Triple Deletion Mutant Research Articles

Cold Shock Proteins Contribute to the Regulation of Listeriolysin O Production inListeria monocytogenes

Cold shock proteins (Csps) are multifunctional nucleic acid binding proteins used to regulate a wide range of gene expression responses in bacteria. We report here that Csps regulate the production of the pore-forming cytolysin listeriolysin (LLO) and hemolysis phenotypes in Listeria monocytogenes. A triple csp gene deletion mutant incapable of producing any Csps, as well as double csp gene deletion mutants only producing either CspA or CspD, caused less hemolysis and produced lower LLO concentration. On the other hand, another double csp gene deletion mutant that produces CspB retained hemolysis and LLO production levels that are similar to the parental wild-type strain. Transcription analysis showed that in absence of all three csp genes or cspB alone, L. monocytogenes cells have decreased levels of hly gene transcripts, which code for the synthesis of LLO proteins. A comparative examination of mRNA stability showed that hly transcripts were more rapidly degraded in L. monocytogenes triple csp gene deletion mutant cells that are not capable of producing Csps. Overall, our results indicate that Csps, in particular CspB, are important components of gene expression regulatory mechanisms that promote efficient LLO production and hence virulence responses of L. monocytogenes.

Trans-Lesion DNA Polymerases May Be Involved in Yeast Meiosis.

Trans-lesion DNA polymerases (TLSPs) enable bypass of DNA lesions during replication and are also induced under stress conditions. Being only weakly dependent on their template during replication, TLSPs introduce mutations into DNA. The low processivity of these enzymes ensures that they fall off their template after a few bases are synthesized and are then replaced by the more accurate replicative polymerase. We find that the three TLSPs of budding yeast Saccharomyces cerevisiae Rev1, PolZeta (Rev3 and Rev7), and Rad30 are induced during meiosis at a time when DNA double-strand breaks (DSBs) are formed and homologous chromosomes recombine. Strains deleted for one or any combination of the three TLSPs undergo normal meiosis. However, in the triple-deletion mutant, there is a reduction in both allelic and ectopic recombination. We suggest that trans-lesion polymerases are involved in the processing of meiotic double-strand breaks that lead to mutations. In support of this notion, we report significant yeast two-hybrid (Y2H) associations in meiosis-arrested cells between the TLSPs and DSB proteins Rev1-Spo11, Rev1-Mei4, and Rev7-Rec114, as well as between Rev1 and Rad30. We suggest that the involvement of TLSPs in processing of meiotic DSBs could be responsible for the considerably higher frequency of mutations reported during meiosis compared with that found in mitotically dividing cells, and therefore may contribute to faster evolutionary divergence than previously assumed.

Isobutanol production fromd-xylose by recombinantSaccharomyces cerevisiae

Simultaneous overexpression of an optimized, cytosolically localized valine biosynthesis pathway together with overexpression of xylose isomerase XylA from Clostridium phytofermentans, transaldolase Tal1 and xylulokinase Xks1 enabled recombinant Saccharomyces cerevisiae cells to complement the valine auxotrophy of ilv2,3,5 triple deletion mutants for growth on D-xylose as the sole carbon source. Moreover, after additional overexpression of ketoacid decarboxylase Aro10 and alcohol dehydrogenase Adh2, the cells were able to ferment D-xylose directly to isobutanol.

Regulation of Inhibition of Neutrophil Infiltration by the Two-Component Regulatory System CovRS in Subcutaneous Murine Infection with Group A Streptococcus

