Experimental antibacterial therapy with puroindolines,lactoferrin and lysozyme in Listeria monocytogenes-infected mice

Puroindoline A and puroindoline B from plant seeds, bovine lactoferrin and chicken eggs lysozyme are antimicrobial proteins of innate immune system that lyse invading organisms. We investigate their potential antibacterial activity against Listeria monocytogenes in a mouse model. Bacteria were isolated from various organs for 7 days after challenge. Livers displayed consistently higher bacterial count (up to 10⁷ cfu/g) than spleens, kidneys and brains. The efficacy of the AMPs was therefore established by measuring the infection level (cfu number) of these organs. Puroindoline A and puroindoline B (5 mg/mouse), lactoferrin and lysozyme (1.25 mg/mouse), intravenously injected individually, inhibited bacterial growth completely. Puroindoline A, puroindoline B and lactoferrin were effective when administered 24 h before infection; lysozyme was effective at the time of infection or 5 days after. Their combined use resulted in the enhancement of individual antibacterial activities. Complete inhibition of bacterial growth was observed using concurrently 0.059 mg/mouse of puroindoline A and 0.019 mg/mouse of puroindoline B, lactoferrin and lysozyme. Individual antimicrobial proteins reduced significantly the expression level of pro-inflammatory cytokines (IL-6, IL-8, INF-γ and TNF-α), acute phase proteins (C-reactive protein and fibrinogen) and the T lymphocyte antigens CD4, CD8a, CD8b and CD25. These results suggest their potential use for the control of L. monocytogenes infections.     

The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes

Pathogenic bacteria have to cope with defence mechanisms mediated by adaptive and innate immunity of the host cells. Cationic antimicrobial peptides (CAMPs) represent one of the most effective components of the host innate immune response. Here we establish the function of Lmo1695, a member of the VirR-dependent virulence regulon, recently identified in Listeria monocytogenes. Lmo1695 encodes a membrane protein of 98 kDa with strong homology to the multiple peptide resistance factor (MprF) of Staphylococcus aureus. Like staphylococcal MprF, we found that Lmo1695 is involved in the synthesis of the membrane phospholipid lysylphosphatidylglycerol (L-PG). In addition, Lmo1695 is also essential for lysinylation of diphosphatidylglycerol (DPG), another phospholipid widely distributed in bacterial membranes. A Deltalmo1695 mutant lacking the lysinylated phospholipids was particularly susceptible to CAMPs of human and bacterial origin. The mutant strain infected both epithelial cells and macrophages only poorly and was attenuated for virulence when tested in a mouse model of infection. Lmo1695 is a member of a growing list of survival factors which enable growth of L. monocytogenes in different environments.     

Association of peptides with heat shock protein gp96 occurs in vivo and not after cell lysis.

Immunization of mice and rats with gp96 preparations isolated from syngeneic cancers has been shown to elicit protective immunity to a number of cancers. The specific immunogenicity of gp96 preparations derives from the antigenic peptides chaperoned by the gp96 molecule and not from gp96 molecules per se. Studies reported here demonstrate that the association of peptides with gp96 occurs in vivo and is not a procedural artifact which occurs in vitro after cell lysis. This demonstration has a bearing on the proposed functional role of HSP peptide association in antigen processing and presentation by MHC I molecules.     

Interaction of Listeria monocytogenes autolysin amidase with glycosaminoglycans promotes listerial adhesion to mouse hepatocytes

Adherence to the cell surface is a key event during infection of pathogenic microorganisms. We have previously reported that autolysin amidase (Ami) of Listeria monocytogenes promotes an efficient listerial adherence to mouse hepatocytes and requires for listerial pathogenicity. Cell wall anchoring (CWA) domain of Ami has been shown to bind lipoteichoic acid on listerial cell wall but the binding of Ami to host cell surface molecules remains to be determined. In this study, we present evidence here that Ami promotes efficient adherence of L. monocytogenes to mouse hepatocytes mediated by glycosaminoglycans (GAGs). The adhesion of L. monocytogenes wild type but not Ami-deficient mutant to the hepatocytes was dramatically attenuated by 4-nitrophenyl-β-D-xylopyranoside, a specific inhibitor of GAG association to cell surface. Full-length and truncated Ami were used to investigate the binding of Ami to GAGs and we found that four-repeated CWA of Ami is sufficient to bind GAGs on the host cell surface. Competitive assay and surface plasmon resonance demonstrated that Ami interacts with sulfated GAGs but not non-sulfated GAGs. The results suggest that Ami acts as an adhesin of L. monocytogenes to hepatocytes by interaction via its four-repeated CWA domain and sulfated GAGs.     

