bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic modulation by Legionella pneumophila upon infection of macrophages
Authors: Abu Kwaik Y , Eisenstein B I , Engleberg N C
Published: January 2020

Since many pathogenic bacteria manifest a coordinate regulation of gene expression in response to different environmental stimuli, we examined the phenotypic response of Legionella pneumophila to infection of macrophage-like U937 cells. Intracellular L. pneumophila was radiolabeled, and cell extracts were subjected to two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At least 35 Legionella proteins were selectively induced during infection of macrophages, and one of these proteins was not detected in organisms grown in vitro. Expression of at least 32 proteins was selectively repressed during infection of macrophages, and 9 of these proteins were undetectable in intracellularly grown organisms. Thirteen of the macrophage-induced proteins were also induced by one or more of several stress conditions in vitro, and two of these proteins were the heat shock GroEL- and GroES-like proteins. Nineteen of the macrophage-repressed proteins were also repressed by one or more of the stress conditions in vitro. Our data showed that intracellular L. pneumophila manifested a phenotypic modulation and a global stress response to the intracellular environment of the macrophage. The data suggested that multiple regulons are involved in this modulation, which may contribute to the survival of L. pneumophila within alveolar macrophages.
Effector role of blood monocytes in experimental visceral leishmaniasis
Authors: Cervia J S , Rosen H , Murray H W
Published: January 2020

In BALB/c mice, liver granulomas provoked by visceral infection with intracellular Leishmania donovani are rapidly populated by influxing blood monocytes. To determine the host defense effector role of these mononuclear phagocytes, we treated three populations of infected animals with 5C6, an anti-type 3 complement receptor monoclonal antibody (MAb), which inhibits monocyte recruitment into inflamed tissues. In naive BALB/c mice, injections of 5C6 impaired the initial acquisition of antileishmanial resistance and arrested the development of mature liver granulomas. In sensitized mice with established immunity, both resistance to rechallenge and accelerated granuloma formation were similarly inhibited by MAb administration. Finally, in naive mice, 5C6 MAb also abolished the antileishmanial activity induced by treatment with the macrophage-activating lymphokine gamma interferon. Together, these results suggest a key effector role for the influxing blood monocyte in both initial and established antileishmanial defense and granuloma assembly and in the infected liver as the mononuclear phagocyte target for the antimicrobial effects of gamma interferon.
Specific and nonspecific responses of murine B cells to membrane blebs of Borrelia burgdorferi
Authors: Whitmire W M , Garon C F
Published: January 2020

Lymphocyte blastogenesis assays and immunoblotting were used to investigate and compare murine B-cell responses to preparations of extracellular membrane blebs (BAg) and spirochetes (Ag) of Borrelia burgdorferi. Immunoblotting BAg, Ag, and medium control preparations with serum from naive and infected C57BL/10 mice revealed that BAg and Ag had similar specific reactivity profiles except that major antigens of 83, 60, and 41 kDa were detected in Ag but not in BAg. It was determined that 1 microgram (dry weight) of Ag contained 0.0051 and 0.0063 microgram of outer surface proteins A (OspA) and OspB, respectively, whereas 1 microgram (dry weight) of BAg contained 0.0024 microgram of OspA and 0.0015 microgram of OspB. Both BAg and Ag caused blastogenesis in cultures of spleen cells from both groups of mice, but BAg-stimulated lymphocytes exhibited significantly greater (P < or = 0.05) blastogenesis after 2 or 6 days of culture than did lymphocytes stimulated by Ag or medium control. Flow cytometry and antibody capture enzyme-linked immunosorbent assays identified responding lymphocytes as B cells which secreted polyclonal immunoglobulin M (IgM) but not IgG or IgA. Treatment of BAg and lipopolysaccharide controls with polymyxin B resulted in as much as 20.7 and 54.3% mean decreases in blastogenesis, respectively. Fractionation of BAg or Ag by ultracentrifugation before culture with spleen cells from naive mice indicated that B-cell blastogenesis was probably associated with spirochetal membranes. The results of this study demonstrate that specific humoral responses are directed towards extracellular membrane blebs which lack the 83-, 60-, and 41-kDa antigens of intact spirochetes and that blebs also possess significant nonspecific mitogenic activity for murine B cells. This activity was not due entirely to typical lipopolysaccharide or OspA and OspB lipoproteins.
Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola
Authors: Egli C , Leung W K , Müller K H , Hancock R E , McBride B C
Published: January 2020

A 53-kDa protein from the outer sheath of the oral spirochete Treponema denticola was purified to homogeneity and shown to reconstitute channels in black lipid bilayer model membranes. The channel had a single-channel conductance of 1.8 nS in 0.1 M KCl, making this the largest porin channel observed to date (estimated diameter, 3.4 nm). Electron micrographs of 53-kDa-protein-containing outer sheaths of T. denticola showed a regular hexagonal array of darker staining pits.
Cytocidal effects of Escherichia coli hemolysin on human T lymphocytes
Authors: Jonas D , Schultheis B , Klas C , Krammer P H , Bhakdi S
Published: January 2020

