bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation or inhibition of the respiratory burst in cultured macrophages in a mycobacterium model: initial stimulation is followed by inhibition after phagocytosis
Authors: Gordon A H , Hart P D
Published: January 2020

Microorganisms cause varying degrees of stimulation of superoxide (O2-) production (respiratory burst [RB]) in macrophages but in some cases apparently inhibit the RB induced in the same monolayers by a conventional stimulator. We have explored these differences. A mycobacterium model, the slowly multiplying mouse pathogen Mycobacterium microti, induced a modest RB in resident macrophage monolayers, compared with the substantial RB induced by opsonized zymosan (Zy). However, if the 1-h M. microti pulse immediately preceded the Zy assay (instead of being concurrent), the RB was consistently less than that elicited by the Zy alone. Cytochalasin (an inhibitor of phagocytosis) enhanced Zy-induced RB, supporting the view that the burst is cell surface mediated, but this agent apparently eliminated the inhibition of the Zy-induced RB caused by prior M. microti exposure, suggesting that this inhibition may have an intracellular origin. The inhibition described extended not only to another mycobacterium (Mycobacterium bovis BCG) but also to a previous application of Zy itself. The general implications for macrophage functions of these observations on timing and sites of initiation are briefly discussed.
Lymphocyte proliferation in response to Brucella abortus 2308 or RB51 antigens in mice infected with strain 2308, RB51, or 19
Authors: Stevens M G , Olsen S C , Pugh G W
Published: January 2020

Lymphocyte proliferation to 22 protein fractions (106 to 18 kDa) of Brucella abortus 2308 or the lipopolysaccharide O-antigen-deficient mutant of 2308, strain RB51, was measured for 20 weeks after infection of mice with strain 2308, RB51, or 19. Throughout the 20-week study, the 22 protein fractions of 2308 and RB51 induced a similar pattern of proliferation when they were incubated with lymphocytes from the infected mice. In addition, during the 20 weeks, lymphocytes from all groups of infected mice exhibited the highest proliferation when the lymphocytes were incubated with 18-kDa or smaller proteins from either 2308 or RB51. Lymphocytes obtained from mice at 6 weeks after infection with strain RB51 or 19 exhibited similar proliferation to the 18-kDa proteins of S2308 or SRB51. Lymphocytes from strain 2308-infected mice did not proliferate to these proteins until 10 weeks after infection, and the responses were similar to those in strain RB51-infected mice but lower than those in strain 19-infected mice. Lymphocytes obtained from mice at 20 weeks after infection with strain 19 or 2308 proliferated to most of the 22 fractions of 2308 or RB51, which contained 106- to 18-kDa proteins. However, lymphocytes obtained from strain RB51-infected mice at 20 weeks did not proliferate to any of these fractions. These results indicate that mice infected with RB51 have less-persistent lymphocyte proliferative responses to 2308 proteins than do mice infected with 2308 or 19. In addition, all 2308 proteins that stimulate lymphocyte proliferation appear to be present in RB51.
Cloning and sequencing of a Bordetella pertussis serum resistance locus
Authors: Fernandez R C , Weiss A A
Published: January 2020

We have characterized a new virulence factor in Bordetella pertussis: serum resistance. Compared with Escherichia coli HB101, wild-type B. pertussis was relatively resistant to classical-pathway, complement-dependent killing by normal human serum. However, a mutant of B. pertussis (BPM2041) which is less virulent in mice and which has Tn5 lac inserted in a previously uncharacterized bvg-regulated gene was found to be at least 10-fold more susceptible to serum killing than the wild type. We have named this locus brk, for Bordetella resistance to killing. We have cloned and sequenced the brk locus, and it encodes two divergently transcribed open reading frames (ORFs), termed BrkA and BrkB. Both ORFs are necessary for serum resistance. Within the 300 bases which separate the two ORFs and upstream of each ORF are putative sites for BvgA binding. BrkA shows 29% identity to pertactin and has two RGD motifs in addition to a conserved proteolytic processing site and an outer membrane targeting signal. Like pertactin, BrkA is involved in adherence and invasion. Despite the similarities, a pertactin mutant was found to be not as sensitive to serum killing as the BrkA or BrkB mutants. BrkB is similar to ORFs in E. coli and Mycobacterium leprae and displays domains of homology to various transporters. On the basis of its hydropathy profile, BrkB is predicted to be a cytoplasmic membrane protein. By Southern blot, brk sequences were found in Bordetella bronchiseptica and Bordetella parapertussis but not in Bordetella avium.
Abrogation of gamma interferon-induced inhibition of Ehrlichia chaffeensis infection in human monocytes with iron-transferrin
Authors: Barnewall R E , Rikihisa Y
Published: January 2020