Hypervirulent invasive group A streptococcus (GAS) isolates inhibit neutrophil infiltration more than pharyngitis isolates do, and the molecular basis of this difference is not well understood. This study was designed to first determine whether natural null mutation of the two-component regulatory system CovRS is responsible for the enhancement of the inhibition of neutrophil recruitment seen in hypervirulent GAS. Next, we examined the role of CovRS-regulated interleukin-8/CXC chemokine peptidase (SpyCEP), C5a peptidase (ScpA), and platelet-activating factor acetylhydrolase (SsE) in the enhanced innate immune evasion. Invasive isolate MGAS5005 induces less neutrophil infiltration and produced a greater lesion area than pharyngitis isolate MGAS2221 in subcutaneous infections of mice. It is known that MGAS5005, but not MGAS2221, has a natural 1-bp deletion in the covS gene. Replacement of covS(Δ1bp) in MGAS5005 with wild-type covS resulted in the MGAS2221 phenotype. Deletion of covS from MGAS2221 resulted in the MGAS5005 phenotype. Tests of single, double, and triple deletion mutants of the MGAS5005 sse, spyCEP, and scpA genes found that SsE plays a more important role than SpyCEP and ScpA in the inhibition of neutrophil recruitment and that SsE, SpyCEP, and ScpA do not have synergistic effects on innate immune evasion by MGAS5005. Deletion of sse, but not spyCEP or scpA, of MGAS2221 enhances neutrophil recruitment. Thus, covS null mutations can cause substantial inhibition of neutrophil recruitment by enhancing the expression of the chemoattractant-degrading virulence factors, and SsE, but not SpyCEP or ScpA, is required for CovRS-regulated GAS inhibition of neutrophil infiltration.

The Live Attenuated Actinobacillus pleuropneumoniae Triple-Deletion Mutant Δ apxIC Δ apxIIC Δ apxIV - ORF1 Strain, SLW05, Immunizes Pigs against Lethal Challenge with Haemophilus parasuis

Haemophilus parasuis and Actinobacillus pleuropneumoniae both belong to the family Pasteurellaceae and are major respiratory pathogens that cause large economic losses in the pig industry worldwide. We previously constructed an attenuated A. pleuropneumoniae serovar 1 live vaccine prototype, SLW05 (ΔapxIC ΔapxIIC ΔapxIV-ORF1), which is able to produce nontoxic but immunogenic ApxIA, ApxIIA, and ApxIVA. This triple-deletion mutant strain was shown to elicit protective immunity against virulent A. pleuropneumoniae. In the present study, we investigated whether immunization with SLW05 could also protect against lethal challenge with virulent H. parasuis SH0165 (serovar 5) or MD0322 (serovar 4). The SLW05 strain was found to elicit a strong humoral antibody response in pigs and to confer significant protection against challenge with a lethal dose of H. parasuis SH0165 or MD0322. IgG subtype analysis revealed that SLW05 induces a bias toward a Th1-type immune response and stimulates interleukin 2 (IL-2) and gamma interferon (IFN-γ) production. Moreover, antisera from SLW05-vaccinated pigs efficiently inhibited both A. pleuropneumoniae and H. parasuis growth in a whole-blood assay. This is the first report that a live attenuated A. pleuropneumoniae vaccine with SLW05 can protect against lethal H. parasuis infection, which provides a novel approach for developing an attenuated H. parasuis vaccine.

Genetic analysis of the metabolic pathways responsible for aroma metabolite production by Saccharomyces cerevisiae

During alcoholic fermentation, higher alcohols, esters, and acids are formed from amino acids via the Ehrlich pathway by yeast, but many of the genes encoding the enzymes have not yet been identified. When the BAT1/2 genes, encoding transaminases that deaminate amino acids in the first step of the Ehrlich pathway are deleted, higher metabolite formation is significantly decreased. Screening yeast strains with deletions of genes encoding decarboxylases, dehydrogenases, and reductases revealed nine genes whose absence had the most significant impact on higher alcohol production. The seven most promising genes (AAD6, BAT2, HOM2, PAD1, PRO2, SPE1, and THI3) were further investigated by constructing double- and triple-deletion mutants. All double-deletion strains showed a greater decrease in isobutanol, isoamyl alcohol, isobutyric, and isovaleric acid production than the corresponding single deletion strains with the double-deletion strains in combination with ∆bat2 and the ∆hom2-∆aad6 strain revealing the greatest impact. BAT2 is the dominant gene in these deletion strains and this suggests the initial transaminase step of the Ehrlich pathway is rate-limiting. The triple-deletion strains in combination with BAT2 (∆bat2-∆thi3-∆aad6 and ∆bat2-∆thi3-∆hom2) had the greatest impact on the end metabolite production with the exception of isoamyl alcohol and isovaleric acid. The strain deleted for two dehydrogenases and a reductase (∆hom2-∆pro2-∆aad6) had a greater effect on the levels of these two compounds. This study contributes to the elucidation of the Ehrlich pathway and its significance for aroma production by fermenting yeast cells.

