No evidence for productive PERV infection of baboon cells in in vivo infection model.

OBJECTIVES: The discovery that pig endogenous retroviruses are infectious for human cells in vitro lead to vehement discussions about the possible risk of infection after clinical xenotransplantation. Since PERV transmission to non-human primate cells in vitro has been observed, similar to human cells, infection studies in non-human primates should represent the best model to analyze a potential PERV transmission after xenotransplantation. However, it is still open to discussion, whether non-human primate cells can be infected productively-similar to human cells- and whether those species are suitable to analyze PERV infection risks in vivo. METHODS: In vitro, only few cell types can be tested for susceptibility. We developed a pig to baboon cell transplantation model with special emphasis on B-cell effective immunosuppression, removal of anti Gal-alpha 1,3-Gal-antibodies, inhibition of the complement cascade and long term survival of transplanted cellular grafts. This model allows us to investigate in vivo, whether any baboon cell types may be permissive for productive PERV infection. The xenograft recipients were investigated for up to 535 days post transplantation. Gal-alpha 1,3-Gal-antibody and complement levels were monitored. Potential PERV transmission was analyzed, not only in PBMC, but in a variety of tissue samples as well as in serum and plasma samples by PCR, RT-PCR and by detection of RT-activity. Moreover, potential PERV specific immune responses were studied by a highly sensitive Western-Blot-assay. RESULTS: Despite several days of extremely low levels of Gal-alpha 1,3-Gal-antibody and complement, and despite of long term xenochimerism, no evidence for PERV infection was obtained in any of the tested tissues or in the tested serum samples. CONCLUSION: This study supplies further evidence for a low susceptibility of baboons towards productive PERV infection after xenotransplantation.

Genome of the bacterium Streptococcus pneumoniae strain R6.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues.

Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.

Analysis of potential porcine endogenous retrovirus (PERV) transmission in a whole-organ xenotransplantation model without interfering microchimerism.

The question whether porcine xenografts can lead to porcine endogenous retrovirus (PERV) infection of recipients is critical for the evaluation of the safety of pig-to-man xenotransplantation. Unfortunately, polymerase chain reaction (PCR)-based analysis of potential PERV infections in nonhuman-primate whole-organ xenotransplantation models is hampered by false positive results due to chimeric porcine cells. To avoid the inherent analytical problem of xenomicrochimerism, we developed a non-life-supporting pig-to-primate kidney xenotransplantation model: porcine kidneys were transplanted, whereas the functioning recipient kidneys remained in situ. Subsequent to rejection (after 2 hours to 15 days), xenografts were removed, and recipients remained alive for up to 287 days. Immunosuppressive therapy based on cyclophosphamide, cyclosporine, and steroids was maintained for 28 days after transplantation. Using appropriate PCR assays, xenochimerism was found in tissue samples and partly even in peripheral blood leukocytes (PBLs) while the porcine kidneys were in situ. After graft removal, xenochimerism was no longer detectable, thus allowing analysis for possible PERV transmission.

The miaA mutator phenotype of Escherichia coli K-12 requires recombination functions.

miaA mutants, which contain A-37 instead of the ms(2)i(6)A-37 hypermodification in their tRNA, show a moderate mutator phenotype leading to increased GC-->TA transversion. We show that the miaA mutator phenotype is dependent on recombination functions similar to, but not exactly the same as, those required for translation stress-induced mutagenesis.

Reduction of GC --> TA transversion mutation by overexpression of MutS in Escherichia coli K-12.

Overexpression of the MutS repair protein significantly decreased the rate of lacZ GC --> TA transversion mutation in stationary-phase and exponentially growing bacteria and in mutY and mutM mutants, which accumulate mismatches between 8-oxoguanine (8-oxoG) and adenine residues in DNA. Conversely, GC --> TA transversion increased in mutL or mutS mutants in stationary phase. In contrast, overexpression of MutS did not appreciably reduce lacZ AT --> CG transversion mutation in a mutT mutant. These results suggest that MutS-dependent repair can correct 8-oxoG:A mismatches in Escherichia coli cells but may not be able to compete with mutation fixation by MutY in mutT mutants.

Productive infection of primary human endothelial cells by pig endogenous retrovirus (PERV).

The potential risk of viral transmission in the setting of xenotransplantation has gained major attention. Different porcine cell types have been shown to release retroviral particles, which are infectious for human cell lines in vitro. However, there are only a few data on whether PERV (pig endogenous retrovirus) is able to infect primary human cells. In this study we have analyzed endothelial cells, vascular fibroblasts, mesangial cells, mononuclear cells, hematopoetic stem cells and bone marrow stromal cells for PERV transmission. We now provide evidence for primary human endothelial cells, vascular fibroblasts, and mesangial cells to be susceptible to PERV transmission. PERV infection was productive in endothelial cells and mesangial cells. Our data confirm and extend former reports concerning the PERV infection of human cells. The PERV infection of different primary human cells represents further significant evidence for a viral risk during xenotransplantation. In this context, special attention should be directed towards productive infection of human endothelial cells: in the setting of xenotransplantation this cell type will have close contact with porcine cells and PERV particles.

