Autolysin amidase of Listeria monocytogenes promotes efficient colonization of mouse hepatocytes and enhances host immune response

International Journal of Medical Microbiology 301 (2011) 480–487

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Autolysin amidase of Listeria monocytogenes promotes efficient colonization of mouse hepatocytes and enhances host immune response Krisana Asano a , Hiroshi Sashinami a , Arihiro Osanai a , Yoshiya Asano b , Akio Nakane a,∗ a b

Dept. of Microbiology and Immunology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562, Japan Dept. of Neuroanatomy, Histology and Cell Biology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562, Japan

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Article history: Received 5 August 2010 Received in revised form 24 December 2010 Accepted 6 January 2011 Keywords: Listeria monocytogenes Autolysin amidase Adhesion Hepatocytes Peptidoglycan hydrolase Host immune response

a b s t r a c t Listeria monocytogenes is an intracellularly growing pathogen which is able to infect and to spread from cells to cells. It produces several virulence factors required for invasion and intracellular niche colonization. Endogenous peptidoglycan hydrolases which are important for survival of bacteria have been shown to be involved in pathogenesis. An autolysin amidase (Ami)-deficient mutant of L. monocytogenes (ami) is attenuated in virulence as evidenced by a reduction in mortality of infected mice. We showed that Ami is not essential for bacterial growth and protein secretion. Histopathological analysis suggests that Ami promotes bacterial colonization of hepatocytes. By using cultured eukaryotic cells, we present evidence that a critical function of Ami in pathogenesis is to promote an efficient listerial adherence and internalization into mouse hepatocytes. Simultaneously, the peptidoglycan hydrolase activity of Ami linked to the release of immunologically active cell wall components enhances production of tumor necrosis factor (TNF)-␣ and interleukin 6. In the early phase of infection, interferon-␥ and TNF-␣ production of ami-infected mice is significantly less than that of wild-type controls, suggesting a contribution of Ami to enhance the host innate immune response to listerial infection. © 2011 Elsevier GmbH. All rights reserved.

Introduction Listeria monocytogenes is a Gram-positive intracellular pathogen that is environmentally widespread and causes severe food-borne infections in humans and animals (Farber and Peterkin, 1991). It is able to invade a wide range of cell types, including macrophages, hepatocytes, enterocytes, epithelial cells, and endothelial cells. After entry into the host cells, L. monocytogenes lyses the phagosomal vacuole and is released into the cytoplasm (Vazquez-Boland et al., 2001). It then replicates and spreads into adjacent cells by mediating actin assembly (Tilney and Portnoy, 1989). Cellular internalization of this bacterium requires invasive proteins, internalin (Inl) A and InlB (Braun et al., 1997; Gaillard et al., 1991). To escape from the phagocytic vacuoles, this bacterium produces listeriolysin O (LLO) (Portnoy et al., 1988) and phospholipase C (Poussin and Goldfine, 2005; Gründling et al., 2003). This bacterium also produces ActA, a protein required for actin-based motility to spread from cell to cell (Kocks et al., 1992). Several autolysins of L. monocytogenes have been identified and characterized such as p60, NamA, Auto, IspC, and Ami (Wang and Lin, 2008; Cabanes et al., 2004; Popowska, 2004). They are peptidoglycan (PG) hydrolases that are capable of cleaving the covalent

∗ Corresponding author. Tel.: +81 172 39 5033; fax: +81 172 39 5034. E-mail address: [email protected] (A. Nakane). 1438-4221/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2011.01.002

bonds in the cell wall PG. Their biological functions include cell division, protein secretion, and cell wall turnover. Autolysins have been shown to be required for bacterial virulence (Lenz et al., 2003). Contribution of autolysins in pathogenesis is tentatively linked to maintenance of bacterial cell surface architecture and release of immunologically active cell wall components (Canvin et al., 1995; Díaz et al., 1992). Some of them are shown to be required for host cell invasion and intracellular motility process (Cabanes et al., 2004; Pilgrim et al., 2003; Hess et al., 1996). Ami is a 99-kDa amidase protein exhibiting N-acetylmuramoyll-alanine amidase activity (McLaughlan and Foster, 1998). Its N-terminal domain is similar to the alanine amidase domain of Alt autolysin of Staphylococcus aureus and responsible for cleavage of the bond between N-acetylmuramic acid and l-alanine in the cell wall PG. Its C-terminal cell wall anchoring (CWA) domain (amino acids 262–917) contains 8 glycine/tryptophan (GW) modules. The C-terminal region of Ami is homologous to the C-terminal domain of InlB, which is made up of 3 GW modules (Braun et al., 1997). Both InlB and Ami associate to the bacterial surface by their conserved GW modules, which bind lipoteichoic acid (Braun et al., 1997; Jonquières et al., 1999). GW modules of the InlB have been shown to bind to cellular matrix proteoglycans (Marino et al., 2002; Jonquières et al., 2001). Thus, it is possible that Ami GW modules exert a similar function. CWA domain of Ami is shown to mediate bacterial adhesion to several human cells (Milohanic et al., 2001). However, the mechanism that

