bluuebunny/crossref_metadata_2025 huggingface dataset:
Calixarenes with host-mediated potency in experimental tuberculosis: further evidence that macrophage lipids are involved in their mechanism of action
Authors: Hart P D , Armstrong J A , Brodaty E
Published: January 2020

Some time ago, it was found that attachment of hydrophilic polyoxyethylene chains to various hydrophobic phenols and alcohols gave water-soluble products which, although inactive in vitro, influenced and experimental tuberculous infection. With short chains the infection was suppressed, and with long chains it was promoted. Later work concentrated on Macrocyclon (short chain) and HOC-60 (long chain), both derived from a hydrophobic, polyphenolic calixarene. Growth of Mycobacterium tuberculosis inside macrophages (M phi) was inhibited by Macrocyclon and stimulated by HOC-60. Also, triglyceride lipase from M phi extracts and an extracellular phospholipase were inhibited by Macrocyclon and stimulated by HOC-60. This suggestion of a mechanism has been strengthened by the finding that M phi cultivated in monolayers and treated with Macrocyclon showed accumulation of lipid and little formation of fatty acid after incubation of killed cells. With HOC-60, lipid was depleted and much fatty acid was found.
Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine-rich Entamoeba histolytica protein induces an antiamebic immune response and protects gerbils from amebic liver abscess
Authors: Zhang T , Stanley S L
Published: January 2020

Attenuated salmonellae represent attractive candidates for the delivery of foreign antigens by oral vaccination. In this report, we describe the high-level expression of a recombinant fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective antigen derived from virulent amebae, and a bacterially derived maltose-binding protein (MBP) in an attenuated strain of Salmonella typhimurium. Mice and gerbils immunized with S. typhimurium expressing SREHP-MBP produced mucosal immunoglobulin A antiamebic antibodies and serum immunoglobulin G antiamebic antibodies. Gerbils vaccinated with S typhimurium SREHP-MBP were protected against amebic liver abscess, the most common extraintestinal complication of amebiasis. Our findings indicate that the induction of mucosal and immune responses to the amebic SREHP antigen is dependent on the level of SREHP-MBP expression in S. typhimurium and establish that oral vaccination with SREHP can produce protective immunity to invasive amebiasis.
Role of YadA in resistance to killing of Yersinia enterocolitica by antimicrobial polypeptides of human granulocytes
Authors: Visser L G , Hiemstra P S , van den Barselaar M T , Ballieux P A , van Furth R
Published: January 2020

The virulence plasmid pYVe of Yersinia enterocolitica codes for the production of the outer membrane protein YadA and the secretion of several proteins, called Yops, which may protect this bacterium against killing by human granulocytes. Granulocytes kill ingested microorganisms by oxygen-dependent and oxygen-independent mechanisms, the latter including antimicrobial polypeptides. The aim of this study was to determine whether virulent (pYVe+) Y. enterocolitica and plasmid-cured avirulent (pYVe-) Y. enterocolitica differ in susceptibility to antimicrobial polypeptides extracted from granules of human granulocytes. The acetic acid granule extract contained several polypeptides with antimicrobial activity against Y. enterocolitica as determined by gel overlay and radial diffusion assays. Two of these polypeptides were identified as lysozyme and defensins. pYVe+ Y. enterocolitica was less susceptible than pYVe- Y. enterocolitica to the antimicrobial activity of granule extract, lysozyme, and defensins as determined in a suspension assay, which indicated that the pYVe plasmid mediates a reduced susceptibility to these polypeptides. The role of YadA in the resistance to antimicrobial polypeptides was analyzed by using mutants of Y. enterocolitica that specifically lack or express YadA. The results demonstrated that YadA conferred resistance to the killing of Y. enterocolitica by the granule extract. Together, these results indicate that the plasmid-encoded factor YadA contributes to the resistance of Y. enterocolitica to the killing by antimicrobial polypeptides of human granulocytes.
Control of natural killer cell-mediated innate resistance against the intracellular pathogen Listeria monocytogenes by gamma/delta T lymphocytes
Authors: Ladel C H , Blum C , Kaufmann S H
Published: January 2020

Listeria monocytogenes is an intracellular bacterium which causes an acute infectious disease in mice. Initial host resistance depends on innate immunity mediated primarily by natural killer (NK) cells followed by specific alpha/beta T cells, which are central to acquired specific immunity. Gamma/delta T lymphocytes seem to provide a link between the innate and the specific immune response. All these lymphocyte populations produce gamma interferon (IFN-gamma), which, because of its macrophage-activating potential, is central to antibacterial protection. IFN-gamma from NK cells not only contributes to early host resistance but also promotes development of protective T-cell responses of helper T type 1 (Th1) type. Here, we show that innate resistance and early IFN-gamma production in listeriosis are markedly impaired in T-cell receptor (TCR)-delta-/- but not TCR-beta-/- gene disruption mutant mice. By two-color cytofluorimetry, we demonstrate that NK cells rather than gamma/delta T lymphocytes are the major cellular source of IFN-gamma in immunocompetent mice and that IFN-gamma production by NK cells is impaired in the TCR-delta-/- mutants. Probably, reduced tumor necrosis factor production in listeria-infected TCR-delta-/- mutants contributed to impaired NK cell activation. Our data reveal a novel function of gamma/delta T cells as regulators of innate resistance against sublethal infection with an intracellular pathogen.
Intracellular survival and replication of Erysipelothrix rhusiopathiae within murine macrophages: failure of induction of the oxidative burst of macrophages
Authors: Shimoji Y , Yokomizo Y , Mori Y
Published: January 2020

