leads to rapid and effective translocation into target cells, providingY. pestiswith the high cytotoxic potential ofY. enterocoliticaWA O:8. We suggest that the relative inferiority ofY. pestisin triggering cell death in macrophages may be advantageous for its in vivo propagation in the early stages of infection.

Bacterial infection of the lungs triggers a swift innate immune response that involves the production of cytokines and chemokines that promote recruitment of immune cells from the bone marrow (BM) into the infected tissue and limit the ability of the pathogen to replicate. Recent in vivo studies of pneumonic plague in animal models indicate that the pulmonary pro-inflammatory response to airway infection with Yersinia pestis is substantially delayed in comparison to other pathogens. Consequently, uncontrolled proliferation of the pathogen in the lungs is observed, followed by dissemination to internal organs and death. While the lack of an adequate early immune response in the lung is well described, the response of BM-derived cells is poorly understood. In this study, we show that intranasal infection (i.n.) of mice with a fully virulent Y. pestis strain is sensed early by the BM compartment, resulting in a reduction in CXCR4 levels on BM neutrophils and their subsequent release into the blood 12 hours post infection. In addition, increased levels of BM-derived hematopoietic stem and progenitor cells (HSPC) were detected in the blood early after infection. Mobilization of both immature and mature cells was accompanied by the reduction of BM SDF-1 (CXCL-12) levels and the reciprocal elevation of SDF-1 in the blood 24 hours post infection. RT-PCR analysis of RNA collected from total BM cells revealed an early induction of myeloid-associated genes, suggesting a prompt commitment to myeloid lineage differentiation. These findings indicate that lung infection by Y. pestis is sensed by BM cells early after infection, although bacterial colonization of the BM occurs at late disease stages, and point on a potential cross-talk between the lung and the BM at early stages of pneumonic plague.

The interrelationship between signal-mediated endoplasmic reticulum retention and control of subunit assembly in secreted complex proteins was examined in recombinant 293 cells expressing human acetylcholinesterase (HuAChE). This was achieved by analyzing the mutual effects of co-residing retention and dimerization signals on enzyme secretion by transfected cells. The function of putative signals within the COOH-terminal tetrapeptide CSDL of HuAChE was examined by site-directed mutagenesis. The CSDL tetrapeptide carries the free cysteine (Cys-580) involved in subunit assembly, yet it fails to function as a KDEL-type retention signal. This was demonstrated by mutations that increase similarity to the canonical retention signal (substitution of CSDL by KSDL) or those that deviate from it (substitution to CSAL). Cells expressing both types of mutants exhibited cell-associated HuAChE levels identical to that of wild type enzyme. Appendage of an engineered KDEL retention signal to a dimerization-impaired HuA-ChE subunit (the C580A mutant) resulted in intracellular retention of large amounts of fully active enzyme not prone to proteolytic degradation. On the other hand, attachment of KDEL to a native, dimerization-competent HuAChE polypeptide did not lead to intracellular retention and allowed efficient secretion of enzyme to the cell growth medium. Yet, appendage of KDEL to the native HuAChE led to some retardation in the transport of enzyme molecules through the Golgi apparatus, as manifested by increase in cellular population of endo H-resistant dimers, when compared with wild type enzyme. Taken together, these results indicate (alpha) that sub-unit dimerization mediated by the COOH-terminal cysteine of HuAChE can reverse the signal-mediated retention by masking recognition of KDEL by its cognate receptor and (b) that the native sequences of the acetylcholinesterase subunit polypeptide do not appear to function as a coupled retention/dimerization signal in the control of secretion of assembled enzyme molecules.

Plague, which is initiated by Yersinia pestis infection, is a fatal disease that progresses rapidly and leads to high mortality rates if not treated. Antibiotics are an effective plague therapy, but antibiotic-resistant Y. pestis strains have been reported and therefore alternative countermeasures are needed. In the present study, we assessed the potential of an F1 plus LcrV-based vaccine to provide protection shortly pre- or post-exposure to a lethal Y. pestis infection. Mice vaccinated up to one day before or even several hours after subcutaneous challenge were effectively protected. Mice immunized one or three days pre-challenge were protected even though their anti-F1 and anti-LcrV titers were below detection levels at the day of challenge. Moreover, using B-cell deficient μMT mice, we found that rapidly induced protective immunity requires the integrity of the humoral immune system. Analysis of the individual contributions of vaccine components to protection revealed that rF1 is responsible for the observed rapid antibody-mediated immunity. Applying anti-F1 passive therapy in the mouse model of bubonic plague demonstrated that anti-F1 F(ab')(2) can delay mortality, but it cannot provide long-lasting protection, as do intact anti-F1 molecules. Fc-dependent immune components, such as the complement system and (to a lesser extent) neutrophils, were found to contribute to mouse survival. Interestingly, T cells but not B cells were found to be essential for the recovery of infected animals following passive anti-F1 mediated therapy. These data extend our understanding of the immune mechanisms required for the development of a rapid and effective post-exposure therapy against plague.