K. Asano et al. / International Journal of Medical Microbiology 301 (2011) 480–487

underlies the involvement of Ami in virulence is not fully understood. In this study, functions of Ami possibly contributing to pathogenesis of L. monocytogenes were investigated including bacterial growth, protein secretion, adhesive and invasive activities to eukaryotic cells, and release of immunologically active cell wall components. We showed that Ami is not necessary for bacterial growth and protein secretion. A critical role of Ami in pathogenesis is to promote bacterial colonization to hepatocytes. Simultaneously, Ami releases immunologically active cell wall components. Ami enhances host immune response to L. monocytogenes infection in mice. Materials and methods Bacterial strains, plasmids, and growth conditions L. monocytogenes 1b 1684 wild-type (WT) (Nakane et al., 1988) and Ami-deficient mutant (ami) were grown in brain heart infusion broth (BD Biosciences, Sparks, MD) or tryptic soy broth (BD Biosciences) at 37 ◦ C. A temperature-sensitive shuttle vector, pAULA, was kindly provided by Trinad Chakraborty, University Teaching Hospital of Giessen, Germany. It was used to construct an ami-inactivated mutant of L. monocytogenes (Chakraborty et al., 1992). Plasmid pJEBAN3 encoding yellow fluorescent protein (YFP) was electroporated into L. monocytogenes to give yellow fluorescent strain (Andersen et al., 2006). L. monocytogenes harboring pAULA and pJEBAN3 plasmid was cultured in tryptic soy broth supplemented with 5 and 10 ␮g/ml erythromycin (Wako Pure Chemical Industries, Osaka, Japan), respectively. Construction of Ami-deletion mutant of L. monocytogenes DNA fragments containing upstream and downstream regions (0.5 kb each) of the ami gene (lmo2558) were amplified by PCR using genomic DNA of L. monocytogenes as template. The upstream and downstream fragments were cloned into the pAULA plasmid using KpnI & BamHI and BamHI & SalI, respectively. To achieve allelic exchange, the plasmid was electroporated into L. monocytogenes at 1250 V/mm with time constant at 5 ms. A deletion mutant was selected from transformants using non-permissive temperature growth as described previously (Schäferkordt and Chakraborty, 1995). Deletion of the ami gene from the erythromycin-sensitive clones was analyzed by PCR. Non-existence of Ami was confirmed by immunodetection using anti-Ami antibodies produced in our laboratory. Mice C57BL/6 mice were purchased from Clea Japan Inc., Tokyo, Japan, and cared for under specific-pathogen-free conditions in the Institute for Animal Experimentation, Hirosaki University Graduate School of Medicine. All animal experiments in this study were performed following the Guidelines for Animal Experimentation of the Hirosaki University. Cell lines and culture conditions Mouse macrophage cell line RAW264.7 cells and mouse hepatocyte cell line NMuLi cells (Dainippon Sumitomo Pharma, Osaka, Japan) were cultured in Dulbecco’s modified Eagle medium (Nissui Pharmaceutical Co., Tokyo, Japan), supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS), and 0.03% of l-glutamine (Wako). Mouse dendritic cell line DC2.4 cells (Shen et al., 1997) were provided by Dr. T. Ohteki, Dept. of Biodefense Research, Medical Research Institute, Tokyo Medical and Dental