Co-expression analysis, proteomic and metabolomic study on the impact of a Deg/HtrA protease triple mutant in Synechocystis sp. PCC 6803 exposed to temperature and high light stress

Members of the DegP/HtrA protease family are widespread in nature and play an important role in proteolysis of misfolded and damaged proteins. The cyanobacterium Synechocystis sp. PCC 6803 contains three Deg proteases, HhoA (Sll1679), HhoB (Sll1427) and HtrA (Slr1204). Using the proteomic or metabolomic approach we investigated a triple deletion mutant (Δdeg) exposed to light or temperature stress. To cope with the stress conditions the triple mutant reduces its energy metabolism and stress-related proteins are induced to protect the cells. Additionally the co-expression of the genes encoding the three proteases with other genes in Synechocystis sp. PCC 6803 was analyzed. While HhoA seems to be involved in house-keeping processes related to protein (re)folding, protein clearance and signaling, the hhoB expression cluster is dominated by genes encoding periplasmic proteins linked to metabolism or signal transduction pathways. The htrA expression pattern is similar to that of genes encoding proteins of the electron transport chain, iron- and nitrogen metabolism. Our integrative approach indicates significant rearrangements in cells depleted of the Deg/HtrA proteases when exposed to stress, both, in the cytoplasmic and extracytoplasmic space.

Genetic analysis of the metabolic pathways responsible for aroma metabolite production by Saccharomyces cerevisiae

During alcoholic fermentation, higher alcohols, esters, and acids are formed from amino acids via the Ehrlich pathway by yeast, but many of the genes encoding the enzymes have not yet been identified. When the BAT1/2 genes, encoding transaminases that deaminate amino acids in the first step of the Ehrlich pathway are deleted, higher metabolite formation is significantly decreased. Screening yeast strains with deletions of genes encoding decarboxylases, dehydrogenases, and reductases revealed nine genes whose absence had the most significant impact on higher alcohol production. The seven most promising genes (AAD6, BAT2, HOM2, PAD1, PRO2, SPE1, and THI3) were further investigated by constructing double- and triple-deletion mutants. All double-deletion strains showed a greater decrease in isobutanol, isoamyl alcohol, isobutyric, and isovaleric acid production than the corresponding single deletion strains with the double-deletion strains in combination with ∆bat2 and the ∆hom2-∆aad6 strain revealing the greatest impact. BAT2 is the dominant gene in these deletion strains and this suggests the initial transaminase step of the Ehrlich pathway is rate-limiting. The triple-deletion strains in combination with BAT2 (∆bat2-∆thi3-∆aad6 and ∆bat2-∆thi3-∆hom2) had the greatest impact on the end metabolite production with the exception of isoamyl alcohol and isovaleric acid. The strain deleted for two dehydrogenases and a reductase (∆hom2-∆pro2-∆aad6) had a greater effect on the levels of these two compounds. This study contributes to the elucidation of the Ehrlich pathway and its significance for aroma production by fermenting yeast cells.

Xer‐cise in Helicobacter pylori: One‐step Transformation for the Construction of Markerless Gene Deletions

Xer-cise is an efficient selectable marker removal technique that was first applied in Bacillus subtilis and Escherichia coli for the construction of markerless gene deletions. Xer-cise marker excision takes advantage of the presence of site-specific Xer recombination in most bacterial species for the resolution of chromosome dimers at the dif site during replication. The identification and functional characterization of the difH/XerH recombination system enabled the development of Xer-cise in Helicobacter pylori. Markerless deletions were obtained by a single natural transformation step of the Xer-cise cassette containing rpsL and cat genes, for streptomycin susceptibility and chloramphenicol resistance respectively, flanked by difH sites and neighboring homologous sequences of the target gene. Insertion/deletion recombinant H.pylori were first selected on chloramphenicol-containing medium followed by selection on streptomycin-containing medium for clones that underwent XerH mediated excision of the rpsL-cat cassette, resulting in a markerless deletion. XerH-mediated removal of the antibiotic marker was successfully applied in three different H.pylori strains to obtain markerless gene deletions at very high efficiencies. An unmarked triple deletion mutant was also constructed by sequential deletion of ureA, vacA and HP0366 and removal of the selectable marker at each step. The triple mutant had no growth defect suggesting that multiple difH sites per chromosome can be tolerated without affecting bacterial fitness. Xer-cise eliminates the need for multiple passages on non selective plates and subsequent screening of clones for loss of the antibiotic cassette by replica plating.

