The role of two phospholipases in the pathogenicity of Listeria monocytogenes.
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Animals↗
Biomedical subjects
D A Portnoy
Publications and source records attributed to D A Portnoy.
Induction of a cellular immune response to a foreign antigen by a recombinant Listeria monocytogenes vaccine.
A recombinant strain of Listeria monocytogenes that stably and constitutively expresses Escherichia coli beta-galactosidase was used as a live vaccine vector. BALB/c mice were immunized orally or parenterally with the recombinant L. monocytogenes, and their cellular and humoral immune responses to beta-galactosidase were measured. Spleen cells taken 1 week after oral inoculation or 5 weeks after oral or parenteral inoculation (with a boost at 4 weeks) showed beta-galactosidase-specific CTL responses. The CTL line derived from mice immunized i.p. was also shown to be class I restricted and Thy-1.2+, CD8+, and TCR alpha beta+. All mice immunized with the recombinant L. monocytogenes had positive delayed-type hypersensitivity responses to heat-killed L. monocytogenes, but only 15% had a positive delayed-type hypersensitivity reaction to beta-galactosidase. Individual serum samples from mice immunized i.p. or i.v. were tested for antibody to beta-galactosidase. Approximately 11% had low positive titers for beta-galactosidase antibodies. These results demonstrate that both oral and parenteral immunization with recombinant L. monocytogenes results in a cellular immune response to the foreign protein, which is primarily a specific CD8+ CTL response.
Animals↗
Cell biology. Devious devices of Salmonella.
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Animals↗
Listeriolysin O is a target of the immune response to Listeria monocytogenes.
The immunologic mechanism of protective immunity to the intracellular parasite Listeria monocytogenes (Lm) is not well understood, however, antilisterial immunity can be adoptively transferred with T lymphocytes from Lm-immune donors. The Lm-immune cells are believed to produce macrophage-activating lymphokines, which leads to the eventual macrophage-dependent eradication of the bacterium. Increasing evidence suggests that immunity to Lm resides exclusively within the CD8+ T cell subset. It is possible that the Lm-immune CD8+ T cells function to release sequestered Lm from nonprofessional phagocytes to awaiting activated macrophage populations. This study was conducted to determine if listeriolysin O (LLO), which is an essential determinant of Lm pathogenicity, is also a target of the antilisterial immune response. We have found that target cells infected with a LLO+ Lm strain are lysed by Lm-immune cytotoxic cells, whereas target cells infected with a LLO- Lm mutant, or pulsed with a heat-killed Lm preparation, are not lysed by the Lm-immune effector cells. We have used a Bacillus subtilis (Bs) construct that expresses the LLO gene product and found that target cells infected with the LLO+ Bs construct are lysed by antilisterial cytotoxic cells. The antilisterial cytotoxic response is targeted against LLO, in that we have also used a Bs construct that expresses the perfringolysin (PLO) gene product and found that target cells infected with the PLO+ Bs are not lysed by antilisterial cytotoxic effector cells. These data strongly suggest that LLO is a target antigen of antilisterial immunity and may represent the dominant target during the expression of the immune response to Lm.
Animals↗
The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization.
The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen capable of rapid movement through the host cell cytoplasm. The biophysical basis of the motility of L. monocytogenes is an interesting question in its own right, the answer to which may shed light on the general processes of actin-based motility in cells. Moving intracellular bacteria display phase-dense 'comet tails' made of actin filaments, the formation of which is required for bacterial motility. We have investigated the dynamics of the actin filaments in the comet tails using the technique of photoactivation of fluorescence, which allows monitoring of the movement and turnover of labelled actin filaments after activation by illumination with ultraviolet light. We find that the actin filaments remain stationary in the cytoplasm as the bacterium moves forward, and that length of the comet tails is linearly proportional to the rate of movement. Our results imply that the motile mechanism involves continuous polymerization and release of actin filaments at the bacterial surface and that the rate of filament generation is related to the rate of movement. We suggest that actin polymerization provides the driving force for bacterial propulsion.
Actins↗
Innate immunity to a facultative intracellular bacterial pathogen.
The murine response to Listeria monocytogenes has long been considered a paradigm of T-cell-mediated immunity. There is, however, substantial evidence that T-cell-deficient mice are capable of surviving a L. monocytogenes challenge. Recently, advances have been made in our understanding of the cell biology and pathogenesis of infection.