The role of N-glycosylation in the function of human acetylcholinesterase (HuAChE) was examined by site-directed mutagenesis (Asn to Gln substitution) of the three potential N-glycosylation sites Asn-265, Asn-350 and Asn-464. Analysis of HuAChE mutants, defective in a single or multiple N-glycosylation sites, by expression in transiently or stably transfected human embryonal 293 kidney cells suggests the following. (a) All three AChE glycosylation signals are utilized, but not all the secreted molecules are fully glycosylated. (b) Glycosylation at all sites is important for effective biosynthesis and secretion; extracellular AChE levels in mutants defective in one, two or all three sites amounted to 20-30%, 2-4% and about 0.5% of wild-type level respectively. (c) Some glycosylation mutants display impaired stability, as reflected by increased susceptibility to heat inactivation; substitution of Asn-464 has the most pronounced effect on thermostability. (d) Abrogation of N-glycosylation has no detectable effect on the enzyme activity of HuAChE; all glycosylation mutants, including the triple mutant, hydrolyse acetylthiocholine efficiently, displaying Km, kcat. and kcat./Km values similar to those of the wild-type enzyme. (e) In most mutants, inhibition profiles with edrophonium and bisquaternary ammonium ligands are identical with those of wild-type enzyme; the Asn-350 mutants, however, exhibit a slight decrease in their affinity towards these ligands. (f) Elimination of oligosaccharide side chains has no detectable effect on the surface-related ‘peripheral-site’ functions; like the wild-type enzyme, all mutants were inhibited by propidium and by increased concentrations of acetylthiocholine.

The well-characterized virulence factors of Yersinia pestis the causative agent of plague are those encoded by the virulence plasmids. Previously we have isolated an attenuated Y. pestis transposon insertion mutant in which the chromosomal pcm gene was disrupted. The pcm gene is located within a putative stress response locus that includes the surE, nlpD, and rpoS genes. In this study, we investigated the expression and the role of pcm locus genes in Y. pestis pathogenesis by constructing a set of isogenic surE, pcm, nlpD and rpoS mutants in the fully virulent Kimberley53 strain. We show that the NlpD lipoprotein is the only factor encoded from the pcm locus that is essential for Y. pestis virulence. A chromosomal deletion of the nlpD gene sequence resulted in a drastic reduction in virulence to an LD50 of at least 107 cfu for subcutaneous and airway routes of infection. The mutant was unable to colonize mouse organs following infection. The unsegmented morphology of the nlpD mutant indicates that NlpD is involved in cell separation; however, deletion of nlpD did not affect in vitro growth rate. Trans-complementation experiments with the Y. pestis nlpD gene restored virulence and all other phenotypic defects. Finally, we demonstrate that the nlpD mutant could be used as a very potent live vaccine against bubonic and pneumonic plague.

A bioinformatic screen of Yersinia pestis genome identified 11 TPS components, carrying the POlypeptide TRansport-Associated (POTRA ) domain, found in other pathogens to be involved in immunogenicity and pathogenicity. All rTpsBs (transporters ) cloned and expressed in Escherichia coli induced a humoral responses in immunized mice. Mice surviving intranasal infection with fully virulent Y. pestis Kimberley53 strain generated sera cross reacting with some recombinants TpsBs. Moreover, at least 3 TpsB polypeptides were directly detected in bacteria isolated following intranasal Y. pestis infection. All in vivo produced TpsBs appear to be truncated, missing about 75–85 amino acids. MALDI-TOF-MS analysis permitted to identify the truncation at a region overlapping a domain of a closely related Bordetella pertussis TpsB (FhaC ), recently proposed to undergo a major conformational change that allows effector translocation.