Escherichia coli hemolysin is the prototype of a large family of pore-forming toxins produced by gram-negative organisms. Besides its known cytotoxic activities against granulocytes, monocytes, endothelial cells, and renal epithelial cells, we now demonstrate that the toxin potently kills human T lymphocytes. Evidence based on different and independent approaches indicates that lymphocidal activity is due to formation of transmembrane pores. Additionally, cells prestimulated with phytohemagglutinin respond to low doses of E. coli hemolysin with DNA fragmentation similar to that observed in cells undergoing programmed cell death. Kinetic considerations lead us to conclude that DNA degradation may, however, represent an epiphenomenon. Killing of T cells is another means through which E. coli hemolysin could directly impair host defense.
The ail gene of Yersinia enterocolitica has a role in the ability of the organism to survive serum killing
Authors: Pierson D E , Falkow S
Published: January 2020

Two Yersinia enterocolitica genes, inv and ail, play a major role in the ability of this microorganism to enter cultured mammalian cells. ail-homologous sequences are present only in pathogenic species and strains of Yersinia. We previously demonstrated (D. E. Pierson and S. Falkow, Infect. Immun. 58:1059-1064, 1990) that four different nonpathogenic isolates of Y. enterocolitica are not able to invade tissue culture cells because they contain functionally inactive variants of the inv gene. When a functional version was introduced into these strains, they became invasive. In this study, we introduced a functional ail gene into the same strains and found that the ail gene was expressed but that these strains neither adhere to nor invade cultured animal cells. However, these recombinant strains became resistant to killing by human serum, whereas their parental strains were not. Using an ail mutant, we also demonstrate that the ail gene has a role in both invasion/adherence and serum resistance in a pathogenic isolate of Y. enterocolitica. These results support a role for Ail in the pathogenesis of Y. enterocolitica infection and disease.
Rickettsial stimulation of endothelial platelet-activating factor synthesis
Authors: Walker T S , Mellott G E
Published: January 2020

Endothelial cells (EC) synthesize platelet-activating factor (PAF) when activated by agents such as ATP or thrombin, and PAF production occurs as a consequence of endothelial phospholipase A activity. Because interactions between Rickettsia prowazekii and a variety of host cells result in the expression of phospholipase A activity, we assessed the relative abilities of uninfected and rickettsia-infected EC to synthesize PAF. Endothelial cells were infected with rickettsiae and examined at 24-h intervals for rickettsial multiplication, EC viability, and PAF synthesis. By 24 h postinfection, 80% of the EC were infected with an average of 10.6 rickettsiae per cell; by 72 h, the rickettsiae were too numerous to count and the numbers of viable EC began to decrease. Both rickettsia-infected and sham-treated EC synthesized PAF when stimulated with either thrombin or ATP, but rickettsia-infected EC synthesized about three times as much PAF in response to cell activation as did their uninfected counterparts. Additionally, unlike their uninfected counterparts, rickettsia-infected EC synthesized significant amounts of PAF in the absence of cell activation; rickettsia-infected EC synthesized as much PAF in the absence of activation as did uninfected EC in response to ATP. In each case, essentially all of the newly synthesized PAF remained with the cell pellet. Finally, EC incubated with high numbers of rickettsiae (1,000 rickettsiae per EC) for 30 min synthesized more PAF when activated with ATP than did their sham-treated activated counterparts.
Immunological and molecular polymorphisms of OspC, an immunodominant major outer surface protein of Borrelia burgdorferi
Authors: Wilske B , Preac-Mursic V , Jauris S , Hofmann A , Pradel I , Soutschek E , Schwab E , Will G , Wanner G
Published: January 2020

The gene of the immunodominant major protein pC of Borrelia burgdorferi was previously cloned and sequenced (R. Fuchs, S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Mol. Microbiol. 6:503-509, 1992). pC is abundantly expressed on the outer surface of B. burgdorferi, as demonstrated by immunoelectron microscopy with monoclonal antibody L22 1F8. Accordingly, pC is renamed OspC, by analogy to the outer surface proteins OspA and OspB. Western immunoblot analysis of 45 B. burgdorferi isolates with monoclonal antibodies revealed that OspC is immunologically heterogeneous. Partial sequence analysis of the ospC gene confirmed the protein heterogeneity at the genetic level. We found that the degree of identity between the ospC partial sequences of five strains representing different OspA serotypes was only 63.3 to 85.4%. Immunological heterogeneity was also observed among representatives of the three newly designated genospecies of B. burgdorferi sensu lato, B. burgdorferi sensu stricto, B. garinii, and group VS461. Heterogeneity was confirmed for B. garinii at the genetic level. The ospC gene was also cloned from strains that did not express OspC, and antibody-reactive OspC was expressed in Escherichia coli. In addition, OspC-expressing variants were obtained from a nonexpressing strain by plating single colonies on solid medium. These findings confirm that the ospC gene is also present in nonexpressing strains. Because OspC is an immunodominant protein for the early immune response in Lyme borreliosis and was effective as a vaccine in an animal model, the immunological and molecular polymorphisms of ospC and OspC have important implications for the development of diagnostic reagents and vaccines.
Role of M protein in pharyngeal colonization by group A streptococci in rats
Authors: Hollingshead S K , Simecka J W , Michalek S M
Published: January 2020