Purification of plasmid DNA by tangential flow filtration.

A simple, scalable method for purification of plasmid DNA is described. The method includes modification of the classical alkaline-lysis-based plasmid extraction method by extending the solubilization step from less than 30 min to 24 h. The extraction is followed by the novel use of tangential flow filtration (TFF) for purification of the remaining contaminants. The method does not include the use of any organic solvents, RNase, high-speed centrifugation, or column chromatography steps. The method typically yields 15 to 20 mg of plasmid DNA per liter of bacterial culture and results in removal of >99% of RNA and >95% of the protein that remains after the modified alkaline lysis procedure. The procedure has been demonstrated to be effective in the isolation of seven different plasmids. Plasmids isolated using this method had comparable transfection capability relative to plasmid isolated using a classical, cesium chloride gradient-based method.

Identification of the miaB gene, involved in methylthiolation of isopentenylated A37 derivatives in the tRNA of Salmonella typhimurium and Escherichia coli.

The tRNA of the miaB2508::Tn10dCm mutant of Salmonella typhimurium is deficient in the methylthio group of the modified nucleoside N(6)-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A37). By sequencing, we found that the Tn10dCm of this strain had been inserted into the f474 (yleA) open reading frame, which is located close to the nag locus in both S. typhimurium and Escherichia coli. By complementation of the miaB2508::Tn10dCm mutation with a minimal subcloned f474 fragment, we showed that f474 could be identified as the miaB gene, which is transcribed in the counterclockwise direction on the bacterial chromosome. Transcriptional studies revealed two promoters upstream of miaB in E. coli and S. typhimurium. A Rho-independent terminator was identified downstream of the miaB gene, at which the majority (96%) of the miaB transcripts terminate in E. coli, showing that the miaB gene is part of a monocistronic operon. A highly conserved motif with three cysteine residues was present in MiaB. This motif resembles iron-binding sites in other proteins. Only a weak similarity to an AdoMet-binding site was found, favoring the idea that the MiaB protein is involved in the thiolation step and not in the methylating reaction of ms(2)i(o)(6)A37 formation.

Mismatch repair is diminished during stationary-phase mutation.

This paper is an invited Response to a recent Commentary [P.L. Foster, Rev. Mut. Res. 436 (1999) 179-184] entitled "Are adaptive mutations due to a decline in mismatch repair? The evidence is lacking". The Commentary argues that no evidence exists supporting the idea that mismatch repair is limiting specifically during stationary-phase mutation. A primary concern of the author is to question the method that we used previously to measure growth-dependent mutation. In this method, mutation rates are calculated using counts of mutant colonies taken at times when those colonies arise, rather than at a predetermined, fixed time. Here we show further data that illustrate why this must be done to ensure accurate mutation measurements. Such accuracy was necessary for our published determination that mismatch repair proteins are not limiting during growth-dependent mutation, but become so during stationary-phase mutation. We review the evidence supporting the idea that stationary-phase reversion of a lac frameshift mutation occurs in an environment of decreased mismatch repair capacity. Those data are substantial. The data presented in the Commentary, in apparent contradiction to this idea, do not justify the conclusion presented there.

Flk couples flgM translation to flagellar ring assembly in Salmonella typhimurium.

The hook-basal body (HBB) is a key intermediate structure in the flagellar assembly pathway in Salmonella typhimurium. The FlgM protein inhibits the flagellum-specific transcription factor sigma28 in the absence of the intact HBB structure and is secreted out of the cell following HBB completion. The flk gene encodes a positive regulator of the activity of FlgM at an assembly step just prior to HBB completion: at the point of assembly of the P- and L-rings. FlgM inhibition of sigma28-dependent class 3 flagellar gene transcription was relieved in P- and L-ring assembly mutants (flgA, flgH, and flgI) by introduction of a null mutation in the flk gene (J. E. Karlinsey et al., J. Bacteriol. 179:2389-2400, 1997). In P- and L-ring mutant strains, recessive mutations in flk resulted in a reduction in intracellular FlgM levels to those seen in wild-type (Fla+) strains. The reduction in intracellular FlgM levels by mutations in the flk gene was concomitant with a 10-fold increase in transcription of the flgMN operon compared to that of the isogenic flk+ strain, while transcription of the flgAMN operon was unaffected. This was true for both direct measurement of the flgAMN and flgMN mRNA transcripts by RNase T2 protection assays and for lac operon fusions to either the flgAMN or flgMN promoter. Loss of Flk did not allow secretion of FlgM through basal-body structures lacking the P- and L-rings. Intracellular FlgM was stable to proteolysis, and turnover occurred primarily after export out of the cell. Loss of Flk did not result in increased FlgM turnover in either P- or L-ring mutant strains. With lacZ translational fusions to flgM, a null mutation in flk resulted in a significant reduction of flgM-lacZ mRNA translation, expressed from the class 3 flgMN promoter, in P- and L-ring mutant strains. No reduction in either flgAMN or flgMN mRNA stability was measured in the absence of Flk in Fla+, ring mutant, or HBB deletion strains. We conclude that the reduction in the intracellular FlgM levels by mutation in the flk gene is only at the level of flgM mRNA translation.