Ehrlichia chaffeensis is an obligate intracellular bacterium which infects cells of the macrophage/monocyte lineage. To test whether gamma interferon (IFN-gamma) inhibits infection of monocytes with E. chaffeensis, human peripheral blood monocytes were incubated with recombinant human IFN-gamma for 3 h and then exposed to E. chaffeensis. With 2,000 U of IFN-gamma per ml, maximal inhibition of infection by E. chaffeensis was observed. THP-1 cells, a human monocyte cell line, pretreated with phorbol myristic acetate or not pretreated, were incubated with various concentrations of IFN-gamma. Maximum inhibition was obtained at 1,000 U of IFN-gamma per ml with phorbol myristic acetate-treated THP-1 cells. However, nontreated cells did not achieve a similar level of anti-ehrlichial activity even with 10,000 U of IFN-gamma per ml. IFN-gamma given within 6 h postinfection was effective in inhibiting E. chaffeensis. Nitric oxide production was not demonstrated in the monocyte medium incubated with IFN-gamma and E. chaffeensis. None of the reactive oxygen intermediate scavengers tested blocked the IFN-gamma-induced anti-ehrlichial activity. Deferoxamine, an intracellular iron chelator, at 15 microM completely inhibited the survival of E. chaffeensis. Iron-saturated transferrin at 1.67 mg/ml completely reversed the IFN-gamma-induced ehrlichial killing. These results indicate that (i) E. chaffeensis is sensitive to intracytoplasmic iron depletion, (ii) E. chaffeensis is sensitive to IFN-gamma-induced killing, and (iii) the anti-ehrlichial activity induced in human monocytes by IFN-gamma is mediated by limitation of available cytoplasmic iron and is not due to the generation of reactive oxygen intermediates or nitric oxide.
Borrelia burgdorferi antigens that are targeted by antibody-dependent, complement-mediated killing in the rhesus monkey
Authors: Aydintug M K , Gu Y , Philipp M T
Published: January 2020

We identified surface antigens of Borrelia burgdorferi that are targeted by antibody-dependent, complement-mediated killing (ADCK) in the rhesus monkey. For this purpose, we had available serum samples from three animals infected with B. burgdorferi JD1 by needle inoculation and from two monkeys that were infected with the same B. burgdorferi strain by Ixodes scapularis tick bite. Sera from monkeys from the first group contained antibodies to OspA and OspB detectable by Western blot (immunoblot) using whole B. burgdorferi antigens, whereas serum samples from animals in the second group did not. The targeting of OspA and OspB by functional antibodies was demonstrated directly by showing that ADCK was partially inhibited when antibodies were preincubated with an excess of soluble recombinant OspA or OspB. Simultaneous addition of OspA and OspB did not result in an additive inhibitory effect on ADCK, a result that suggests that the epitopes on OspA and that on OspB targeted by antibody in this mechanism are the same, or at least cross-reacting. The targeting of non-OspA, non-OspB surface antigens was inferred from the fact that sera from tick-inoculated animals, which did not contain detectable anti-OspA or anti-OspB antibodies, were able to effect ADCK. This killing effect was not inhibitable by the addition of recombinant OspA or OspB or both proteins together. We also showed that both immunoglobulin G and M antibodies participate in the ADCK mechanism in the rhesus monkey. Rhesus complement does not kill B. burgdorferi in vitro in the absence of antibody, and antibody alone is effective in killing only at serum dilutions lower than 1:15. However, such "complement-independent" antibodies were not present in all bleeds. Two main conclusions may be drawn from the analysis of our results. First, both OspA and OspB are targeted by the ADCK mechanism in the rhesus monkey. Second, one or more B. burgdorferi surface antigens that are neither OspA nor OspB also participate in ADCK.
Deficient induction of the immune response to oral immunization with cholera toxin in malnourished rats during suckling
Authors: Fló J , Roux M E , Massouh E
Published: January 2020

