Inhibition of macrophage-mediated antigen presentation by hemolysin-producing Listeria monocytogenes

T lymphocytes and macrophages from Listeria-infected mice were used to evaluate the processing and presentation of live Listeria monocytogenes in vitro. Antigen presentation to T cells was quantitated by interleukin-2 production. In contrast to inert antigens such as heat-killed Listeria, live bacteria were processed and presented poorly. To evaluate the role of hemolysin (Hly), we used isogenic pairs of Hly+ and Hly- Listeria as antigens. In contrast to live Hly- bacteria, which were presented as well as heat-killed Listeria, live Hly+ bacteria were presented poorly. Hly+ bacteria also inhibited the presentation of heat-killed Listeria. This effect was apparent with as few as 10 bacteria/macrophage and was not due to loss of macrophage viability or decreased Ia expression after exposure to the live bacteria. With respect to murine listeriosis, the LD50 values for the Hly- strains were at least 1000 times higher than those for the Hly+ strains. These results suggest that the ability of Hly+ bacteria to inhibit antigen processing and presentation may be an important determining factor in Listeria infection and immunity.     

CD40 signaling converts a minimally immunogenic antigen into a potent vaccine against the intracellular pathogen Listeria monocytogenes

Conventional vaccination strategies have failed for numerous pathogens, and the development of novel approaches to vaccine development is a major public health priority. Killed or subunit vaccines represent an attractive approach due to their safety, but they suffer from low immunogenicity and generally require adjuvants. In this study, the possibility of harnessing CD40 signaling for enhancing the immunogenicity of killed vaccines was investigated. Intravenous immunization of C57BL/6 mice with heat-killed Listeria monocytogenes (HKL) induced minimal immunity, but HKL administered together with an agonistic anti-CD40 mAb induced high levels of both CD4(+) and CD8(+) T cells capable of producing IFN-gamma following in vitro HKL stimulation. HKL/anti-CD40 vaccination elicited robust protection against subsequent Listeria challenge. Approximately 1000-fold fewer bacteria were detected in the liver and spleen of vaccinated mice, and vaccinated mice were also able to resist a normally lethal Listeria challenge. CD40-mediated adjuvant activity required endogenous IL-12 at the time of vaccination, and protection was mediated by both CD8(+) and CD4(+) T cells. Thus, CD40 signaling can deliver potent adjuvant activity for vaccination against intracellular pathogens and is particularly effective for pathogens requiring both CD4(+) and CD8(+) T cells for effective control.     

Upregulation of vascular endothelial growth factor by heat-killed Listeria monocytogenes in macrophages

We investigated the effect of heat-killed Listeria monocytogenes (HKLM) on the expression of vascular endothelial growth factor (VEGF) in RAW264.7 macrophage-like cells. The expression of VEGF was induced in RAW264.7 cells treated with HKLM. Pretreatment of cells with cycloheximide, a protein synthesis inhibitor, inhibited the induction of VEGF mRNA by HKLM. Induction of VEGF by HKLM was partially inhibited by treatment of cells with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, or a neutralizing antibody against tumor necrosis factor-alpha (TNF-alpha). In addition, HKLM induced phosphorylation of p38 MAPK. These results suggest that p38 MAPK and TNF-alpha are involved in the VEGF expression induced by HKLM in RAW264.7 cells. We confirmed that increased VEGF expression is immunohistochemically detected in splenic macrophages of mice infected with L. monocytogenes (L. monocytogenes). VEGF is thought to be involved in inflammatory reactions induced by L. monocytogenes infection.     

Biochemistry and biology of antigen presentation by macrophages

The presentation of antigen by macrophages has been studied. We have shown that globular proteins must be processed in endocytic vesicles of low pH prior to presentation. Some of the structural requirements of one such processed peptide have been determined, as has the affinity for Ia of that peptide. Finally, we have shown that a membrane-associated form of interleukin-1 is also required for presentation of processed antigen to T cells.     

