MYCOBACTERIUM-BOVIS BACILLE CALMETTE-GUERIN STRAINS SECRETING LISTERIOLYSIN OF LISTERIA-MONOCYTOGENES

Recombinant (r) Mycobacterium bovis strains were constructed that secr ete biologically active listeriolysin (Hly) fusion protein of Listeria monocytogenes, The r-BCG strains pAT261:Hly or pMV306:Hly expressed p lasmid multicopies or chromosomal single copies of the hly gene, respe ctively, Human and murine macrophage-like cell lines were infected wit h r-BCG pAT261:Hly and pMV306:Hly strains. Interestingly, intracellula r persistence of both r-BCG strains was reduced in macrophages as comp ared with the parental BCG strain, By immunogold labeling Hly was dete cted in membrane structures and within the phagosomal space of macroph ages, In addition, Hly was localized within cytoplasmic vacuoles outsi de the mycobacteria-containing phagosome of host cells infected with r -BCG pAT261:Hly or r-BCG pMV306:Hly, Hly fusions consistently colocali zed with a lysosome-associated membrane glycoprotein, suggesting that membrane-attack conformation of Hly was not altered, Although r-BCG pA T261:Hly and r-BCG pMV306:Hly microorganims apparently did not egress into the cytoplasmic compartment of host cells, they both improved maj or histocompatibility complex class I presentation of cophagocytosed s oluble protein as compared with wild-type BCG microbes. These data sug gest that Hly secretion endows BCG with an improved capacity to stimul ate CD8 T cells. Because CD8 T cells play a major role in protection a gainst tuberculosis such Hly secreting r-BCG constructs are antituberc ulosis vaccine candidates.