Malnourished rats during suckling were orally immunized with cholera toxin (CT) after different periods of refeeding. Intestinal fluids, sera, and supernatant fluids from cultured mesenteric lymph node (MLN) cells were obtained after rats were given three doses of CT and analyzed by enzyme-linked immunosorbent assay (ELISA) to evaluate the specific antibody response. Serum-specific immunoglobulin G (IgG), IgA, and IgM were severely diminished in malnourished rats immunized with three doses of CT after 1 week of refeeding when compared with those of controls. Also, a decreased IgA ELISA titer of the intestinal fluids and abrogation of the capacity to neutralize the CT in the intestinal ligated loop test were found. When a booster was given at 113 days of age, the immune response continued to be affected in the serum and the intestinal fluid. The results from the analysis of the supernatant fluids from cultured MLN cells were coincident with those mentioned above. When one dose of CT was administered into Peyer's patches (PP) after 1 week of refeeding, an impaired immune response was found in the intestinal fluid of malnourished rats during suckling compared with that of controls. This result together with the analysis of supernatant from MLN and PP cell cultures suggests that antigen triggering in the PP was affected. When the refeeding period was extended to 30 days and then the first dose of CT was administered, the antibody immune responses in intestinal fluid serum and supernatant fluid approached control values. These observations reinforce the fact that the gut-associated lymphoid tissue immaturity of the rats when they received the first CT dose (at 28 days old) was the main reason for the decreased immune response observed in the experimental group.
Purified Shiga-like toxins induce expression of proinflammatory cytokines from murine peritoneal macrophages
Authors: Tesh V L , Ramegowda B , Samuel J E
Published: January 2020

Infections with Shiga toxin-producing Shigella dysenteriae type 1 and Shiga-like toxin (SLT)-producing Escherichia coli cause outbreaks of bloody diarrhea in which patients are at risk for developing life-threatening complications involving the renal and central nervous systems. Histopathology studies and in vitro experiments suggested that the toxins damage toxin receptor-expressing endothelial cells (EC) lining glomerular and central nervous system capillaries. In the presence of inducible host factors (cytokines), EC sensitivity to SLT toxicity was increased approximately 1 million-fold. We hypothesized that to manifest the vascular lesions characteristic of infection with toxin-producing bacteria, two signals were needed: systemic toxins and elevated proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 1 [IL-1], and IL-6). Human EC do not secrete these cytokines when stimulated with SLTs in vitro, suggesting that additional cells may be involved in pathogenesis. Therefore, we carried out comparative analyses of the capacity of purified (endotoxin-free) SLTs and lipopolysaccharides (LPS) to induce cytokine mRNA and proteins from murine macrophages. The cells were essentially refractory to SLT cytotoxicity, expressing low to undetectable levels of toxin receptor. SLTs and LPS induced TNF activity and IL-6 expression from macrophages, although dose response and kinetics of cytokine induction differed. LPS was a more effective inducing agent than SLTs. SLT-I-induced TNF activity and IL-6 expression were delayed compared with induction mediated by LPS. IL-1 alpha production required approximately 24 h of exposure to SLTs or LPS. Macrophages from LPS-hyporesponsive C3H/HeJ mice produced low levels of TNF activity when treated with SLT-I, suggesting that LPS and SLTs may utilize separate signaling pathways for cytokine induction.
Further characterization of the PhoP regulon: identification of new PhoP-activated virulence loci
Authors: Belden W J , Miller S I
Published: January 2020

Salmonella typhimurium survival within macrophages is an essential virulence property necessary to enteric fever pathogenesis. This survival requires coordinate transcriptional activation of virulence genes within acidified macrophage phagosomes. Virulence gene transcription is regulated by a two-component system comprising the PhoP (transcriptional activator) and PhoQ (sensor-kinase) proteins. Thirteen new PhoP-activated loci (designated pagD to pagP) encoding membrane or secreted proteins have been identified by use of the transposon TnphoA. Three of these loci have a chromosomal location that was linked to the previously identified pagC locus. Strains with TnphoA insertions in pagD, pagJ, pagK, and pagM were significantly attenuated for mouse virulence (50% lethal dose greater than 1,000 times that of wild-type bacteria). No strains with pag::TnphoA insertions were found to have altered sensitivity to the cationic antimicrobial peptide NP-1 defensin. PhoP and PhoQ are pleotropic regulators of membrane or secreted proteins, suggesting that the ability to effect a global change in the expression of these proteins is required for S. typhimurium survival within acidified macrophage phagosomes.
Production of a hemolytic factor by Candida albicans
Authors: Manns J M , Mosser D M , Buckley H R
Published: January 2020