As the initial step in infection, group A streptococci (GAS) colonize either the nasopharyngeal mucosa or the skin of humans. A number of virulence factors have been implicated in the colonization phase of pathogenesis based upon their in vitro activities, but the in vivo data supporting their role in colonization of the host tissues is lacking. In this investigation, the potential requirement for M protein in pharyngeal colonization by GAS was explored by using near-isogenic strains in experimental animals studies. Fischer rats were infected by intranasal and oral inoculation with both M-positive and M-negative Streptococcus pyogenes strains. Colonization of the pharyngeal area by the streptococci was monitored at various time intervals. Both M-positive and M-negative strains colonized during the first week following infection, indicating that M protein was not necessary for this initial colonization. Two M-positive strains of S. pyogenes were recovered from the rats up to 23 weeks following inoculation, while the colonization levels for M-negative strains decreased rapidly in the second and third weeks, becoming negligible by the fourth week. This indicates a potential role for M protein in the persistence of colonization at this mucosal surface. Colonization of rats with either M-positive strain of S. pyogenes also resulted in the appearance of salivary and serum antibody responses. This in vivo model should allow further investigation into factors required for GAS disease, including the examination of the potential role of the host immune response both in modulation of the pharyngeal surface and in modulation of antigenic changes in M protein or other surface factors.
Immunogenicity and efficacy of oral or intranasal Shigella flexneri 2a and Shigella sonnei proteosome-lipopolysaccharide vaccines in animal models
Authors: Orr N , Robin G , Cohen D , Arnon R , Lowell G H
Published: January 2020

Immunity against shigellosis has been shown to correlate with the presence of antibodies specific for Shigella lipopolysaccharide (LPS). We here propose a new candidate vaccine for shigellosis composed of purified Shigella flexneri 2a or Shigella sonnei LPS hydrophobically complexed with group C type 2b Neisseria meningitidis outer membrane protein proteosomes. Immunization of mice either orally or intranasally with this complex induced specific homologous anti-LPS antibodies in both intestinal and respiratory secretions as well as in sera. Strong anamnestic responses were found after two or three immunizations. LPS alone, alkaline-detoxified LPS, or alkaline-detoxified LPS complexed with proteosomes was not effective. Oral or intranasal immunization of guinea pigs with two or more doses of this proteosome-LPS vaccine elicited homologous protection against Shigella keratoconjunctivitis (Serény test). These data demonstrate that proteosomes can be used as an effective mucosal vaccine delivery system and that orally or intranasally administered acellular vaccines can protect against Shigella infections.
In vivo production of exotoxin A and its role in endogenous Pseudomonas aeruginosa septicemia in mice
Authors: Hirakata Y , Furuya N , Tateda K , Kaku M , Yamaguchi K
Published: January 2020

We have examined the production of Pseudomonas aeruginosa exotoxin A (ETA) and its role in endogenous bacteremia in mice. Mice given P. aeruginosa D4 orally died of bacteremia between days 10 and 13 following cyclophosphamide-induced leukocytopenia. In this model, serum endotoxin was detected beginning on day 7 by the Limulus assay and P. aeruginosa was cultured from blood beginning on day 9. ETA and tumor necrosis factor alpha (TNF) were also detected in serum by enzyme-linked immunosorbent assay beginning on day 9. Purified ETA did not stimulate the production of TNF in normal mice primed with a synthetic derivative of muramyl dipeptide in the absence of endotoxin. However, ETA enhanced and primed endotoxin-induced TNF production in mice. The mortality rate of mice given ETA mutant PAO-PRI (5.0%) was significantly lower than that of mice given the parent strain (78.8%). These data indicate that ETA may be an important factor in the occurrence of P. aeruginosa bacteremia and/or the death of mice. Also, ETA may be responsible for enhancing the production of a lethal dose of TNF in the presence of endotoxin in P. aeruginosa bacteremia.
Pathogenesis of tuberculosis: interaction of Mycobacterium tuberculosis with macrophages
Authors: McDonough K A , Kress Y , Bloom B R
Published: January 2020

Central to understanding the pathogenesis of tuberculosis is the interaction between the pathogen and mononuclear phagocytes. A key question about that interaction is whether Mycobacterium tuberculosis exerts an effect on phagolysosome fusion. We have reexamined the dynamics of phagolysosome fusion and its effect on intracellular bacterial replication in M. tuberculosis-infected macrophages by performing an extensive study at the electron microscopic level. Thoria-labelled murine and human macrophages were infected with a virulent (H37Rv) or avirulent (H37Ra) strain of M. tuberculosis or with Mycobacterium bovis BCG vaccine for times ranging from 2 h to 7 days. In all cases, by 2 h postinfection, approximately 85% of the bacteria clearly resided in fused vacuoles. However, at 4 days postinfection, fusion levels for viable H37Rv and H37Ra were reduced by half, whereas the fusion profiles of BCG and of heat-killed H37Rv and H37Ra were unchanged. A comparison of the numbers of bacteria per fused and nonfused vacuoles suggests both a net transfer of bacteria out of fused vacuoles and preferential bacterial multiplication in nonfused vacuoles. H37Rv and H37Ra appeared to bud from the phagolysosomes into tightly apposed membrane vesicles that did not fuse with secondary lysosomes. In some cases, no such membrane was seen and the bacteria appeared to be free in the cytoplasm. Only viable H37Rv showed a significant increase in bacterial counts during the course of infection. Thus, both of the attenuated strains we examined differed from the virulent strain H37Rv in their abilities to replicate successfully within macrophages, but each diverged from H37Rv at a different point in the process. Viable tubercle bacilli H37Rv and H37Ra had the capacity to escape from fused vesicles as the infection progressed; BCG did not. After extrusion from the phagolysosome, H37Rv, but not H37Ra, was able to multiply. These results suggest a novel mechanism by which virulent M. tuberculosis eludes the microbicidal mechanisms of macrophages by escaping from fused phagolysosomes into nonfused vesicles or the cytoplasm.
Identification of a carbohydrate recognition domain in filamentous hemagglutinin from Bordetella pertussis
Authors: Prasad S M , Yin Y , Rodzinski E , Tuomanen E I , Masure H R
Published: January 2020

