Impact of either elevated or decreased levels of cytochrome bd expression on Shigella flexneri virulence.

Shigella spp. are the major cause of bacillary dysentery worldwide. The pathogenic process involves bacterial invasion and lysis of the phagocytic vacuole, followed by replication and movement within the cell cytoplasm and, ultimately, spread directly into adjacent cells. This study demonstrates that S. flexneri cytochrome bd expression is necessary for normal intracellular survival and virulence. Cytochrome bd is one of two terminal oxidases in the bacterial respiratory chain that reduce molecular oxygen to water, utilizing intermediates shuttled through the electron transport chain. S. flexneri mutants that contain a disruption in the cydC locus, which leads to defective cytochrome bd expression, or in the riboflavin (ribE) or ubiquinol-8 (ubiH) biosynthetic pathway, which leads to elevated cytochrome bd expression, were evaluated in intracellular survival and virulence assays. The cydC mutant formed significantly smaller plaques, had significantly decreased intracellular survival, and had a 100-fold increase in lethal dose for mice compared with the wild type. The ribE and ubiH mutants each formed significantly larger plaques and had a 10-fold decrease in lethal dose for mice compared with the wild type. The data indicate that expression of cytochrome bd is required for S. flexneri intracellular survival and virulence.

Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin-based motility.

Shigella pathogenesis involves bacterial invasion of colonic epithelial cells and movement of bacteria through the cytoplasm and into adjacent cells by means of actin-based motility. The Shigella protein IcsA (VirG) is unipolar on the bacterial surface and is both necessary and sufficient for actin-based motility. IcsA is inserted into the outer membrane as a 120-kDa polypeptide that is subsequently slowly cleaved, thereby releasing the 95-kDa amino-terminal portion into the culture supernatant. IcsP, the major Shigella protease that cleaves IcsA, was identified and cloned. It has significant sequence similarity to the E. coli serine proteases, OmpP and OmpT. Disruption of icsP in serotype 2a S. flexneri leads to a marked reduction in IcsA cleavage, increased amounts of IcsA associated with the bacterium and altered distribution of IcsA on the bacterial surface. The icsP mutant displays significantly increased rates of actin-based motility, with a mean speed 27% faster than the wild-type strain; moreover, a significantly greater percentage of the icsP mutant moves in the cytoplasm. Yet, plaque formation on epithelial monolayers by the mutant was not altered detectably. These data suggest that IcsA, and not a host protein, is limiting in the rate of actin-based motility of wild-type serotype 2a S. flexneri.

A subclass of cell surface carbohydrates revealed by a CHO mutant with two glycosylation mutations.

A novel lectin-resistance phenotype was displayed by a LEC10 Chinese hamster ovary (CHO) cell mutant that was selected for resistance to the erythroagglutinin, E-PHA. Biochemical and genetic analyses revealed that the phenotype results from the expression of two glycosylation mutations, LEC10 and lec8. The LEC10 mutation causes the appearance of N-acetylglucosaminyltransferase III (GlcNAc-TIII) activity and the production of N-linked carbohydrates with a bisecting GlcNAc residue. The lec8 mutation inhibits translocation of UDP-Gal into the Golgi lumen and thereby dramatically reduces galactosylation of all glycoconjugates. This reduction in galactose addition does not, however, cause Lec8 mutants to be very resistant to the galactose-binding lectin, ricin. By contrast, the double mutant LEC10.Lec8 behaved like a LEC10 mutant and was highly resistant to ricin. Based on structural studies of cellular glycopeptides as well as glycopeptides of the G glycoprotein of vesicular stomatitis virus grown in mutant cells, it appears that the ricin resistance of LEC10.Lec8 cells is due to the presence of a small number of Gal residues on branched, N-linked carbohydrates that also carry the bisecting GlcNAc residue. Labelling of N-linked cellular carbohydrates with [3H]galactose was found to occur at a low level for a wide spectrum of cellular glycoproteins in independent Lec8 mutants. Studies of the LEC10.Lec8 mutant have, therefore, led to the identification of a subset of structures that are acceptors for Gal when intra-Golgi UDP-Gal levels are limiting. This mutant also illustrates the potential for regulating cell surface recognition by carbohydrate-binding proteins by altering the expression of a single glycosyltransferase such as GlcNAc-TIII.

Lectin-resistant CHO cells: selection of seven new mutants resistant to ricin.

