ASM News Vol 63, Num 11, November, 1997

Journal Highlights

Enterococcus faecalis Yields Potential Antigens

Enterococcus faecalis is an eemerging pathogen that is highly resistant to antibiotics. It is an important source of nosocomial infections such as endocarditis, which is life threatening. George M. Weinstock and Barbara E. Murray of the University of Texas Medical School at Houston and coworkers have cloned many antigen-encoding genes. The ultimate goal of the research is to find antigens that would make suitable targets for development of antimicrobials or vaccine or passive immunization treatments. Sequencing and comparison with gene databases shows that "a very high fraction are surface or secreted proteins, as we had hoped," says Weinstock.

"We are systematically making mutants that knock out each of the genes we have cloned," says Weinstock. "We are going to test those mutants to see whether they are reduced in infectivity in animal models or tissue culture. That would identify which of these many genes are potentially important."

(Y. Xu, L. Jiang, B.E. Murray, and G. M. Weinstock. 1997. Enterococcus faecalis antigens in human infections. Infect. Immun. 65:4207-4215.)

DNA Probe Detects Rifampin Resistance--Fast!

Resistance to rifampin, a key drug in TB treatment and a marker for multidrug resistance, is an increasing problem. Rudi Rossau of Innogenetics N.V., Ghent, Belgium, and colleagues have developed a rapid molecular diagnostic test for simultaneous detection of Mycobacterium tuberculosis and mutations in the gene encoding rifampin resistance.

The DNA probe assay is unique in that "it provides the means for reliable and very rapid detection--one day instead of weeks--of resistance to rifampin," say Rossau. "Our test also shows that, as a general principle, reliable detection of drug resistance is indeed possible using DNA probes." Finally, the test's high performance opens the possibility for other DNA assays to determine drug resistance, and may also position DNA probe testing among the valid alternatives to laborious culture-dependent diagnostic methods, says Rossau.

(R. Rossau, H. Traore, H. De Beenhouwer, W. Mijs, G. Jannes, P. De Rijk, and F. Portaels. 1997. Evaluation of the INNO-LiPA Rif TB assay, a reverse hybridization assay for the simultaneous detection of Mycobacterium tuberculosis complex and its resistance to rifampin. Antimicrob. Agents Chemother. 41:2093-2098.)

Syphilis Pathogen Yields a Few Secrets in Outer Membrane Studies

The best way to eradicate syphilis would be to develop a vaccine. Unfortunately, Treponema pallidum cannot be cultivated on articficial medium. Furthermore, its outer membranes, proteins of which would be vaccine targets, are among the most inscrutable structures of any bacterial pathogen.

Justin D. Radolf and colleagues of the University of Texas Southwestern Medical Center, Dallas, have developed techniques for isolating T. Pallidum outer membranes and have characterized proteins found therein. However, none of four candidates appeared to be outer membrane proteins, says Radolf. He suggests they may be contaminants from the periplasm or the cytoplasmic membrane, and warns that "this ... sounds a cautionary note about interpreting protein and gene sequence information obtained from outer membrane preparations." Nonetheless, "the proteins illuminated the microbe's heretofore poorly understood physiology," and substantial sequencing "provided novel insights into the genetic composition of T. pallidum." Further, the study results raise important questions about the use of outer membrane preparations for identifying rare outer membrane proteins of T. pallidum.

(D.W. Shevchenko, D.R. Akins, E.J. Robinson, M. Li, V. Shevchenko, and J.D. Radolf. 1997. Identification of homologs for thioredoxin, peptidyl prolyl cis-trans isomerase, and glycerophosphodiester phosphodiesterase in outer membrane fractions of Treponema pallidum, the syphilis spirochete. Infect. Immun. 65:4179-4189.)

New Method Concentrates Bacteria--in Minutes!

The chronic problem of foodborne illness has recently received great attention. Gene probe and immunological tests have dramatically shortened test time over tradtional methods, but several hours of enrichment are still required.

Hydroxyapatite, a major component of tooth enamel, is widely used for purification of biochemical substances. Elaine D. Berry and Gregory Siragusa of the U.S. Department of Agiculture, Clay Center, Neb., found that many species of bacteria also adhere to the compound in large numbers. "Our work shows that hydroxyapatite is useful for simple and fast--5 minutes--removal and concentration of bacteria from suspensions, including meat sample homogenates and animal carcass sponge samples," says Berry. Adherence is usually greater than 95% of initial concentrations. The researchers are now seeking to link hydroxyapatite concentration with PCR, says Berry, in order to enhance detection of low levels of specific bacteria in meat without prior cultural enrichment.

(E.D. Berry and G.R. Siragusa. 1997. Hydroxyapatite adherence as a means to concentrate bacteria. Appl. Environ. Microbiol. 63:4069-4074.)

Component of Chromatin Linked to Cancer Development

Failure of mechanisms that maintain differentiated identities of cells over many cell divisions can lead to disorders like cancer. The polycomb (PcG) gene family are part of such a mechanism in Drosophila melanogaster. Aire P. Otte of the University of Amsterdam, the Netherlands, and colleagues have identified a human homolog, hPc2, which is part of chromatin. The researchers found that overexpression of a mutant hPc2, which cannot repress gene activity, generates transformed cells in which expression of the c-myc proto-oncogene is strongly enhanced, but that overexpression of the wild-type hPc2 generates cells with decreased c-myc expression.

"Our data suggest that hPc2 is a repressor of proto-oncogene activity," says Otte. "This strengthens the barely explored link between changes in chromatin structure and oncogenesis. We plan to employ chemical cross-link methods to study whether c-myc is indeed a direct target of the polycomb protein complex." Also, Otte plans to investigate if one of both alleles of hPc2 are deleted in several human cancers, which could help determine if it is a tumor suppressor gene.

(P.E. Satijn, D.J. Olson, J. van der Vlag, K.M. Hamer, C. Lambrechts, H. Masselink, M. J. Gunster, R.G.A.B. Sewalt, R. van Driel, and A.P. Otte. 1997. Interference with the expression of a novel human polycomb protein, hPc2, results in cellular transformation and apoptosis. Mol. Cell. Biol. 17:6076-6086.)

Antiviral DNA Vaccines Protect Mouse Pups, Adults

Although antiviral vaccines are generally safe and effective, two problems interfere with their use in young children. First, maternal antibodies inactivate conventional vaccines by attacking viral vaccine proteins, even after levels have fallen off to the point where they no longer protect an infant. Second, many modes of immunization simply don't work in the immature neonatal immune system.

DNA vaccines circumvent the former problem, as they contain no viral proteins. "For a variety of reasons we though that DNA immunization might also address the second problem," says coauther J. Lindsay Whitton, of the Scripps Research Institute, La Jolla, Calif. Vaccination at birth with DNA encoding a protein from lymphocytic chroiomeningitis virus (LCMV), a natural pathogen of mice, protected pups from virus challenge, and maternal antibodies did not diminish efficacy. "We plan to extend these studies using other viruses and other antigens," says Whitton. "In particular, most of our studies focused on induction of cytotoxic T cell immunity; we now want to more precisely determine how well antiviral antibodies are induced in the baby mice by plasmid DNA."

(D.E. Hassett, J. Zhang, and J.L. Whitton. 1997. Neonatal DNA immunization with a plasmid encoding an internal viral protein is effective in the presence of maternal antibodies and protects against subsequent viral challenge. J. Virol. 71: 7881-7888.)

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