INFECTION AND IMMUNITY, Apr. 1992, p. 1625-1632 0019-9567/92/041625-08$02.00/0 Copyright X 1992, American Society for Microbiology

Vol. 60, No. 4

Pathogenicity and Immunogenicity of Listeria monocytogenes Small-Plaque Mutants Defective for Intracellular Growth and Cell-to-Cell Spread RONALD A. BARRY,1* H. G. ARCHIE BOUWER,"12 DANIEL A. PORTNOY,3 AND DAVID J. HINRICHS"12 Immunology Research, Veterans Administration Medical Center, Portland, Oregon 97207'; Earle A. Chiles Research Institute, Portland, Oregon 972132; and Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 191043

Received 8 November 1991/Accepted 22 January 1992

Listeria monocytogenes strains previously generated by transposon mutagenesis were examined with respect to virulence and induction of protective immunity in BALB/c mice. The phenotypic defects observed in these mutant L. monocytogenes strains included decreased hemolysin (listeriolysin 0 [LLO]) production, phospho-

lipase C activity, intracellular growth, and/or cell-to-cell spread in vitro. While 50%o lethal dose determinations performed with these mutant strains indicated reduced virulence for BALB/c mice, sublethal infection with the majority of these mutant strains provided protection against a subsequent challenge with the fully virulent L. monocytogenes parent strain. In addition, in vitro infection of the J774 cell line with most of these mutant strains converted these phagocytic cells to targets of L. monocytogenes-immune cytotoxic cells. The exceptions to these findings were two LLO-negative, avirulent mutant strains which were unable to immunize mice against a secondary challenge with virulent L. monocytogenes. One of these mutants contained a transposon insertion within the structural gene for LLO, and the other contained a transposon insertion in the structural gene for the transcriptional activator of the LLO gene. These two LLO-negative mutant strains also were unable to escape phagolysosomes in infected J774 cells and could not transform these phagocytic cells into targets of L. monocytogenes-immune cytotoxic cells. These findings confirm the importance of LLO in the induction of antilisterial immunity and suggest that a cytoplasmic localization of these pathogenic bacteria is required for the development of protective immunity. The development of specific cell-mediated immunity to Listeria monocytogenes is dependent on sublethal infection with viable forms of this bacterial pathogen. Immunization with inactivated (killed) forms of virulent strains does not provide protection against virulent strains (35, 38, 52). This form of immunity is dependent on the ability of the host to withstand the intracellular replication of bacteria and is mediated by immune T lymphocytes, which function to activate macrophages to increased bactericidal levels (2, 33, 35, 36, 38, 42). This activation apparently is mediated by lymphokines, especially gamma interferon, released following the recognition of listerial antigens associated with restrictive major histocompatibility complex molecules on antigen-presenting cells (26, 27, 32). Adoptive transfer studies have implicated both antigenspecific CD4+ and CD8+ T lymphocytes as cellular components in the expression of cell-mediated antilisterial immunity in experimental hosts (13, 14, 16, 30, 31, 40). Recent studies have indicated that the expression of delayed-type hypersensitivity to listerial antigens is primarily dependent on CD4+ immune T cells, while protection against viable L. monocytogenes challenge is primarily dependent on CD8+ immune T cells (1, 7, 14, 16). A cytotoxic role for the CD8+ immune T cell subset has been implicated in recent studies demonstrating cytotoxic activity of (28) or lymphokine (gamma interferon) release by (10) these cells following the recognition of L. monocytogenes-infected target cells. These findings suggest that this immune T cell subset also may play a vital role in the recognition of infected host cells, which

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serve as a reservoir for L. monocytogenes replication in vivo; this recognition event may be essential in resolving infection with virulent strains of L. monocytogenes. In contrast, specific humoral (antibody-mediated) responses play little or no role in the development and expression of immunity to this pathogen (22, 35, 38, 44). Several determinants of pathogenicity have been associated with virulent strains of L. monocytogenes (reviewed in reference 45). One important determinant is the elaboration of a sulfhydryl-activated hemolysin, called listeriolysin 0 (LLO) (20, 21, 23, 46). The production of this pore-forming hemolysin has proven to be an essential determinant of L. monocytogenes pathogenicity (15). In vitro studies have indicated that this molecule is required for the escape of bacteria from phagolysosomes (6), which is required for subsequent intracellular replication and direct cell-to-cell spread of this pathogen in cultured cell monolayers (17, 19, 39, 51). Recently, a protocol involving transposon mutagenesis was used to select mutant strains of L. monocytogenes which are defective in intracellular growth and/or cell-to-cell spread in cultured cell monolayers (50). This protocol produced several classes of mutant strains which are defective in LLO production, the cytoplasmic induction of actin polymerization, and/or phospholipase activity. The development of these mutants allows for investigation of the influence of potential virulence factors on the development and expression of an antilisterial immune response. In this report, we evaluated the various mutants as to in vivo pathogenicity, immunogenicity, and the ability to infect mammalian cells in vitro and render them recognizable by L. monocytogenes-immune cytotoxic cells. Our results suggest

Corresponding author. 1625

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BARRY ET AL.

INFEcr. IMMUN.

TABLE 1. Summary of characteristics and virulence of selected L. monocytogenes mutant strains

Strain Lm1O403S DP-L967 DP-L1154 DP-L10349 DP-L1044 DP-L758 DP-L793 DP-L973 DP-L995 DP-L1049 DP-L1054 DP-L867 DP-L1107

LLO

Classa

(U/mlp)

Phospholipase C

activityc

growthd

1 1 2 2 3 3 3 3 4 5

80 80 80

Pathogenicity and immunogenicity of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread.

Listeria monocytogenes strains previously generated by transposon mutagenesis were examined with respect to virulence and induction of protective immu...
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