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Listeria monocytogenes is a food-borne pathogen able to cause serious disease in human and animals. Listeriolysin O (LLO), a major virulence factor secreted by this bacterium, is a vacuole-specific lysin that facilitates bacterial entrance into the host cytosol. Thus, LLO plays a key role in the translocation and intracellular spread of L. monocytogenes. To study the effect of LLO on virulence and immunopotency, a LLO-deficient L. monocytogenes mutant was constructed using a shuttle vector followed by homologous recombination. The mutant strain had lost hemolytic activity, which resulted in an extremely reduced virulence, 5 logs lower than that of the parent strain, yzuLM4, in BALB/c mice. The number of bacteria detected in the spleens and livers of mice infected with the mutant was greatly reduced, and the bacteria were rapidly eliminated by the host. Kinetics studies in this murine model of infection showed that the invasion ability of the mutant strain was much lower than that of the parent strain. Moreover, immunization with the mutant strain conferred protective immunity against listerial infection. In particular, stimulation with Ag85B240-259, strong specific Th1 type cellular immunity was elicited by vaccination C57BL/6 mice with hly deficient strain delivering Mycobacterium tuberculosis fusion antigen Ag85B-ESAT-6 via intravenous inoculation. These results clearly show that highly attenuated LLO-deficient L. monocytogenes is an attractive vaccine carrier for delivering heterologous antigens.
L. monocytogenes is an important food-borne intracellular pathogen that infects many types of animals and more than 17 avian species. Listeriosis can be life-threatening in neonates, pregnant women, elderly persons, and the immunosuppressed patients (1). About 2,500 cases of food-borne disease caused by L. monocytogenes are reported annually in the USA (2), and L. monocytogenes has become a major public health concern in developing countries as well. Consequently, research efforts have been aimed at developing an attenuated live vaccine to prevent listeriosis. Listeria has the unusual ability to escape from the phagosome of infected cells and to proceed with replication in the cytosol. Since both CD4+ and CD8+ T-cells are key elements in Listeria-specific immunity (3), there has been much interest in exploiting the bacteria as a highly attractive vaccine carrier for the presentation of passenger antigens to the class I and II pathways of the major histocompatibility complex (MHC) (4, 5).
As a food-borne pathogen transported through the gastrointestinal tract, L. monocytogenes crosses the intestinal barrier and, via the lymph and blood, is disseminated hematogenously throughout the body. Each of these activities is dependent upon the production of bacterial virulence factors. The major virulence genes (hly, plcA, plcB, mpl, actA, inlA, and inlB) cluster in two distinct loci on the bacterial chromosome (6, 7). Among these genes, hly encodes the major virulence factor listeriolysin O (LLO), a 529-amino-acid protein with hemolytic activity and a member of the cholesterol-dependent cytolysin family of pore-forming proteins. LLO plays a crucial role in the escape of L. monocytogenes from both primary and secondary phagosomes and in multiplication of the bacterium in the host cytosol (8).
In the present study, we used L. monocytogenes serotype1/2a strain yzuLM4, isolated from goat, to generate a mutant strain with a defined in-frame deletion in hly. The virulence of this mutant, designated yzuLMΔhly, was attenuated compared to the wild-type strain. Interestingly, however, the mutant retained the ability to induce immune responses in mice challenged with the homologous strain yzuLM4 or the heterologous strain yzuF36. The attenuated mutant strain could have very important applications in the prevention of listeriosis and provides a solid foundation for the construction of a vaccine vector to prevent human and animal diseases. Furthermore, it can be used to elucidate the mechanisms of pathogenesis of L. monocytogenes virulence factors and of the host's immune response to the bacterium.
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- MATERIALS AND METHODS
Listeriosis is primarily an infectious disease of pregnant women, neonates and infants, elderly individuals, and the immunocompromised patients. Thus, it is paramount that Listeria vaccine strains to be used in these susceptible population should be sufficiently attenuated so as not to pose a risk of infection (18). A number of highly attenuated mutant strains of L. monocytogenes have been developed for potential use as vaccines. Virulence genes, including prfA, actA, hly, plcB, plcA, plcA/plcB, inlA, inlB, and inlAB, have been deleted from the chromosome to generate the corresponding mutant strains (6). While the LD50 values of these mutants are lower than those of the wild-type strain, the virulence of the mutants differs. Hohmann deleted two genes (actA and plcB) from L. monocytogenes strain10403S, and the lgLD50 of this mutant was 7.60 (19). Jiang deleted actA and hly and the lgLD50 of the resulting mutant strains were 7.44 and 4.19, respectively (20). The whole hly gene deleted strain named DP-L2161 was reported by D.A. Portnoy in 1994 (LD50 to BALB/c mice is 2 × 109) (21), and by using this strain Harty JT's research work verified that the LLO-deficient strain can produce long-term protective immunity(22). Another whole gene deleted strain named Δhly L.monocytogenes was reported by Mitsuyama M, however, the result of their study revealed only transient protection (23). In the current study, a different construction strategy from other reports is in that the mutant strain yzuLM4Δhly was part-gene deleted, but the virulence is highly reduced (LD50 to BALB/c mice is 2.15 × 109), which is in accordance with the results of Portnoy (21, 24). The significant decrease in bacterial toxicity means increased safety to human and animals (25).
