Improving live attenuated bacterial carriers for vaccination and therapy

Live attenuated bacteria are well established as vaccines. Thus, their use as carriers for prophylactic and therapeutic macromolecules is a logical consequence. Here we describe several experimental applications of bacteria to carry heterologous macromolecules into the murine host. First, Listeria monocytogenes are described that are able to transfer eukaryotic expression plasmids into host cells for gene therapy. High multiplicities of infection are still required for efficient gene transfer and we point out some of the bottlenecks that counteract a more efficient transfer and application in vivo. Then, we describe Salmonella enterica serovar Typhimurium (S. typhimurium) as an expression plasmid transfer vehicle for oral DNA vaccination of mice. We demonstrate that the stabilization of the plasmid transformants results in an improved immune response. Stabilization was achieved by replacing the origin of replication of the original high-copy-number plasmid by a low-copy-number origin. Finally, we describe Salmonella carriers for the improved expression of heterologous proteins. We introduce a system in which the plasmid is carried as a single copy during cultivation but is amplified several fold upon infection of the host. Using the same in vivo inducible promoter for both protein expression and plasmid amplification, a substantial increase in antigen expression in vivo can be achieved. A modification of this approach is the introduction of inducible gene expression in vivo with a low-molecular-weight compound. Using P_BAD promoter and l-arabinose as inducer we were able to deliberately activate genes in the bacterial carrier. No background activity could be observed with P_BAD such that an inducible suicide gene could be introduced. This is adding an important safety feature to such live attenuated carrier bacteria.     

Control of the expression of the ADE2 gene of the yeast Saccharomyces cerevisiae

The ADE2 gene encodes AIR-carboxylase which catalyzes the sixth step of the purine biosynthetic pathway in Saccharomyces cerevisiae. We have analyzed the effect of deletions in the promoter region of this gene on the expression of the enzyme using a fusion of the ADE2 gene promoter to the bacterial lacZ gene. Adenine added to the growth medium repressed the expression of the fusion at the level of mRNA. The ADE2-lacZ fusion expression can be slightly activated in response to amino-acid starvation, but only in Gcn4 ⁺ strains and in an adenine-supplemented medium. In the absence of adenine in the medium ADE2 gene expression is derepressed, and neither starvation for histidine nor a gcd1 general control regulatory mutation leads to additional derepression. Our experiments indicate that the ADE2 gene of the purine biosynthetic pathway is under both specific adenine control and the general amino-acid control system. The cis-acting promoter elements mediating both modes of regulation overlap each other and are located around the proximal TGACTC sequence.     

RNAi knock-down of the Litopenaeus vannamei Toll gene (LvToll) significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi

In this study, we used real-time PCR to simultaneously monitor the responses of 12 key genes of the shrimp innate immune system in Litopenaeus vannamei after challenge with Vibrio harveyi. In the proPO activating system, we found that proPO was up-regulated (3.3× control at 36 hpi). The hemolymph clotting genes transglutaminase (TGase) and clotting protein were also up-regulated, as were 5 genes in the antimicrobial peptide system (ALF, Crustin, Lyz, PEN2 and PEN4), with only PEN3 showing no significant changes. In the antioxidant defense system, SOD was slightly elevated while GPx was substantially down-regulated. In the pattern recognition receptor system, at 24 hpi, the Toll gene (LvToll) showed the highest relative increase in expression level of all the investigated genes (15× greater than the sterile seawater control). In the second part of this study, when LvToll was knocked down by RNAi silencing, there was no effect on either survival rates or bacterial number in unchallenged shrimp. There was also no difference in mortality rates between control shrimp and LvToll-silenced shrimp when these two groups were challenged with a viral pathogen (white spot syndrome virus; WSSV). However, when LvToll-silenced shrimp were challenged by V. harveyi, there was a significant increase in mortality and bacterial CFU counts. We note that the increase in bacterial CFU count occurred even though treatment with EGFP dsRNA had the opposite effect of reducing the CFU counts. We conclude that LvToll is an important factor in the shrimp innate immune response to acute V. harveyi infection, but not to WSSV.     

