Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.     

Indigenous Vibrio cholerae strains from a non-endemic region are pathogenic

Of the 200+ serogroups of Vibrio cholerae, only O1 or O139 strains are reported to cause cholera, and mostly in endemic regions. Cholera outbreaks elsewhere are considered to be via importation of pathogenic strains. Using established animal models, we show that diverse V. cholerae strains indigenous to a non-endemic environment (Sydney, Australia), including non-O1/O139 serogroup strains, are able to both colonize the intestine and result in fluid accumulation despite lacking virulence factors believed to be important. Most strains lacked the type three secretion system considered a mediator of diarrhoea in non-O1/O13 V. cholerae. Multi-locus sequence typing (MLST) showed that the Sydney isolates did not form a single clade and were distinct from O1/O139 toxigenic strains. There was no correlation between genetic relatedness and the profile of virulence-associated factors. Current analyses of diseases mediated by V. cholerae focus on endemic regions, with only those strains that possess particular virulence factors considered pathogenic. Our data suggest that factors other than those previously well described are of potential importance in influencing disease outbreaks.     

Microbial community composition and function beneath temperate trees exposed to elevated atmospheric carbon dioxide and ozone

We hypothesized that changes in plant growth resulting from atmospheric CO₂ and O₃ enrichment would alter the flow of C through soil food webs and that this effect would vary with tree species. To test this idea, we traced the course of C through the soil microbial community using soils from the free-air CO₂ and O₃ enrichment site in Rhinelander, Wisconsin. We added either ¹³C-labeled cellobiose or ¹³C-labeled N-acetylglucosamine to soils collected beneath ecologically distinct temperate trees exposed for 3 years to factorial CO₂ (ambient and 200 µl l^-1 above ambient) and O₃ (ambient and 20 µl l^-1 above ambient) treatments. For both labeled substrates, recovery of ¹³C in microbial respiration increased beneath plants grown under elevated CO₂ by 29% compared to ambient; elevated O₃ eliminated this effect. Production of ¹³C-CO₂ from soils beneath aspen (Populus tremuloides Michx.) and aspen-birch (Betula papyrifera Marsh.) was greater than that beneath aspen-maple (Acer saccharum Marsh.). Phospholipid fatty acid analyses (¹³C-PLFAs) indicated that the microbial community beneath plants exposed to elevated CO₂ metabolized more ¹³C-cellobiose, compared to the microbial community beneath plants exposed to the ambient condition. Recovery of ¹³C in PLFAs was an order of magnitude greater for N-acetylglucosamine-amended soil compared to cellobiose-amended soil, indicating that substrate type influenced microbial metabolism and soil C cycling. We found that elevated CO₂ increased fungal activity and microbial metabolism of cellobiose, and that microbial processes under early-successional aspen and birch species were more strongly affected by CO₂ and O₃ enrichment than those under late-successional maple.     

Evolution of major histocompatibility complex class I genes in Cetartiodactyls

Previous studies of cattle MHC have suggested the presence of at least four classical class I loci. Analysis of haplotypes showed that any combination of one, two or three genes may be expressed, although no gene is expressed consistently. The aim of this study was to examine the evolutionary relationships among these genes and to study their phylogenetic history in Cetartiodactyl species, including cattle and their close relatives. A secondary aim was to determine whether recombination had occurred between any of the genes. MHC class I data sets were generated from published sequences or by polymerase chain reaction from cDNA. Phylogenetic analysis revealed that MHC class I sequences from Cetartiodactyl species closely related to cattle were distributed among the main cattle gene "groups", while those from more distantly related species were either scattered (sheep, deer) or clustered in a species-specific manner (sitatunga, giraffe). A comparison between gene and species trees showed a poor match, indicating that divergence of the MHC sequences had occurred independently from that of the hosts from which they were obtained. We also found two clear instances of interlocus recombination among the cattle MHC sequences. Finally, positive natural selection was documented at positions throughout the alpha 1 and 2 domains, primarily on those amino acids directly involved in peptide binding, although two positions in the alpha 3 domain, a region generally conserved in other species, were also shown to be undergoing adaptive evolution.     

Ganglioside GD1a restores infectibility to mouse cells lacking functional receptors for polyomavirus

Recent investigations on the pathway of cell entry by polyomavirus (Py) and simian virus 40 (SV40) have defined specific gangliosides as functional receptors mediating virus binding and transport from the plasma membrane to the endoplasmic reticulum (B. Tsai et al., EMBO J. 22:4346-4355, 2003; Gilbert and Benjamin, in press). These studies were carried out with C6 rat glioma cells, a heterologous host chosen for its known deficiency in ganglioside biosynthesis. Here, a cell genetic approach was undertaken to identify components required for the early steps of infection using mouse cells as the natural host for Py. Receptor-negative (R-) mouse cells, screened based on resistance to Py infection, were shown to bind Py but failed to allow entry of the virus. R- cells were also found to be resistant to SV40. Infectibility was restored or enhanced by the addition of the same specific gangliosides found in earlier studies with C6 cells. In one R- line, overexpression of caveolin-1 also increased infectibility. These results support and extend findings on gangliosides in lipid rafts as functional receptors and mediators of internalization for Py and SV40.     

Mutations in the extra sex combs and Enhancer of Polycomb genes increase homologous recombination in somatic cells of Drosophila melanogaster

We found that heterozygous mutant alleles of E(Pc) and esc increased homologous recombination from an allelic template in somatic cells in a P-element-induced double-strand break repair assay. Flies heterozygous for mutant alleles of these genes showed increased genome stability and decreased levels of apoptosis in imaginal discs and a concomitant increase in survival following ionizing radiation. We propose that this was caused by a genomewide increase in homologous recombination in somatic cells. A double mutant of E(Pc) and esc had no additive effect, showing that these genes act in the same pathway. Finally, we found that a heterozygous deficiency for the histone deacetylase, Rpd3, masked the radiation-resistant phenotype of both esc and E(Pc) mutants. These findings provide evidence for a gene dosage-dependent interaction between the esc/E(z) complex and the Tip60 histone acetyltransferase complex. We propose that esc and E(Pc) mutants enhance homologous recombination by modulating the histone acetylation status of histone H4 at the double-strand break.     

Genetic reincarnation of workers as queens in the Eastern honeybee Apis cerana

Thelytokous parthenogenesis, or the asexual production of female offspring, is rare in the animal kingdom, but relatively common in social Hymenoptera. However, in honeybees, it is only known to be ubiquitous in one subspecies of Apis mellifera, the Cape honeybee, A. mellifera capensis. Here we report the appearance of queen cells in two colonies of the Eastern honeybee Apis cerana that no longer contained a queen or queen-produced brood to rear queens from. A combination of microsatellite genotyping and the timing of the appearance of these individuals excluded the possibility that they had been laid by the original queen. Based on the genotypes of these individuals, thelytokous production by natal workers is the most parsimonious explanation for their existence. Thus, we present the first example of thelytoky in a honeybee outside A. mellifera. We discuss the evolutionary and ecological consequences of thelytoky in A. cerana, in particular the role thelytoky may play in the recent invasions by populations of this species.     

Design, synthesis, and SAR of heterocycle-containing antagonists of the human CCR5 receptor for the treatment of HIV-1 infection

Replacement of the large hydantoin-indole moiety from our previous work with a variety of smaller heterocyclic analogues gave rise to potent CCR5 antagonists having binding affinity comparable to the hydantoin analogues. The synthesis, SAR, and biological profiles of this class of antagonists are described.