Faculty Bibliography

An S678P substitution in Fks1p, the major subunit of glucan synthase, was sufficient to confer echinocandin resistance in Aspergillus fumigatus. The equivalent mutation in Candida spp. has been implicated in echinocandin resistance. This work demonstrates that modification of Fks1p is a conserved mechanism for echinocandin resistance in pathogenic fungi.

A Candida krusei strain from a patient with acute myelogenous leukemia that displayed reduced susceptibility to echinocandin drugs contained a heterozygous mutation, T2080K, in FKS1. The resulting Phe655-->Cys substitution altered the sensitivity of glucan synthase to echinocandin drugs, consistent with a common mechanism for echinocandin resistance in Candida spp.

Antifungal efficacies of the echinocandin drugs caspofungin, micafungin, and anidulafungin were reduced significantly in the presence of 50% human serum, which yielded nearly equivalent MICs or minimum effective concentrations against diverse Candida spp. and Aspergillus spp. Consistent with a direct drug interaction, serum decreased the sensitivity of glucan synthase to echinocandin drugs.

Insertion and deletion (indel)-based analyses have great potential for rooting the tree of life, but their use has been limited because they require ubiquitous sequences that have not been horizontally/laterally transferred. Very few such sequences exist. Here we describe and demonstrate a new algorithm that can use nonubiquitous sequences for rooting. This algorithm, top-down indel rooting, uses the traditional logical framework of indel rooting, but by considering gene gains and losses in addition to indel gains and losses, it is able to analyze incomplete data sets. The method is demonstrated using theoretical examples and incomplete gene sets. In particular, it is applied to the well-studied Hsp70/MreB indel, a sequence set thought to have been compromised by gene transfers from Firmicutes to archaebacteria. By sequentially assigning all observable character states, including gene absences, to the questionable archaebacterial Hsp70 and MreB sequences, we demonstrate that this gene set robustly excludes the root of the tree of life from the Gram-negative, double-membrane prokaryotes independently of the archaeal character states. There are very few ubiquitous paralog gene sets, and most of them contain compromised data. The ability of top-down rooting to use incomplete and/or compromised gene sets promises to make rooting analyses more robust and to greatly increase the number of useful indel sets.

Transcuprein is a high-affinity copper carrier in the plasma that is involved in the initial distribution of copper entering the blood from the digestive tract. To identify and obtain cDNA for this protein, it was purified from rat plasma by size exclusion and copper-chelate affinity chromatography, and amino acid sequences were obtained. These revealed a 190-kDa glycosylated protein identified as the macroglobulin alpha(1)-inhibitor III, the main macroglobulin of rodent blood plasma. Albumin (65 kDa) copurified in variable amounts and was concluded to be a contaminant (although it can transiently bind the macroglobulin). The main macroglobulin in human blood plasma (alpha(2)-macroglobulin), which is homologous to alpha(1)-inhibitor III, also bound copper tightly. Expression of alpha(1)I3 (transcuprein) mRNA by the liver was examined in rats with and without copper deficiency, using quantitative polymerase chain reaction methodology and Northern blot analysis. Protein expression was examined by Western blotting. Deficient rats with 40% less ceruloplasmin oxidase activity and liver copper concentrations expressed about twice as much alpha(1)I3 mRNA, but circulating levels of transcuprein did not differ. Iron deficiency, which increased liver copper concentrations by threefold, reduced transcuprein mRNA expression and circulating levels of transcuprein relative to what occurred in rats with normal or excess iron. We conclude that transcupreins are specific macroglobulins that not only carry zinc but also carry transport copper in the blood, and that their expression can be modulated by copper and iron availability.

Directed indels, insertions, and deletions within paralogous genes, have the potential to root the tree of life. Here we apply a newly developed rooting algorithm, top-down rooting, to indels found in informational and operational gene sets, introduce new computational tools for indel analyses, and present evidence (P < .01) that the root of the tree of life is not present in its traditional location, between the Eubacteria and the Archaebacteria. Using indels contained in the dihydroorotate dehydrogenase/uroporphyrinogen decarboxylase gene pair and in the ribosomal protein S12/beta prime subunit of the RNA polymerase gene pair, we exclude the root from within the clade consisting of the Firmicutes plus the Archaebacteria and their most recent common ancestor. These results, plus previous directed indel studies excluding the root from the eukaryotes, restrict the root to just four possible sites. One potential root is on the branch leading to the double-membrane prokaryotes, another is on the branch leading to the Actinobacteria, another is within the Actinobacteria, and the fourth is on the branch leading to the Firmicutes-Archaea clade. These results imply (1) that the cenancestral population was not hyperthermophilic, but moderate thermophily cannot be excluded for the root on the branch leading to the Firmicutes-Archaea clade, (2) that the cenancestral population was surrounded by ester lipids and a peptidoglycan layer, and (3) that parts of the mevalonate synthesis pathway were present in the population ancestral to the Bacilli and the Archaebacteria, including geranylgeranylglyceryl phosphate synthase, an enzyme thought to be partially responsible for the unique sn-1 stereochemistry of the archaeal glycerol phosphate backbone.

Conditional targeted cell ablation in zebrafish would greatly expand the utility of this genetic model system in developmental and regeneration studies, given its extensive regenerative capabilities. Here, we show that, by combining chemical and genetic tools, one can ablate cells in a temporal- and spatial-specific manner in zebrafish larvae. For this purpose, we used the bacterial Nitroreductase (NTR) enzyme to convert the prodrug Metronidazole (Mtz) into a cytotoxic DNA cross-linking agent. To investigate the efficiency of this system, we targeted three different cell lineages in the heart, pancreas, and liver. Expression of the fusion protein Cyan Fluorescent Protein-NTR (CFP-NTR) under control of tissue-specific promoters allowed us to induce the death of cardiomyocytes, pancreatic beta-cells, and hepatocytes at specific times. Moreover, we have observed that Mtz can be efficiently washed away and that, upon Mtz withdrawal, the profoundly affected tissue can quickly recover. These findings show that the NTR/Mtz system is effective for temporally and spatially controlled cell ablation in zebrafish, thereby constituting a most promising genetic tool to analyze tissue interactions as well as the mechanisms underlying regeneration.

Cell infection with Trypanosoma cruzi, the agent of Chagas' disease, begins with the uptake of infective trypomastigotes within phagosomes and their release into the cytosol, where they transform into replicating amastigotes; the latter, in turn, differentiate into cytolytically released and infective trypomastigotes. We ask here if the T. cruzi infection program can develop in enucleated host cells. Monolayers of L929 cells, enucleated by centrifugation in the presence of cytochalasin B and kept at 34 degrees C to extend the survival of cytoplasts, were infected with parasites of the CL strain. Percent infection, morphology, stage-specific markers, and numbers of parasites per cell were evaluated in nucleated and enucleated cells, both of which were present in the same preparations. Parasite uptake, differentiation and multiplication of amastigotes, development of epimastigote- and trypomastigote-like forms, and initial cytolytic release of parasites were all documented for cytoplasts and nucleated cells. Although the doubling times were similar, parasite loads at 48 and 72 h were significantly lower in the cytoplasts than in nucleated cells. Similar results were obtained with the highly virulent strain Y as well as with strains CL-14 and G, which exhibit low virulence for mice. Cytoplasts could also be infected with the CL strain 24 or 48 h after enucleation. Thus, infection of cells by T. cruzi can take place in enucleated host cells, i.e., in the absence of modulation of chromosomal and nucleolar gene transcription and of RNA modification and processing in the nucleus.