Regulation of Wolbachia ankyrin domain encoding genes in Drosophila gonads

The maternally inherited obligatory intracellular bacterium Wolbachia is a reproductive parasite of many insect species. Wolbachia evades the host immune system, uses the mitotic apparatus to ensure infection of daughter cells, migrates through the host to the gonads and causes reproductive phenotypes, most commonly cytoplasmic incompatibility (CI), i.e. incompatibility of sperm from infected males and eggs from uninfected females. Due to the interconnected facts that Wolbachia is not ex vivo culturable and that no established transformation system exists, virtually nothing is known about Wolbachia-host interactions at the macromolecular level. Intriguingly, the Wolbachia genome codes for an unusually high number of ankyrin repeat (ANK) proteins. ANKs mediate protein–protein interactions in many different contexts. More common in eukaryotes, they also occur in prokaryotes. Some intracellular pathogenic bacteria export ANK effector proteins to the host cytoplasm. This makes the Wolbachia ANK genes candidates for mediating interactions with host cells. We quantified expression of ANK genes of Wolbachia strain wMel in adult gonads and detected host sex-specific regulation of two wMel ANK genes in the gonads in two different backgrounds. Regulation was tissue-specific and independent of host background. We further analyzed expression of their homologues in strains wAu and wRi and found regulation only in wAu. Regulation was tissue-specific and there was no correlation between regulation of these genes and the ability of a strain to induce CI.     

Transient expression of firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea

Summary We report here on the development of a transient expression system for Chlorella ellipsoidea using a heterologous gene, firefly luciferase. Cells of this unicellular green alga were converted to protoplasts and treated with plasmid pDO432, which bears luciferase under the control of the CaMV 35S promoter. This treatment resulted in detectable luciferase activity in cell extracts. Expression required Cellulysin treatment, active cell metabolism, and the addition of carrier DNA and polyethylene glycol. Linearization of the luciferase plasmid did not significantly alter the activity. A time course of expression showed that luciferase is made rapidly, within about 7 h after addition of DNA, but that the activity disappears over the course of a few days. These experiments represent an important first step in the development of a Chlorella transformation system.     

Expression of <Emphasis Type="Italic">HSPF1</Emphasis> and <Emphasis Type="Italic">LIM</Emphasis> in the lymphoblastoid cells derived from patients with bipolar disorder and schizophrenia

We have previously reported the altered expressions of HSPF1 and LIM in the lymphoblastoid cell lines (LCLs) derived from Japanese patients with bipolar disorder (bipolar I disorder). The altered expression at the LCL level would be useful for developing diagnostic markers as well as a cellular model for bipolar disorder. In this study, we extended our previous study by measuring their expressions using the following samples: (1) larger number of LCLs from Japanese subjects, (2) LCLs from Caucasian subjects, and (3) LCLs from patients with bipolar II disorder or schizophrenia. We confirmed the increased expression of HSPF1 (P=0.009) and decreased expression of LIM (P=0.001) in the LCLs from patients with Japanese bipolar I disorder. These altered expressions were also observed in those from patients with Japanese bipolar II disorder (P=0.002 for HSPF1 and P=0.072 for LIM). We also found the altered expressions of HSPF1 in LCLs from Caucasian patients with bipolar II disorder (P=0.011) and LIM in those from patients with schizophrenia (P=0.001).     

Distinction Between Lipoma and Liposarcoma by MDM2 Alterations: A Case Report of Simultaneously Occurring Tumors and Review of the Literature

We investigated a lipoma and a well-differentiated/dedifferentiated liposarcoma (WD/DDL), occurring simultaneously in one patient for the possible role of p53 and mdm2 in the molecular oncogenesis of liposarcoma and tumor progression. The hypothesis tested was if there is a continuum in the development from lipoma to liposarcoma. Lipoma was characterized by a lack of p53 mutation, p53 LOH and p53 protein expression, as well as by mdm2 amplification and mdm2 protein expression. p53 mutation and p53 LOH were found neither in the well-differentiated nor in the dedifferentiated parts of the liposarcoma. In contrast, mdm2 amplification and an increase in mdm2 protein expression were found to be associated with malignancy and dedifferentiation, whereas p53 protein expression was only slightly increased. These findings indicate that mdm2 constitutes one of the most common targets for molecular aberration in WD/DDL. We suggest that mdm2 is a marker distinguishing between ordinary lipoma and well-differentiated liposarcoma, and that the genesis of these tumors is different.     