Characterization of triglyceride lipase genes of fission yeast Schizosaccharomyces pombe

Triglycerides (TG) are major storage lipids for eukaryotic cells. In this study, we characterized three genes of fission yeast Schizosaccharomyces pombe, SPCC1450.16c, SPAC1786.01c, and SPAC1A6.05c, that show high homology to Saccharomyces cerevisiae TG lipase genes, TGL3, TGL4, and TGL5. Deletion of each gene increased TG content by approximately 1.7-fold compared to the parental wild-type strain, and their triple deletion mutant further increased TG content to 2.7-fold of the wild-type strain, suggesting that all three genes encode TG lipase and are functioning in S. pombe. The triple deletion mutant showed no growth defect in rich and synthetic medium, but its growth was sensitive to cerulenin, an inhibitor of fatty acid synthesis. This growth defect by cerulenin was restored by adding oleic acid in media, suggesting that these genes were involved in the mobilization of TG in S. pombe. When ricinoleic acid was produced in the triple mutant by introducing CpFAH12 fatty acid hydroxylase gene from Claviceps purpurea, percent composition of ricinoleic acid increased by 1.1-fold compared to the wild-type strain, in addition to a 1.6-fold increase in total fatty acid content per dry cell weight (DCW). In total, the ricinoleic acid production per DCW increased by 1.8-fold in the triple deletion mutant.

Heparan Sulfate Chains of Syndecan-1 Regulate Ectodomain Shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.

SoxS Increases the Expression of the Zinc Uptake System ZnuACB in an Escherichia coli Murine Pyelonephritis Model

Paralogous transcriptional regulators MarA, Rob, and SoxS act individually and together to control expression of more than 80 Escherichia coli genes. Deletion of marA, rob, and soxS from an E. coli clinical isolate prevents persistence beyond 2 days postinfection in a mouse model of pyelonephritis. We used microarray analysis to identify 242 genes differentially expressed between the triple deletion mutant and its parent strain at 2 days postinfection in the kidney. One of these, znuC of the zinc transport system ZnuACB, displayed decreased expression in the triple mutant compared to that in the parental strain, and deletion of znuC from the parental strain reduced persistence. The marA rob soxS triple deletion mutant was less viable in vitro under limited-Zn and Zn-depleted conditions, while disruption of znuC caused a reduction in the growth rates for the parental and triple mutant strains to equally low levels under limited-Zn or Zn-depleted conditions. Complementation of the triple mutant with soxS, but not marA or rob, restored the parental growth rate in Zn-depleted medium, while deletion of only soxS from the parental strain led to low growth in Zn-depleted medium. Both results suggested that SoxS is a major regulator responsible for growth under Zn-depleted conditions. Gel shift experiments failed to show direct binding of SoxS to the znuCB promoter, thus suggesting indirect control of znuCB expression by SoxS. While SoxS expression in the triple mutant fully restored persistence, increased expression of znuACB via a plasmid in this mutant only partially restored wild-type levels of persistence in the kidney. This work implicates SoxS control of znuCB expression as a key factor in persistence of E. coli in murine pyelonephritis.

A Non-Marker Mutagenesis Strategy to Generate Poly- hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzicola (Xoc), the critical pathogen causing bacterial leaf streak in rice, possesses a hrp cluster that is responsible for triggering hypersensitive response (HR) in non-host tobacco and pathogenicity in host rice, and is considered to be one of the model pathogens in the rice model plant. Here, we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1. It was used to generate single or poly-gene mutants of hpa1, hpa2, hrcV, hrpE, hpaB, and hrpF gene for functional analysis. In total, five single, four double, and two triple hrp gene mutants were constructed. The double and triple hrp gene deletion mutants triggered novel phenotypes in planta. Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.