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University, Tokyo, Japan, after approval from Dr. K. Rock, Dept. of Pathology and Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA, USA. They were cultured in RPMI1640 medium (Nissui) supplemented with 10% fetal bovine serum, 0.03% of l-glutamine, and 50 ␮M 2-mercaptoethanol (Wako). All cells were grown at 37 ◦ C under 5% CO2 . L. monocytogenes infection Mice were infected intravenously with 5 × 106 CFU of L. monocytogenes WT or ami. Survival of mice was observed, or the number of viable bacteria in liver and spleen was determined as previously described (Sashinami et al., 2005). RAW264.7 cells and DC2.4 cells were infected with L. monocytogenes WT or ami at MOI 10, and NMuLi cells were infected with the bacteria at MOI 100. After incubation for 30 min, the extracellular bacteria were eliminated with 50 ␮g/ml gentamicin (Wako), and the intracellular bacteria were observed or enumerated. Actin tail formation Cells were infected with L. monocytogenes YFP-WT or YFPami for 30 min, and the extracellular bacteria were eliminated by gentamicin. At 6 h after infection, the cells were fixed with 4% paraformaldehyde (Wako) for 15 min at room temperature and washed with phosphate-buffered saline (PBS). Cells were then permeabilized with 0.5% Triton X-100 (Sigma–Aldrich Japan, Tokyo, Japan) in PBS. Actin filaments were stained with 100 nM rhodamine-conjugated phalloidin (Cytoskeleton Inc., Denver, CO). Fluorescent bacteria and actin tails were observed under a confocal microscope (Nikkon Eclipse C1si, Nikkon, Tokyo, Japan). Electron microscopy Macrophage cell line RAW264.7 cells were cultivated on sterilized glass slides and infected with L. monocytogenes. At 0, 2, and 6 h after infection, the cells were fixed with 4% paraformaldehyde, 1% glutaraldehyde (Wako) in PBS, and then post-fixed with 1% osmium tetroxide (Heraeus Chemicals, Port Elisabeth, South Africa) in 0.1 M phosphate buffer (pH 7.4). The samples were dehydrated through a graded series of ethanol (Wako) and propylene oxide (Wako) at room temperature and embedded in Epon 812 resin (TAAB Laboratories Equipment Ltd., Berkshire, UK). They were then polymerized with resin in gelatin capsules (No. 0; Eli Lilly Co., Indianapolis, IN) at 60 ◦ C for 48 h. After polymerization, the samples on glass slides were transferred to the resin block. Ultrathin sections (70–80 nm) were cut with a diamond knife, stained with Sato’s lead citrate (Sato, 1968) and uranyl acetate (Merck, Darmstadt, Germany) and observed under a transmission electron microscope JEM 1250 (JEOL Ltd., Tokyo, Japan) at 80 kV. Immunodetection Ami, InlB, and LLO were detected by Western blotting using specific antibodies which were produced in our laboratory. Whole-cell protein extracts were used for detection of Ami and InlB. Cell supernatants precipitated with 10% trichloroacetic acid was used for LLO detection. Protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride membrane (Immobilon-P; Millipore, Billerica, MA). After blocking with 0.5% skim milk in PBS and incubation of the membrane with specific antibodies and horseradish peroxidaseconjugated goat anti-rabbit immunoglobulin G (MP Biomedicals, Illkirch, France), the signal was detected by ECL detection reagents (GE Healthcare, Piscataway, NJ) on Hyperfilm ECL (GE Healthcare).

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Histopathology Mice were infected intravenously with 5 × 106 CFU of L. monocytogenes WT or ami. Perfusion-fixation and dissection were performed on day 3 after infection. Organs were collected and fixed with 4% paraformaldehyde. The tissues were embedded in paraffin (Wako), and the sections were stained with hematoxylin (Wako) and eosin (Wako) according to the standard protocol. Bacteria localized in the tissue sections were observed by Gram stain (Engbaek et al., 1979).

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L. monocytogenes WT or ami was added to the confluent layer of cells. After 15 min of incubation, non-adherent bacterial cells were removed by washing 3 times with PBS. The cells were fixed with 4% paraformaldehyde for 15 min and washed twice with PBS. Adherent bacteria were sequentially labeled with rabbit anti-Listeria spp. antibodies (ViroStat Inc., Portland, ME) and rhodamine-conjugated goat anti-rabbit immunoglobulin G (MP Biomedicals) and then observed under fluorescent microscope. The fluorescent pixels were quantitatively analyzed using ImageJ version 1.40 g (National Institutes of Health).