Epigallocatechin gallate inhibits intracellular survival of Listeria monocytogenes in macrophages

Epigallocatechin gallate (EGCg), the major tea catechin, is known as a potent anti-microbial and anti-tumor compound. The effects of EGCg on host defense mechanisms against Listeria monocytogenes infection were examined in vitro using mouse peritoneal exudate cells. The study showed that EGCg inhibited the intracellular growth of L. monocytogenes in macrophages. The enhancement of in vitro anti-L. monocytogenes activity by EGCg is not due to the modulation of reactive oxygen intermediates or the production of reactive nitrogen intermediates but due to the inhibition of its escaping from the phagosome into cytosolic space. Anti-L. monocytogenes of EGCg is through the inhibition of hemolytic and cholesterol-binding activity of listeriolysin O, which usually disrupts the phagosomal membrane in the escaping phase of L. monocytogenes.     

Listeria monocytogenes infection overcomes the requirement for CD40 ligand in exogenous antigen presentation to CD8(+) T cells.

In vivo priming of CD8(+) T lymphocytes against exogenously processed model Ags requires CD4(+) T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4(+) T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8(+) T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8(+) T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8(+) T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8(+) T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immunized with L. monocytogenes and OVA-loaded splenocytes, cross-priming of OVA-specific naive CD8(+) T cells occurred. No significant decline in cross-priming against OVA was measured when either TNF or IFN-gamma was neutralized in L. monocytogenes-infected animals, demonstrating that multiple signals exist to overcome CD40L blockade of CD8(+) T cell cross-priming during bacterial infection. These data support a model in which DCs can be stimulated in vivo through signals other than CD40, becoming APC that can effectively stimulate CD8(+) T cell responses against exogenous Ags during infection.     

The sites in the I-Ak histocompatibility molecule photoaffinity labeled by an immunogenic lysozyme peptide

The class II histocompatibilty molecule I-Ak was photoaffinity labeled by NH2- and COOH-terminal photoreactive conjugates of an immunogenic hen egg white lysozyme (HEL) peptide. The labeled alpha and beta chains were digested with protease from Staphylococcus aureus strain V-8 (protease V-8) and/or trypsin, and the proteolytic fragments were separated by high performance liquid chromatography (HPLC) (peptide mapping). Reproducible peptide maps containing a major labeled component were obtained from the three conjugates reported here whose photoreactive group was attached via short spacers of limited flexibility. The COOH-terminal conjugate N-acetyl HEL-(49-61)-iodo-4-azidosalicyloyl thioester (compound 1) labeled hydrophilic tryptic digest fragments on both chains of I-Ak. The labeled digest fragments were homogeneous in reverse-phase and anion-exchange HPLC, indicating that the photoaffinity labeling was site-specific. Conversely, the NH2-terminal conjugate iodo-4-azidosalicyloyl HEL-(46-61) (compound 2: IASA-(46-61)) labeled exceptionally hydrophobic sequences on both chains of I-Ak. The labeling was also site-specific because reverse-phase HPLC of primary digests with protease V-8 and secondary digests with trypsin showed single major labeled components. The labeling of I-Ak by IASA-(46-61) was fully inhibitible by HEL-(46-61). In contrast, IASA attached to the smallest immunogenic peptide 52-61 (compound 3) labeled a distinctly different hydrophilic tryptic fragment. The site of the I-Ak molecule that was photoaffinity labeled by IASA-(46-61) (compound 2) was determined. IASA-(46-61) labeled selectively at Pro-118 of a primary alpha chain fragment most likely encompassing residues 115-134. It labeled Thr-121 of a primary beta chain fragment most likely encompassing residues 109-138. We also obtained evidence that IASA-(46-61) occupied the antigen-specific site; the conjugate stimulated a T-cell hybridoma that recognizes the sequence 52-61 and also competed for the binding of this smaller peptide to I-Ak. Thus, peptides that bind to the allele-specific binding site and are long enough to extend beyond it can interact with a hydrophobic area of class II molecules. This area is formed by sequences of the first halves of the second domain of both alpha and beta chains.     