LytR-CpsA-Psr proteins in Staphylococcus aureus display partial functional redundancy and the deletion of all three severely impairs septum placement and cell separation

Staphylococcus aureus contains three members of the LytR-CpsA-Psr (LCP) family of membrane proteins: MsrR, SA0908 and SA2103. The characterization of single-, double- and triple-deletion mutants revealed distinct phenotypes for each of the three proteins. MsrR was involved in cell separation and septum formation and influenced β-lactam resistance; SA0908 protected cells from autolysis; and SA2103, although displaying no apparent phenotype by itself, enhanced the properties of msrR and sa0908 mutants when deleted. The deletion of sa0908 and sa2103 also further attenuated the virulence of msrR mutants in a nematode-killing assay. The severely defective growth phenotype of the triple mutant revealed that LytR-CpsA-Psr proteins are essential for optimal cell division in S. aureus. Growth could be rescued to varying degrees by any one of the three proteins, indicating some functional redundancy within members of this protein family. However, differing phenotypic characteristics of all single and double mutants and complemented triple mutants indicated that each protein played a distinct role(s) and contributed differently to phenotypes influencing cell separation, autolysis, cell surface properties and virulence.

Genetic and biochemical analysis of the serine/threonine protein kinases PknA, PknB, PknG and PknL of Corynebacterium glutamicum: evidence for non-essentiality and for phosphorylation of OdhI and FtsZ by multiple kinases.

We previously showed that the 2-oxoglutarate dehydrogenase inhibitor protein OdhI of Corynebacterium glutamicum is phosphorylated by PknG at Thr14, but that also additional serine/threonine protein kinases (STPKs) can phosphorylate OdhI. To identify these, a set of three single (ΔpknA, ΔpknB, ΔpknL), five double (ΔpknAG, ΔpknAL, ΔpknBG, ΔpknBL, ΔpknLG) and two triple deletion mutants (ΔpknALG, ΔpknBLG) were constructed. The existence of these mutants shows that PknA, PknB, PknG and PknL are not essential in C. glutamicum. Analysis of the OdhI phosphorylation status in the mutant strains revealed that all four STPKs can contribute to OdhI phosphorylation, with PknG being the most important one. Only mutants in which pknG was deleted showed a strong growth inhibition on agar plates containing glutamine as carbon and nitrogen source. Thr14 and Thr15 of OdhI were shown to be phosphorylated in vivo, either individually or simultaneously, and evidence for up to two additional phosphorylation sites was obtained. Dephosphorylation of OdhI was shown to be catalysed by the phospho-Ser/Thr protein phosphatase Ppp. Besides OdhI, the cell division protein FtsZ was identified as substrate of PknA, PknB and PknL and of the phosphatase Ppp, suggesting a role of these proteins in cell division.

Cytochrome c4 Can Be Involved in the Photosynthetic Electron Transfer System in the Purple Bacterium Rubrivivax gelatinosus

Three periplasmic electron carriers, HiPIP and two cytochromes c8 with low- and high-midpoint potentials, are present in the purple photosynthetic bacterium Rubrivivax gelatinosus. Comparison of the growth rates of mutants lacking one, two, or all three electron carrier proteins showed that HiPIP is the main electron donor to the photochemical reaction center and that high-potential cytochrome c8 plays a subsidiary role in the electron donation in photosynthetically growing cells. However, the triple deletion mutant was still capable of photosynthetic growth, indicating that another electron donor could be present. A new soluble cytochrome c, which can reduce the photooxidized reaction center in vitro, was purified. Based on amino acid sequence comparisons to known cytochromes, this cytochrome was identified as a diheme cytochrome c of the family of cytochromes c4. The quadruple mutant lacking this cytochrome and three other electron carriers showed about three times slower growth than the triple mutant under photosynthetic growth conditions. In conclusion, cytochrome c4 can function as a physiological electron carrier in the photosynthetic electron transport chain in R. gelatinosus.