Modulation of host cell death during infection is a prevalent virulence strategy developed by many bacterial pathogens. Yersinia pestis, the causative agent of the fatal plague disease was found to exert limited cytotoxicity towards target immune cells, mediated by type III secretion system effector YopJ. In contrast, the highly cytotoxic closely related Y. enterocolitica O:8 causes a self limited gastrointestinal disease. This phenomenon led us to suggest that the reduced cytotoxic potency of Y. pestis is related to its increased virulence potential. Generation of Y. pestis strain expressing YopP instead of YopJ, enhanced its cytotoxic potency towards macrophages in-vitro and mouse spleen target cells in-vivo. The highly cytotoxic Y. pestis strain demonstrated a reduced ability to colonize internal organs of mice infected subcutaneously, and most strikingly was avirulent in a mouse model of bubonic plague. These results indicate inverse relationship between cytotoxic potency and in-vivo virulence. Still, the same YopP-expressing Y. pestis strain remained fully virulent to mice upon intravenous or intranasal infections, indicating retention of virulence potential. In addition, it was found that subcutaneous administration of the highly cytotoxic Y. pestis strain activated extremely rapid, potent and systemic protective response against concomitant challenges with a virulent strain via the subcutaneous, intravenous or airway routes. These findings may have important implications on the design of future plague vaccine/therapies and contribute to our understanding of virulence strategies of Y. pestis in nature.

Plague, initiated by Yersinia pestis infection, is a fatal disease that progresses rapidly and leads to high mortality rates if not treated within a short period of time after onset of symptoms. Antibiotics are effectively used for plague therapy yet antibiotic resistance Y. pestis strains have been reported and therefore alternative therapies are needed. In this study we demonstrate the ability of rabbit polyclonal antibodies directed against Y. pestis F1 and LcrV antigens to confer protection in mouse models of bubonic and pneumonic plague.

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.

Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.

Yersinia pestis is the causative agent of plague. Previously we have isolated an attenuated Y. pestis transposon insertion mutant in which the pcm gene was disrupted. In the present study, we investigated the expression and the role of pcm locus genes in Y. pestis pathogenesis using a set of isogenic surE, pcm, nlpD and rpoS mutants of the fully virulent Kimberley53 strain. We show that in Y. pestis, nlpD expression is controlled from elements residing within the upstream genes surE and pcm. The NlpD lipoprotein is the only factor encoded from the pcm locus that is essential for Y. pestis virulence. A chromosomal deletion of the nlpD gene sequence resulted in a drastic reduction in virulence to an LD(50) of at least 10(7) cfu for subcutaneous and airway routes of infection. The mutant was unable to colonize mouse organs following infection. The filamented morphology of the nlpD mutant indicates that NlpD is involved in cell separation; however, deletion of nlpD did not affect in vitro growth rate. Trans-complementation experiments with the Y. pestis nlpD gene restored virulence and all other phenotypic defects. Finally, we demonstrated that subcutaneous administration of the nlpD mutant could protect animals against bubonic and primary pneumonic plague. Taken together, these results demonstrate that Y. pestis NlpD is a novel virulence factor essential for the development of bubonic and pneumonic plague. Further, the nlpD mutant is superior to the EV76 prototype live vaccine strain in immunogenicity and in conferring effective protective immunity. Thus it could serve as a basis for a very potent live vaccine against bubonic and pneumonic plague.

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica Oratio8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10(7)-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD(50) of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.

Plague is a life-threatening disease caused by Yersinia pestis, for which effective-licensed vaccines and reliable predictors of in vivo immunity are lacking. V antigen (LcrV) is a major Y. pestis virulence factor that mediates translocation of the cytotoxic Yersinia protein effectors (Yops). It is a well-established protective antigen and a part of currently tested plague subunit vaccines. We have developed a highly sensitive in vitro macrophage cytotoxicity neutralization assay which is mediated by anti-LcrV antibodies; and studied the potential use of these neutralizing antibodies as an in vitro correlate of plague immunity in mice. The assay is based on a Y. pestis strain with enhanced cytotoxicity to macrophages in which endogenous yopJ was replaced by the more effectively translocated yopP of Y. enterocolitica O:8. Mice passively immunized with rabbit anti-LcrV IgG or actively immunized with recombinant LcrV were protected against lethal doses of a virulent Y. pestis strain, in a mouse model of bubonic plague. This protection significantly correlated with the in vitro neutralizing activity of the antisera but not with their corresponding ELISA titers. In actively immunized mice, a cutoff value for serum neutralizing activity, above which survival was assured with high degree of confidence, could be established for different vaccination regimes. The impact of overall findings on the potential use of serum neutralizing activity as a correlate of protective immunity is discussed.