Specific immunogenicity of heat shock protein gp96 derives from chaperoned antigenic peptides and not from contaminating proteins

The peptide-binding property of MHC is central to adaptive immunological functions. A similar property of heat shock proteins (HSPs) hsp70 and hsp90 has been implicated in Ag presentation by MHC and in cross-priming. The peptide-binding pocket of hsp70 has been characterized structurally and functionally and a peptide-binding site in gp96 (of hsp90 family) has been defined. Nonetheless, questions persist whether the specific immunogenicity of HSP preparations derives from the peptides chaperoned by the HSPs or by proteins contaminating the HSP preparations. Because absolute purity of a protein preparation is a metaphysical concept, other approaches are necessary to address the question. In this study, we demonstrate that the specific immunogenicity of gp96 preparations isolated from cells expressing beta-galactosidase derives from the MHC I epitope precursors associated with the gp96 and not from contaminating beta-galactosidase protein nor unassociated fragments derived from it. Although the observations here are limited to a single HSP and antigenic peptides chaperoned by it, they can be extended broadly.     

Sequential production of alpha and beta interferons and gamma interferon in the circulation of Listeria monocytogenes-infected mice after stimulation with bacterial lipopolysaccharide

Sequential production of interferon (IFN)-alpha/beta and IFN-gamma in the circulation of mice which had been previously infected with viable Listeria monocytogenes was induced by injection of lipopolysaccharide (LPS) derived from Salmonella typhimurium. IFN-alpha/beta production occurred 2 hr after injection of LPS, thereafter IFN-gamma appeared and the maximum titer was demonstrated at 6 hr. At that time, almost all of the IFN was IFN-gamma. IFN-gamma production in response to LPS was observed from the 5th through the 11th day after infection with Listeria, but it was not demonstrated in either mice infected with lower doses of viable Listeria or mice immunized with heat-killed bacteria. IFN-alpha/beta production was not drastically affected by treatment with hydrocortisone, cyclophosphamide, carrageenan, antithymocyte serum, or anti-asialo GM1 antibody, whereas IFN-gamma production was suppressed by administration of all those agents. Noteworthily, IFN-alpha/beta, but not IFN-gamma, was produced even 6 hr after stimulation with LPS in cyclophosphamide- or antithymocyte serum-treated mice. IFN-gamma induction by LPS was markedly suppressed in mice in which IFN-alpha/beta produced by Listeria infection itself had been depleted by treatment with anti-mouse IFN-alpha/beta antibody, but it was not inhibited in mice when IFN-alpha/beta induced not by Listeria infection but by LPS had been depleted by treatment with anti-mouse IFN-alpha/beta antibody.     

Cell surface expression of the endoplasmic reticular heat shock protein gp96 is phylogenetically conserved.

In mammals, the heat shock protein gp96 complexed to antigenic peptides elicits T cell adaptive immunity. By itself, however, gp96 can evoke responses that are characteristic of innate immunity. Interestingly, this protein, which resides in the endoplasmic reticulum, is expressed on the surface of certain mouse tumor cells. Given that heat shock proteins are highly conserved, we investigated whether the cell surface expression of gp96 is also evolutionarily conserved. Our data reveal that gp96, most likely containing the endoplasmic reticulum retention motif (KDEL), is expressed on the surface of three different Xenopus lymphoid tumor cell lines, each derived from a different spontaneously arising thymic tumor. Levels of expression differ among the tumor lines tested, with more immunogenic tumors expressing greater amounts of surface gp96. Moreover, a high level of gp96 surface expression is detectable on a subset of Xenopus normal nontransformed splenic lymphocytes (mainly surface IgM+ B cells) but not on other normal cells, including macrophages and nucleated erythrocytes. Surface expression of a gp96 protein homologue occurs also on some, but not all, T and B cell clones derived from peripheral blood cells of the channel catfish, as well as on lymphocyte-like cells, but not on erythrocytes from the hagfish, a primitive agnathan vertebrate lacking markers of an adaptive immune system. gp96 is actively directed to and retained on the plasma membrane of Xenopus lymphocytes and tumor cells and hagfish lymphocyte-like cells by a process that requires vesicular transport. This selective surface expression of gp96 on some immune cells from different vertebrate classes is consistent with an ancestral immunological role of gp96 as danger-signaling molecule.     

Primary listerial infections are exacerbated in mice administered neutralizing antibody to macrophage colony-stimulating factor.