We investigated the ability of a virulent wild-type parent strain and acapsular avirulent transposon mutants to enter and survive intracellularly within murine peritoneal macrophages. In the presence of normal or immune serum, the parent and mutant strains were both ingested; however, the number of ingested bacteria was three- to fourfold greater in the case of mutant strains than in the case of the parent strain. The parent strain, but not the mutant strains, survived and replicated intracellularly when ingested in the presence of normal serum, whereas both the parent and the mutant strains were readily killed when ingested in the presence of immune serum. To further investigate the mechanism by which the parent strain can survive and replicate within macrophages, we studied the oxidative burst response of macrophages to these strains by measuring chemiluminescence and intracellular reduction of Nitro Blue Tetrazolium dye. Challenge exposure of macrophages with either the parent strain preopsonized with immune serum or the mutant strains preopsonized with normal or immune serum induced a strong oxidative burst, whereas the level was very low when the parent strain was preopsonized with normal serum. Phagocytosis of either the parent strain, in the presence of immune serum, or the mutant strains, in the presence of normal or immune serum, by macrophages reduced large amounts of intracellular Nitro Blue Tetrazolium, whereas minimal amounts were reduced by the parent strain in the presence of normal serum. These results suggest that virulent E. rhusiopathiae can survive and subsequently replicate within murine macrophages when ingested in the presence of normal serum and that the reduced production of reactive oxidative metabolites by macrophages may, in part, be responsible for this occurrence.
Elevated levels of Legionella pneumophila stress protein Hsp60 early in infection of human monocytes and L929 cells correlate with virulence
Authors: Fernandez R C , Logan S M , Lee S H , Hoffman P S
Published: January 2020

Legionella pneumophila 2064 was selectively radiolabelled in mouse L929 cells and human monocytes to identify proteins expressed early in the course of infection. Polypeptide profiles (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography) of adherent or intracellular bacteria indicated that a 60-kDa stress protein (Hsp60) was preferentially synthesized. Hsp60 synthesis was not induced by medium alone. The synthesis of many polypeptides, including OmpS (major outer membrane protein), diminished over the 1-h period postinfection. However, by 17 h postinfection OmpS and Hsp60 were the dominant proteins synthesized by 2064. To establish whether induction of Hsp60 was a correlate of virulence, an isogenic avirulent strain (2064M) of 2064 was isolated following selection on a nonpermissive medium. 2064M did not exhibit a stress response when adherent or intracellular in L929 cells or in human monocytes and failed to abrogate phagosome-lysosome fusion. When grown in vitro, 2064M exhibited no deficiencies in the heat shock response and its polypeptide profile resembled that of 2064. Immunogold electron microscopy was used to localize Hsp60 in L. pneumophila-infected L929 cells. There was an increase in the number of gold particles associated with phagosomes for phagosomes harboring single 2064 bacteria compared with those harboring 2064M. Moreover, by 1 h postinfection, a sixfold increase in the number of gold spheres associated with the membranes of phagosomes was observed for phagosomes harboring 2064 compared with those harboring 2064M. These studies indicate that virulent, but not NaCl-tolerant avirulent, strains of L. pneumophila respond to host-cell-associated environmental signals early in the course of infection. This response includes increased synthesis and possibly extracellular secretion of Hsp60 concomitant with repression of the expression of other genes, including ompS.
CR3-dependent resistance to acute Toxoplasma gondii infection in mice
Authors: Johnson L L , Gibson G W , Sayles P C
Published: January 2020

Studies were performed to determine whether resistance to acute Toxoplasma gondii infection in mice depends on a mechanism involving CR3, the type 3 complement receptor. Nineteen of 22 mice (86%) given multiple injections of the anti-CR3 monoclonal antibody, 5C6, prior to and after intraperitoneal inoculation of cysts of the ordinarily mildly virulent ME49 strain of T. gondii died within 8 to 12 days, whereas control antibody-treated mice survived. All (five of five) anti-CR3-treated BALB/c mice infected via the natural peroral route died within 8 days of infection. Flow cytometric analysis of cells recovered from peritoneal lavages of anti-CR3-treated T. gondii-infected mice revealed that the percentage of Thy-1+ CD4- CD8- cells was reduced to about 50% of that of control antibody-treated mice and to about 20% of the number of such cells in controls. The numbers of macrophages, polymorphonuclear leukocytes, and lymphocytes recovered from the peritoneal cavities of T. gondii-infected mice were all reduced in anti-CR3-treated mice to about 40% of those of controls. In addition, anti-CR3-treated mice had less than 25% of the induced NK cell activity of the controls, and gamma interferon was reduced to undetectable levels. Thus, the rapid death of anti-CR3-treated mice was probably caused by impaired preimmune defenses. Histological examination of anti-CR3-treated T. gondii-infected mice revealed extensive liver pathology compared with that of infected mice given a control antibody or uninfected mice given anti-CR3. The inflammation, degeneration, and necrosis in most of the anti-CR3-treated mice were severe enough to account for the observed mortalities.
Hyper-immunoglobulin G2 production by B cells from patients with localized juvenile periodontitis and its regulation by monocytes
Authors: Zhang J B , Quinn S M , Rausch M , Gunsolley J C , Schenkein H A , Tew J G
Published: January 2020

