Internalin B promotes the replication of Listeria monocytogenes in mouse hepatocytes

The uptake of Listeria monocytogenes by a variety of cell types in vitro is facilitated by the protein products of the inlAB (internalin) operon expressed by the organism. In the case of mouse hepatocytes, the extent to which inlAB expression influenced the uptake of Listeria in vitro was markedly dependent upon the ratio of bacteria to cells. At a ratio of 100:1, greater than 40-fold fewer transposon-induced inl4B mutant listeriae entered hepatocytes compared to the isogenic wild-type control; the difference was only fourfold, however, in cultures inoculated at a 1:1 ratio. Similarly, the uptake of in-frame inlB or inlAB deletion mutants differed only fourfold from the uptake of wild-type or inlA mutant Listeria at a 1:1 multiplicity of infection. Mutations affecting inlB or inlAB, on the other hand, resulted in a marked decrease in the capacity of Listeria to proliferate within mouse hepatocytes in vivo and in vitro. Electron micrographs of Listeria-infected hepatocytes demonstrated the impaired capacity of inlB mutants to escape from endocytic vacuoles and to enter the cytoplasm where proliferation occurs. These findings indicate that the protein product of inlB exerts a significant effect on the intracellular replication of Listeria.     

Internalin of Listeria monocytogenes with an intact leucine-rich repeat region is sufficient to promote internalization

Listeria monocytogenes can use two different surface proteins, internalin (InlA) and InlB, to invade mammalian cells. The exact role of these invasiveness factors in vivo remains to be determined. In cultured cells, InlA is necessary to promote Listeria entry into human epithelial cells, such as Caco-2 cells, whereas InlB is necessary to promote Listeria internalization in several other cell types, including hepatocytes, fibroblasts, and epithelioid cells, such as Vero, HeLa, CHO, or Hep-2 cells. We have recently reported that the InlA receptor on Caco-2 cells is the cell adhesion molecule E-cadherin and demonstrated that nonpermissive fibroblasts become permissive for internalin-mediated entry when transfected with the gene coding for LCAM, the chicken homolog of the human E-cadherin gene. In this study, we demonstrate for the first time that the internalin protein alone is sufficient to promote internalization into cells expressing its receptor. Indeed, internalin confers invasiveness to both Enterococcus faecalis and internalin-coated latex beads. As shown by transmission electron microscopy, these beads were phagocytosed via a "zipper" mechanism similar to that observed during the internalin-E-cadherin-mediated entry of Listeria. Moreover, a functional analysis of internalin demonstrates that its amino-terminal region, encompassing the leucine-rich repeat (LRR) region and the inter-repeat (IR) region, is necessary and sufficient to promote bacterial entry into cells expressing its receptor. Several lines of evidence suggest that the LRR region would interact directly with E-cadherin, whereas the IR region would be required for a proper folding of the LRR region.     

How does gamma-interferon mediate resistance to Listeria monocytogenes?

Gamma-interferon (IFN-gamma) seems to be required for resistance to Listeria monocytogenes in vivo, but acts early in infection, before apparent T cell involvement. The early role of IFN-gamma is unknown, but might include enhancing antigen presentation, inducing secretion of tumor necrosis factor alpha and helping early precursor macrophages to express nonspecific listericidal activity.     

Peptide epitopes from noncytosolic Listeria monocytogenes can be presented by major histocompatibility complex class I molecules

Listeria monocytogenes is an intracellular pathogen which escapes the phagosome and resides in the cytosol of the host cell. Using Listeria innocua and a mutant strain of L. monocytogenes (listeriolysin O negative), which do not enter the cytosol of the host cell, we demonstrate class I presentation of an epitope of p60, a protein secreted by L. monocytogenes, to a class I-restricted CD8+ cytotoxic T lymphocyte clone.     

荧光定量PCR检测单核细胞增生性李斯特氏菌

[目的]建立一种快速检测单核细胞增生性李斯特氏菌的实时荧光定量PER方法.[方法]针对单核细胞增生性李斯特氏菌的特异基因hlyA,结合锁定寡核苷酸(LNA),设计引物和探针,利用阳性质粒和模拟标本建立单核细胞增生性李斯特氏菌实时定量荧光PCR检测方法.[结果]以单核细胞增生性李斯特氏菌为模板克隆了hlyA基因,得到阳性质粒,并进一步建立了荧光定量PCR方法,得到标准曲线Y=-3.273 X+37.640,相关系数R<'2>=1.000;该方法的灵敏度可以达到1.2×10 CFU/ml;人工污染猪肉检出限可以达到3.2×10<'2>CFU/ml.[结论]建立了快速检测单核细胞增生性李斯特氏菌的实时荧光定量PCR方法,为实现食品中单核细胞增生性李斯特氏菌的快速检测构建了一个技术平台.     

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.     