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DC2.4 cells were prepared and seeded into a 24-well tissue culture plate (Greiner Labortechnik, Frickenhausen, Germany) with 1 × 106 cells/well. They were stimulated with non-degraded cell wall, lysosyme-degraded cell wall, or Ami-degraded cell wall for 24 h. The supernatant was collected and cytokine titers were determined by enzyme-linked immunosorbent assay (ELISA). Mice were infected intravenously with 5 × 106 CFU of L. monocytogenes WT or ami, and cytokine titers in spleens and sera of infected mice were determined 24 and 72 h after infection. Titers of tumor necrosis factor (TNF)-␣ and interleukin (IL)-6 were determined with immunoassay kits according to the manufacturer’s instructions (Biosource USA, Camarillo, CA). Interferon (IFN)-␥ production was determined by double-sandwich ELISA as previously described (Nakane et al., 1996). Statistical analysis Data were expressed as means ± standard deviations, and Student’s t test was used to determine the significance of the differences between control and experimental group.

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Fig. 1. Lack of Ami results in attenuated virulence of L. monocytogenes. (A) Survival of mice infected with 5 × 106 CFU of L. monocytogenes WT or ami. Four independent experiments were performed (n = 14). (B) Determination of the bacterial number in liver and spleen after infection with 5 × 106 CFU of L. monocytogenes on day 1 and day 3. The results are expressed as the means ± SD of 3 independent experiments (*; p < 0.01, **; p < 0.05).

plays a role in the virulence of L. monocytogenes and prompted us to further investigate the role of Ami in pathogenesis.

Results Ami is required for virulence of L. monocytogenes ami, an Ami-deficient mutant, was constructed from the parental L. monocytogenes 1b 1684 strain via homologous recombination using the pAULA-based integration-excision procedure (Schäferkordt and Chakraborty, 1995). Mice were infected intravenously with 5 × 106 CFU of L. monocytogenes WT or ami, and survival of mice was observed up to 15 days after infection. All WT-infected mice died within 13 days, whereas 79% of the amiinfected mice survived up to 15 days (Fig. 1A). The number of viable bacteria in liver and spleen was determined. On day 1, the bacterial load in spleens of ami-infected mice was not different from that of WT, whereas the bacterial load in livers of ami-infected mice was less than that of WT. On day 3, the bacterial numbers in both organs from the ami-infected mice were significantly lower than those of WT (Fig. 1B). These results clearly confirmed that Ami

Lack of Ami has no effect on bacterial growth, protein secretion, and actin-based motility Characteristics of ami were investigated. The ami had no growth defect in culture medium (Fig. 2A) or in eukaryotic cells (Fig. 2B and Fig. S1A). Electron micrographs of L. monocytogenesinfected macrophages showed that the morphologies of the WT and ami were indistinguishable (Fig. 2C). To determine whether the lack of Ami affected protein secretion, the secretory and surfaceassociated proteins of ami were analyzed by SDS–PAGE (Fig. S1B) and two-dimensional gel electrophoresis (data not shown). Except for the band or spot of the Ami protein (99 kDa and pI = 9.89), the secretory and surface-associated protein patterns between WT and ami were similar. Western blot analysis revealed that the expression of InlB and LLO in ami was not impaired (Fig. 2D). ami has no defect in actin tail formation (Fig. 2E). These results suggest that Ami is not necessary for bacterial growth, protein secretion, or actin-based motility.

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Fig. 2. Ami is not required for bacterial growth, bacterial morphology, protein secretion, and actin-based motility. (A) Growth curve of L. monocytogenes WT and ami cultured in brain heart infusion broth at 37 ◦ C under aerobic conditions. (B) Intracellular growth of L. monocytogenes WT and ami in macrophages. RAW264.7 cells were infected with WT or ami at MOI 10. After incubation for 30 min, the extracellular bacteria were eliminated with gentamicin and the intracellular bacteria were enumerated. (C) Electron micrographs of RAW264.7 cells infected with WT or ami at 0, 2, and 6 h. No significant differences in morphology of ami were found in comparison to WT. (D) Immunological detection of Ami, InlB, and LLO in ami. Bacteria were grown in tryptic soy broth for 16 h. Protein samples were separated by SDS–PAGE and detected by Western blotting using anti-Ami, anti-InlB, or anti-LLO antibodies. (E) Actin tail formation of WT and ami in cells. Cells were infected with YFP-WT and YFP-ami for 6 h. After fixing and permeabilizing the cells, actin tails were stained with rhodamine-phalloidin. The fluorescent signal was observed under confocal microscope. Black and white arrowheads indicate bacterial cell and actin tail, respectively.