Localized juvenile periodontitis (LJP) runs in families, and a predisposition to develop disease appears to be inherited in an autosomal dominant fashion. Patients with LJP have elevated levels of serum immunoglobulin G2 (IgG2), and this is most striking in black LJP patients. We hypothesized that the markedly elevated serum IgG2 levels related to LJP status and race may be attributable to a fundamental difference in the response of black LJP leukocytes. To test this possibility, leukocytes from black LJP patients, black non-periodontitis (NP) controls, and white NP controls were cultured with a nonspecific mitogen (pokeweed mitogen) which stimulates immunoglobulin production. The levels of IgG2 produced were measured using an enzyme-linked immunosorbent assay. The results revealed that the serum IgG2 level differences among black LJP patients and white and black NP subjects were reproducible in peripheral blood leukocytes in vitro. Analysis revealed that B cells from the LJP patients appeared to be predisposed to produce high levels of IgG2. Further analysis supported the concept that the high IgG2 responses of B cells from black LJP patients were regulated by monocytes. Replacing the monocytes in cultures from white NP subjects with LJP monocytes from black patients resulted in production of IgG2 at levels that were comparable with those produced by the LJP B cells from black patients. In short, B cells from black LJP patients produce elevated levels of IgG2 in vitro, and at least part of this elevation appears to be attributable to regulation via the LJP monocytes.
Identification of a domain in Rck, a product of the Salmonella typhimurium virulence plasmid, required for both serum resistance and cell invasion
Authors: Cirillo D M , Heffernan E J , Wu L , Harwood J , Fierer J , Guiney D G
Published: January 2020

Rck is encoded on the Salmonella typhimurium virulence plasmid and is a member of a family of related 17- to 19-kDa outer membrane proteins of Enterobacteriaceae, including Ail (Yersinia enterocolitica) and PagC (S. typhimurium). Structural models for these proteins predict eight membrane-spanning domains alternating with hydrophilic inner and outer loops. When expressed in Escherichia coli, Rck and Ail, but not PagC, confer high-level resistance to the bactericidal activity of complement as well as the ability to adhere to and invade mammalian cell lines. To identify functional domains of Rck, we made and screened random mutations in Rck for decreased bioactivity. We found that a single amino acid substitution (glycine to aspartic acid) in the putative third outer loop greatly reduced Rck-mediated serum resistance and eukaryotic cell invasion. We then constructed two chimeric proteins between Rck and PagC. Substitution of the C-terminal half of Rck with the corresponding PagC fragment containing both the third and the fourth outer loops abolishes the Rck-mediated serum resistance and invasion phenotypes. Substitution of Rck with a smaller C-terminal portion of PagC containing the fourth outer loop did not affect the invasive phenotype or serum resistance. These data reveal that the third putative outer membrane loop region is important for the virulence-associated properties of the Rck protein and suggest a similarity between the mechanism of serum resistance and epithelial cell invasion involving the same domain of Rck.
An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence
Authors: Wayne L G , Hayes L G
Published: January 2020

It was demonstrated previously that abrupt transfer of vigorously aerated cultures of Mycobacterium tuberculosis to anaerobic conditions resulted in their rapid death, but gradual depletion of available O2 permitted expression of increased tolerance to anaerobiosis. Those studies used a model based on adaptation of unagitated bacilli as they settled through a self-generated O2 gradient, but the model did not permit examination of homogeneous populations of bacilli during discrete stages in that adaptation. The present report describes a model based on culture of tubercle bacilli in deep liquid medium with very gentle stirring that keeps them in uniform dispersion while controlling the rate at which O2 is depleted. In this model, at least two stages of nonreplicating persistence were seen. The shift into first stage, designated NRP stage 1, occurred abruptly at a point when the declining dissolved O2 level approached 1% saturation. This microaerophilic stage was characterized by a slow rate of increase in turbidity without a corresponding increase in numbers of CFU or synthesis of DNA. However, a high rate of production of glycine dehydrogenase was initiated and sustained while the bacilli were in this state, and a steady ATP concentration was maintained. When the dissolved O2 content of the culture dropped below about 0.06% saturation, the bacilli shifted down abruptly to an anaerobic stage, designated NRP stage 2, in which no further increase in turbidity was seen and the concentration of glycine dehydrogenase declined markedly. The ability of bacilli in NRP stage 2 to survive anaerobically was dependent in part on having spent sufficient transit time in NRP stage 1. The effects of four antimicrobial agents on the bacilli depended on which of the different physiologic stages the bacilli occupied at a given time and reflected the recognized modes of action of these agents. It is suggested that the ability to shift down into one or both of the two nonreplicating stages, corresponding to microaerophilic and anaerobic persistence, is responsible for the ability of tubercle bacilli to lie dormant in the host for long periods of time, with the capacity to revive and activate disease at a later time. The model described here holds promise as a tool to help clarify events at the molecular level that permit the bacilli to persist under adverse conditions and to resume growth when conditions become favorable. The culture model presented here is also useful for screening drugs for the ability to kill tubercle bacilli in their different stages of nonreplicating persistence.
Construction and characterization of a Yersinia enterocolitica O:8 high-temperature requirement (htrA) isogenic mutant
Authors: Li S R , Dorrell N , Everest P H , Dougan G , Wren B W
Published: January 2020