The adherence of Bordetella pertussis to ciliated cells and macrophages is critical to colonization and infection of the respiratory tract. Adherence to both types of cells involves the recognition of eukaryotic carbohydrates by the bacterial adhesin filamentous hemagglutinin (Fha). The carbohydrate recognition domain (CRD) of Fha is considered an important antigen for subcomponent vaccines to maximize the generation of antiadherence antibodies capable of protecting against colonization. For identification of the CRD of Fha, a bank of eight monoclonal antibodies (MAbs) that mapped to four contiguous regions were tested for their ability to block Fha binding to lactosylceramide or to block bacterial binding to ciliated cells. Only MAb 12.5A9, which maps to amino acid residues 1141 to 1279, blocked both Fha binding to lactosylceramide and bacterial binding to ciliated cells. An 18-kDa polypeptide corresponding to this region was expressed in Escherichia coli. Cell lysates containing this protein bound to lactosylceramide in a manner identical to that of native Fha. Mutant strains of B. pertussis that contained an in-frame deletion of the coding sequence for this region produced a truncated Fha that showed negligible cross-reactivity with MAb 12.5A9. In an adherence assay, these mutant strains failed to bind efficiently to either ciliated cells or macrophages. The numbers of adherent bacteria for these strains were reduced to the number obtained with a nonadherent strain. We conclude that the region defined by residues 1141 to 1279 of Fha constitutes a CRD critical for bacterial adherence and represents a potential candidate for a subcomponent vaccine.
Localization of the ActA polypeptide of Listeria monocytogenes in infected tissue culture cell lines: ActA is not associated with actin "comets"
Authors: Niebuhr K , Chakraborty T , Rohde M , Gazlig T , Jansen B , Köllner P , Wehland J
Published: January 2020

The ActA protein of the gram-positive pathogen Listeria monocytogenes is a 90-kDa polypeptide required for interaction of the bacteria with components of the host cell microfilament system to generate intra- and intercellular movement. To study the localization, distribution, and expression of the ActA polypeptide in L. monocytogenes grown either in broth culture or in infected tissue culture cells, we first isolated ActA by monoclonal antibody-based immunoaffinity chromatography. Polyclonal rabbit antisera raised against purified ActA revealed that ActA was associated with the cell wall and exposed on the surface of the bacteria, readily accessible to ActA antibodies. In contrast, a C-terminally truncated ActA1 polypeptide expressed by the isogenic actA1 mutant was detected only in the supernatant fluids. Immunofluorescence microscopy and electron microscopic studies using immunogold labeling showed that ActA was present on the surface of the bacteria infecting PtK2 and J774 cells at all stages of the infection cycle and was not found to be associated with the actin "tail" of individual bacteria. For the isogenic actA1 mutant strain, which grew as microcolonies within infected cells, only diffuse staining of the secreted ActA1 polypeptide in the host cytoplasm was observed. The ActA polypeptide therefore appears to be required in the initiation of actin accumulation by the bacterium and is apparently not directly involved in the generation of the actin tail. Analysis of strains of several L. monocytogenes serotypes indicated microheterogeneity in the molecular weights of the ActA polypeptides of individual strains and led to the detection of a serotype 3a strain that does not produce ActA.
Endogenous gamma interferon is essential in granuloma formation induced by glycolipid-containing mycolic acid in mice
Authors: Asano M , Nakane A , Minagawa T
Published: January 2020

The present study examined the role of endogenous gamma interferon (IFN-gamma) in the formation of granulomas in mice which had been given a single intravenous injection of glycolipid-containing mycolic acid (trehalose 2,3,6'-trimycolate) purified from cell walls of Rhodococcus aurantiacus (Gordona aurantiaca) (GaGM) in the form of liposome. The histological status of granuloma formation in the livers, spleens, and lungs of GaGM-injected mice was studied at weeks 1 through 5, and the titers of endogenous IFN-gamma in all of these organ extracts and in the sera were determined by enzyme-linked immunosorbent assay. The granulomas, composed of epithelioid cells, developed until 3 weeks postinjection, and thereafter the granulomas regressed. The production of endogenous IFN-gamma was biphasic, with an early phase detected at days 1 through 3 and a late phase detected at weeks 1 through 5. The latter peak of endogenous IFN-gamma production proceeded in parallel with granuloma formation. Both the areas of granulomas and titers of IFN-gamma in these organs were dependent on the doses of GaGM used for injection. The cells which produce endogenous IFN-gamma in the spleens appear within the granulomas. To study the role of endogenous IFN-gamma in granuloma formation, the in vivo administration of rat anti-mouse IFN-gamma monoclonal antibody was carried out. Anti-mouse IFN-gamma monoclonal antibody neutralized endogenous IFN-gamma and resulted in the suppression of the number of granulomas and the size of each granuloma. These findings suggest that biphasic production of endogenous IFN-gamma in the local lesions may be crucial to the formation and development of the granulomas.
Contact of lymphocytes with Helicobacter pylori augments natural killer cell activity and induces production of gamma interferon
Authors: Tarkkanen J , Kosunen T U , Saksela E
Published: January 2020