Animals↗
Molecular determinants of Listeria monocytogenes pathogenesis.
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Amino Acid Sequence↗
Pathogenicity and immunogenicity of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread.
Listeria monocytogenes strains previously generated by transposon mutagenesis were examined with respect to virulence and induction of protective immunity in BALB/c mice. The phenotypic defects observed in these mutant L. monocytogenes strains included decreased hemolysin (listeriolysin O [LLO]) production, phospholipase C activity, intracellular growth, and/or cell-to-cell spread in vitro. While 50% lethal dose determinations performed with these mutant strains indicated reduced virulence for BALB/c mice, sublethal infection with the majority of these mutant strains provided protection against a subsequent challenge with the fully virulent L. monocytogenes parent strain. In addition, in vitro infection of the J774 cell line with most of these mutant strains converted these phagocytic cells to targets of L. monocytogenes-immune cytotoxic cells. The exceptions to these findings were two LLO-negative, avirulent mutant strains which were unable to immunize mice against a secondary challenge with virulent L. monocytogenes. One of these mutants contained a transposon insertion within the structural gene for LLO, and the other contained a transposon insertion in the structural gene for the transcriptional activator of the LLO gene. These two LLO-negative mutant strains also were unable to escape phagolysosomes in infected J774 cells and could not transform these phagocytic cells into targets of L. monocytogenes-immune cytotoxic cells. These findings confirm the importance of LLO in the induction of antilisterial immunity and suggest that a cytoplasmic localization of these pathogenic bacteria is required for the development of protective immunity.
Animals↗
Capacity of listeriolysin O, streptolysin O, and perfringolysin O to mediate growth of Bacillus subtilis within mammalian cells.
The Listeria monocytogenes hemolysin listeriolysin O (LLO) plays a major role in mediating the escape of L. monocytogenes from a vacuolar compartment. In a previous report, it was shown that Bacillus subtilis expressing LLO could escape from a host vacuolar compartment and grow in the cytoplasm (J. Bielecki, P. Youngman, P. Connelly, and D. A. Portnoy, Nature [London] 345:175-176, 1990). In the present study, two related thiol-activated hemolysins, streptolysin O (SLO) and perfringolysin O (PFO), were expressed in B. subtilis and their ability to mediate intracellular growth was monitored by visual inspection and by assaying for CFU. Like LLO, PFO was active within the vacuolar environment, whereas SLO showed negligible activity. However, expression of PFO seemed to damage the host cells. The pH of the vacuole probably had little to do with these results, since all three hemolysins showed full or enhanced activity at pH 5.5, although LLO showed greatly reduced activity at pH 7. In addition, neutralization of the pH within host vacuoles by using weak bases had little effect on the lysis of the vacuole. The lack of SLO activity is probably caused by its lower specific activity; the purified protein had 10-fold less activity on a molar basis. These results suggest that LLO is not unique in its capacity to mediate intracellular growth of B. subtilis.
Animals↗
Transcriptional activation of the Listeria monocytogenes hemolysin gene in Bacillus subtilis.
The prfA gene of Listeria monocytogenes was recently reported to be required for expression of hly, which encodes a pore-forming hemolysin essential for pathogenicity (M. Leimeister-Wachter, C. Haffner, E. Domann, W. Goebel, and T. Chakraborty, Proc. Natl. Acad. Sci. USA 87:8336-8340, 1990). We demonstrate here that a hly-lacZ fusion introduced into Bacillus subtilis is strongly activated when the prfA gene product is supplied in trans under the control of an isopropyl-beta-D-thiogalactopyranoside-inducible promoter, Pspac. Moreover, the PrfA-dependent activation of hly is abolished by point mutations in a 14-bp DNA palindromic sequence present in the 5' upstream region of hly. This indicates that PrfA is both necessary and sufficient for hly transcriptional activation and establishes the palindrome as the likely target sequence for PrfA interaction. The presence of a palindrome in the upstream regions of three additional L. monocytogenes genes clustered near hly suggests that PrfA may serve as a transcriptional activator for a major virulence regulon of L. monocytogenes. In addition, the ability of PrfA to activate its target promoters effectively in B. subtilis suggests that further analysis of this regulon and perhaps other aspects of L. monocytogenes gene regulation might be carried out in part through reconstruction experiments in B. subtilis.