The high-temperature requirement (HtrA) family of stress response proteins are induced by different environmental stress conditions in a variety of bacteria and have been shown to contribute to the pathogenicity of some of these species. In this study, the htrA gene from Yersinia enterocolitica O:8 was amplified, cloned, and sequenced. Analysis of the deduced amino acid sequence predicted that the putative HtrA homolog contains a serine protease active site and a catalytic triad characteristic of trypsin-like serine proteases, structural features characteristic of previously described HtrA proteins. In order to evaluate the biological functions of Y. enterocolitica HtrA, an isogenic mutant was constructed by a reverse-genetics PCR-based approach. Characterization of the mutant provided evidence supporting a stress response function for the Y. enterocolitica htrA gene product. In contrast to the parent strain, the mutant showed increased sensitivity to killing by H2O2, O2- and temperature stress (50 degrees C). The mutant was avirulent in the murine yersiniosis injection model and offered partial protection to mice challenged with the parent strain. Further studies with the Y. enterocolitica htrA mutant should increase our knowledge of the host-pathogen interactions which occur during Yersinia infections.
DNA sequencing and gene expression of the emm gene cluster in an M50 group A streptococcus strain virulent for mice
Authors: Yung D L , Hollingshead S K
Published: January 2020

The strain B514, an M serotype 50 strain, is capable of causing a natural upper respiratory infection leading to death in mice, as reported by Hook et al. in 1960 (E. W. Hook, R. R. Wagner, and R. C. Lancefield, Am. J. Hyg. 72:111-119, 1960). Thus, this strain was of interest for use in developing an animal model for group A streptococcal colonization and disease. The emm gene cluster for this strain was examined by PCR mapping and found to contain three emm family genes and cluster pattern 5. PCR-generated fragments corresponding to the SF4 (mrp50), SF2 (emmL50), and SF3 (enn50) genes were cloned and the entire gene cluster was sequenced. The gene cluster has greater than 97% DNA identity to previously sequenced regions of the gene cluster of the M2 strain T2/44/RB4 if two small divergent regions that encode the mature amino terminus of the SF-2 and SF-3 gene products are not included. If expressed, the genes encode proteins which bind human immunoglobulin G (Mrp50 and EmmL50) or immunoglobulin A (Enn50). However, in isolates taken directly after passage in mice, the surface proteins arising from these genes were barely detectable. The transcription of each gene in the B514 strain was investigated by Northern (RNA) hybridization, and mRNA transcripts were detected and quantitated relative to those of the recA gene, a housekeeping gene. Transcription of all three emm family genes was found to be over 30-fold attenuated relative to transcription of the same genes in strain T2/44/RB4. This suggests that the positive regulator, Mga, either is not expressed in this strain or has a different requirement for activation; it also suggests that the capsule may be sufficient to inhibit phagocytosis under these circumstances.
The O4 specific antigen moiety of lipopolysaccharide but not the K54 group 2 capsule is important for urovirulence of an extraintestinal isolate of Escherichia coli
Authors: Russo T , Brown J J , Jodush S T , Johnson J R
Published: January 2020

Group 2 capsules and lipopolysaccharides are regarded as important virulence factors in extraintestinal isolates of Escherichia coli, but their specific contributions to bladder and renal infections, if any, are unknown. Proven isogenic derivatives deficient in the K54 antigen alone (CP9.137), the O4 antigen alone (CP921), or both the K54 and O4 antigens (CP923) were compared with their wild-type parent (CP9 [O4/K54JH5]) for growth in human urine in vitro and for virulence in vivo in a mouse model of ascending urinary tract infection (UTI). Growth of CP9.137 and CP921 was equivalent to that of CP9 in human urine. CP923 demonstrated a small but reproducible decrease in log-phase growth but achieved the same plateau density. In the mouse model of UTI, the isogenic mutant deficient in the 04 antigen alone (CP921) and, to a greater degree, the derivative deficient in both the K54 and O4 antigens (CP923) were significantly less virulent in nearly all parameters measured. In contrast, the K54 knockout derivative was as virulent as its parent, CP9, in causing bladder infection and nearly as virulent in causing renal infection. These results demonstrate an important role for the O4 antigen moiety of lipopolysaccharide in the pathogenesis of UTI. The possibility that the K54 antigen also plays a minor role cannot be excluded.
Neutralizing monoclonal antibodies against listeriolysin: mapping of epitopes involved in pore formation
Authors: Darji A , Niebuhr K , Hense M , Wehland J , Chakraborty T , Weiss S
Published: January 2020

Six different mouse monoclonal antibodies (MAbs) and a specific rabbit polygonal antibody were raised against listeriolysin. Four of the MAbs also recognized seeligeriolysin, and five cross-reacted with ivanolysin. The hemolytic activity could be neutralized by the polygonal antibody as well as by five of the MAbs. None of the neutralizing antibodies interfered with the binding of listeriolysin to the cellular membrane. The epitopes recognized by the MAbs were localized by using overlapping synthetic peptides between positions 59 and 279, a region hitherto not implicated in mediating hemolytic activity.
Deletion of amino acids 29 to 81 in adhesion protein YadA of Yersinia enterocolitica serotype O:8 results in selective abrogation of adherence to neutrophils
Authors: Roggenkamp A , Ruckdeschel K , Leitritz L , Schmitt R , Heesemann J
Published: January 2020