Candida albicans exhibits hemolytic activity when grown on glucose-enriched blood agar. This activity is present on intact organisms, and it is secreted into the culture medium. Hemoglobin released from lysed erythrocytes can restore the transferrin-inhibited growth of C. albicans. We conclude that C. albicans expresses a hemolytic factor which allows it to acquire iron from host erythrocytes.
Administration of antigranulocyte monoclonal antibody RB6-8C5 prevents expression of acquired resistance to Listeria monocytogenes infection in previously immunized mice
Authors: Czuprynski C J , Brown J F , Wagner R D , Steinberg H
Published: January 2020

Recent studies have established the importance of neutrophils in innate resistance to Listeria monocytogenes infection in mice. The purpose of this study was to determine the importance of neutrophils in acquired resistance to L. monocytogenes infection. Previously immunized mice that were depleted of neutrophils by administration of the antigranulocyte monoclonal antibody RB6-8C5 demonstrated less resistance to L. monocytogenes challenge than did nonimmunized control mice. In contrast, immunized control mice exhibited a heightened resistance to rechallenge, as expected. These results suggest that neutrophils make previously unrecognized contributions to acquired immunity to a facultative intracellular pathogen.
Enhancement of anti-Shigella lipopolysaccharide (LPS) response by addition of the cholera toxin B subunit to oral and intranasal proteosome-Shigella flexneri 2a LPS vaccines
Authors: Orr N , Arnon R , Rubin G , Cohen D , Bercovier H , Lowell G H
Published: January 2020

Addition of the cholera toxin B subunit to oral and intranasal proteosome-Shigella flexneri 2a lipopolysaccharide vaccines improved their immunogenicities. Enhancement of anti-O-Shigella immunoglobulin A levels was most evident in lung lavages following oral immunization and in lung and intestinal fluids when suboptimal doses were used with either immunization route.
Virulence of Aspergillus fumigatus double mutants lacking restriction and an alkaline protease in a low-dose model of invasive pulmonary aspergillosis
Authors: Smith J M , Tang C M , Van Noorden S , Holden D W
Published: January 2020

To investigate the pathogenicity of Aspergillus fumigatus mutants lacking putative virulence factors, we have developed a new murine model of invasive pulmonary aspergillosis based on neutropenia, the major factor predisposing patients to this infection. Mice were treated with cyclophosphamide and inoculated by the intranasal route with 5 x 10(3) conidia, a significant reduction from inoculum levels used in previous models. Evidence for the production of the extracellular alkaline protease (Alp) in lung tissue was obtained by using a fungal transformant harboring an alp::lacZ reporter gene fusion. The pathogenicities of single mutant strains lacking either Alp or the ribotoxin restrictocin and of a double mutant strain lacking both proteins were assessed in this infection model. There were no significant differences between the mutant and the wild-type strains in terms of mortality or histological-features. Inoculations with mixtures of conidia showed that the double mutant strain is slightly less virulent than the wild-type strain. We conclude that Alp and restrictocin are not important virulence determinants in pulmonary infection.
GTP-binding proteins are involved in the modulated activity of human neutrophils treated with the Panton-Valentine leukocidin from Staphylococcus aureus
Authors: Hensler T , Köller M , Prévost G , Piémont Y , König W
Published: January 2020

Significant amounts of leukotriene B4 (LTB4) are generated by human polymorphonuclear neutrophils (PMNs) after incubation with the Panton-Valentine leukocidin (Luk-PV) from Staphylococcus aureus V8 strains. We showed that GTP-binding proteins (G proteins) are involved in the Luk-PV-activated signal transduction of PMNs. ADP-ribosylation of heterotrimeric G proteins by cholera and pertussis toxins decreased the Luk-PV-induced LTB4-generation. In contrast, ADP-ribosylation of the low-molecular-weight G proteins rho and rac by Clostridium botulinum exoenzyme C3 increased the Luk-PV-induced LTB4 synthesis. The subsequent stimulation of Luk-PV-treated PMNs by either calcium ionophore A23187, sodium fluoride, or formylmethionyl-leucyl-phenylalanine was significantly inhibited. This decrease was paralleled by a loss of G-protein functions, including GTPase activity and GTP-binding capacity. An increase of G-protein functions was obtained with low amounts of Luk-PV. In addition to the modulated G-protein functions, ADP-ribosylation of 24-, 40-, and 45-kDa proteins by Luk-PV was detected. As shown in control experiments, the ADP-ribosylated 24-kDa proteins were not substrates for C. botulinum exoenzyme C3. Introduction of ras p21 into digitonin-permeabilized PMNs was without effect on subsequent Luk-PV stimulation. In addition, the translocation of ras p21, ras GAP, and 5-lipoxygenase into the membrane of Luk-PV-treated PMNs, as well as the expression of chemotactic membrane receptors for LTB4 and formylmethionyl leucyl phenylalanine, was significantly diminished.
Expression of the L8 lipopolysaccharide determinant increases the sensitivity of Neisseria meningitidis to serum bactericidal activity
Authors: Moran E E , Brandt B L , Zollinger W D
Published: January 2020