Defensins enable macrophages to inhibit the intracellular proliferation of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that infects a large diversity of host cells, including macrophages. To avoid the phagosome microbicidal environment, L. monocytogenes secretes a pore-forming toxin (listeriolysin O, LLO) that releases the bacterium into the cytoplasm. We hypothesized that the α-defensins (HNPs) and/or humanized θ-defensin (RC-1) peptides produced by human and non-human primate neutrophils, respectively, cooperate with macrophages to control L. monocytogenes infection. Our results establish that HNP-1 and RC-1 enable macrophages to control L. monocytogenes intracellular growth by inhibiting phagosomal escape, as a consequence, bacteria remain trapped in a LAMP-1-positive phagosome. Importantly, HNP-1 interaction with macrophages and RC-1 interaction with bacteria are required to prevent macrophage infection. In accordance with these results, RC-1 is a more potent anti-listerial peptide than HNP-1 and HNP-1 is acquired by macrophages and trafficked to the phagocytosed bacteria. Finally, HNP-1 and RC-1 antimicrobial activity is complemented by their ability to prevent LLO function through two mechanisms, blocking LLO-dependent perforation of macrophage membranes and the release of LLO from the bacteria. In conclusion, at the site of infection the cooperation between antimicrobial peptides, such as HNP-1, and macrophages likely plays a critical role in the innate immune defence against L. monocytogenes.     

Low-level iron-dependent mutants of Listeria monocytogenes and their virulence in macrophages

Listeria monocytogenes is an opportunistic intracellular pathogen capable of growth that requires iron for growth within phagocytic cells and virulence expression. In the presence of an appropriate concentration tropolone, an iron-chelating agent, growth of L. monocytogenes is completely inhibited. However, this inhibition can be relieved by addition of dopamine, norepinephrine, or ferric citrate. By selection on streptonigrin medium supplemented with tropolone and norepinephrine, we have obtained two spontaneous mutants, Lm-8 and Lm-15, with the same iron dependence but lower iron dependence than the wild-type Lm-B38. The association between iron requirement and virulence of the two mutants and the wild type was studied in the J774 macrophage cell line. One hour after phagocytosis by the J774 macrophage cell line, the two mutants and the parental strain displayed no difference in the number of phagocytosed bacteria. Twenty-four hours after phagocytosis, the number of bacteria within the surviving macrophages was identical for the wild strain and the two clones. However, only 40% of macrophage cells infected with Lm-8 and 90% of those infected with Lm-15 were alive after 24 h in comparison with macrophage cells infected with the parental strain Lm-B38. These data demonstrate that there is no direct correlation between iron requirement and virulence of L. monocytogenes in the J774 macrophage cell line.     

Cytolysin-dependent delay of vacuole maturation in macrophages infected with Listeria monocytogenes

The bacterial pathogen Listeria monocytogenes (Lm) evades the antimicrobial mechanisms of macrophages by escaping from vacuoles to the cytosol, through the action of the cytolysin listeriolysin O (LLO). Because of heterogeneities in the timing and efficiency of escape, important questions about the contributions of LLO to Lm vacuole identity and trafficking have been inaccessible. Expression of cyan fluorescent protein (CFP)-labelled endocytic membrane markers in macrophages along with a yellow fluorescent protein (YFP)-labelled indicator of Lm entry to the cytosol identified compartments lysed by bacteria. Lm escaped from Rab5a-negative, lysosome-associated membrane protein-1 (LAMP1)-negative, Rab7-positive, phosphatidylinositol 3-phosphate [PI(3)P]-positive vacuoles. Lm vacuoles did not label with YFP-Rab5a unless the bacteria were first opsonized with IgG. Wild-type Lm delayed vacuole fusion with LAMP1-positive lysosomes, relative to LLO-deficient Lm. Bacteria prevented from expressing LLO until their arrival into LAMP1-positive lysosomes escaped inefficiently. Thus, the LLO-dependent delay of Lm vacuole fusion with lysosomes affords Lm a competitive edge against macrophage defences by providing bacteria more time in organelles they can penetrate.     