The serum and tissue levels of macrophage colony-stimulating factor (M-CSF) are elevated in mice during a primary immunologic response to infection by Listeria monocytogenes. Experiments were performed to determine the specific role of M-CSF in the resolution of listerial infections. The bulk of Listeria injected into a mouse i.v. is deposited in the liver. The expression of M-CSF mRNA in the liver increased markedly within 2 h postinfection. Maximum expression was dependent upon the dose of Listeria inoculated. The administration of anti-M-CSF mAb reduced the percentage of Mac-1+ mononuclear phagocytes subsequently found in the livers of infected animals. This reduction correlated inversely with an increase in the number of Listeria associated with both the parenchymal and NPC populations. These results suggest that M-CSF may play an important role in the primary immunologic response to Listeria in the liver by stimulating the production, mobilization, and/or biologic activity of Mac-1+ mononuclear phagocytes.     

Heat-induced oligomerization of gp96 occurs via a site distinct from substrate binding and is regulated by ATP

Among three different isoforms of non-muscle myosin heavy chains (NMMHCs), only NMMHCA is associated with inherited human disease, called MYH9 disorders, characterized by macrothrombocytopenia and characteristic granulocyte inclusions. Here targeted gene disruption was performed to understand fundamental as well as pathological role of the gene for NMMHCA, MYH9. Heterozygous intercrosses yielded no homozygous animals among 552 births, suggesting that MYH9 expression is required for embryonic development. In contrast, MYH9+/- mice were viable and fertile without gross anatomical, hematological, and nephrological abnormalities. Immunofluorescence analysis also showed the normal cytoplasmic distribution of NMMHCA. We further measured the auditory brainstem response and found two of six MYH9+/- mice had hearing losses, whereas the remaining four were comparable to wild-type mice. Such observation may parallel the diverse expression of Alport's manifestations of human individuals with MYH9 disorders and suggest the limited requirement of the gene for maintenance and function of specific organs.     

Flagellin from Listeria monocytogenes is glycosylated with beta-O-linked N-acetylglucosamine

Glycan staining of purified flagellin from Listeria monocytogenes serotypes 1/2a, 1/2b, 1/2c, and 4b suggested that the flagellin protein from this organism is glycosylated. Mass spectrometry analysis demonstrated that the flagellin protein of L. monocytogenes is posttranslationally modified with O-linked N-acetylglucosamine (GlcNAc) at up to six sites/monomer. The sites of glycosylation are all located in the central, surface-exposed region of the protein monomer. Immunoblotting with a monoclonal antibody specific for beta-O-linked GlcNAc confirmed that the linkage was in the beta configuration, this residue being a posttranslational modification commonly observed in eukaryote nuclear and cytoplasmic proteins.     

A conformational switch is associated with receptor affinity in peptides derived from the CD4-binding domain of gp120 from HIV I

A 15-residue region within the CD4-binding domain of gp120 from HIV I was identified with use of folding algorithms as conserving the potential for forming a particular secondary structure throughout 11 sequenced HIV strains. The region chosen has a potential for forming both beta-sheet and alpha-helix; the helical form would be amphipathic with the five hydrophobic residues all totally or functionally conserved. Five peptides were synthesized corresponding to this region in strain LAV and the strain most highly divergent from it in primary structure (Z3) plus three additional peptides with critical substitutions in the LAV sequence. The conformation of these five peptides was examined under various conditions with circular dichroism, and the results were compared with the ability of each peptide to bind to a CD4-expressing strain of HeLa cells (HeLa T4). In solution, the unmodified peptides exhibit a bistable structure, existing as beta-sheet in dilute buffer and converting to alpha-helix under more apolar conditions. The transition is reversible and sharp, occurring at a particular point in the polar/apolar gradient with virtually no intermediate state. The ability to undergo this bistable flip is closely associated with binding ability, amino acid substitutions that eliminate binding ability also eliminating the switch, and vice versa. The transition thus may reflect conformational changes occurring in this region of gp120 as it binds to the CD4 receptor.     

Water is required for proton transfer from aspartate-96 to the bacteriorhodopsin Schiff base.

During the M in equilibrium with N----BR reaction sequence in the bacteriorhodopsin photocycle, proton is exchanged between D96 and the Schiff base, and D96 is reprotonated from the cytoplasmic surface. We probed these and the other photocycle reactions with osmotically active solutes and perturbants and found that the M in equilibrium with N reaction is specifically inhibited by withdrawing water from the protein. The N----BR reaction in the wild-type protein and the direct reprotonation of the Schiff base from the cytoplasmic surface in the site-specific mutant D96N are much less affected. Thus, it appears that water is required inside the protein for reactions where a proton is separated from a buried electronegative group, but not for those where the rate-limiting step is the capture of a proton at the protein surface. In the wild type, the largest part of the barrier to Schiff base reprotonation is the enthalpy of separating the proton from D96, which amounts to about 40 kJ/mol. We suggest that in spite of this D96 confers an overall kinetic advantage because when this residue becomes anionic in the N state its electric field near the cytoplasmic surface lowers the free energy barrier of the capture of a proton in the next step. In the D96N protein, the barrier to the M----BR reaction is 20 kJ/mol higher than what would be expected from the rates of the M----N and N----BR partial reactions in the wild type, presumably because this mechanism is not available.     