In attempts to isolate new CHO glycosylation mutants, selection protocols using plant lectins that bind galactose residues of cell surface carbohydrates were applied to mutagenized CHO populations. The lectins were used alone or in combination to obtain seven ricin-resistant phenotypes. Each mutant had distinctive properties compared with previously described ricin-resistant CHO cells. One of the new phenotypes was dominant in somatic cell hybrids, and the others were recessive. Complementation analyses between related lectin-resistant (LecR) phenotypes indicated that each new isolate represented a novel genotype. Five of the mutants had properties typical of new CHO glycosylation mutants. The remaining two mutants were not readily categorized. Although they did not appear to be ricin-internalization or protein-synthesis mutants, they also did not display the marked alterations in sensitivity to several lectins of different sugar specificity expected for glycosylation mutants. The seven new LecR mutants described in these studies brings the total number of different LecR CHO mutants isolated by this and other laboratories to about 40. Criteria for identifying new LecR mutations in CHO cells are discussed.

Isolation of Chinese hamster ovary ribosomal mutants differentially resistant to ricin, abrin, and modeccin.

The molecular action of ricin A chain involves cleavage of the N-glycosidic bond between ribose and the adenine 4324 nucleotides from the 5' end of mammalian 28 S rRNA (Endo, Y., and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130). In this paper, four ricin- and abrin-resistant Chinese hamster ovary cell mutants that possess ribosomes resistant to this N-glycosidase action are described. Three of the mutant phenotypes, Lec26, Lec27, and Lec28, were recessive in somatic cell hybrids and define at least two new lectin-resistant complementation groups. The most extensively characterized mutant type, LEC17, was dominant in such hybrids. None of the mutants were cross-resistant to modeccin. Post-mitochondrial supernatants from each of the four mutants were resistant to inhibition of cell-free protein synthesis by ricin, ricin A chain, and abrin. In addition, polysomes isolated from mutant cells were resistant to cleavage of the adenine-ribose N-glycosidic bond by ricin A chain or abrin, as assayed by the release of an approximately 470-nucleotide fragment following aniline treatment of ribosomal RNA extracted from toxin-treated polysomes. The unique lectin-resistance properties of the different mutants suggests that the accessibility of adenine 4324 to each toxin differs. It seems likely that the recessive Chinese hamster ovary ribosomal mutants reflect structural changes in different ribosomal proteins while the dominant phenotype may be due to the modification of protein(s) or rRNA involved in toxin-ribosome interaction. Further analysis of these cell lines should provide new insights into the structure/function relationships of eukaryotic ribosomes.

Novel genetic instability associated with a developmentally regulated glycosyltransferase locus in Chinese hamster ovary cells.

LEC10 is a dominant glycosylation mutant of Chinese hamster ovary (CHO) cells that expresses a developmentally regulated glycosyltransferase (GlcNAc-TIII) not detectable in parental CHO cells. Several mutagens were found to increase the frequency of LEC10 mutants up to 10-fold over the spontaneous frequency of less than or equal to 10(-7), while 5azaC treatment had no effect. Revertants were obtained at high frequency (approximately 10(-4)) and were found to belong to two classes. Three independent revertants gave rise to new LEC10 mutants at high frequency (approximately 10(-4)) while seven others gave new LEC10 mutants at the low frequency typical of unmutagenized parental CHO cells. No evidence of a general mutator phenotype was found in the revertant lines with a high rereversion frequency. The combined data suggest a novel form of genomic instability at the LEC10 locus in CHO cells. Genetic events that affect the expression of developmentally regulated glycosyltransferases may be identified by further studies of LEC10 and other dominant CHO glycosylation mutants.

DNA transfection of Mammalian cells using polybrene.

Many techniques have been developed to transfect mammalian cells with DNA (1-2). The most commonly used method is to expose cells to a coprecipitate of DNA and calcium phosphate (3). This technique works very well with both genomic and recombinant DNA sequences for some cell lines, e.g., mouse L-cells, but less well with other cell types such as Chinese hamster ovary (CHO) cells (2-4). Since the ability to rescue mutant phenotypes by exogenous DNA provides a means to identify gene products and to clone their genes (5), the reduced ability to transform CHO cells by the calcium phosphate technique has slowed the use of the large number of CHO cell mutants (6) in transfection and rescue experiments. In this chapter we will describe a simple, reproducible method for DNA transfection (7) that is a modification of a method originally developed for the introduction of viral sequences into chick embryo fibroblasts (8). The method involves exposing the cells to DNA in the presence of the polycation polybrene, followed by a DMSO shock. The polybrene apparently interacts with the charge on the cell and the DNA, allowing the DNA to absorb more easily to the cell membrane.

High-frequency transfection of CHO cells using polybrene.

High-frequency transfection of CHO cells has been achieved for several plasmids, a cosmid library, and genomic DNA using Polybrene and dimethyl sulfoxide. All plasmid transfectants examined were stable and exhibited plasmid sequences in genomic DNA. The method is simple, reproducible, and succeeded with several independent CHO clones in the presence or the absence of carrier DNA, even at very low concentrations of plasmid DNA.