Plasmid R6K contains inverted repeats of an approximately 100-base-pair sequence separated by 5.5 kilobases. These long inverted repeats (LIRs) occur within the alpha and beta origins of replication and are essential for origin function. In this study, primer-extension analysis of DNA modified in vivo by dimethyl sulfate or KMnO4 revealed that both alpha and beta LIRs acquire similar structural distortions of the DNA helix in a functional R6K replicon. These distortions were not seen in plasmids containing isolated LIR sequences. In the functional replicon, the dimethyl sulfate and KMnO4 hyperreactive sites appear on complementary strands and are located to one side of an internal palindromic sequence within the LIRs. This asymmetry coincides with the primary direction of DNA replication from alpha and beta origins in vivo. We also observed two intermediate structures when certain R6K cis- or trans-acting elements are missing. Sequences near the alpha origin are required for generation of the dimethyl sulfate hyperreactive sites, whereas sequences near the beta origin are responsible for the appearance of KMnO4 hyperreactive sites. We suggest that these structures represent a hierarchy that leads to a "locked" preinitiation complex, which functions to synchronize and determine the direction of replication from the alpha and beta replication origins in vivo.

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.

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.

YopP in Y. enterocolitica and YopJ in Y. pseudotuberculosis, have been shown to exert a variety of adverse effects on cell signaling leading to suppression of cytokine expression and induction of programmed cell death. A comparative in vitro study with Y. pestis and Y. enterocolitica 0:8 virulent strains shows some critical disparity in YopJ/YopP-related effects on immune cells. Involvement of yopJ in virulence was evaluated in mouse model of bubonic plague.

In a search for novel attenuated vaccine candidates for use againstYersinia 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 virulentY. pestisKimberley53 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 10−5to 10−7to 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, andgreA) or for hypothetical polypeptides, as well as for the virulence regulator genelcrF. One of the avirulent mutant strains (LD50, >107CFU) was found to be disrupted in thepcmlocus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcmmutant 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.

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.

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.

The wide use of pesticides is a threat to the public health. An attractive tool for bioremediation of pesticides pollution is the bacterial pesticides degrading enzyme, phosphotriesterase (OPH), which is able to hydrolyze a wide range of organophosphate compounds. Yet, engineering of a more potent hydrolytic machinery into this enzyme could yield an advantageous bioremediation tool.

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein that activates transcription by catalyzing isomerization of closed complexes between sigma54-holoenzyme and a promoter to open complexes. To catalyze this reaction, NtrC must be phosphorylated and form an appropriate oligomer so that it can hydrolyze ATP. NtrC can also repress transcription by sigma70-holoenzyme. In this paper we characterize "repressor" mutant forms of NtrC from Salmonella typhimurium, forms that have lost the ability to activate transcription by sigma54-holoenzyme (in vitro activity at least 1000-fold lower than wild-type) but retain the ability to repress transcription by sigma70-holoenzyme. The amino acid substitutions in NtrCrepressor proteins that were obtained by classical genetic techniques alter residues in the central domain of the protein, the domain directly responsible for transcriptional activation. Commensurate with this, phosphorylation and the autophosphatase activities of NtrCrepressor proteins, which are functions of the amino-terminal regulatory domain of NtrC, are normal. In addition, these proteins have essentially normal DNA-binding, which is a function of the C-terminal region of NtrC and bind cooperatively to enhancers. (The NtrC(G219K) protein has "improved" DNA-binding, which is discussed.) We previously presented evidence that several NtrCrepressor proteins have impaired ATPase activity. We now show that two other repressor proteins, NtrC(A216V) and NtrC(A220T), have as much ATPase activity as wild-type NtrC when they are phosphorylated and bound to an enhancer and that they have considerably more activity than an unphosphorylated NtrC(constitutive) protein, which is capable of activating transcription. These results demonstrate that NtrC(A216V) and NtrC(A220T) fail in a function of the central domain other than ATPase activity. Although they may fail in contact with sigma54-holoenzyme per se, the fact that alanine is the amino acid normally found at these positions leads us to speculate that these proteins fail in coupling energy to a change in conformation of the polymerase.

Three novel R6K genes which are responsible for expression of DNA distortion polypeptides (DDP) were identified. The DDPs act in vivo in concert to induce similar stepwise DNA helix distortions within two long inverted repeats (alpha LIR and beta LIR), which are essential elements for the two distally located R6K alpha and beta DNA replication origins. DDP1 and DDP2 are encoded by two tandem genes located at the 5' end of alpha LIR, whereas a gene coding for DDP3 is located at the 3' end of beta LIR. DDP1 and DDP2 are required for primary DNA distortion within alpha LIR or beta LIR, while DDP3 is essential for generation of secondary DNA distortion in these LIR sequences. Creation of DNA distortion within alpha LIR depends on its specific interaction with DDP1 and on the presence of the R6K primase DNA-binding site. The possible relevance of these findings to R6K replication is discussed.