Liver damage of ami-infected mice is attenuated Histopathological analysis of WT- and ami-infected mice was examined on day 3 after infection. The results indicated that liver damage of ami-infected mice was dramatically attenuated compared to WT (Fig. 3A and B), whereas no significant difference in tissue damage of spleen, brain, lung, and kidneys was found between WT- and ami-infected mice (data not shown). Liver-damaged tissues showed infiltration of neutrophils forming microabscesses surrounded with normal hepatocytes. Gram stain demonstrated that WT bacterial cells spread throughout the microabscesses (Fig. 3C). At the peripheries of microabscesses, WT bacterial cells were around and inside the hepatocytes. In contrast, ami bacterial cells were visible only in the middle of microabscesses. In fact, very few ami bacterial cells could be found surrounding hepatocytes. This observation suggests that Ami promotes listerial colonization of hepatocytes.

Ami is required for efficient hepatocyte colonization In the cultured cells, ami adhered to phagocytic RAW264.7 and DC2.4 cells as well as WT, but showed less adhesion to mouse hepatocytes, NMuLi, than the WT strain (Fig. 4A). Related results were obtained from invasion assay. The ami exhibited a reduc-

tion in internalization into NMuLi compared to the WT strain while its entry into the RAW264.7 and DC2.4 cells was as efficient as WT (Fig. 4B). These results suggest that Ami plays a role in bacterial adhesion, a favorable process for bacterial invasion into mouse hepatocytes.

PG hydrolase activity of Ami generates immunologically active components PG hydrolases are responsible for cell wall turnover which possibly generates immunologically active components. To examine whether PG hydrolase activity of Ami affects immune response, cytokine production was preliminarily determined from DC2.4 cells stimulated with cell wall extracted from WT and ami. The result showed that the production of TNF-␣ and IL-6 from cells stimulated with ami cell wall was less than those from WT cell wall-stimulated cells (Fig. S2A). The cytokine production was then determined from cells stimulated with Ami-predigested cell wall. Recombinant Ami was prepared, and its peptidoglycan hydrolase activity was confirmed (Fig. S2B and C). Cell wall was freshly prepared from the WT strain and adjusted to be 3.0 OD450 nm units. It was then digested with 10 ␮g/ml of recombinant Ami or lysosyme at 37 ◦ C for 24 h and used to stimulate the cells. TNF-␣ and IL-6 production from the cells stimulated with Ami-degraded cell wall was

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Fig. 3. ami is attenuated in the liver of the mouse infection model. Histopathology of liver from mice infected with L. monocytogenes WT or ami was examined. (A) Hematoxylin and eosin stain of liver sections from WT- and ami-infected mice. Arrowheads indicate microabscesses. (B) Quantitative analysis of microabscess area per liver section area. The results are expressed as the means ± SD of 10 random sections from 2 independent experiments (*; p < 0.01). (C) Gram stain of L. monocytogenes in liver sections from WT- and ami-infected mice. Dotted lines indicate area of microabscesses. Bacterial cells are stained to be dark blue (N; nucleus of hepatocyte). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

significantly higher than those stimulated with non-degraded cell wall and lysozyme-degraded cell wall (Fig. 5A and B). It should be noted that recombinant Ami at the same concentration could not stimulate either TNF-␣ or IL-6 production. These results suggest that PG hydrolase activity of Ami releases immunologically active components from the listerial cell wall. Ami enhances host immune response to listerial infection In the mouse infection model, the titers of IFN-␥ and TNF-␣ in spleens and sera of the WT and ami-infected mice were determined. Both IFN-␥ and TNF-␣ production from WT-infected mice were significantly higher than that of ami (Fig. 6A and B), even though TNF-␣ production in the sera could not be detected. On day 3, the cytokine production from L. monocytogenes-infected mice correlated to the amount of bacterial number in the spleens (Fig. 1B). On the other hand, on day 1 in which the bacterial load in the spleens of WT and ami was similar (Fig. 1B), reduction of IFN-␥ and TNF-␣ production by ami infection suggests that Ami enhances the immune response in the early phase of listerial infection. Discussion L. monocytogenes is an intracellular pathogen which can invade a wide range of cell types. It produces several endogenous PG hydrolases or autolysins which are important for its survival (Popowska, 2004; Carroll et al., 2003). Some of the L. monocytogenes autolysins

