Recombinant Listeria monocytogenes Expressing a Cell Wall-Associated Listeriolysin O Is Weakly Virulent but Immunogenic

Listeriolysin O (LLO) is an essential virulence factor for the gram-positive bacterium Listeria monocytogenes. Our goal was to determine if altering the topology of LLO would alter the virulence and toxicity of L. monocytogenes in vivo. A recombinant strain was generated that expressed a surface-associated LLO (sLLO) variant secreted at 40-fold-lower levels than the wild type. In culture, the sLLO strain grew in macrophages, translocated to the cytosol, and induced cell death. However, the sLLO strain showed decreased infectivity, reduced lymphocyte apoptosis, and decreased virulence despite a normal in vitro phenotype. Thus, the topology of LLO in L. monocytogenes was a factor in the pathogenesis of the infection and points to a role of LLO secretion during in vivo infection. The sLLO strain was cleared by severe combined immunodeficient (SCID) mice. Despite the attenuation of virulence, the sLLO strain was immunogenic and capable of eliciting protective T-cell responses.Listeria monocytogenes is a gram-positive facultative intracellular pathogen extensively used to understand host-pathogen interactions (44, 51, 53). It expresses the highly conserved pore-forming toxin listeriolysin O (LLO), a member of a large family of cholesterol-dependent cytolysins found in many important pathogens (11, 33, 50). LLO is required for L. monocytogenes virulence both in vivo and vitro. L. monocytogenes genetically deficient in LLO (Δhly) is incapable of any growth in vivo or inducing protective immunity except at extremely high infectious doses (27). This finding supports the relationship between virulence and development of strong protective immunity.The effect of LLO on cells is multifaceted. It induces calcium fluxes at the cell surface (16, 46), cytokine production by splenocytes (40), apoptosis of dendritic cells and T lymphocytes (8, 26), and Toll-like receptor 4 signaling (43). LLO is also a source of T-cell epitopes of L. monocytogenes in multiple major histocompatibility complex (MHC) haplotypes (22, 52). Most studies on LLO have focused on its ability to perforate the phagolysosome at acidic pH, which allows L. monocytogenes to enter the cytosol of the infected phagocytic cell (24, 45). The Δhly strain does not cross from the phagosome to the cytosol and is effectively killed (20, 45). L. monocytogenes constructed to express variable levels of LLO protein in vitro has shown a dose threshold that must be reached for entry into the cytosol (14). There is recent work that suggests that low levels of LLO secretion can lead to maintenance of L. monocytogenes in vacuoles, suggesting a possible mechanism for granuloma formation and persistent infection in immunocompromised strains of mice (6).During infection with L. monocytogenes, there is a phase of lymphocyte apoptosis in the spleen that peaks at the second day of the infection (30, 34). Apoptosis is dependent on the level of infection with L. monocytogenes. High doses of LLO-deficient or heat-killed L. monocytogenes do not induce the lesions, demonstrating that live, virulent infection is required for their development. Induction of lymphocyte apoptosis is immunomodulatory and a major determinant in the virulence of L. monocytogenes infection (11). For example, mice deficient in type I interferon (IFN) receptor signaling are more resistant to listeriosis and have decreased levels of lymphocyte and macrophage apoptosis (2, 10, 41). Tumor necrosis factor (TNF)-related apoptosis-inducing ligand^−/− (TRAIL) mice are also more resistant to infection and have less apoptosis (54), while liver X receptor^−/− (LXR) mice have increased macrophage apoptosis and are more susceptible to listeriosis (32). And pointedly, mice deficient in lymphocytes have no detectable splenic apoptosis and are more resistant early after infection than conventional mice (3, 9).Our interpretation on the pathogenesis of the lesions is that LLO released extracellularly during the strong exponential phase of growth is central for their development. Treatment of cultured T cells at neutral pH with sublytic nanomolar and subnanomolar doses of LLO can induce apoptosis (8). The apoptosis induced by LLO on activated T cells is highly dependent on granzyme expression within the affected cell (12). Nonactivated lymphocytes can be sensitized to the apoptotic effect of LLO by treatment with type I IFN (10).To examine the possible role of LLO as an extracellular protein, we engineered a recombinant strain of L. monocytogenes in which the LLO protein is covalently linked to the bacterial cell wall (surface-associated LLO [sLLO]). The sLLO strain expresses wild-type levels of bacterially associated LLO but secretes 40-fold less total protein. The growth, pathogenicity, and inflammatory properties of the sLLO strain are severely attenuated in vivo. Importantly, there is reduced apoptosis in the infectious foci following infection with the sLLO mutant. This strain is also capable of eliciting memory T-cell responses and protective immunity.