Macrophages activated by Listeria monocytogenes induce organ-specific autoimmunity

We have previously reported an experimental autoimmune model induced by the local infection of Listeria monocytogenes. The unilateral inoculation of virulent Listeria into a testis of a normal mouse induced a delayed-type hypersensitivity response against testicular antigen and caused autoimmune orchitis in the contralateral testis. The orchitis was transferred to naive mice by T cells from the intratesticularly infected mice. In this paper, we demonstrated that avirulent Listeria, which lacks the expression of listeriolysin O, failed to induce any anti-testicular responses or contralateral orchitis even when it was inoculated at a high dose into the testis. Furthermore, the intraperitoneal inoculation of virulent Listeria with testicular antigen induced the anti-testicular responses and orchitis although intraperitoneal inoculation of testicular antigen with avirulent Listeria failed to induce them. The difference between virulent and avirulent Listeria in the induction of anti-testicular responses was supposed to be dependent on the difference in macrophage activation by the two bacterial strains because, first, the anti-testicular responses were elicited in normal mice when macrophages from virulent Listeria-infected mice were intraperitoneally transferred with testicular antigen although no viable bacteria were detected from the macrophages, and secondly, in contrast, the intraperitoneal co-inoculation of macrophages from avirulent Listeria-infected mice and testicular antigen failed to elicit any anti-testicular responses. Finally, we found that the virulent Listeria-induced macrophages expressed a higher level of CD80 (B7-1) and CD86 (B7-2) molecules than did the avirulent Listeria-induced macrophages and naive peritoneal macrophages. These results thus suggest that virulent Listeria activates macrophages to induce autoreactive T cells while avirulent Listeria does not. The up-regulation of B7 molecules by virulent Listeria infection is a candidate of the mechanism for the activation of autoreactive T cells.     

Listeria monocytogenes arthritis of several joints

A patient with rheumatoid arthritis (RA) developed an infection caused by Listeria monocytogenes in her left knee and both shoulder joints. The clinical presentation of the disease was rather indolent with relatively moderate joint symptoms. Moreover, the synovial fluid sample was only slightly turbid with a white blood cell count of 23.5 x 10(9)/1. As compared to the earlier reported cases of L. monocytogenes septic arthritis, our patient is unique because she had infection in several joints. The polyarticular joint involvement combined with the clinical symptoms resembling the activation of RA posed us diagnostic difficulties.     

The molecular mechanisms of actin-based intracellular motility by Listeria monocytogenes

A rapid, sensitive method was developed for the quantification of the R- and S-enantiomers of ketoprofen and their acyl glucuronide conjugates in the plasma and dialysate of hemodialysis-dependent anephric patients. Unconjugated R- and S-ketoprofen plasma concentrations were determined directly by liquid chromatography using a S,S-Whelk-O1 chiral stationary phase. R- and S-Ketoprofen glucuronide for use as standard were resolved using a C18 reversed-phase HPLC column with a mobile phase containing the ion-pair reagent tetrabutylammonium hydrogen sulfate. Plasma glucuronides, however, could not be directly quantified due to matrix interference. Therefore, the glucuronides were isolated using reversed-phase HPLC and quantified after alkaline hydrolysis using the S,S-Whelk-O1 chiral stationary phase column.     

Inactivation of Listeria monocytogenes in milk by pulsed electric field

Pasteurized whole, 2%, and skim milk were inoculated with Listeria monocytogenes Scott A and treated with high-voltage pulsed electric field (PEF). The effects of milk composition (fat content) and PEF parameters (electric field strength, treatment time, and treatment temperature) on the inactivation of the bacterium were studied. No significant differences were observed in the inactivation of L. monocytogenes Scott A in three types of milk by PEF treatment. With treatment at 25 degrees C, 1- to 3-log reductions of L. monocytogenes were observed. PEF lethal effect was a function of field strength and treatment time. Higher field strength or longer treatment time resulted in a greater reduction of viable cells. A 4-log reduction of the bacterium was obtained by increasing the treatment temperature to 50 degrees C. Results indicate that the use of a high-voltage PEF is a promising technology for inactivation of foodborne pathogens.     

Mature macrophage cell lines exhibit variable responses to LPS

Bacterial lipopolysaccharide (LPS) is a potent activator of cells of the macrophage/monocyte lineage. Two mature macrophage cell lines, P388D1 and RAW264.7, exhibit very different biological responses to LPS. Although RAW264.7 cells release arachidonic acid from phospholipid in response to LPS stimulation, P388D1 cells do not respond in this manner. However, LPS primes P388D1 cells to release arachidonic acid in response to other stimuli. The goal of this work is to contrast the biochemical events that occur in LPS-treated P388D1 and RAW264.7 macrophages. Enzyme assays indicate that LPS treatment induces the activation of cytosolic PLA2 in RAW264.7, but not in P388D1 cells. Phorbol ester (PMA), a receptor-independent stimulus, also fails to induce arachidonic acid release from P388D1 cells, suggesting that these cells may have a defect in the signal transduction machinery that is common to LPS and PMA. This hypothesis is supported by the observation that the expression of the LPS receptors CD14 and CD11b/CD18 is similar on P388D1 and RAW264.7 cells. Western blot analyses indicate that the erk kinases are activated upon LPS treatment of RAW264.7 but not P388D1 cells. LPS-induced arachidonic acid release is reduced in cells treated with the MEK inhibitor PD98059, suggesting that activated erk kinases mediate the phosphorylation and activation of cPLA2 in this system. Interestingly, the p42 isoform of erk (erk2) appears to be activated in resting P388D1 cells. This observation indicates that the MAP kinase cascade may be constitutively activated in P388D1 cells which may in turn limit their ability to respond to LPS. Together, these data provide evidence that mature macrophages from different sources can exhibit variable responses to LPS and highlight the danger of making generalizations regarding the effects of LPS on macrophages.     