We studied the capacity of glutaraldehyde-fixed Helicobacter pylori to stimulate natural killer (NK) cell activity. Bacteria were incubated overnight with peripheral blood lymphocytes enriched for large granular lymphocytes (LGL), the mediators of non-major histocompatibility complex-restricted cellular cytotoxicity. Then, the cytolytic activity of LGL was tested against various tumor target cells. We observed that efficient cytolytic activity was generated against resistant and nonresistant tumor target cell lines. Nine local clinical isolates of H. pylori and the reference strain NCTC 11637 were tested, and they all were equally effective in inducing NK cell activity. However, flagellin antigen, glycine extract, urease, and lipopolysaccharide prepared from H. pylori NCTC 11637 all failed to induce significant NK cell activity. The supernatants which were collected after coincubation of bacteria with LGL contained a factor(s) which could activate resting LGL into efficient cytolytic activity. The supernatants were also analyzed for interferon (IFN) activity. We observed that high titers of IFN were produced and that IFN activity was neutralized with anti-gamma interferon (IFN-gamma) antiserum, but not with anti-IFN-alpha antiserum. Thus, contact of lymphocytes with H. pylori leads to efficient stimulation of NK cell activity and the production of IFN-gamma.
Mechanism of protective immunity induced by porin-lipopolysaccharide against murine salmonellosis
Authors: Muthukkumar S , Muthukkaruppan V R
Published: January 2020

Investigations were undertaken to characterize the protective immunity induced by porin-lipopolysaccharide (LPS) against Salmonella typhimurium infection in mice. Mice immunized with porin-LPS showed higher levels of antiporin immunoglobulin G than mice which received porin alone. Further, T cells from porin-LPS-immunized mice showed an augmented proliferative response to porin in vitro compared with the response of T cells from porin-injected animals. The passive transfer of anti-LPS antibodies conferred significant protection (17%), while antiporin serum failed to protect mice against lethal challenge, indicating the protective ability of anti-LPS antibodies. However, the transfer of serum obtained from porin-LPS-immunized mice resulted in better protection (30%) than did anti-LPS or antiporin antibodies alone. In contrast to LPS, monophosphoryl lipid A completely failed to induce protection against lethal infection. However, comparable to the effect of LPS, injection of porin with monophosphoryl lipid A enhanced antibody response and the protective ability of porin (81.25%). The transfer of T cells from porin-LPS-immunized mice provided higher levels of protection (47%) against lethal challenge than did T cells from porin-immunized mice (23%). The combination of T cells and serum from porin-immunized mice transferred 36% protection. However, a combination of T cells and serum from porin-LPS-immunized mice conferred the highest level of protection (92%), which was reflected by the number of survivors (100%) in the porin-LPS-immunized group. These results demonstrate that besides the protective effect of anti-LPS antibodies, the ability of LPS to augment humoral and cell-mediated immune responses to porin confers effective protection against Salmonella infection.
Antimicrobial proteins of murine macrophages
Authors: Hiemstra P S , Eisenhauer P B , Harwig S S , van den Barselaar M T , van Furth R , Lehrer R I
Published: January 2020

Three murine microbicidal proteins (MUMPs) were purified from cells of the murine macrophage cell line RAW264.7 that had been activated by gamma interferon. Similar proteins were also present in nonactivated RAW264.7 cells, in cells of the murine macrophage cell line J774A.1, and in resident and activated murine peritoneal macrophages. MUMP-1, MUMP-2, and MUMP-3 killed Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Mycobacterium fortuitum, and Cryptococcus neoformans in vitro. MUMP-1 resembled an H1 histone but was unusual because its N-terminal residue (serine) was not N acetylated. Although MUMP-2 was N terminally blocked, its high lysine/arginine ratio and its reactivity with an antibody to H1 histones suggested that it also belonged to the H1 histone family. MUMP-3 was identical to histone H2B in 30 of 30 amino-terminal residues. Although the antimicrobial properties of histones have been recognized for decades, this is the first evidence that such proteins may endow the lysosomal apparatus of macrophages with nonoxidative antimicrobial potential. Other MUMPs, including some with a more restricted antimicrobial spectrum and one that appeared to be induced in RAW264.7 cells after gamma interferon stimulation, were noted but remain to be characterized.
Osmolarity and growth phase overlap in regulation of Salmonella typhi adherence to and invasion of human intestinal cells
Authors: Tartera C , Metcalf E S
Published: January 2020

The study of the effects of osmolarity and growth phase on Salmonella typhi adherence to and invasion of Henle 407 epithelial cells provides the first evidence of a clear overlap between these two environmental stimuli. High-osmolarity conditions are required in the late-log phase for optimum induction of the adherent and invasive phenotypes.
Role of the YadA protein in prevention of opsonization of Yersinia enterocolitica by C3b molecules
Authors: China B , Sory M P , N'Guyen B T , De Bruyere M , Cornelis G R
Published: January 2020

When mixed with normal human serum, wild-type pathogenic Yersinia enterocolitica, previously incubated at 37 degrees C, fixed less C3b than its variant cured of the virulence plasmid pYV. Mutants unable to secrete the Yop proteins were still protected against C3b deposition. By contrast, mutants deficient in the production of outer membrane protein YadA fixed more C3b than their YadA+ parent. Gene yadA, cloned as a minimal polymerase chain reaction fragment and introduced in trans, complemented the mutations. Production of YadA by recombinant Escherichia coli LK111 also resulted in a reduction of the amount of C3b deposited on the bacterial surface. The reduction of C3b at the surface of Y. enterocolitica YadA+ compared with YadA- cells correlated with an increase of the amount of factor H fixed at the bacterial surface. The YadA monomer separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane was able to bind factor H. We conclude that factor H bound to YadA reduces the C3b deposition on the bacterial surface, probably by a rapid inactivation of C3b.
Specificity of rabbit antisera against the rough lipopolysaccharide of Salmonella minnesota R4 (chemotype Rd2P-)
Authors: Swierzko A , Brade L , Paulsen H , Brade H
Published: January 2020