Bacillus subtilis↗
The interaction of bacteria with mammalian cells.
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Actin Cytoskeleton↗
Listeria monocytogenes mutants lacking phosphatidylinositol-specific phospholipase C are avirulent.
A number of bacterial species secrete phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we show that the facultative intracellular bacterial pathogen, Listeria monocytogenes, contains a gene, plcA, predicting a polypeptide with 31% amino acid identity to a Bacillus thuringiensis PI-PLC. Accordingly, L. monocytogenes secretes PI-PLC activity, while a mutant with a transposon insertion in plcA lacks detectable PI-PLC activity. In addition, expression of plcA in B. subtilis resulted in secretion of PI-PLC activity. The L. monocytogenes PI-PLC-defective mutant was three logs less virulent for mice and failed to grow in host tissues. The mutant was also defective for in vitro growth in mouse peritoneal macrophages. These results strongly suggest that PI-PLC is an essential determinant of L. monocytogenes pathogenesis. Whether the PI-PLC acts on a bacterial or host substrate remains to be determined.
Animals↗
Acute aortic occlusion secondary to Aspergillus endocarditis in an intravenous drug abuser.
A 55-year-old black man, an intravenous substance abuser who had an acute arterial embolus to the distal aorta originating from his mitral valve, was noted on pathologic examination of the clot to have aspergillosis emboli. The infective endocarditis also resulted in emboli to the brain with subsequent death.
Acute Disease↗
Presentation of Listeria monocytogenes to CD8+ T cells requires secretion of hemolysin and intracellular bacterial growth.
Cell-mediated immunity to Listeria monocytogenes (LM) involves both CD4+ and CD8+ T cell responses. An important virulence factor in the pathogenesis of infection and development of protective immunity to LM is secretion of the sulfhydryl-activated hemolysin (Hly), listeriolysin. Listeria secretion of Hly allows LM to escape the endosomal compartment and enter the cytosol of the cell where intracellular growth can occur. We developed a system using bone marrow macrophages cultured in CSF-1 or IFN-gamma for comparing the response of CD4+ or CD8+ T cells to heat-killed, live Hly-, and live Hly+ LM. Macrophages grown in CSF were permissive for intracellular growth of Hly+ but not Hly- LM. CD8+ T cells recognized Hly+ LM but not HK or Hly- LM pulsed macrophages. However, CD4+ T cells recognized all three Listeria preparations fed to IFN-gamma-treated macrophages. These results suggest that both heat-killed LM and live LM efficiently enter the exogenous pathway for class II Ag processing and presentation. In contrast, only Hly+ LM activates the class I pathway, probably as a result of Hly+ bacterial replication within the cytosolic compartment.
Animals↗
Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells.
Intracellular parasites can be classified into those that reside within a host vacuole and those which grow directly in the host cytoplasm. Members of the latter group, which includes Rickettsia, Shigellae, Trypanosoma cruzi, and Listeria monocytogenes, possess haemolytic activity associated with the ability to enter the host cytoplasm. Therefore mutants of L. monocytogenes lacking a pore-forming haemolysin, listeriolysin O, do not escape from the endosomal compartment and consequently fail to become established in the cytoplasm. To examine the role of listeriolysin O, we cloned the structural gene for the L. monocytogenes haemolysin, hlyA, into an asporogenic mutant of Bacillus subtilis under the control of an IPTG-inducible promoter. After being internalized by the macrophage-like cell line J774, haemolytic B. subtilis disrupted the phagosomal membrane and grew rapidly within the macrophage cytoplasm. These results show that a single gene product is sufficient to convert a common soil bacterium into a parasite that can grow in the cytoplasm of a mammalian cell.
Bacillus subtilis↗
Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly.