The influence of lipopolysaccharide (LPS) expression on the sensitivity of Neisseria meningitidis to serum bactericidal activity was investigated by using a series of variants that expressed different LPS determinants. For each of two different strains, a series of three LPS variants that expressed L3,7, L8, or both were analyzed. LPS variants were identified and monitored by colony blotting with murine monoclonal antibodies specific for the L8 or the L3,7,9 immunotype determinants. Differences in LPS expression were verified by analysis of proteinase K lysates of whole cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then by silver staining or immunoblotting. The variants were used as test strains in bactericidal assays with human sera and with murine sera and monoclonal antibodies. Expression of the L8 LPS epitope was correlated with increased sensitivity to serum bactericidal activity. The geometric mean bactericidal titers of 12 to 16 sets of pre- and postvaccination sera were determined for each variant. Mean serum titers increased progressively with increased expression of L8 on the target strain. The bactericidal titers of anti-outer membrane protein monoclonal antibodies also increased with increased L8 expression.
Alteration of protective and serologic responses in BALB/c mice vaccinated with chemically modified versus nonmodified proteins of Brucella abortus 19
Authors: Pugh G W , Tabatabai L B
Published: January 2020

A study was conducted to determine whether the covalent chemical modification of Brucella abortus 19 salt-extractable proteins (BCSP) and BCSP derivatives would modulate the immune responses in BALB/c mice. Salt-extractable proteins BCSP 0-70 and BCSP 70-100 were modified with acetoacetic anhydride, and recombinant proteins rBCSP20 (20 kDa), rBCSP31 (31 kDa), and rBCSP45 (45 kDa) were modified with succinic and dodecanoyl anhydrides. Four weeks after mice were vaccinated with the different preparations, principal and control mice were challenge exposed with a virulent culture of B. abortus 2308, and mice were necropsied 2 weeks later. Serum samples were obtained immediately before mice were challenge exposed and at necropsy. Sera were tested for specific immunoglobulin M (IgM) and G (IgG) antibodies by using an enzyme-linked immunosorbent assay. Acylation decreased the immune responses (increased IgG antibodies and reduced spleen CFU and splenomegaly) induced by both BCSP 0-70 and BCSP 70-100. Modification of the recombinant proteins by dodecanoyl and succinic anhydrides had no effect on the protection induced; however, the IgG serologic responses to the homologous and heterologous proteins were altered. Monophosphoryl lipid A markedly enhanced the immunogenicity of BCSP 0-70.
Saccharomyces cerevisiae virulence phenotype as determined with CD-1 mice is associated with the ability to grow at 42 degrees C and form pseudohyphae
Authors: McCusker J H , Clemons K V , Stevens D A , Davis R W
Published: January 2020

Saccharomyces cerevisiae isolates have been shown previously to exhibit a high degree of variation in their ability to proliferate and persist in CD-1 mice (K.V. Clemons, J.H. McCusker, R. W. Davis, and D.A. Stevens, J. Infect. Dis. 169:859-867, 1994). Isolate origin was not a firm predictor of virulence phenotype, since the virulence phenotypes of clinical and nonclinical isolates ranged from virulent to avirulent and from intermediate to avirulent, respectively. Therefore, it was important to determine if there was any association between putative virulence traits and virulence that might help explain the variation in virulence phenotypes. S. cerevisiae isolates spanning a range of virulence phenotypes in experimental infections were examined for putative virulence traits: the ability to grow at supraoptimal temperatures (42, 39, and 37 degrees C), gelatin liquefaction, casein utilization, and pseudohyphal formation. Gelatin liquefaction appeared to be unrelated to pseudohyphal formation on casein or to virulence. Significant differences in the ability to grow at 39 and 42 degrees C were observed when the virulent and intermediate classes were compared with the avirulent class. Less extreme but still significant differences in pseudohyphal formation were observed when the virulent and intermediate classes were compared with the avirulent class. Therefore, two virulence traits, similar to those identified in other pathogenic fungi, the ability to grow at elevated temperatures and pseudohyphal formation, have been identified in S. cerevisiae.
Aeromonas salmonicida resistance to complement-mediated killing
Authors: Merino S , Albertí S , Tomás J M
Published: January 2020