have been shown to be involved in pathogenesis. Auto is required for entry into eukaryotic cells (Bublitz et al., 2009; Cabanes et al., 2004). p60-deficient mutant leads to abnormal cell division and lose of actin-base motility (Pilgrim et al., 2003). IspC-deficient mutant impairs the surface expression of virulence proteins (Wang and Lin, 2008). Although Ami is shown to adhere to several human cells via its CWA domain (Milohanic et al., 2001), the contribution of Ami in pathogenesis is not fully understood. Milohanic et al. (2001) showed that inactivation of Ami in mutants lacking InlA, InlB, or both internalins, results in a marked reduction of adhesion to eukaryotic cell line, and expression of Ami CWA domain restores the adhesion capacity of their mutants. However, cell invasion of their mutants is not impaired. In contrast, expression of CWA domain in the mutants inhibits entry of bacteria into the host cells (Milohanic et al., 2001). These data cannot explain attenuation of the virulence of Ami-inactivated mutants. In our study, an Ami-deficient mutant of L. monocytogenes 1b 1684 was constructed, and a role of Ami in virulence was confirmed. In vivo studies demonstrated a marked attenuation of virulence of our ami mutant in mice (Fig. 1A and B). Functions of Ami which possibly contribute in pathogenesis were then investigated. We clearly demonstrated that a lack of Ami in ami did not affect bacterial growth, bacterial morphology, protein secretion, or actin-based motility (Fig. 2A–E and Fig. S1). Immunological detection and actin tail formation confirmed that the amount of important virulence factors, InlB, LLO, and ActA, in ami was not altered. These results suggest that Ami is not necessary for survival and architecture of bacterial cells.

K. Asano et al. / International Journal of Medical Microbiology 301 (2011) 480–487

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Fig. 5. Ami releases immunologically active compounds. DC2.4 cells were prepared and seeded into 24-well tissue culture plate with 1 × 106 cells/well. Cell wall of L. monocytogenes WT (OD450 nm unit of 3.0) was digested with 10 ␮g/ml recombinant Ami or lysozyme by incubating at 37 ◦ C for 24 h. The reactions without cell wall or enzymes were used as controls. The reaction mixtures were then diluted for 100 times in culture medium and used to stimulate the cells for 24 h. The titers of TNF-␣ (A) and IL-6 (B) in culture supernatant were determined by ELISA (*; p < 0.01).

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Fig. 4. Ami promotes bacterial adhesion and invasion into hepatocytes. (A) Adhesion assay of WT and ami mutant. The bacteria were incubated with the cells for 15 min. After washing, adherent bacterial cells were labeled with rabbit anti-Listeria spp. antibodies and rhodamine-conjugated goat anti-rabbit immunoglobulin G. Quantitative analysis of adhesion of the ami mutant is shown as % relative to the WT set as 100%. The results are expressed as the means ± SD of 3 independent experiments (*; p < 0.01). (B) Invasion assay of WT and ami. Cells were infected with the bacteria for 30 min. After eliminating the extracellular bacteria with gentamicin, intracellular bacteria were enumerated on tryptic soy agar plates. Invasion of the ami is shown as % relative to the WT set as 100%. The results are expressed as the means ± SD of 3 independent experiments (*; p < 0.01).

To further investigate the role of Ami in pathogenesis, we compared histopathological differences between WT- and amiinfected mice. Mice were dissected on day 3 after the lethal-dose infection because WT-infected mice showed the most severe pathophysiology. We found that WT- and ami-infected mice showed no histopathologic difference in spleen, brain, lung, and kidneys (data not shown), whereas the livers from amiinfected mice had a significant attenuation in severity (Fig. 3A and B). Gram stain demonstrated that only WT, but not ami bacterial cells were present around and inside the hepatocytes

(Fig. 3C), suggesting that Ami promotes listerial colonization to the hepatocytes. The liver is one of the mainly infected organs involved in listeriosis. Three patterns of L. monocytogenes infection in the liver have been described: solitary liver abscess, multiple liver abscesses, and acute hepatitis (Braun et al., 1993). In the case of multiple liver abscesses, the outcome involves increased morbidity and mortality (López-Prieto et al., 2000). A reduction in liver microabscesses in ami-infected mice implied that Ami is responsible for severity during listeriosis. In the study of cultured cells, the ami exhibited a reduction in bacterial adhesion and internalization to mouse hepatocytes (Fig. 4A and B). These results correlated to the bacterial number of the ami-infected mice which showed a reduced bacterial number in the liver (Fig. 1B). On the other hand, the adhesion and entry of the mutant into mouse phagocytes were as efficient as WT (Fig. 4A and B). It correlates with the bacterial uptake in the spleen, an organ that contains a robust population of phagocytic cells. As shown in Fig. 1B, the bacterial numbers of WT and ami on day 1 in the spleen were not significantly different. On day 3, on which the bacterial numbers in both liver and spleen of WT-infected mice were significantly higher than that of ami (Fig. 1B), enormous WT bacterial cells in liver may spread to the overall body and affect the bacterial load in the spleen. It should be noted that the histopathology of spleens from WT- and ami-infected mice showed no significant difference (data not shown) in spite of a higher bacterial burden in the spleen of WT-infected mice compared to ami on day 3 (Fig. 1B). Tissue damages and the enormous bacterial numbers in