In order to analyze the multiple functions of the yersinia adhesin YadA in more detail, we constructed an N-terminally truncated YadA protein (deletion of amino acids [aa] 29 to 81) of Yersinia enterocolitica serotype 0:8. The region aa 29 to 81 of YadA is located between the signal sequence and the amino-terminal hydrophobic domain (aa 80 to 101), which is involved in surface polymerization and collagen binding. The deletion of aa 29 to 81 (resulting in YadADelta29-81) had no effect on the well-known features of YadA such as autoagglutination, serum resistance, HEp-2 cell adherence, binding of collagen, and binding of the complement-inhibiting factor H. In contrast to this, mutant WA(pYVO8-A-Delta29-81), producing the truncated YadADelta29-81 had lost the ability to adhere to polymorphonuclear leukocytes and to induce an oxidative burst. This functional deficiency was comparable to that of a yadA-null mutant (K. Ruckdeschel, A. Roggenkamp, S. Schubert, and J. Heesemann, Infect. Immun. 64:724-733, 1996). Moreover, mutant WA(pYVO8-ADelta29-81) turned out to be attenuated in virulence comparably to the yadA-null mutant, as demonstrated with orogastrically and intravenously infected mice. In summary, this study shows that specific functions of YadA (i) can be impaired by designed mutations and (ii) are important in distinct stages of the infection process.
Macrophages exposed to Borrelia burgdorferi induce Lyme arthritis in hamsters
Authors: Du Chateau B K , England D M , Callister S M , Lim L C , Lovrich S D , Schell R F
Published: January 2020

The mechanism(s) by which Lyme arthritis is induced has not been elucidated. In this study, we showed that macrophages have a direct, effector role in the pathogenesis of Lyme arthritis. Severe destructive arthritis was induced in recipients of macrophages obtained from Borrelia burgdorferi-vaccinated and nonvaccinated hamsters exposed to Formalin-inactivated B. burgdorferi in vitro and then challenged with the Lyme spirochete. Swelling of the hind paws was detected within 8 h of infection, increased rapidly, and peaked at 21 h. This initial swelling decreased, and by day 4 only slight swelling was detected. Severe swelling of the hind paws was detected 8 days after infection and increased rapidly, with peak swelling occurring on day 11. Histopathologic examination affirmed that macrophages exposed to Formalin-inactivated spirochetes induced a severe destructive Lyme arthritis. The onset and severity of the severe destructive arthritis were dependent on the number of macrophages transferred. By contrast, macrophages not exposed to Formalin-inactivated B. burgdorferi failed to induce severe destructive arthritis in recipients after challenge with B. burgdorferi. Similarly, severe destructive arthritis was not detected in recipients of macrophages injected with spirochetal growth medium. Our results also showed that transferred macrophages could not protect hamsters from infection with B. burgdorferi, as spirochetes were readily recovered from their tissues when cultured. These findings demonstrate that macrophages exposed to B. burgdorferi are directly involved in the induction of Lyme arthritis.
Acidic pH changes receptor binding specificity of Helicobacter pylori: a binary adhesion model in which surface heat shock (stress) proteins mediate sulfatide recognition in gastric colonization
Authors: Huesca M , Borgia S , Hoffman P , Lingwood C A
Published: January 2020

The gastric pathogen helicobacter pylori is one of a number of bacteria which bind specifically to gangliotetraosylceramide, gangliotriaosylceramide, and phosphatidylethanolamine in vitro at neutral pH. Since this organism encounters an acid pH during initial infection of the stomach, we have monitored the effect of pH on receptor binding specificity and found induction of specific binding to sulfoglycolipids (sulfatide) following brief treatment at low pH. We have previously shown that heat shock proteins (hsps) bind to sulfatide, and the suspicion that this was a stress-induced response is supported by the fact that a similar change in H. pylori binding specificity was observed if the organisms were briefly exposed to heat shock treatment. Following the stress stimulus, the change in glycolipid binding specificity was prevented by the inclusion of inhibitors of protein synthesis or by incubation with anti-hsp antibodies. Expression of hsps in the surface extract and surface reactivity with anti-hsp antibodies correlated with the change in glycolipid binding specificity. Despite the presence of high levels of H. pylori cell surface urease activity which may neutralize the microenvironmental pH, the acid-induced change in binding specificity was enhanced in the presence of urea. These studies suggest that cell surface hsps mediate sulfatide recognition by this organism under stress conditions. A binary receptor model is proposed for gastric colonization by H. pylori.
The major surface glycoprotein of Trypanosoma cruzi amastigotes are ligands of the human serum mannose-binding protein
Authors: Kahn S J , Wleklinski M , Ezekowitz R A , Coder D , Aruffo A , Farr A
Published: January 2020