Involvement of the gapA- and epd (gapB)-encoded dehydrogenases in pyridoxal 5'-phosphate coenzyme biosynthesis in Escherichia coli K-12.

We show that epd (gapB) mutants lacking an erythrose 4-phosphate (E4P) dehydrogenase are impaired for growth on some media and contain less pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) than their epd+ parent. In contrast to a previous report, we found that gapA epd double mutants lacking the glyceraldehyde 3-phosphate and E4P dehydrogenases are auxotrophic for pyridoxine. These results implicate the GapA and Epd dehydrogenases in de novo PLP and PMP coenzyme biosynthesis.

Expression, purification, and characterization of recombinant Escherichia coli pyridoxine 5'-phosphate oxidase.

A previously cloned pdxH gene from Escherichia coli coding for pyridoxine 5'-phosphate oxidase was transferred to a pET22b vector and expressed in E. coli HMS174(DE3) cells. The soluble overexpressed enzyme was rapidly purified in high yield using two chromatography columns with an overall purification of about 2.8-fold. The purified enzyme contained tightly bound FMN. The enzyme exhibited the same spectral properties and similar kinetic constants to those previously reported by G. Zhao and M. E.Winkler (J. Bacteriol. 177, 883, 1995), but differed from the properties reported by other investigators. A rapid procedure was developed for preparing apoPNP Ox in high yield. Both the holo- and apoenzymes were homodimers. The molar absorbtivity coefficient for the protein was determined for the fully active apoPNP Ox from is amino acid composition. Using this value and the spectral properties of the bound FMN it was shown by three different methods that the dimeric enzyme contains two molecules of bound FMN per dimer and not one FMN as previously reported.

Identification and function of the pdxY gene, which encodes a novel pyridoxal kinase involved in the salvage pathway of pyridoxal 5'-phosphate biosynthesis in Escherichia coli K-12.

pdrK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5'-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY+ overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette into pdxY and crossed the resulting pdxY::omegaKan(r) mutation into the bacterial chromosome of a pdrB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts of pdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6 vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show that pdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNA(Tyr) synthetase) in a multifunctional operon. pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in the tyrS-pdxY intercistronic region.

Negative regulation of mutS and mutH repair gene expression by the Hfq and RpoS global regulators of Escherichia coli K-12.

The MutS, MutL, and MutH proteins play major roles in several DNA repair pathways. We previously reported that the cellular amounts of MutS and MutH decreased by as much as 10-fold in stationary-phase cultures. Consequently, we tested whether the amounts of MutS, MutL, and MutH were regulated by two global regulators, RpoS (sigma38) and Hfq (HF-I [putative RNA chaperone]), which are involved in stationary-phase transition. We report here that mutations in hfq and rpoS reversed the stationary-phase down-regulation of the amounts of MutS and MutH. hfq regulation of the amount of MutS in stationary-phase cultures was mediated by RpoS-dependent and -independent mechanisms, whereas hfq regulation of the amount of MutH was mediated only through RpoS. Consistent with this interpretation, the amount of MutS but not MutH was regulated by Hfq, but not RpoS, in exponentially growing cells. The amount of MutL remained unchanged in rpoS, hfq-1, and rpoS+, hfq+ strains in exponentially growing and stationary-phase cultures and served as a control. The beta-galactosidase activities of single-copy mutS-lacZ operon and gene fusions suggested that hfq regulates mutS posttranscriptionally in exponentially growing cultures. RNase T2 protection assays revealed increased amounts of mutS transcript that are attributed to increased mutS transcript stability in hfq-1 mutants. Lack of Hfq also increased the amounts and stabilities of transcripts initiated from P(miaA) and P1hfqHS, two of the promoters for hfq, suggesting autoregulation, but did not change the half-life of bulk mRNA. These results suggest that the amounts of MutS and MutH may be adjusted in cells subjected to different stress conditions by an RpoS-dependent mechanism. In addition, Hfq directly or indirectly regulates several genes, including mutS, hfq, and miaA, by an RpoS-independent mechanism that destabilizes transcripts.

Mismatch repair protein MutL becomes limiting during stationary-phase mutation.

Postsynthesis mismatch repair is an important contributor to mutation avoidance and genomic stability in bacteria, yeast, and humans. Regulation of its activity would allow organisms to regulate their ability to evolve. That mismatch repair might be down-regulated in stationary-phase Escherichia coli was suggested by the sequence spectrum of some stationary-phase ("adaptive") mutations and by the observations that MutS and MutH levels decline during stationary phase. We report that overproduction of MutL inhibits mutation in stationary phase but not during growth. MutS overproduction has no such effect, and MutL overproduction does not prevent stationary-phase decline of either MutS or MutH. These results imply that MutS and MutH decline to levels appropriate for the decreased DNA synthesis in stationary phase, whereas functional MutL is limiting for mismatch repair specifically during stationary phase. Modulation of mutation rate and genetic stability in response to environmental or developmental cues, such as stationary phase and stress, could be important in evolution, development, microbial pathogenicity, and the origins of cancer.