Rabbit polyclonal antibodies against the rough mutant lipopolysaccharide (LPS) of Salmonella minnesota R4 (chemotype Rd2P-) were serologically characterized by using R4 LPS, deacylated LPS, dephosphorylated LPS, and synthetic partial structures, including compounds comprising the core region of Rd2P- LPS bound to the beta 1-->6-linked glucosamine disaccharide with two amide-linked 3-hydroxytetradecanoic acid residues or coupled to bovine serum albumin. By using a passive hemolysis assay and an enzyme immunoassay and absorption and inhibition experiments, the antibody specificities present could be determined. One group of antibodies required components of the core oligosaccharide (with or without the side chain 3-deoxy-D-manno-octulosonic acid [Kdo]) and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The phosphate-independent antibodies were directed against the core oligosaccharide, recognizing an epitope consisting of one terminal heptose linked to Kdo or to the reducing moiety of the alpha 2-->4-linked Kdo disaccharide. Antibodies requiring the presence of acyl residues and those reacting with a single heptose or Kdo residue were not detected.
Effects of Escherichia coli and E. coli lipopolysaccharides on the function of human ureteral epithelial cells cultured in serum-free medium
Authors: Elgavish A
Published: January 2020

Escherichia coli is the microorganism most commonly isolated from human urinary tract infections. Earlier studies by others have shown that bacterial attachment and production of toxins (e.g., lipopolysaccharides [LPS]) enhance recruitment of leukocytes to the infection site and mucosal inflammation. The mechanisms by which these changes occur have not been completely defined. In the present study, epithelial cell cultures isolated from the human ureter (UT cells) (A. Elgavish, J. J. Wille, F. Rahemtulla, and L. Debro, Am. J. Physiol. 261:C916-C926, 1991; J. J. Wille, J. Park, and A. Elgavish, J. Cell. Physiol. 150:52-58, 1992) served as a model system with which to explore the mechanisms of action of Escherichia coli and E. coli LPS in UT cells. E. coli adhered to UT cells and inhibited carrier-mediated sulfate uptake to half of that in untreated UT cells, suggesting that the intracellular pool of sulfate available for sulfation may be lower in infected cells and may lead to the production of undersulfated glycoconjugates. Incubation of UT cells with E. coli LPS inhibited carrier-mediated sulfate uptake to an extent similar to that caused by whole E. coli, indicating that the effect of E. coli on sulfate uptake may be mediated by LPS. LPS caused an increase in Na+ content in rapidly proliferating UT cells but not in quiescent cells. We postulated that this change in the intracellular ionic environment or changes coupled to it (e.g., pH or Ca2+ levels) may serve as a transducing signal. This possibility was supported by the fact that LPS stimulated clustering of ICAM-1 on the cell surface of rapidly proliferating but not quiescent UT cells. This study suggests that, in vivo, LPS stimulation of ICAM-1 clustering on the surface of the urothelium may allow more effective binding of leukocytes. This may be the mechanism underlying earlier findings in vivo indicating a role for LPS in the recruitment of leukocytes to the urinary tract as a host defense mechanism following urinary tract infection.
Chylomicrons enhance endotoxin excretion in bile
Authors: Read T E , Harris H W , Grunfeld C , Feingold K R , Calhoun M C , Kane J P , Rapp J H
Published: January 2020

Chylomicrons prevent endotoxin toxicity and increase endotoxin uptake by hepatocytes. As a consequence, less endotoxin is available to activate macrophages, thereby reducing tumor necrosis factor secretion. To determine whether the chylomicron-mediated increase in hepatocellular uptake of endotoxin results in increased endotoxin excretion into bile, we examined bile after endotoxin administration. A sublethal dose (7 micrograms/kg) of 125I-endotoxin was incubated with either rat mesenteric lymph containing nascent chylomicrons (500 mg of chylomicron triglyceride per kg of body weight) or an equal volume of normal saline (controls) for 3 h and then infused into male Sprague-Dawley rats. Bile samples were collected via a common bile duct catheter for 24 h. Infusion of endotoxin incubated with chylomicrons increased biliary excretion of endotoxin by 67% at 3 h (P < or = 0.006) and by 20% at 24 h (P < or = 0.01) compared with infusion of endotoxin incubated in saline. Endotoxin activity, as measured by the Limulus assay, was not detected in the bile of test animals. However, endotoxin activity was detected after hot phenol-water extraction of bile, demonstrating that endotoxin is inactive in the presence of bile but retains bioactivity after hepatic processing. Since the majority of an intravenous endotoxin load has been shown to be cleared by the liver, acceleration of hepatocyte clearance and biliary excretion of endotoxin may represent a component of the mechanism by which chylomicrons protect against endotoxin-induced lethality.
Stimulation of DNA synthesis in osteoblast-like MC3T3-E1 cells by Bordetella bronchiseptica dermonecrotic toxin
Authors: Horiguchi Y , Sugimoto N , Matsuda M
Published: January 2020