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Fig. 6. Ami enhances the host immune response of listerial infection. Mice were infected with 5 × 106 CFU/mouse of L. monocytogenes WT or ami. On day 1 and day 3 after infection, the cytokine titers in the spleens and sera were determined by ELISA. The results are expressed as the means ± SD of 3 independent experiments (*; p < 0.01, **; p < 0.05).

the liver of WT-infected mice indicate that Ami-mediated hepatocyte colonization is critical in the virulence of listerial infection intravenously. As mentioned above, Ami promotes bacterial adhesion to mouse hepatocytes (Fig. 4A). It correlates with the results obtained in a previous study (Milohanic et al., 2001). Adherence of bacterial cells to the host cells is a key event during listerial infection which is preferable for later bacterial invasion. We also found that the effect of Ami on bacterial adhesion to hepatocytes correlates with bacterial invasiveness (Fig. 4B). It is inconsistent with the results obtained by Milohanic and colleagues, which demonstrated that host invasion of Ami-inactivated mutants is not impaired (Milohanic et al., 2001). Correlation between Ami-mediated cell adhesion and invasion in our study is reasonable to explain the attenuation in virulence of ami. L. monocytogenes has several mechanisms to evade host defense. Its intracellular niche allows it to evade several aspects of the innate and adaptive immune systems. Simultaneously, L. monocytogenes infection induced a robust immune response. Immune systems are crucial to recognize several molecules of L. monocytogenes and eliminate this pathogen from the host. Cell wall is one of pathogenassociated molecular patterns that are recognized by cells of the innate immune system (Boneca, 2005; Inohara et al., 2003). L. monocytogenes modifies PG in its cell wall by N-deacetylation resulting in the resistance to the PG hydrolase activity of host lysozyme. This resistance increases bacterial viability in phagosomes and leads to evasion of this bacterium from host immune response by preventing the release of cell wall components (Boneca et al., 2007). On the other hand, L. monocytogenes produces several autolysins including Ami (Bierne and Cossart, 2007). In this study, we also determined the role of PG hydrolase activity of Ami in the host immune response. We demonstrated that the activity of Ami linked to release cell wall components enhances cytokine production in vitro (Fig. 5A and B). The mouse infection model also showed that the IFN-␥ and TNF-␣ production of WT-infected mice was higher than that of ami (Fig. 6A and B). On day 3, the cytokine response correlates with the bacterial load in the organs (Fig. 1B). On the other hand, the bacterial load of WT in the spleen on day 1 was not

significantly different from that of ami (Fig. 1B). The data on day 1 suggest that an induction of cytokine production in the spleen of WT-infected mice could be due to the presence of Ami on the bacterial surface. However, the bacterial load of WT in the liver was higher than that of ami (Fig. 1B). The difference in cytokine production in the spleen on day 1 may also possibly come from the liver after diffusion through the blood. Our results indicate that Ami enhances the host immune response in the early phase of listerial infection, although it is still unclear whether Ami is involved in the cytokine response directly in the release of cell wall components or indirectly in the bacterial load in the liver. Generally, a reduction of bacterial numbers is mediated by a cytokine-dependent inflammatory response, primarily IFN-␥ and TNF-␣, that results in the recruitment of additional activated macrophages and neutrophils primed for bacterial destruction (Pamer, 2004; Bancroft et al., 1991). Cytokine response in WT-infected mice would be an effective way for hosts to eliminate this pathogen. However, the stimulation of host responses by potent biological effectors is also responsible for severe pathology. Therefore, the enhancement of the cytokine response by Ami also contributes to the pathogenesis of listerial infection. In this study, the role of Ami in pathogenesis of listerial infection was investigated. We demonstrated that Ami-mediated hepatocyte colonization is critical for the virulence of listerial infection. Ami promotes bacterial adhesion to the hepatocytes, and this adhesion correlates with the entry of bacterial cells into hepatocytes and the bacterial number in the liver. These data are supported by histopathology which demonstrated here for the first time that ami is significantly attenuated in the liver of mice infected intravenously. In addition, PG hydrolase activity of Ami simultaneously releases immunologically active components. Ami enhances the host immune response and contributes to the pathogenesis of L. monocytogenes in vivo. Acknowledgements We thank T. Chakraborty, Institute of Medical Microbiology, University Teaching Hospital of Giessen, Germany, and Jens Bo