Trypanosoma cruzi, an obligate intracellular protozoan parasite, chronically infects mammals and causes Chagas' disease in humans. T. cruzi evasion of the mammalian immune response and establishment of chronic infection are poorly understood. During T. cruzi infection, amastigotes and trypomastigotes disseminate in the mammalian host and invade multiple cell types. Parasite surface carbohydrates and mammalian lectins have been implicated in the invasion of mammalian cells. A recent study has demonstrated that the human mannose-binding protein and the macrophage mannose receptor, two mammalian C-type lectins, bind to T. cruzi (S. J. Kahn, M. Wleklinski, A. Aruffo, A. Farr, D. Coder, and M. Kahn, J. Exp. Med. 182:1243-1258,1995). In this report we identify the major surface glycoproteins, including the SA85-1 glycoproteins, as T. cruzi ligands of the mannose-binding protein. Further characterization of the interaction between the mannose-binding protein and T. cruzi demonstrates that (i) the SA85-1 glycoproteins are expressed by amastigotes and trypomastigotes but only amastigotes express the mannose-binding protein ligand, (ii) treatment of amastigotes with alpha-mannosidase inhibits the binding of mannose-binding protein, and (iii) amastigote binding of mannose-binding protein is stable despite the spontaneous shedding of some glycoproteins from its surface. Together, the data indicate that developmentally regulated glycosylation of surface glycoproteins controls the expression of ligands that affect the interactions between T. cruzi and mannose-binding protein. It has been established that the binding of mannose-binding protein to microorganisms facilitates their uptake into phagocytic cells. Preferential opsonization of amastigotes with mannose-binding proteins may account for their clearance from the circulation and may contribute to the parasite's ability to invade different cell types.
Monocytes of individual human subjects display heterogeneous bacterial uptake and antilisterial activity
Authors: Zerlauth G , Chehadeh H E , Maier E , Schaff Z , Eibl M M , Mannhalter J W
Published: January 2020

Peripheral blood monocytes (Mo) of normal human donors simultaneously exhibit two subsets differing in their functional activity towards the facultative intracellular bacterium Listeria monocytogenes. One subset (on average, 25% of total Mo) was characteristically able to ingest a large number of L. monocytogenes bacteria and permitted intracellular growth of these bacteria. The other Mo subpopulation (on average, 75% of total Mo) was far less active in phagocytosing L. monocytogenes and restricted intracellular L. monocytogenes growth. Electron microscopy revealed that the Listeria-permissive Mo subset allowed the bacteria to escape to the cytosol, a mechanism by which these bacteria evade the lethal attack of phagocytes. The Listeria-restrictive Mo subset, on the other hand, confined the bacteria to the phagolysosomes, where they were exposed to the killing mechanisms of the Mo. Permissiveness for L. monocytogenes growth was further associated with differences in the capacity of the Mo subsets to synthesize tumor necrosis factor alpha TNF-alpha), an important mediator in the defense against intracellular bacteria. Following challenge with L. monocytogenes, the Listeria-restrictive Mo subset secreted two to six times more TNF-alpha than did the Listeria-permissive Mo subset. Enhanced TNF-alpha secretion was paralleled by increased accumulation of TNF-alpha mRNA as assessed by quantitative PCR. Despite these functional differences, the two Mo subsets were indistinguishable with respect to expression of cell surface markers known to be involved in adherence and phagocytosis of microbes. A speculative physiological role of the two Mo subsets may lie in the dual function of Mo as microbicidal effector cells and accessory cells for antigen-specific immune reactions.
Specificity of human bactericidal antibodies against PorA P1.7,16 induced with a hexavalent meningococcal outer membrane vesicle vaccine
Authors: van der Voort E R , van der Ley P , van der Biezen J , George S , Tunnela O , van Dijken H , Kuipers B , Poolman J
Published: January 2020

A set of isogenic strains was constructed from the meningococcal reference strain H44/76 (B:15:P1.7,16) which differed only in their outer membrane protein (OMP) compositions. First, three isogenic strains lacking the expression of either class 3 (PorB) or class 4 (RmpM) OMP or both were obtained. Second, three isogenic class 1 OMP loop-deficient strains of H44/76 lacking the predicted loop 1 or 4 or both of class 1 OMP (PorA) were obtained. Third, three isogenic class 1 OMP strains which differed by point mutations in the predicted loop 4 of subtype P1.16 were constructed. Strains were constructed through transformation with gene constructs made in Escherichia coli and their homologous recombination into the meningococcal chromosome. This study describes the contribution of one of the six class 1 OMPs, PorA P1.7,16, in the development of bactericidal antibodies after a single immunization of adult volunteers with 50 or 100 micrograms of protein within a hexavalent PorA outer membrane vesicle vaccine. PorA-, PorB-, and RpmM-deficient isogenic strains were used to define the human immune response against PorA. The loop-deficient isogenic strains were used to define the contribution of loops 1 and 4 of PorA in the development of bactericidal anti-PorA antibodies. The isogenic strains carrying a point mutation in loop 4 were used to study the cross-reactivity of the induced bactericidal antibodies against target strains showing microheterogeneity. The results indicate that a single immunization with the hexavalent PorA vaccine induced a dose-dependent bactericidal immune response, which is directed mainly against PorA. The epitope specificity of antibodies is directed mostly against loop 1, although loop 4 and as-yet-unidentified epitopes of PorA P1.7,16 are also involved.
Role of calcium during lipopolysaccharide stimulation of neutrophils
Authors: Rodeberg D A , Babcock G F
Published: January 2020

This study investigated the role of intracellular calcium concentration ([Ca]i) as a possible intermediate in the lipopolysaccharide (LPS) second messenger pathway for the activation of neutrophils (polymorphonuclear leukocytes [PMNs]). Isolated PMNs were loaded with the calcium-sensitive fluorescent dye fura-2. The PMNs were stimulated with either LPS or the positive control formyl-Met-Leu-Phe (fMLP). As expected, PMN exposure to fMLP increased [Ca]i. However, LPS stimulation did not induce any detectable changes. Depletion of intracellular Ca stores with thapsigargin, or extracellular Ca with EGTA, significantly inhibited the upregulation of the CD11b/CD18 integrin in response to fMLP but not LPS. We conclude that [Ca]i is not an early intermediate in the second-messenger pathway for the activation of PMNs by LPS.
Repertoire of transcribed peripheral blood T-cell receptor beta chain variable-region genes in acute rheumatic fever
Authors: Abbott W G , Skinner M A , Voss L , Lennon D , Tan P L , Fraser J D , Simpson I J , Ameratunga R , Geursen A
Published: January 2020