Mobilization of steryl esters from lipid particles of the yeast Saccharomyces cerevisiae

In the yeast as in other eukaryotes, formation and hydrolysis of steryl esters (SE) are processes linked to lipid storage. In Saccharomyces cerevisiae, the three SE hydrolases Tgl1p, Yeh1p and Yeh2p contribute to SE mobilization from their site of storage, the lipid particles/droplets. Here, we provide evidence for enzymatic and cellular properties of these three hydrolytic enzymes. Using the respective single, double and triple deletion mutants and strains overexpressing the three enzymes, we demonstrate that each SE hydrolase exhibits certain substrate specificity. Interestingly, disturbance in SE mobilization also affects sterol biosynthesis in a type of feedback regulation. Sterol intermediates stored in SE and set free by SE hydrolases are recycled to the sterol biosynthetic pathway and converted to the final product, ergosterol. This recycling implies that the vast majority of sterol precursors are transported from lipid particles to the endoplasmic reticulum, where sterol biosynthesis is completed. Ergosterol formed through this route is then supplied to its subcellular destinations, especially the plasma membrane. Only a minor amount of sterol precursors are randomly distributed within the cell after cleavage from SE. Conclusively, SE storage and mobilization although being dispensable for yeast viability contribute markedly to sterol homeostasis and distribution.

Dissecting sensor functions in cell wall integrity signaling in Kluyveromyces lactis

KlWSC1, KlWSC2/3 and KlMID2, which encode putative plasma membrane sensors for cell wall integrity signaling in Kluyveromyces lactis, were cloned and characterized. Double and triple deletion mutants show severe cell integrity defects, indicating overlapping functions. The Klwsc1 Klmid2 double deletion phenotype can be suppressed by overexpression of the downstream components KlROM2, KlPKC1 and KlBCK1. KlWsc1 sensor domain analyses showed that an amino-terminal elongation as well as an extension within the cytoplasmic domain are dispensable for function. Heterologous complementation by KlMID2 and KlWSC1 in Saccharomyces cerevisiae is only achieved upon overexpression. In contrast to ScMID2, ScWSC1 complements in K. lactis. Functional studies with chimeric Mid2 constructs indicate that species specificity is mainly conferred by the extracellular domain. Sensor-GFP fusions localize to the plasma membrane, with a cell cycle dependent distribution of KlWsc1-GFP. Both Wsc-type sensors concentrate in discrete spots within the plasma membrane.

An Escherichia coli undecaprenyl-pyrophosphate phosphatase implicated in undecaprenyl phosphate recycling

Undecaprenyl phosphate (Und-P) is a universal lipid carrier of glycan biosynthetic intermediates for carbohydrate polymers that are exported to the bacterial cell envelope. Und-P arises from the dephosphorylation of undecaprenyl pyrophosphate (Und-PP) molecules produced by de novo synthesis and also from the recycling of released Und-PP after the transfer of the glycan component to other acceptor molecules. The latter reactions take place at the periplasmic side of the plasma membrane, while cytoplasmic enzymes catalyse the de novo synthesis. Four Und-PP pyrophosphatases were recently identified in Escherichia coli. One of these, UppP (formerly BacA), accounts for 75 % of the total cellular Und-PP pyrophosphatase activity and has been suggested to participate in the Und-P de novo synthesis pathway. Unlike UppP, the other three pyrophosphatases (YbjG, YeiU and PgpB) have a typical acid phosphatase motif also found in eukaryotic dolichyl-pyrophosphate-recycling pyrophosphatases. This study shows that double and triple deletion mutants in the genes uppP and ybjG, and uppP, ybjG and yeiU, respectively, are supersensitive to the Und-P de novo biosynthesis inhibitor fosmidomycin. In contrast, single or combined deletions including pgpB have no effect on fosmidomycin supersensitivity. Experimental evidence is also presented that the acid phosphatase motifs of YbjG and YeiU face the periplasmic space. Furthermore, the quadruple deletion mutant DeltauppP-DeltaybjG-DeltayeiU-DeltawaaL has a growth defect and abnormal cell morphology, suggesting that accumulation of unprocessed Und-PP-linked O antigen polysaccharides is toxic for these cells. Together, the results support the notion that YbjG, and to a lesser extent YeiU, exert their enzymic activity on the periplasmic side of the plasma membrane and are implicated in the recycling of periplasmic Und-PP molecules.