Degradation of endothelial cell matrix collagen is correlated with induction of stromelysin by an activated ras oncogene

A conditional expression system was established whereby the human K-ras, v-src, and v-mos genes were cloned into a conditional expression vector downstream of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Rat-1 fibroblasts were transfected with these constructs and selected in medium containing G418. Cloned transfectants were isolated and characterized for absolute dependence on dexamethasone for expression of oncogene products and anchorage-independent growth in soft agar. Expression of activated p21^K-ras(val12) enabled the fibroblasts to degrade extracellular matrix collagen secreted by murine microvessel endothelial cells. Concurrent with p21^K-ras(val12) induction a proteinase with the characteristic size and substrate specificity of transin, the murine homologue of the human matrix metalloproteinase stromelysin, was expressed and secreted. Induction of v-mos and v-src oncogenes resulted in little or no detectable transin expression respectively coinciding with a relative or absolute failure to increase degradation of extracellular matrix collagen. This study suggests that in this system the expression of the ras oncogene can contribute to the in vitro invasive behavior of tumor cells by upregulating the production of a metalloproteinase capable of degrading collagen synthesized by vascular endothelial cells.     

MRG1-1, a dominant allele that confers methomyl resistance in yeast expressing the cytoplasmic male sterility T-urf13 gene from maize

We have previously described a eukaryotic heterologous expression system, with the urf13TW gene in yeast, which mimics the disease susceptibility associated with the Texas cytoplasmic male sterility in maize. This yeast model was used to isolate yeast nuclear mutants conferring methomyl resistance. The genetic strategy we have developed focused on screening for nuclear dominant yeast mutations which restore methomyl resistance. MRG1-1, a yeast nuclear dominant allele, was identified as a methomyl-resistance restorer. We have shown that methomyl resistance co-segregated with a pleiotropic phenotype in the heterozygous MRG1-1/MRG1 diploids, detectable even in the absence of the maize-derived mitochondrial protein and/or methomyl. We observed an increase in oxygen uptake, a significant decrease of the levels of cytochrome aa₃, and a decrease in the growth yield. This phenotype is influenced by the carbon source and the results suggest a defect in the adaptation to the respiratory pathway in MRG1-1 yeast cells.     

Investigations into the regulation and function of the SH2 domain-containing protein-tyrosine phosphatase,SHP-1

Our laboratory is interested in identifying genes relevant to diseases. Our approach is to use spontaneous mouse mutants with immunological defects and decipher the molecular basis of the phenotypes. In the early 1990s, our attention was focused on the motheaten and viable motheaten mouse mutants. We used these mutant mice as a model system for elucidating the genetic and cellular events contributing to expression of normal hematopoietic and immune function. Our initial goal was to identify the gene responsible for the motheaten and viable motheaten phenotype. In 1993, we and others reported that both motheaten and viable motheaten mice have mutations in the SHP-1 gene. Currently, there are more than 600 publications involving SHP-1. In this review, rather than summarizing all these studies, we highlight work involving SHP-1 that were/are carried out in our and our collaborators' laboratories.     

<Emphasis Type="Italic">Pax7</Emphasis> and superior collicular polarity: insights from <Emphasis Type="Italic">Pax6 (Sey)</Emphasis> mutant mice

Pax genes are important modulators of CNS development. Pax7 and Pax6 polarise the neural tube and regionalise the brain. Pax7 is pivotal in specifying the superior colliculus/tectum, an important centre for integration of visuomotor responses and a target for Pax6 ⁺ retinal ganglion cell axons during retinocollicular mapping. Whilst initial Pax7-specification of the mesencephalon is well-established, a role in regulating polarity within the maturing mouse superior colliculus is yet to be defined, although already detailed for the chick tectum. We therefore quantified Pax7 cellular distribution and expression levels at three functionally distinct stages of superior collicular development, and analysed Pax7 expression in response to aberrant axonal input and altered forebrain/midbrain boundary placement in Pax6 mutant mice. Comparative expression profiles of ephrin-A2 and its co-localisation with Pax7 were determined in wildtype and Pax6 mutant mice. Results indicate that graded Pax7 expression in wildtype mice is perturbed in Pax6 mutant mice; changes manifest as a shift in polarity, loss of graded expression and dramatically reduced protein levels during RGC synaptogenesis. Ephrin-A2 expression is similarly altered. These results implicate Pax7 as an important determinant of polarity within the mouse superior colliculus, and suggest a role in retinotopic mapping.     