Differential antigen presentation by cloned populations of mouse splenic macrophages

Soft agar colonies of mouse splenic macrophages differ in their ability to process and present complex Ag to T cell hybridomas. To determine if the basis for this differential activity was the synthesis of molecules that might interfere with the activity of either the hybridoma or the indicator cells used for the bioassay of IL-2, culture supernatants were compared from Ag-presenting and nonpresenting cultures for their content of suppressor activity, using mitogen-treated mouse SC. No correlation was found between a colony's Ag-presenting activity and its secretion of suppressor factors, nor did colonies unable to present Ag release factors that interfered with the detection of IL-2. In a second approach, paired subcultures from individual colonies were tested for their ability to present, to the same hybridoma, both native Ag and the "preprocessed" peptide of the Ag. The presentation of native Ag was restricted to the progeny of a minority of the cloned macrophage progenitors, but all of the progeny cultures presented the peptide. Together, these results suggest that the basis for differential Ag presentation may be in the manner in which the cloned macrophages degrade and process ingested Ag.     

Acquired cellular immunity: extracellular killing of Listeria monocytogenes by a product of immunologically activated macrophages

A listericidal factor was released by monolayers of peritoneal cells from BCG-immune guinea pigs following incubation with PPD in cell culture. Significantly less listericidal factor was released by monolayers of BCG-immune cells incubated without PPD, or by monolayers of nonimmune cells incubated in the presence or absence of antigen. Culture supernatants containing listericidal factor were active at a dilution of 1:10, but supernatants diluted 1:100 were not listericidal. Dialysis of supernatants against fresh tissue culture medium did not affect their ability to kill Listeria, although heating at 60 °C for 30 min destroyed all activity. Supernatants from cultures of guinea pig fibroblasts or ascites-form hepatoma were not listericidal.     

The Listeria monocytogenes DnaK chaperone is required for stress tolerance and efficient phagocytosis with macrophages

Listeria monocytogenes is a facultative intracellular pathogen which can escape bactericidal mechanisms and grow within macrophages. The intracellular environment of macrophages is one of the most stressful environments encountered by an invading bacterium during the course of infection. To study the role of the major stress protein, DnaK, of L. monocytogenes in survival under intracellular stress induced by macrophage-phagocytosis as well as under extracellular environmental stresses, we cloned, sequenced, and analyzed the dnaK locus from L. monocytogenes. Then we constructed an insertional mutation in the dnaK gene by homologous recombination and characterized it. Sequencing has revealed that the dnaK locus consists of four open reading frames in the order hrcA-grpE-dnaK-dnaJ. The mutant grows neither at temperatures above 39 degrees C nor under acidic conditions e.g. pH 3.0. Using the macrophage cell line JA-4, the ability of the dnaK mutant to grow intracellularly was examined. Immediately after phagocytosis, the number of viable dnaK mutant bacteria found within macrophages was significantly lower compared to that of intracellular wild type bacteria. However, following a 1-3 h latency period, the mutant multiplied in a similar fashion to the wild type within macrophage cells. A quantitative analysis of intracellular bacteria in macrophage cells by microscope and a binding assay of bacteria to the surface of macrophages by ELISA revealed that the lower number of viable dnaK mutant in macrophages after phagocytosis is due to the low efficiency of phagocytosis resulting from the reduced binding capacity of the dnaK mutant. These results demonstrate that DnaK of L. monocytogenes is essentially required for survival under high temperatures and acidic conditions. Though it does not largely contribute to the survival of L. monocytogenes in macrophage cells, it is essential for efficient phagocytosis. This is the first evidence that DnaK is required for the efficient phagocytosis of a facultative intracellular pathogen with macrophages.     

PCR技术检测单核细胞增生李斯特氏菌研究进展

单核细胞增生李斯特氏菌(Listeria monocytogenes)是危害公共卫生和食品行业的一种重要人畜共患病致病菌.PCR技术是近年来被广泛地运用到食源性致病菌快速检测的分子生物学方法之一.就PCR技术检测单核细胞增生李斯特氏菌中的特异性鉴别基因、模板DNA制备等关键因素及多重PCR、巢式PCR、反转录PCR与定量PCR等主要技术进行了综述.     

Listeria monocytogenes protein p60 affects hemolytic activity and uptake of bacteria by macrophages

Bacillus subtilis strains expressing listeriolysin O (LLO) and simultaneously LLO and p60 protein were constructed. The effect of p60 protein on hemolytic activity and on the invasion of professional phagocytes was demonstrated in the absence of other virulence factors of L. monocytogenes. The hemolytic activity of LLO in the presence of p60 protein decreased which indicates that p60 promoted adhesion and subsequent invasion of professional phagocytes.