Nitrogen regulatory protein C(NtrC) is a bacterial enhancer-binding protein that activates transcription by the σ 54 -holoenzyme. To activate transcription, NtrC must hydrolyze ATP, a reaction that depends upon its being phosphorylated and forming an appropriate oligomer, In this paper we characterize "constitutive" mutant forms of the NtrC protein from Salmonella typhimurium ; unlike wild-type NtrC, these forms are able to hydrolyze ATP and activate transcription in vitro without being phosphorylated. The amino acids altered in NtrC constitutive proteins are located in both the N-terminal regulatory domain and the central domain, which is directly responsible for transcriptional activation. The residues that are altered are not conserved among activators of the σ 54 -holoenzyme, and are not identical even among NtrC proteins from members of different subgroups of the proteobacteria (purple bacteria). NtrC constitutive proteins are phosphorylated normally; phosphorylation increases their ability to hydrolyze ATP and activate transcription. Moreover, the oligomerization of these proteins that occurs when they bind to an enhancer also increases the ATPase activity of both unmodified and phosphorylated forms. Removal of the N-terminal regulatory domain from two NtrC constitutive proteins with amino acid substitutions in the central domain (NtrC S160F and NtrC V288I ) leaves them active, indicating that essential oligomerization determinants lie outside the regulatory domain. This conclusion is confirmed by the observation that the ATPase activity of ΔN-NtrC S160F is greatly stimulated when it binds to an enhancer, and by the ability of this protein to activate transcription synergistically with a form of NtrC incapable of DNA-binding. Together with previous results indicating that oligomerization determinants do not lie in the C-terminal DNA-binding domain of NtrC; these results provide evidence that they lie in the central domain. f2

Acetylcholinesterase (AChE) occurs in multiple molecular forms in different tissues of vertebrates and invertebrates (for recent reviews see Taylor, 1991; Massoulie et al 1993). This heterogeneity is generated through associations of various catalytic subunits (T or H) and structural subunits. The T subunit is involved in formation of several multimenc AChE ectoenzyme configurations: Secreted AChE is usually composed of soluble homooligomers, whereas cell-bound AChEs consist of tetramers attached to cell-associated structural subunits. The biogenesis of these various molecular forms is believed to be a regulated process involving various control mechanisms.

Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.

Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.

Sequence conservation analysis relates the cholinesterases to a superfamily of polypeptides (Myers et al., 1988; Krejci et al., 1991), including enzymes such as microsomal carboxyesterase, cholesterol esterase, lysophospholipase, Geotrichum lipase and Drosophila esterase-6, as well as several noncatalytic polypeptides. AChE is the best characterized enzyme in this superfamily. Kinetic studies have indicated that the active site of AChE consists of two subsites: an anionic subsite to which the trimethylammonium group of acetylcholine binds and an esteratic subsite which interacts with the ester-bond region and mediates catalysis. Evidence also exists for an allosteric regulation of AChE activity by ligand binding to an anionic site(s) physically remote from the active site (Changeux, 1966).

Acetylcholinesterase (abbreviated AChE) occurs in multiple molecular forms in different tissues of vertebrates and invertebrates (reviewed in Massoulie and Bon, 1982; (Silman and Futerman, 1987; Chatonnet and Lockridge, 1989). This heterogeneity is generated through tissue-specific associations of various catalytic and structural subunits. Characterized catalytic subunits are divided into two major types, the T-type and the H-type, both derived from a single gene by alternative splicing Schumacher et al., 1988; Sikarov et al., 1988). Structural subunits include the collagen-like structure (Krejci et al., 1991) that allows attachment to the basal lamina and the 20kD lipid-linked hydrophobic subunit (Inestrosa et al., 1987) which is associated with the mammalian brain enzyme.

Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.

Fermentation Expression of F1 by E. coli MC1060 (pBRK-F1) was studied in a 4-liter computer-controlled fermentor using a rich medium devoid of animal products: soy protein extract, trace elements and glycerol as carbon source (SY). This medium enabled efficient bacterial growth to a high cell density (60 OD600, Fig 1A). No loss of the expression plasmid was observed under these conditions. Unexpectedly, high and constant level of F1 expression was observed throughout the fermentation (up to 24 hours) even at 28°C (no temperature shift to 37°C was required, Fig. 1B). Moreover, at a high cell density (>10 OD600), F1 was released from the encapsulated E. coli cells and accumulated up to 0.9 g/L in the culture medium (Fig. 1C).