Mycobacterium bovis Bacille Calmette-Guérin strains secreting listeriolysin of Listeria monocytogenes.
Recombinant (r) Mycobacterium bovis strains were constructed that secrete biologically active listeriolysin (Hly) fusion protein of Listeria monocytogenes. The r-BCG strains pAT261:Hly or pMV306:Hly expressed plasmid multicopies or chromosomal single copies of the hly gene, respectively. Human and murine macrophage-like cell lines were infected with r-BCG pAT261:Hly and pMV306:Hly strains. Interestingly, intracellular persistence of both r-BCG strains was reduced in macrophages as compared with the parental BCG strain. By immunogold labeling Hly was detected in membrane structures and within the phagosomal space of macrophages. In addition, Hly was localized within cytoplasmic vacuoles outside the mycobacteria-containing phagosome of host cells infected with r-BCG pAT261:Hly or r-BCG pMV306:Hly. Hly fusions consistently colocalized with a lysosome-associated membrane glycoprotein, suggesting that membrane-attack conformation of Hly was not altered. Although r-BCG pAT261:Hly and r-BCG pMV306:Hly microorganims apparently did not egress into the cytoplasmic compartment of host cells, they both improved major histocompatibility complex class I presentation of cophagocytosed soluble protein as compared with wild-type BCG microbes. These data suggest that Hly secretion endows BCG with an improved capacity to stimulate CD8 T cells. Because CD8 T cells play a major role in protection against tuberculosis such Hly secreting r-BCG constructs are antituberculosis vaccine candidates.
Hess
J
J
Max-Planck-Institute for Infection Biology, Monbijoustrasse 2, D-10117 Berlin, Germany. hess@mpiibberlin.mpg.de
Miko
D
D
Catic
A
A
Lehmensiek
V
V
Russell
D G
DG
Kaufmann
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology; D001500:BCG Vaccine / Q000276:immunology*; D001427:Bacterial Toxins; D002463:Cell Membrane Permeability; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics*; D006460:Hemolysin Proteins; D006461:Hemolysis; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D008089:Listeria monocytogenes; D008264:Macrophages / Q000276:immunology* / Q000382:microbiology; D051379:Mice; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000276:immunology; D010047:Ovalbumin / Q000276:immunology; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
D001500:BCG Vaccine; D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D015395:Histocompatibility Antigens Class I; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010047:Ovalbumin; C038705:hlyA protein, Listeria monocytogenes