Patients with severe group A streptococcal infections have abnormalities in the Vbeta repertoire of peripheral blood T cells that are consistent with superantigen stimulation by cytoplasmic membrane proteins. The purpose of this study was to determine whether similar changes in Vbeta repertoire could be found for patients with acute rheumatic fever (ARF). The mean Vbeta repertoire of peripheral blood T cells in nine hospitalized ARF patients was similar to that of 34 controls and did not change during 6 months of follow-up in 6 of the ARF subjects. We were unable to detect changes in the Vbeta repertoire of peripheral blood T cells from patients with ARF that could be attributed to the influence of a superantigen.
Transient and persistent experimental infection of nonhuman primates with Helicobacter pylori: implications for human disease
Authors: Dubois A , Berg D E , Incecik E T , Fiala N , Heman-Ackah L M , Perez-Perez G I , Blaser M J
Published: January 2020

Helicobacter pylori can establish chronic infection in the human gastric mucosa, and it is a major cause of peptic ulcer disease and a principal risk factor for gastric cancer. This creates a need for H. pylori infection models that mimic the human condition. To test the suitability of rhesus monkeys as infection models, H. pylori-free animals were inoculated intragastrically with mixtures of H. pylori strains, bacteria recovered from colonized animals were typed by arbitrarily primed PCR, and host inflammatory and immunologic responses were monitored. Among five H. pylori-free animals inoculated with a mixture of two human strains plus one monkey strain, one became persistently infected and one became only transiently infected. The recovered bacteria matched the monkey input strain in DNA fingerprint. A subsequent trial using two new human isolates and three animals that had resisted colonization by the monkey strain resulted in persistent infection in one animal and transient infection in two others. Antral gastritis, anti-H. pylori serum immunoglobulin G, and atrophy all increased, but with patterns that differed among animals. We conclude that (i) rhesus monkeys can be infected experimentally with H. pylori, (ii) individuals differ in susceptibility to particular bacterial strains, (iii) infections may be transient, and (iv) the fitness of a particular strain for a given host helps determine the consequences of exposure to that strain.
Brucella abortus as a potential vaccine candidate: induction of interleukin-12 secretion and enhanced B7.1 and B7.2 and intercellular adhesion molecule 1 surface expression in elutriated human monocytes stimulated by heat-inactivated B. abortus
Authors: Zaitseva M , Golding H , Manischewitz J , Webb D , Golding B
Published: January 2020

Development of a vaccine which is capable of generating a strong cellular immune response associated with gamma interferon (IFN-gamma) production and cytotoxic T-cell development requires that the immunogen be capable of inducing the secretion of interleukin-12 (IL-12), which is a pivotal factor for the differentiation of Th1 or Tc1 cells. We have previously shown that the heat-inactivated gram-negative bacterium Brucella abortus can induce IFN-gamma secretion by T cells. In the present study, we demonstrate that B. abortus and lipopolysaccharide (LPS) from B. abortus can induce IL-12 p40 mRNA expression and protein secretion by human elutriated monocytes (99% pure). p40 mRNA was detected within 4 h, and p40 protein could be measured at 24 h. This induction was abrogated by anti-CD14 monoclonal antibody, suggesting that monocytes recognize B. abortus via their receptor for LPS. The biological activity of IL-12 secreted by B. abortus-stimulated monocytes was demonstrated by its ability to upregulate IFN-gamma mRNA expression in T cells separated from monocytes and B. abortus by a transwell membrane. The B. abortus-induced IL-12 also enhanced NK cytolytic activity against K562 target cells. B. abortus was shown to rapidly increase the expression of the costimulatory molecules B7.1 and B7.2 and intercellular adhesion molecule 1 on human monocytes. Together, these data indicate that B. abortus can directly activate human monocytes and provide the cytokine milieu which would direct the immune response towards Th1-Tc1 differentiation.
Characterization of elastase-deficient clinical isolates of Pseudomonas aeruginosa
Authors: Hamood A N , Griswold J , Colmer J
Published: January 2020

Elastase production in Pseudomonas aeruginosa is regulated by the lasR, lasI, rhlR, and rhlI genes. Recently, we have analyzed several clinical isolates of P. aeruginosa for the production of elastase and other extracellular virulence factors. Four of these isolates (CIT1, CIW5, CIW7, and CIW8) produced no elastolytic activity. We have characterized these isolates with respect to their elastase-deficient phenotype. Elastase was detected by immunoblotting experiments using elastase-specific antiserum. We also determined the presence of IasB and IasR mRNAs by Northern (RNA) blot hybridization experiments using lasB and lasR internal probes, respectively. None of the four elastase-deficient strains produced either the elastase protein or the lasB mRNA. Complementation experiments (using plasmids carrying either the lasB or the lasR gene) were conducted to determine if the isolates carry defective lasB or lasR genes. The presence of either a lasB or a lasR plasmid in CIW7 and CIW8 resulted in the production of very low levels of elastase and lasB mRNA. Neither elastase nor lasB mRNA was detected in CIT1 and CIW5 carrying the lasB plasmid. The presence of the lasR plasmid in CIT1 and CIW5 resulted in the production of lasB mRNA and elastase protein in CIW5 only. All elastase-deficient strains produced detectable levels of lasR mRNA which were enhanced in the presence of the lasR plasmid. The Pseudomonas autoinducer (which is encoded by lasI) was also produced by all strains. CIT1 produced both hemolysin and alkaline protease but was defective in pyocyanin production. These results suggest that (i) CIT1 may contain a defect in a lasB-regulatory gene, (ii) CIW5 carries a defect within lasR, and (iii) the defect in isolates CIW7 and CIW8 affects the efficiency of lasB transcription.
Induction of cytokine gene expression by listeriolysin O and roles of macrophages and NK cells
Authors: Nishibori T , Xiong H , Kawamura I , Arakawa M , Mitsuyama M
Published: January 2020

