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J D Cirillo

Publications and source records attributed to J D Cirillo.

22 records · Page 2Linked to original sources
PubMed1994

Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria.

The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority. meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor. The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the construction of in vivo selection systems. We have isolated the asd, dapA, dapB, dapD, and dapE genes involved in the DAP biosynthetic pathway of Mycobacterium bovis BCG. These genes were isolated by complementation of Escherichia coli mutations with an expression library of BCG DNA. Our analysis of these genes suggests that BCG may use more than one pathway for biosynthesis of DAP. The nucleotide sequence of the BCG dapB gene was determined. The activity of the product of this gene in Escherichia coli provided evidence that the gene may encode a novel bifunctional dihydrodipicolinate reductase and DAP dehydrogenase.

Amino Acid Sequence↗
PubMed1991

A novel transposon trap for mycobacteria: isolation and characterization of IS1096.

In the course of developing strategies to obtain a mutation in the aspartate semialdehyde dehydrogenase (asd) gene of Mycobacterium smegmatis, an efficient transposon trap was constructed which may be generally useful for the identification of transposable elements in mycobacteria. A DNA fragment containing the asd gene was replaced with an aminoglycoside phosphotransferase gene (aph) to generate a delta asd::aph allele. Attempts to replace the wild-type asd gene with the delta asd::aph allele were unsuccessful, suggesting that this deletion was lethal to the growth of M. smegmatis. The plasmid, pYUB215, which contains beta-galactosidase expressed from a mycobacteriophage promoter and delta asd::aph, was integrated into the chromosome of M. smegmatis by a homologous, single-crossover, recombination event. Visual screening for inactivation of the beta-galactosidase gene in the resulting strain allowed the isolation of a novel mycobacterial insertion element from M. smegmatis. This insertion element, which is unique to M. smegmatis, was designated IS1096 and transposes at a frequency of 7.2 x 10(-5) per cell in an apparently random fashion. IS1096 is 2,275 bp in length and contains two open reading frames which are predicted to encode proteins involved in transposition. This insertion element exhibits several characteristics that suggest it may be a useful tool for genetic analysis of mycobacteria, possibly including the study of mechanisms of pathogenesis.

Amino Acid Sequence↗
PubMedDate not supplied

Recombinant BCG as a candidate oral vaccine vector.

Bacille Calmette-Guerin (BCG), currently the most widely used vaccine in the world, was originally administered for many years as an oral vaccine. The low frequency of serious complications, inexpensive production, and adjuvanticity make BCG an ideal candidate for a recombinant vaccine vehicle. Although mycobacteria are slow growing and not yet well characterized genetically, we have recently developed technology for the genetic manipulation of BCG and other mycobacteria. Phage and plasmid systems based on a shuttle strategy to manipulate DNA in Escherichia coli and transfer it to mycobacteria have been developed. We have established that the aminoglycoside phosphotransferase gene can be used as an effective selectable marker in the mycobacteria and that a foreign antigen from Mycobacterium leprae can be expressed in BCG. Furthermore, a thorough analysis of mycobacterial expression sequences has been undertaken to optimize the expression of foreign antigens in BCG. We constructed an expression probe shuttle plasmid with beta-galactosidase as reporter gene, and have used it successfully to identify multiple mycobacteriophage DNA sequences with varying levels of constitutive or regulable promoter activity. Further genetic advances required for development of recombinant BCG into an effective recombinant vaccine vehicle, including possibilities for oral administration, are adumbrated.

Administration, Oral↗