We investigated the effects of Bordetella bronchiseptica dermonecrotic toxin on DNA synthesis in MC3T3-E1 cells. The rate of [methyl-3H]thymidine incorporation increased in the toxin-treated cells more than 24 h after addition of the toxin under the serum-starved conditions. This effect was dependent on the toxin concentration ranging from 0.3 to 3 ng/ml and was eliminated by aphidicolin and hydroxyurea, inhibitors for DNA replication. In the toxin-treated culture, the number of cells did not increase but polynucleated cells appeared and their number increased to ca. 50% of the total number of cells 6 days after the toxin addition. From these results, we concluded that the toxin stimulates DNA replication in MC3T3-E1 cells without cell proliferation.
OmpC is involved in invasion of epithelial cells by Shigella flexneri
Authors: Bernardini M L , Sanna M G , Fontaine A , Sansonetti P J
Published: January 2020

Osmoregulation of the Shigella flexneri ompC gene and the role of OmpC in Shigella virulence have been investigated. OmpC was highly expressed when bacteria were grown in medium of either low or high osmolarity. This constitutive expression is in contrast with the regulation observed in Escherichia coli, in which the expression of OmpC is repressed at low osmolarity and induced at high osmolarity. In addition, the Shigella ompC gene was barely expressed by a delta ompB (delta ompR and delta envZ) mutant. We described in a previous report that such a mutant was severely impaired in virulence both in vitro and in vivo. Starting from this observation, and in order to assess which gene(s) regulated by ompR and envZ are involved in virulence, we constructed an S. flexneri delta ompC mutant. Three S. flexneri mutants, ompF'-lacZ, delta ompC, and delta ompB, were compared for virulence. The ompF'lacZ mutant behaved like the S. flexneri serotype 5 wild-type strain M90T in all in vitro and in vivo virulence tests. On the contrary, the delta ompB and delta ompC strains were considerably impaired in their virulence phenotypes. The ability of these two mutants to spread from cell to cell and to kill epithelial cells was severely affected. Consequently delta ompC, as previously described for delta ompB, was unable to elicit a positive Sereny test. The delta ompB mutant was restored to virulence by introducing a recombinant multicopy plasmid carrying the cloned E. coli ompC gene, indicating that a functional OmpC protein was necessary and sufficient to restore virulence to this mutant of S. flexneri.
Involvement of the stage-specific 82-kilodalton adhesion molecule of Trypanosoma cruzi metacyclic trypomastigotes in host cell invasion
Authors: Ramirez M I , Ruiz R de C , Araya J E , Da Silveira J F , Yoshida N
Published: January 2020

This study provides several pieces of evidence indicating that 3F6-Ag, identified by monoclonal antibody (MAb) 3F6 as a stage-specific glycoprotein of approximately 82 kDa on the surface of metacyclic trypomastigotes of different Trypanosoma cruzi strains, promotes the entry of parasites into host cells through a ligand-receptor type interaction. First, invasion of Vero cells by metacyclic trypomastigotes of both CL and Tulahuen strains was significantly inhibited by MAb 3F6 or its Fab fragments. Second, purified 3F6-Ag bound to Vero cells in a dose-dependent and saturable fashion. Third, soluble 3F6-Ag reduced the infection of Vero cells by metacyclic forms of CL and Tulahuen strains by 90 to 97 and 50%, respectively. Unrelated proteins, as well as extracellular matrix components, such as heparan sulfate and collagen, had no effect. Our studies also show that in the Tulahuen strain, 10D8-Ag, a 35/50-kDa glycoprotein identified by MAb 10D8, participates in target cell invasion, confirming previous observations, but the variant form of 10D8-Ag expressed by highly invasive CL strain metacyclic trypomastigotes appears to be irrelevant. Overall, our results indicate that the surface components of T. cruzi metacyclic trypomastigotes involved in the process of host cell penetration are developmentally regulated molecules, such as 3F6-Ag and 10D8-Ag, that have no counterpart in blood- or tissue culture-derived trypomastigotes.
Virulence of enterohemorrhagic Escherichia coli O91:H21 clinical isolates in an orally infected mouse model
Authors: Lindgren S W , Melton A R , O'Brien A D
Published: January 2020

Escherichia coli K-12 strains producing high levels of Shiga-like toxin type II (SLT-II) but not SLT-I were previously shown to be virulent in an orally infected, streptomycin-treated mouse model. In this investigation, we tested the virulence of several SLT-II-producing enterohemorrhagic E. coli (EHEC) isolates from patients with hemorrhagic colitis or hemolytic uremic syndrome. All of the strains tested were able to colonize the mouse intestine. However, only two strains were consistently virulent for mice: O91:H21 strain B2F1 (Strr), which was previously shown to carry two copies of slt-II-related toxins, and O91:H21 strain H414-36/89 (Strr), which was found in this study to contain three genes from the slt-II group. The oral 50% lethal doses of strains B2F1 (Strr) and H414-36/89 (Strr) when fed to streptomycin-treated mice were less than 10 bacteria. Histological sections from moribund mice fed the O91:H21 strains demonstrated extensive renal tubular necrosis; however, hematological results were not consistent with a diagnosis of hemolytic uremic syndrome. The central role of SLT in the virulence of the O91:H21 EHEC strains was supported by the finding that streptomycin-treated mice preinoculated with monoclonal antibody specific for SLT-II survived oral challenge with either B2F1 (Strr) or H414-36/89 (Strr). The basis for the variation in virulence among the SLT-II-producing EHEC strains tested was not determined. However, a correlation between the capacity of an EHEC strain to grow in small intestinal mucus and lethality in the streptomycin-treated mice was observed.
Shiga toxin-associated hemolytic uremic syndrome: interleukin-1 beta enhancement of Shiga toxin cytotoxicity toward human vascular endothelial cells in vitro
Authors: Kaye S A , Louise C B , Boyd B , Lingwood C A , Obrig T G
Published: January 2020