Listeria monocytogenes is an intracellular parasite that can readily infect the macrophage-like cell line J774 and the kidney epithelial cell PtK2. After being ingested, the organism escapes from the phagolysosome into the host-cell cytoplasm. N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin, a specific stain for actin filaments (F-actin), demonstrates that within 1 hr of initiation of infection, the bacteria become surrounded by host-cell cytoplasmic actin filaments. By 3 hr, long projections of F-actin begin to form at one end of the bacteria. These actin structures colocalize with the actin-bundling protein alpha-actinin as well as with tropomyosin. Microinjection of fluorescently labeled alpha-actinin in living cells demonstrates that the formation of these F-actin projections is associated with bacterial movement, actin filaments rapidly assembling behind the bacteria as they migrate through the cytoplasm. These F-actin tails attain lengths up to 40 microns. The movement of the bacteria through the cytoplasm is rapid, 0.12-1.46 microns/sec. Within 2 min of cytochalasin D (0.5 micrograms/ml) treatment, all bacterial intracellular movement stops, and additional bacteria-associated actin assembly is blocked. A nonmotile Listeria mutant induces comparable actin assembly and moves at speeds similar to the wild type, indicating that the forces required for intracellular bacterial movement are generated by the host cell. L. monocytogenes can dramatically stimulate host-cell actin assembly in a directional manner, which serves to rapidly propel the bacteria through the cytoplasm, allowing the organisms to move to peripheral membranes and spread to uninfected cells.
Actinin↗
Actin filament nucleation by the bacterial pathogen, Listeria monocytogenes.
Shortly after Listeria is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. 1 h later, actin filaments coat the Listeria and then become rearranged to form a tail with which the Listeria moves to the macrophage surface as a prelude to spreading. If infected macrophages are treated with cytochalasin D, all the actin filaments associated with the Listeria break down leaving a fine, fibrillar material that does not decorate with subfragment 1 of myosin. This material is associated with either the surface of the Listeria (the cloud stage) or one end (the tail stage). If the cytochalasin-treated infected macrophages are detergent extracted and then incubated in nuclei-free monomeric actin under polymerizing conditions, actin filaments assemble from the fine, fibrillar material, the result being that each Listeria has actin filaments radiating from its surface like the spokes of a wheel (cloud form) or possesses a long tail of actin filaments formed from the fine, fibrillar material located at one end of the Listeria. Evidence that the fine fibrillar material is involved in nucleating actin assembly comes from a Listeria mutant. Although the mutant replicates at a normal rate in macrophages, actin filaments do not form on its surface (cloud stage) or from one end (tail stage), nor does the bacterium spread. Furthermore it does not form the fine fibrillar material. Evidence that the nucleating material is a secretory product of Listeria and not the macrophage comes from experiments using chloramphenicol, which inhibits protein synthesis in Listeria but not in macrophages. If chloramphenicol is applied 1 h after infection, a time before actin filaments are found attached to the Listeria in untreated macrophages, actin filaments never assemble on the Listeria even when fixed 3 h later. Furthermore the fine fibrillar material is absent, although there is a coat of dense granular material.
Actin Cytoskeleton↗
Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread.
To dissect the regulatory and structural requirements for Listeria monocytogenes intracellular growth and cell-to-cell spread, we designed a protocol based on transposon mutagenesis and the isolation of mutants which form small plaques in monolayers of mouse L2 cell fibroblasts. Two different transposable elements were used to generate libraries of insertion mutants: Tn916 and a derivative of Tn917-lac, Tn917-LTV3. Ten classes of mutants were isolated and evaluated for growth and cell-to-cell spread in J774 mouse macrophagelike cells, Henle 407 human epithelial cells, and mouse bone marrow-derived macrophages. Mutants were also evaluated for secretion of hemolysin and phospholipase (assayed by egg yolk opacity) and association with F-actin in the cytoplasm of cells, using NBD-phallacidin staining. The ten classes of mutants included (i) mutants showing abortive intracellular and extracellular growth; (ii) mutants showing abortive intracellular growth; (iii) rough mutants; (iv) mutants showing greatly reduced hemolysin and phospholipase secretion but showing normal growth in cells and little or no association with F-actin; (v) mutants with mutations mapping to an open reading frame (ORF) adjacent to hlyA and referred to as ORF U, lacking phospholipase activity, and with 50% normal hemolysin activity; (vi) mutants with reduced secretion of both hemolysin and phospholipase; (vii) nonhemolytic mutants with mutations mapping to the structural gene, hlyA; (viii) mutants with 25% normal hemolysin secretion and absolutely no association with F-actin; (ix) mutants with mutations mapping to ORF U, lacking phospholipase activity, and with normal hemolysin activity; and (x) mutants showing a mixed-plaque morphology but normal for all other parameters.
Actins↗