In the present study, a 1,150-bp DNA fragment encoding the first three domains of hly had been deleted by using homologous recombination method, so the remaining sequence of hly was unable to express a truncated LLO-d123, but the preserved signal peptide of LLO can still lead to the expression of the last domain LLO-d4. Dubail I's research work verified that this domain is able to bind to cell membrane (26), but whether this truncted protein is helpful to protective immunity is unknown. Our study showed that the hemolytic activity of LLO in supernatant obtained from mutant L. monocytogenes was lost, with subsequent effects on pathogenicity. This was verified by assaying invasion and multiplication in vitro and in vivo. The LD50 for △hly strain infection in mice reflected the significant decrease in the mutant's toxicity, while the kinetics of infection confirmed the reduction in invasiveness. LLO-d123 has been suggested to participate in lysis of the phagosomal membrane (26), therefore, this domain of the protein plays a crucial role in invasion, multiplication, and translocation of L. monocytogenes.
Food-borne listeriosis has become a great concern to public health and to the food industry due to a number of outbreaks and sporadic cases of L. monocytogenes infections arising from foods contaminated with the bacterium. Serotypes 4b, 1/2a, 1/2b and 3b have been found to be responsible for more than 90% of the listeriosis outbreaks that have occurred in the past 2 decades, with serotype 4b strains accounting for approximately 40% of the listeriosis (27, 28). In our study, mice immunized with the mutant strain had 100% protective immunity after challenge with a lethal dose of the homologous strain (yzuLM4) or of the heterologous strain yzuF36. This result suggests the LLO-deficient strain can be used as a vaccine to induce potent protective immunity. The result is in accordance with Koenig's study (29), which showed protective immunity between different L. monocytogenes serotypes. Specifically, mice experimentally infected with L. monocytogenes serotypes 1/2b, 3a, 4b, and 4d were protected against a lethal challenge with the most virulent serotype 4b. The mechanism that the LLO-deficient strain can confer protective immunity is worth studying in the future.
Ag85B contians a large number of B and T cell epitopes of Mycobacterium tuberculosis, which can elicit protective immunity, so it is an attractive antigen target for the development of new TB vaccine (30). In this study the peptide Ag85B240-259, an immune dominant H-2b antigen from Mycobacterium tuberculosis (31), was used to evaluate the triggered protective antigen-specific T cell response. Because antigen specific T-cells can occur at a low frequency in vivo, increased sensitivity of ELISPOT is particularly advantageous when studying T-cell-mediated responses in pathogen infection. Our result showed that following the peptide Ag85B240-259 stimulation, higher level of IFN-γ-secreting cells than IL-4-secreting cells was induced (P < 0.01) in the spleen of mice immunized with recombinant strain LM(Ag85B-ESAT-6). This indicated that a predominantly Th1 response was elicited. Th1 cells produce IFN-γ, TNF-β and IL-2, and are responsible for directing cell-mediated immune responses leading to the eradication of intracellular pathogens. We found that LLO deficient L.monocytogenes still keeps the ability to trigger secondary expansion of antigen specific T cells and confer long-term protection to challenge by wild type L. monocytogens. Our results are also consistent with those from Harty JT et al. (22) and Way S.S et al. (32), which suggest that highly attenuated LLO-deficient L. monocytogenes is an attractive vaccine carrier for delivering heterologous antigens and can be used in susceptible populations to prevent listeriosis and potentially, tumors or diseases caused by other infectious pathogens.
In conclusion, we have successfully constructed a mutant strain yzuLMΔhly with much lower virulence and thus greater safety than the wild-type strain. The mutant retains immunopotency with respect to inducing protective immunity against homologous and heterologous L. monocytogenes infections and is thus of potential interest as a candidate vaccine to prevent listeriosis, and as a vaccine vector in the delivery of heterologous antigens. Furthermore, it provides several possibilities to elucidate the mechanisms of pathogenesis by L. monocytogenes virulence factors and of the immune response to them.