鼻腔接种伤寒杆菌Fe-SOD对鼠伤寒杆菌攻击小鼠的免疫保护作用

为探讨鼻腔接种伤寒杆菌Fe-SOD对鼠伤寒杆菌攻击小鼠的交叉免疫保护作用,用IL－1作为佐剂,将伤寒杆菌Fe-SOD经鼻腔接种小鼠,再以不同剂鼠伤寒杆菌攻击,观察小鼠的存活情况,当用IL－1作为佐剂时,经鼻腔接种伤寒杆菌Fe-SOD的小鼠在2LD50鼠伤寒杆菌攻击后,3天和7天的存活率均明显高于对照组（P＜0．01或P＜0．05）,当以5LD50鼠直菌攻击时,小鼠7天的存活率明显高于对照组（P＜0．01）。结果说明伤寒杆菌Fe-SOD经鼻腔接种后对鼠伤寒杆菌的攻击可产生一定的免疫保护作用,也进一步说明了Fe-SOD是沙门氏菌的共同保护性抗原。     

Selective functional depletion of HIV gp120 peptides complexed with MHC from antigen-presenting cells engaged with specific T lymphocytes.

Human T cell lines specific for different peptides of HIV envelope glycoprotein gp120 have been used as probes to identify the availability of functional MHC-peptide complexes on APC. MHC-peptide complexes recognized by T cells specific for peptide 24 (amino acids 225-240) are no longer available on the surface of APC after interaction with irradiated (binding nonproliferating) T cells with the same fine specificity. On the contrary, MHC-peptide complexes recognized by T cells specific for peptide 30 (amino acids 285-300) were functionally available and could stimulate T cells with such a specificity. The reciprocal experiment yielded similar results. The same data were also reproduced with another pair of gp120 peptides. These data demonstrate that upon clustering of peptide-specific T cells with presenting cells presentation of the same peptide to a second cohort of T cells with identical specificity is abolished, suggesting that a selective functional depletion of the MHC-peptide complexes engaged with specific T cells occurs at the surface of the presenting cells. The depletion does not affect other MHC molecules complexed with unrelated peptides.     

Chimeric synthetic peptides containing two immunodominant epitopes from the envelope gp46 and the transmembrane gp21 glycoproteins of HTLV-I virus.

Two chimeric synthetic peptides incorporating immunodominant sequences from HTLV-I virus were synthesized. Monomeric peptides P7 and P8 represent sequences from transmembrane protein (gp21) and envelope protein (gp46) of the virus. The peptide P7 is a gp21 (374-400) sequence and the peptide P8 is a gp46 (190-207) sequence. Those peptides were arranged in a way that permits one to obtain different combinations of chimeric peptides (P7-GG-P8 and P8-GG-P7), separated by two glycine residues as spacer arms. The antigenic activity of these peptides were evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HTLV-I-positive sera (n = 22), anti-HTLV-I/II-positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 2), and anti-HTLV-II-positive sera (n = 11), while specificity was evaluated with anti-HIV-positive samples (n = 19) and samples from healthy blood donors (n = 30). The efficacy of the chimeric peptides in solid-phase immunoassays was compared with the monomeric peptides and monomeric peptides together. The chimeric peptide P7-GG-P8 proved to be the most reactive with anti-HTLV-I-positive sera. These results may be related to a higher peptide adsorption capacity to the solid surface and for epitope accessibility to the antibodies. This chimeric peptide would be very useful for HTLV-I diagnostics.     

Infertility in a line of mice with the high growth mutation is due to luteal insufficiency resulting from disruption at the hypothalamic-pituitary axis.