K. Asano et al. / International Journal of Medical Microbiology 301 (2011) 480–487

Andersen, Dept. of Microbiology and Risk Assessment, National Food Institute, DTU, Technical University of Denmark, for providing a thermosensitive shuttle vector, pAULA and a YFP-expressing plasmid, pJEBAN3, respectively. We also thank T. Ohteki, Dept. of Biodefense Research, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan, for providing mouse dendritic cell line, DC2.4, after approval from K. Rock, Dept. of Pathology and Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA, USA. This study was supported in part by Grants-in-Aid for Challenging Exploratory Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (21659107 to A.N.) and Grant for Hirosaki University Institutional Research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ijmm.2011.01.002. References Andersen, J.B., Roldgaard, B.B., Lindner, A.B., Christensen, B.B., Licht, T.R., 2006. Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes. BMC Microbiology 6, 86. Bancroft, G.J., Schreiber, R.D., Unanue, E.R., 1991. Natural immunity: a T-cellindependent pathway of macrophage activation, defined in the scid mouse. Immunol. Rev. 124, 5–24. Bierne, H., Cossart, P., 2007. Listeria monocytogenes surface proteins: from genome predictions to function. Microbiol. Mol. Biol. Rev. 71, 377–397. Boneca, I.G., 2005. The role of peptidoglycan in pathogenesis. Curr. Opin. Microbiol. 8, 46–53. Boneca, I.G., Dussurget, O., Cabanes, D., Nahori, M.A., Sousa, S., Lecuit, M., Psylinakis, E., Bouriotis, V., Hugot, J.P., Giovannini, M., Coyle, A., Bertin, J., Namane, A., Rousselle, J.C., Cayet, N., Prévost, M.C., Balloy, V., Chignard, M., Philpott, D.J., Cossart, P., Girardin, S.E., 2007. A critical role for peptidoglycan N-deacetylation in Listeria evasion from the host innate immune system. Proc. Natl. Acad. Sci. USA 104, 997–1002. Braun, L., Dramsi, S., Dehoux, P., Bierne, H., Lindahl, G., Cossart, P., 1997. InlB: an invasion protein of Listeria monocytogenes with a novel type of surface association. Mol. Microbiol. 25, 285–294. Braun, T.I., Travis, D., Dee, R.R., Nieman, R.E., 1993. Liver abscess due to Listeria monocytogenes: case report and review. Clin. Infect. Dis. 17, 267–269. Bublitz, M., Polle, L., Holland, C., Heinz, D.W., Nimtz, M., Schubert, W.D., 2009. Structural basis for autoinhibition and activation of Auto, a virulenceassociated peptidoglycan hydrolase of Listeria monocytogenes. Mol. Microbiol. 71, 1509–1522. Cabanes, D., Dussurget, O., Dehoux, P., Cossart, P., 2004. Auto, a surface-associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence. Mol. Microbiol. 51, 1601–1614. Canvin, J.R., Marvin, A.P., Sivakumaran, M., Paton, J.C., Boulnois, G.J., Andrew, P.W., Mitchell, T.J., 1995. The role of pneumolysin and autolysin in the pathology of pneumonia and septicemia in mice infected with a type 2 pneumococcus. J. Infect. Dis. 172, 119–123. Carroll, S.A., Hain, T., Technow, U., Darji, A., Pashalidis, P., Joseph, S.W., Chakraborty, T., 2003. Identification and characterization of a peptidoglycan hydrolase, MurA, of Listeria monocytogenes, a muramidase needed for cell separation. J. Bacteriol. 185, 6801–6808. Chakraborty, T., Leimeister-Wächter, M., Domann, E., Hartl, M., Goebel, W., Nichterlein, T., Notermans, S., 1992. Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. J. Bacteriol. 174, 568–574. Díaz, E., López, R., García, J.L., 1992. Role of the major pneumococcal autolysin in the atypical response of a clinical isolate of Streptococcus pneumoniae. J. Bacteriol. 174, 5508–5515. Engbaek, K., Johansen, K.S., Jensen, M.E., 1979. A new technique for Gram staining paraffin-embedded tissue. J. Clin. Pathol. 32, 187–190. Farber, J.M., Peterkin, P.I., 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55, 476–511.

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