256. Novel Therapeutic Strategy for Ischemic Stroke by Bone Marrow Stromal Cells and Ex Vivo HGF and FGF2 Genes Transfer with Replication-Incompetent HSV-1 Vector

The bone marrow stromal cells (MSCs), including the primitive pluriopotent mesenchymal stem cells (PMSCs) and the multipotent adult progenitor cells (MAPCs), are attractive targets for cell and gene therapy for various central nervous system disorders.Material and method: For gene modification of MSCs, we used a replication-incompetent HSV-1 vector “1764/4-”, triple-deletion mutant of the ICP4, ICP34.5, and VP16. This vector has very high ability of gene transduction to MSC, 70-75% at MOI 10 and 95% at MOI 50, and its expression can carry on at least one month at high level. Two kinds of vectors expressing HGF and FGF-2 were prepared for this study. FGF-2 vector contains the signal sequence of Interleukin-2 (IL-2ss) ahead of FGF-2 gene to enhance its extracellular secretion, because native FGF2 gene don't have the excretion signal. Native MSCs, HGF-MSCs, IL2ss+FGF-2-MSCs and PBS were administered directly into the lesioned brain 24 hours after transient middle cerebral artery occlusion (MCAO) of adult Wister rats.Results: At day 14, each gene modified MSC group showed significant improvement in modified neurological severity score (mNSS) and apparent decreasing of infarct volume compared with native MSC or sham-operated group. (p<0.05). HGF and FGF-2 expression in vivo were strongly kept through the experimental period. In HGF modified MSC-treated group, the MAP-2 positive cell% tends to increase and the TUNEL positive cell% decrease in the ischemic boundary zone (IBZ). The transdifferentiation of transplanted MSCs to central nervous system cell phenotypes was also checked. Most of the transdifferentiated phenotype was GFAP- positive, and the Nestin-positive cells were scarcely able to find out in the host brain.Discussion and Conclusion: By using the replication- incompetent HSV-1 vector, MSCs can be efficiently engineered to secrete a series of various cytokines in large quantities and in the long term in vitro and in vivo that could potentially treat the ischemic stroke of the brain. HGF and FGF-2 gene-transferred MSCs, achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native MSCs in rat MCAO model. It would be appeared that the main mechanism of these therapeutic effects is the neuroprotection (anti-apoptotic effect) in IBZ by various abundant cytokines secreted from gene engineered MSCs. We will present our detail data of the study and the perspective of this therapeutic strategy. The bone marrow stromal cells (MSCs), including the primitive pluriopotent mesenchymal stem cells (PMSCs) and the multipotent adult progenitor cells (MAPCs), are attractive targets for cell and gene therapy for various central nervous system disorders. Material and method: For gene modification of MSCs, we used a replication-incompetent HSV-1 vector “1764/4-”, triple-deletion mutant of the ICP4, ICP34.5, and VP16. This vector has very high ability of gene transduction to MSC, 70-75% at MOI 10 and 95% at MOI 50, and its expression can carry on at least one month at high level. Two kinds of vectors expressing HGF and FGF-2 were prepared for this study. FGF-2 vector contains the signal sequence of Interleukin-2 (IL-2ss) ahead of FGF-2 gene to enhance its extracellular secretion, because native FGF2 gene don't have the excretion signal. Native MSCs, HGF-MSCs, IL2ss+FGF-2-MSCs and PBS were administered directly into the lesioned brain 24 hours after transient middle cerebral artery occlusion (MCAO) of adult Wister rats. Results: At day 14, each gene modified MSC group showed significant improvement in modified neurological severity score (mNSS) and apparent decreasing of infarct volume compared with native MSC or sham-operated group. (p<0.05). HGF and FGF-2 expression in vivo were strongly kept through the experimental period. In HGF modified MSC-treated group, the MAP-2 positive cell% tends to increase and the TUNEL positive cell% decrease in the ischemic boundary zone (IBZ). The transdifferentiation of transplanted MSCs to central nervous system cell phenotypes was also checked. Most of the transdifferentiated phenotype was GFAP- positive, and the Nestin-positive cells were scarcely able to find out in the host brain. Discussion and Conclusion: By using the replication- incompetent HSV-1 vector, MSCs can be efficiently engineered to secrete a series of various cytokines in large quantities and in the long term in vitro and in vivo that could potentially treat the ischemic stroke of the brain. HGF and FGF-2 gene-transferred MSCs, achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native MSCs in rat MCAO model. It would be appeared that the main mechanism of these therapeutic effects is the neuroprotection (anti-apoptotic effect) in IBZ by various abundant cytokines secreted from gene engineered MSCs. We will present our detail data of the study and the perspective of this therapeutic strategy.