Animals with extreme body growth frequently experience poor reproductive performance, but the cause for this association has not been clearly established. A line of mice homozygous for the high growth (hg) mutation, a mutation that has a major effect on post-weaning growth, exhibits several reproductive deficits including an abnormally high incidence of luteal failure. The objective of the present study was to investigate luteal failure in high-growth (HG) mice during pregnancy and to determine whether the cause of the apparent luteal failure resides in the ovary or the hypothalamic-pituitary unit. In HG females with a demonstrated history of infertility, progesterone injections (1 mg s.c. daily) beginning on Day 1 postcoitum (p.c.) increased the proportion of animals pregnant at Day 17 of gestation. Twice-daily injections of 100 microgram of ovine prolactin (PRL) in alkaline saline given to another group of females beginning on Day 1 p.c. increased the proportion of HG females that were pregnant on Day 6 of gestation compared with saline-injected HG females, although PRL did not increase the pregnancy rate in HG females when compared with a group of noninjected control females. When ovaries from HG females were transplanted into ovariectomized congenic C57 hosts, the C57 graft hosts displayed normal estrous cycles, and upon mating the majority of graft hosts became pregnant. In contrast, when ovaries from C57 females were transplanted into ovariectomized HG hosts, the HG graft hosts displayed normal estrous cycles, but upon mating most were unable to maintain pregnancy. These results suggest that the hypothalamic-pituitary unit of the HG female provides an inadequate signal to the ovaries to maintain pregnancy. Luteal failure in HG females may be due to insufficient PRL as required for establishment and early maintenance of the CL during pregnancy in mice.     

Deferoxamine increases the susceptibility of beta-thalassemic, iron-overloaded mice to infection with Listeria monocytogenes.

The effect of the iron chelator deferoxamine (DFO) on resistance to infection with Listeria monocytogenes in mice with a condition analogous to human beta-thalassemia was studied. Intraperitoneal injection of 10 mg DFO resulted in significantly increased mortality when given one, three and six days before infection with L. monocytogenes (for all three time points, p less than 0.02). There were no significant differences in hematocrit, plasma iron, or splenic iron content between the two groups of mice during these time periods. In addition, splenic counts of L. monocytogenes were not significantly higher in DFO-treated compared to saline-treated mice three days after infection. Moreover, background C57Bl/6J mice were not more susceptible to Listeria infection after receiving DFO than were saline-treated controls. In conclusion, acute administration of DFO increases the susceptibility of beta-thalassemic mice to L. monocytogenes. The effect is not seen in background mice and suggests that DFO increases susceptibility to Listeria infection only in animals with iron overload.     

Abortion in mice with excessive erythrocytosis is due to impaired arteriogenesis of the uterine arcade

We postulate that repeated pregnancy loss, intrauterine growth restriction, and preeclampsia are caused by impaired elevation of uterine blood flow due to disturbed arteriogenesis of the uterine arcade. This hypothesis is based on the observation that pregnant human erythropoietin-overexpressing (plasma levels elevated 12-fold) mice (termed tg6 mice) suffering from excessive erythrocytosis generally abort at midgestation unless their hematocrit of 0.85 is drastically lowered. Transgenic mice show placental malformations that parallel those observed in pregnant women suffering from impaired uterine perfusion. Shear stress, a key factor inducing arteriogenesis, was 5-fold lower in tg6 mice compared with wildtype (WT) littermates. Consequently, uterine artery growth was reduced, and dramatically fewer viable pups (1.63 +/- 2.20 vs. 8.10 +/- 0.74 in WT) of lower weight (1.29 +/- 0.07 g vs. 1.62 +/- 0.12 g in WT) were delivered in first pregnancies. Only in subsequent pregnancies did tg6 deliver approximately the expected number of pups. Birth weights of tg6 offspring, however, remained reduced. As the spleen is a major site of extramedullary erythropoiesis in tg6 animals, splenectomy reduced the hematocrit to 0.6-0.7. In turn, shear stress increased to normal values, and splenectomized primiparous tg6 showed normal uterine artery growth and delivery of pups similar in number and weight compared with WT. We conclude that poor arteriogenesis is a previously unappreciated cause for clinically important pregnancy complications.     

Protection by dexamethasone from a lethal infection with Listeria monocytogenes in mice

Abstract The effects of dexamethasone (DEX) on a lethal infection with Listeria monocytogenes were studied in mice. Mice were completely protected against the lethal infection when treated with 3.3 mg per kg of DEX. The effect was observed only when DEX was injected before infection. The control mice died from day 3 to day 5 of infection, whereas DEX-treated mice could eliminate L. monocytogenes cells from the organs by day 11 of infection. High titres of endogenous tumour necrosis factor (TNF), interleukin-6 (IL-6) and gamma interferon (IFN-γ) were induced in the bloodstreams and organs of the drug-free mice. DEX suppressed IL-6 production, but augmented TNF and IFN-γ production within 24 h of infection, whereas production of all three endogenous cytokines was suppressed in the DEX-treated mice on day 3 of infection when the control mice began to die. These results suggest that DEX shows a protective effect on a lethal infection with L. monocytogenes in mice and that regulation of production of endogenous cytokines might be involved in the effect of DEX.