To determine the role of listeriolysin O (LLO) of Listeria monocytogenes in the host response at the initial stage of infection, cytokine gene expression in mouse peritoneal exudate macrophages and spleen cells was examined by reverse transcription-PCR. Expression of various cytokine mRNAs, especially those of interleukin-1 (IL-1), tumor necrosis factor alpha, gamma interferon (IFN-gamma), and IL-12, was observed to occur in spleen cells after direct stimulation with an LLO preparation purified to a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Induction of mRNA expression by LLO was not blocked by cholesterol, which abrogated the hemolytic activity of LLO. After the depletion of NK cells in spleen cells by treatment with anti-asialo GM1 antibody plus complement, LLO-induced expression of IFN-gamma was decreased, indicating that NK cells were the main source of IFN-gamma. After depletion of macrophages by passing spleen cells over a Sephadex G-10 column, expression of macrophage-derived cytokines, including IL-1alpha, tumor necrosis factor alpha, and IL-12, was diminished. In addition, IFN-gamma mRNA expression was impaired, indicating that IFN-gamma mRNA expression from NK cells required signaling from macrophages. It is suggested that LLO is capable of inducing endogenous cytokines of mice, and both NK cells and macrophages are involved in the host cytokine response to LLO.
Babesia bovis rhoptry-associated protein 1 is immunodominant for T helper cells of immune cattle and contains T-cell epitopes conserved among geographically distant B. bovis strains
Authors: Brown W C , McElwain T F , Ruef B J , Suarez C E , Shkap V , Chitko-McKown C G , Tuo W , Rice-Ficht A C , Palmer G H
Published: January 2020

The ability of rhoptry-associated protein 1 (RAP-1) of Babesia bovis and Babesia bigemina to confer partial protective immunity in cattle has stimulated interest in characterizing both B-cell and T-cell epitopes of these proteins. It was previously shown that B. bovis RAP-1 associates with the merozoite surface as well as rhoptries and expresses B-cell epitopes conserved among otherwise antigenically different B. bovis strains. An amino-terminal 307-amino-acid domain of the molecule that is highly conserved in the B. bigemina RAP-1 homolog did not contain cross-reactive B-cell epitopes. The studies reported here demonstrate that B. bovis RAP-1 is strongly immunogenic for T helper (Th) cells from B. bovis-immune cattle and that like B-cell epitopes, Th-cell epitopes are conserved in different B. bovis strains but not in B. bigemina RAP-1. Lymphocytes from cattle immune to challenge with the Mexico strain of B. bovis proliferated against recombinant B. bovis RAP-1 protein derived from the Mexico strain. T-cell lines established by stimulating lymphocytes with recombinant RAP-1 protein responded against B. bovis, but not B. bigemina, merozoites. T-cell lines established by repeated stimulation of lymphocytes with B. bovis membrane antigen proliferated strongly against RAP-1, demonstrating the immunodominant nature of this protein. RAP-1-specific CD4+ T cell clones recognized Mexico, Texas, Australia, and Israel strains of B. bovis but neither B. bigemina merozoites nor recombinant B. bigemina RAP- 1. Analysis of cytokine mRNA in RAP-1-specific Th cell clones revealed strong expression of gamma interferon but little or no expression of interleukin-2 (IL-2), IL-4, or IL-10. Gamma interferon production was confirmed by enzyme-linked imunosorbent assay. These results indicate the potential to use selected B. bovis RAP-1 peptides as immunogens to prime for strong, anamnestic, strain-cross-reactive type 1 immune responses upon exposure to B. bovis.
Microtubule-disrupting agents inhibit nitric oxide production in murine peritoneal macrophages stimulated with lipopolysaccharide or paclitaxel (Taxol)
Authors: Kirikae T , Kirikae F , Oghiso Y , Nakano M
Published: January 2020

Paclitaxel (Taxol), a yew-derived antimitotic agent which binds to microtubules, can mimic certain effects of lipopolysaccharide (LPS) on macrophages from LPS responder mouse strains. The production of nitric oxide (NO) by the peritoneal macrophages of LPS responder C3H/HeN mice stimulated with taxol or LPS was partially, but not completely, suppressed by microtubule-disrupting agents, such as colchicine, podophyllotoxin, vinblastine, and nocodazole, but not by lumicolchicine, an inactive derivative of colchicine. Inducible NO synthase protein expression induced by taxol and LPS in the macrophages was also suppressed by colchicine, but colchicine did not suppress the transcription of iNOS mRNA in the macrophages after stimulation with taxol or LPS. These findings suggest that microtubules function in the posttranscriptional processes of iNOS protein expression rather than in the transcriptional process of iNOS mRNA and the synthetic process of NO molecules.