ASM News Vol 63, Num 11, November, 1997

Journal Highlights

New Assay Promises to Aid Anti-Toxoplasma Drug Discovery

Toxoplasma gondii is an obligate intracellular parasite that infects immunodeficient patients, as well as the developing fetuses of humans and many animal species. There are no known drug combinations for treating Toxoplasma infection that combine the desired properties of potency and low toxicity.

John C. Boothroyd of Stanford University, Stanford, Calif., and colleagues have developed an easy colorimetric assay that should aid in drug discovery. The authors showed that beta-galactosidase, an enzyme from Escherichia coli, can be efficiently expressed in T. gondii and that growth of parasites expressing this enzyme can easily be monitored in vitro. "The use of a vital indicator means that the assay can be performed in the growing culture itself, recuding manipulations to almost none," says Boothroyd.

"As a research tool, parasites expressing beta-galactosidase will be very useful for tagging strains in mixed infection experiments and for quantifying parasite numbers in vitro and in vivo," says Boothroyd.

(D.C. McFadden, F. Seeber, and J.C. Boothroyd. 1997 Use of Toxoplasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro. Antimicrob. Agents Chemother. 41:1849-1853.)

Alternative Mechanism for Telomere Elongation

Most eukaryotes maintain their chromosome ends via telomerase, an enzyme system that generates short, tandem repeats on the ends of chromosomes, but some use other mechanisms. Telomere elongation, maintenance gone awry, has been implicated in tumor proliferation.

Harald Biessmann and colleagues of the University of California, Irvine, analyzed telomere regulation in the mosquito Anopheles gambiae by using a plasmid marker at the end of chromosome 2. They found that extension of the end of chromosome 2 occurred by unequal crossing over between homologous chromosome 2 ends. Similar alternative mechanisms that prevent telomere shortening might still function in humans and may play a role in tumor growth, says Biessman.

Besides more detailed studies of telomere structure and dynamics, biessmann seeks to understand how a transgene became attached to the end of a telomere of A. gambiae. "Transgenic mosquitoes could be used to interfere with the transmission of the malaria parasite," says Biessmann.

(C.W. Roth, F. Kobeski, M.F. Walter and H. Biessmann. 1997. Chromosome end elongation by recombination in the mosquito Anopheles gambiae. Mol. Cell. Biol. 17:5176-5183.)

Solvent-Tolerant Escherichia coli Mutants Have High Lipopolysaccharide Levels

Anhydrous biocatalysis could mitigate problems of costly treatment of copious waste water from when biocatalysis is performed in water. Microbes that tolerate organic solvents are needed, but most strains with high tolerance belong to the genus Pseudomonas, the biochemistry of which is not as well mapped as that of Escherichia coli.

Rikizo Aono and Hideki Kobayashi of the Tokyo Institute of Technology in Yokohama studied the cell surface properties of E. coli mutants that exhibit high tolerance for organic solvents. Cell surfaces of the mutants were less hydrophobic than those of their wild-type parents. The authors think that this reflected higher levels of lipopolysaccharide (LPS), and possibly reduced porins (e.g., OmpF). They conclude that organic solvents bind to E. coli cells as a function of solvent polarity and cell hydrophobicity. These findings support the development of LPS and/or porin mutants for use in industrial applications requiring biocatalysis in anhydrous media.

(R. Aono and H. Kobayashi. 1997. Cell surface porperties of organic solven-tolerant mutants of Escherichia coli K-12. Appl. Environ. Microbiol. 63:3637-3642.)

Alternating Current Makes Slime Dance

Biofilms growing on indwelling devices, such as catheters and joint prostheses, have been implicated in persistent infections. Since biofilms are more difficult to eradicate with antibiotics than their planktonic counterparts, often the device must be replaced, causing trauma to the patient.

The reason for antibiotic resistance in unclear. Slime may block of neutralize the antibiotic. But in a weak electrical current, antibiotic susceptibility increases 10 to 1,000-fold. Paul Stoodley of the University of Exeter, United Kingdom, and colleagues, collaborating with the Center for Biofilm Engineering, Bozeman, Montana, found that the biofilm rapidly expands and contracts in an alternating electric field, primarily as a result of changes in pH.

"Such rapid expansions and contractions may pump antibiotics into the biofilm," says Stoodley. "This phenomenon could be used to control biofilms on conductive surfaces in industrial plants, or in bioremediation reactors." Stoodley plans to test the phenomenon in gram-positive bacteria and to confirm increased transport from the water phase into biofilms.

(P. Stoodley, D. deBeer, and H.M. Lappin-Scott. 1997. Influence of electric fields and pH on biofilm structure as related to bioelectric effect. Antimicrob. Agents Chemother. 41:1876-1879.)

New Export Pathway Can Store Foreign Proteins in Quantity

Bacterial proteins which pass through the so-called general secretory pathway usually contain "leader" sequences, which are required for secretion. Desmond Mascarenhas and colleagues of the Santa Clara, California, biotechnology firm Celtrix have discovered that when leader sequences are deleted by mutation, certain of these proteins are sequestered in an as-yet-unknown cellular compartment, where they can accumulate to very high levels. They can be extracted from these compartments by osmotic shock.

"Scientifically, these mutants intrigue us because they behave just like mammalian interleukin-1 (known to be secreted by novel pathways in animal cells) when it is expressed in bacteria, suggesting mechanism conservation across wide evolutionary space," says Mascarenhas. "Through protein fusions, such sequestration could also be used to accumulate foreign proteins in bacteria at much higher levels than was previously possible. We will focus our future research on these questions."

(Y.R. Thorstenson, Y. Zhang, P.S. Olson, and D. Mascarenhas. 1997. Leaderless polypeptides efficiently extracted from whole cells by osmotic shock. J. Bacteriol. 179:5333-5339.)

Biofilms Succumb to Benign but Expensive Concoction

Biofilms plague ships' hulls, piping, contact lenses, teeth, and medical implants and are hard to remove without harming either the substrate or the environment. Charlotte Johansen of Novo Nordisk, Bagsvaerd, Denmark, and colleagues report that they achieved both killing and nondestructive removal of biofilms from various substrates by combining oxydoreductases with various polysaccharide-hydrolyzing enzymes. The agent the authors developed, Pectinex Ultra, is too expensive for most applications, says Johansen, but it is a useful step towards better biofilm removers.

(C. Johansen, P. Falholt, and L. Gram. 1997. Enzymatic removal and disinfection of bacterial biofilms. Appl. Environ. Microbiol. 63:3724-3728.)

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