Development of hemolytic uremic syndrome (HUS) after infection by Shigella dysenteriae 1 or enterohemorrhagic Escherichia coli has been associated with the production of Shiga toxins (verotoxins). The putative target of Shiga toxins in HUS is the renal microvascular endothelium. This report shows that preincubation of human umbilical vein endothelial cells (HUVEC) with interleukin-1 beta (IL-1 beta) enhances the cytotoxic potency of Shiga toxin toward HUVEC. A preincubation of HUVEC with IL-1 beta is required for sensitization of HUVEC to Shiga toxin. Sensitization of HUVEC to Shiga toxin is IL-1 beta dose dependent. Development of the IL-1 beta response is time dependent, beginning within 2 h of IL-1 beta preincubation and increasing over the next 24 h. That these responses were due to IL-1 beta was demonstrated by heat inactivation of IL-1 beta, by neutralization of IL-1 beta by specific antibody, and by the ability of an IL-1 beta receptor antagonist to inhibit the effect of IL-1 beta. Shiga toxin-related inhibition of HUVEC protein synthesis preceded loss of cell viability. IL-1 beta incubation with HUVEC induced the receptor for Shiga toxin, globotriaosylceramide. Lipopolysaccharide included during IL-1 beta preincubation with HUVEC increased sensitivity to Shiga toxin in an additive manner. We conclude that IL-1 beta may induce Shiga toxin sensitivity in endothelial cells and contribute to the development of HUS.
Lipoteichoic acid induces secretion of interleukin-8 from human blood monocytes: a cellular and molecular analysis
Authors: Standiford T J , Arenberg D A , Danforth J M , Kunkel S L , VanOtteren G M , Strieter R M
Published: January 2020

Invasion by gram-positive and gram-negative bacterial organisms is characterized immunopathologically by the activation of mononuclear phagocytic cells, leading to the elaboration of macrophage-derived regulatory and chemotactic factors, and the resultant influx of inflammatory leukocytes. Little is known regarding the mechanisms by which gram-positive organisms initiate macrophage activation and subsequent inflammation. In this investigation, we postulated that lipoteichoic acid (LTA) purified from two different gram-positive bacterial species was an important signal for the expression of chemotactic cytokines from human peripheral blood monocytes (PBM). In initial experiments, we demonstrated that cell-associated interleukin-8 (IL-8) was expressed by mononuclear phagocytes present in inflamed areas of endocardium in cases of acute Staphylococcus aureus endocarditis. We next demonstrated that LTA purified from either Staphylococcus aureus or Streptococcus pyogenes induced the time- and dose-dependent expression of IL-8 mRNA and protein from human PBM. The expression of IL-8 mRNA from LTA- but not lipopolysaccharide (LPS)-treated PBM was superinduced by concomitant treatment with cycloheximide, indicating that the expression of IL-8 mRNA from LTA-treated PBM was negatively controlled by repressor proteins. Furthermore, mRNA stability studies indicated that IL-8 mRNA was less stable in the presence of LTA than in the presence of LPS. Our findings indicate that LTA can induce the secretion of the polymorphonuclear leukocyte chemotactic factor IL-8 and that LTA may be an important cellular mediator of inflammatory cell recruitment that characterizes immune responses to gram-positive bacterial infections.
Necessity and sufficiency of beta interferon for nitric oxide production in mouse peritoneal macrophages
Authors: Zhang X , Alley E W , Russell S W , Morrison D C
Published: January 2020

Bacterial lipopolysaccharide and some cytokines can activate macrophages to secrete nitric oxide. Macrophage-derived nitric oxide is a key cytotoxic factor for microbicidal and tumoricidal processes. We report here that a monoclonal antibody specific for beta interferon inhibited lipopolysaccharide-induced nitric oxide production in thioglycolate-elicited C3HeB/FeJ peritoneal macrophages and macrophage-like cell line RAW 264.7. In addition, exogenous added beta interferon enabled lipopolysaccharide-hyporesponsive thioglycolate-elicited C3H/HeJ peritoneal macrophages to produce nitric oxide in response to lipopolysaccharide. These data support the concept that beta interferon provides an essential signal(s) for lipopolysaccharide-triggered nitric oxide production by mouse macrophages. Heat-killed Staphylococcus aureus, a gram-positive bacterium which was unable to initiate nitric oxide production in thioglycolate-elicited C3HeB/FeJ peritoneal macrophages in vitro, promoted nitric oxide formation in the presence of beta interferon, suggesting that beta interferon may be a general cofactor necessary for bacterium-derived stimulus-induced nitric oxide production in these macrophages. However, neither beta interferon nor tumor necrosis factor alpha, alone or in combination, triggered nitric oxide production in thioglycolate-elicited mouse peritoneal macrophages, demonstrating that these macrophage-derived cytokines, while necessary, were not sufficient by themselves for the induction of nitric oxide production in these cells. On the other hand, gamma interferon and tumor necrosis factor alpha acted together to induce nitric oxide production in vitro in the absence of lipopolysaccharide in thioglycolate-elicited mouse peritoneal macrophages, indicating that these two types of interferons provided different signals during the activation of these macrophages.