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High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets.

ABSTRACT: Mycobacterium tuberculosis, the primary etiologic agent of tuberculosis, is the world's leading cause of death from a single infectious agent, and new vaccines and drugs to combat it are urgently needed. The major extracellular proteins of M. tuberculosis, which are released into its phagosome in macrophages, its host cells in humans, are leading candidates for a vaccine and prime targets for new drugs. However, the development of these biologicals has been hampered by the unavailability of large quantities of recombinant extracellular proteins identical to their native counterparts. In this report, we describe the heterologous expression and secretion of four major M. tuberculosis extracellular proteins (the 30-, 32, 16-, and 23.5-kDa proteins--the first, second, third, and eighth most abundant, respectively) in rapidly growing, nonpathogenic mycobacterial species. Multiple attempts to obtain secretion of the proteins by using Escherichia coli- and Bacillus subtilis-based expression systems were unsuccessful, suggesting that high-level expression and secretion of these Mycobacterium-specific proteins require a mycobacterial host. All four recombinant proteins were stably expressed from the cloned genes' own promoters at yields that were 5- to 10-fold higher than those observed for the native proteins. The four proteins were purified to apparent homogeneity from culture filtrates by ammonium sulfate precipitation and ion-exchange and molecular sieve chromatography. The recombinant proteins were indistinguishable from their native counterparts by multiple criteria. First, N-terminal amino acid sequence determination demonstrated that processing of the leader peptides was highly accurate. Second, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed identical migration patterns. Third, mass spectrometry analysis confirmed that differences in mass were < or = 5 Da. A homolog of the M. tuberculosis 30-kDa protein was identified in M. smegmatis by means of DNA analyses and immunoscreening. This is the first time that secretion of recombinant M. tuberculosis extracellular proteins in their native form has been achieved. This study opens the door to mass production of correctly processed and secreted extracellular proteins of M. tuberculosis in a heterologous host and allows ready evaluation of their biologic and immunologic function.

SUBMITTER: Harth G

PROVIDER: S-EPMC175322 | biostudies-other | 1997 Jun

REPOSITORIES: biostudies-other

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High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets.

Harth G G Lee B Y BY Horwitz M A MA

Infection and immunity 19970601 6

Mycobacterium tuberculosis, the primary etiologic agent of tuberculosis, is the world's leading cause of death from a single infectious agent, and new vaccines and drugs to combat it are urgently needed. The major extracellular proteins of M. tuberculosis, which are released into its phagosome in macrophages, its host cells in humans, are leading candidates for a vaccine and prime targets for new drugs. However, the development of these biologicals has been hampered by the unavailability of larg ...[more]

PMID: 9169770

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Project description:Nonpigmented and late-pigmenting rapidly growing mycobacteria (RGM) are increasingly isolated in clinical microbiology laboratories. Their accurate identification remains problematic because classification is labor intensive work and because new taxa are not often incorporated into classification databases. Also, 16S rRNA gene sequence analysis underestimates RGM diversity and does not distinguish between all taxa. We determined the complete nucleotide sequence of the rpoB gene, which encodes the bacterial beta subunit of the RNA polymerase, for 20 RGM type strains. After using in-house software which analyzes and graphically represents variability stretches of 60 bp along the nucleotide sequence, our analysis focused on a 723-bp variable region exhibiting 83.9 to 97% interspecies similarity and 0 to 1.7% intraspecific divergence. Primer pair Myco-F-Myco-R was designed as a tool for both PCR amplification and sequencing of this region for molecular identification of RGM. This tool was used for identification of 63 RGM clinical isolates previously identified at the species level on the basis of phenotypic characteristics and by 16S rRNA gene sequence analysis. Of 63 clinical isolates, 59 (94%) exhibited <2% partial rpoB gene sequence divergence from 1 of 20 species under study and were regarded as correctly identified at the species level. Mycobacterium abscessus and Mycobacterium mucogenicum isolates were clearly distinguished from Mycobacterium chelonae; Mycobacterium mageritense isolates were clearly distinguished from "Mycobacterium houstonense." Four isolates were not identified at the species level because they exhibited >3% partial rpoB gene sequence divergence from the corresponding type strain; they belonged to three taxa related to M. mucogenicum, Mycobacterium smegmatis, and Mycobacterium porcinum. For M. abscessus and M. mucogenicum, this partial sequence yielded a high genetic heterogeneity within the clinical isolates. We conclude that molecular identification by analysis of the 723-bp rpoB sequence is a rapid and accurate tool for identification of RGM.

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Dataset Information

High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets.

Publications

High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets.

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