Exercise-induced stimulation of murine macrophage phagocytosis may be mediated by thyroxine

The present study was designed to test the hypothesis that changes in plasma concentrations of hormones may be responsible for the exercise-induced macrophage phagocytic stimulation. The effect of 30-min incubation of macrophages with plasma from mice previously exposed to swimming until exhaustion (with or without previous training) was studied, and the results showed a similar stimulation of the phagocytic capacity (attachment and ingestion) of these cells to that found in previous studies after exercise. Also, changes in plasma concentration of both thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3) after exercise were measured, and their effect on phagocytic capacity after in vitro incubation with peritoneal macrophages was investigated. Results indicated that, after exercise, plasma concentrations of T3 and T4 increased. Incubation of peritoneal macrophages for 30 min with a concentration of T3 similar to that observed in the plasma immediately after exercise (1.5 ng/ml) induced no modifications in the phagocytic capacity. However, a physiological concentration of T4 after exercise (75 ng/ml) stimulated the phagocytic capacity of peritoneal macrophages. In addition, a 10,000-fold greater concentration of these thyroid hormones did not modify the macrophage function. It is concluded that physiological concentration of T4 may be a mediator of the stimulation of the phagocytic function in macrophages induced by exercise.     

Alphav integrins mediate adhesion and migration of breast carcinoma cell lines

Integrins with the alphav subunit are involved in cell adhesion and cellular signaling. Some alphav integrins have been associated with tumor progression and dissemination. The objective of this study was to assess the contribution of alphav integrins to the adhesive and migratory behavior of cells derived from breast carcinoma (BCA). The expression and function of alphav integrins was characterized in three BCA cell lines which exhibit different metastatic potentials. These include MCF-7 cells which metastasize inefficiently, MDA-MB-231 cells, which have a moderate metastatic potential, and MDA-MB-435 cells, which metastasize extensively. Each cell type displays a different repertoire of alphav integrins on the cell surface. The complement of alphav integrins on each cell type influences their ability to adhere and migrate. The most striking difference among these cell lines was the expression of the alphavbeta3 integrin. The highly metastatic MDA-MB-435 cells express substantial levels of this receptor, whereas MDA-MB-231 and MCF-7 cells do not. The MDA-MB-435 cells showed a greater ability to adhere and to migrate and this functional difference can largely be attributed to the expression of alphavbeta3 integrin. This characterization is a first step toward determining the role of alphav integrins in animal models of BCA metastasis, and lends support to the hypothesis that the alphavbeta3 integrin can be a contributing factor in metastatic disease.     

A proposed nonpathogenic biological indicator for thermal inactivation of Listeria monocytogenes

Listeria innocua M1 was developed as a thermal processing indicator organism for L. monocytogenes by selection of a rifampin- and streptomycin-resistant mutant. zetaD values were 5.6 and 5.8 degrees C, and D (68 degrees C) values were 3.8 and 4.9 s for L. monocytogenes and L. innocua, respectively, in skim milk. The advantages of easy selection, similar heat resistance, and nonpathogenicity make L. innocua M1 appropriate for challenge studies designed to evaluate process lethality with respect to L. monocytogenes.     

Selective activation of an apoptotic retinoid precursor in macrophage cell lines

Advances in the understanding of the retinoid signaling mechanism has allowed the discovery of highly selective retinoids that activate only one specific receptor class, subtype, or signaling pathway. These novel compounds lack certain of the common retinoid toxicities and therefore suggest promising new approaches for therapeutic applications. We describe here a new compound, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid methyl ester (MX84), that is selectively activated in macrophages, leading to killing of only macrophage monocyte type cells in vitro. We provide evidence that MX84 is an inactive precursor that is converted into an active apoptosis-inducing retinoid in macrophages. The macrophage activity is also secreted, and our data suggest that the secreted activity is a phospholipase D type activity. Our observation may lead to the development of molecules that are highly macrophage-selective apoptosis inducers in vivo and that could represent important novel therapeutics against diseases caused by excessive macrophage activity.     

Dexamethasone enhances colonization of soft agar by tumorigenic mouse lung-derived cell lines

When cultured on plastic, tumorigenic mouse lung-derived cell lines exhibit different proliferative responses to glucocorticoids; some lines are inhibited while others are stimulated or unaffected. In contrast to the variable dexamethasone responses when cells are cultured on plastic, soft agar colonization by each of these cell lines is enhanced by dexamethasone. Enhanced soft agar growth is unlikely to result from expression of a mutant glucocorticoid receptor, since dexamethasone also enhanced colony formation in two cell lines that stably express a transfected normal glucocorticoid receptor gene. Thus, cell attachment influences the effect of glucocorticoids on cell cycle progression.