Functional and structural characterisation of <Emphasis Type="Italic">Ag</Emphasis>MNPV <Emphasis Type="Italic">ie1</Emphasis>

We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computer assisted analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana defective NPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, the ie1 upstream activating region (UAR) was molecularly dissected and cloned upstream of the E. coli lacZ ORF. The results obtained, after transfection of UFL-AG-286 insect cells, leading us to find that the −492 and −357 versions contains sequence motifs important for the level of the lacZ reporter gene expression. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. The GenBank accession number of the sequence reported in this paper is AF368905.     

Consecutive gene deletions in Aspergillus nidulans: application of the Cre/loxP system

The ability to perform multiple gene deletions is an important tool for conducting functional genomics. We report the development of a sequential gene deletion protocol for the filamentous fungus Aspergillus nidulans using the Cre/loxP recombinase system of bacteriophage P1. A recyclable genetic marker has been constructed by incorporating loxP direct repeats either side of the Neurospora crassa pyr-4 gene (encodes orotidine 5′-monophosphate decarboxylase) which is able to complement the A. nidulans pyrG89 mutation. This construct can be directed to delete specific genomic regions by attaching flanking sequences corresponding to the desired target. The pyr-4 marker can subsequently be eliminated by Cre-catalysed recombination between the loxP sites. The recombinase gene (cre), which has been placed under the control of the A. nidulans xlnA (xylanase A) gene promoter thus providing a means to switch on (xylose induction) or off (glucose repression) recombinase expression, has been integrated into the genome of an A. nidulans mutant strain defective in orotidine 5′-monophosphate decarboxylase activity (pyrG89). We demonstrate the effectiveness of our deletion system by sequentially deleting two genes, yellow (yA) and white (wA), involved in the synthesis of conidial pigment.     

Age-related neuronal loss in the cochlea is not delayed by synaptic modulation

Age-related synaptic change is associated with the functional decline of the nervous system. It is unknown whether this synaptic change is the cause or the consequence of neuronal cell loss. We have addressed this question by examining mice genetically engineered to over- or underexpress neuregulin-1 (NRG1), a direct modulator of synaptic transmission. Transgenic mice overexpressing NRG1 in spiral ganglion neurons (SGNs) showed improvements in hearing thresholds, whereas NRG1 −/+ mice show a complementary worsening of thresholds. However, no significant change in age-related loss of SGNs in either NRG1 −/+ mice or mice overexpressing NRG1 was observed, while a negative association between NRG1 expression level and survival of inner hair cells during aging was observed. Subsequent studies provided evidence that modulating NRG1 levels changes synaptic transmission between SGNs and hair cells. One of the most dramatic examples of this was the reversal of lower hearing thresholds by “turning-off” NRG1 overexpression. These data demonstrate for the first time that synaptic modulation is unable to prevent age-related neuronal loss in the cochlea.     

Association analysis of HSP90B1 with bipolar disorder

Pathophysiological role of endoplasmic reticulum (ER) stress response signaling has been suggested for bipolar disorder. The goal of this study was to test the genetic association between bipolar disorder and an ER chaperone gene, HSP90B1 (GRP94/gp96), which is located on a candidate locus, 12q23.3. We tested the genetic association between bipolar disorder and HSP90B1 by case-control studies in two independent Japanese sample sets and by a transmission disequilibrium test (TDT) in NIMH Genetics initiative bipolar trio samples (NIMH trios). We also performed gene expression analysis of HSP90B1 in lymphoblastoid cells. Among the 11 SNPs tested, rs17034977 showed significant association in both Japanese sample sets. The frequency of the SNP was lower in NIMH samples than in Japanese samples and there was no significant association in NIMH trios. Gene expression analysis of HSP90B1 in lymphoblastoid cells suggested a possible relationship between the associated SNP and mRNA levels. HSP90B1 may have a pathophysiological role in bipolar disorder in the Japanese population, though further study will be needed to understand the underlying functional mechanisms.     

Transient expression in Physarum of a chloramphenicol acetyltransferase gene under the control of actin gene promoters

Summary We cloned and sequenced two actin promoters from Physarum, and constructed plasmids carrying these promoters upstream of a bacterial chloramphenicol acetyltransferase (cat) gene. We then tested the plasmids for their ability to express cat in Physarum amoebae. We present reliable methods for introducing plasmid DNA into Physarum amoebae by electroporation, and show that expression of the cat gene in amoebae occurs in the presence, but not the absence, of one or the other Physarum actin promoter.