The resistance of Aeromonas salmonicida to complement-mediated killing was investigated by using different strains and their isogenic mutants that had been previously characterized for their surface components. We found that the classical complement pathway is involved in serum killing of susceptible A. salmonicida strains, while the alternative complement pathway seems not to be involved. All of the A. salmonicida strains are able to activate complement, but the smooth strains (with or without the A-layer) are resistant to complement-mediated killing. The reasons for this resistance are that C3b may be bound far from the cell membrane and that it is rapidly degraded; therefore, the lytic final complex C5b-9 (membrane attack complex) is not formed. Isogenic rough mutants are serum sensitive because they bind more C3b than the smooth strains, and if C3b is not completely degraded, then the lytic complex (C5b-9) is formed.
Evidence for naturally occurring recombination in the gene encoding the major outer membrane protein of lymphogranuloma venereum isolates of Chlamydia trachomatis
Authors: Hayes L J , Yearsley P , Treharne J D , Ballard R A , Fehler G H , Ward M E
Published: January 2020

The nucleotide sequence of the major outer membrane protein gene (omp1) was determined for three geographically distinct lymphogranuloma venereum isolates which were serologically untypeable. The three omp1 sequences were hybrids of serovars L1 and L2, containing a putative DNA recombination site in variable segment 2. Efforts to manipulate the chlamydial genome in vitro by recombination should be intensified.
Bacterial heat shock proteins directly induce cytokine mRNA and interleukin-1 secretion in macrophage cultures
Authors: Retzlaff C , Yamamoto Y , Hoffman P S , Friedman H , Klein T W
Published: January 2020

Bacterial heat shock proteins (hsp) have been shown to be important immunogens stimulating both T cells and B cells. However, little is known concerning the direct interactions between hsp and macrophages. In this study, we demonstrated that treatment of macrophage cultures with purified bacterial hsp, including Legionella pneumophila hsp60, Escherichia coli GroEL, Mycobacterium tuberculosis hsp70, Mycobacterium leprae hsp65, and Mycobacterium bovis BCG hsp65, increased the steady-state levels of cytokine mRNA for interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor as well as supernatant IL-1 secretion. This effect was shown not to be due to contamination of the hsp preparations with bacterial lipopolysaccharide. However, not all hsp induced cytokines; M. tuberculosis hsp10 showed minimal activity in our study. These results suggest that bacterial hsp might modulate immunity by rapidly and directly increasing cytokine production in macrophages.
Identification of a gene essential for piliation in Haemophilus influenzae type b with homology to the pilus assembly platform genes of gram-negative bacteria
Authors: Watson W J , Gilsdorf J R , Tucci M A , McCrea K W , Forney L J , Marrs C F
Published: January 2020

Haemophilus influenzae type b (Hib) pili are complex filamentous surface structures consisting predominantly of pilin protein subunits. The gene encoding the major pilin protein subunit of Hib adherence pili has been cloned and its nucleotide sequence has been determined. In order to identify specific accessory genes involved in pilus expression and assembly, we constructed isogenic Hib mutants containing insertional chromosomal mutations in the DNA flanking the pilin structural gene. These mutants were screened for pilin production, pilus expression, and hemagglutination. Pili and pilin production were assessed by immunoassays with polyclonal antisera specific for pilin and pili of Hib strain Eagan. Hemagglutination was semiquantitatively evaluated in a microtiter plate assay. Six Hib mutants produced proteins immunoreactive with antipilin antiserum but no longer produced structures reactive with antipilus antiserum. In addition, the mutants were unable to agglutinate human erythrocytes. Nucleotide sequence analysis localized the insertion sites in the six mutants to 2.5-kb open reading frame upstream of the pilin structural gene and immediately downstream of an Hib pilin chaperone gene. The amino acid sequence encoded by this open reading frame has significant homology to members of the pilus assembly platform protein family, including FhaA of Bordetella pertussis, MrkC of Klebsiella pneumoniae, and the Escherichia coli assembly platform proteins FimD and PapC. This open reading frame, designated hifC, appears to represent a gene essential to Hib pilus biogenesis that has genetic and functional similarity to the pilus platform assembly genes of other gram-negative rods.
Modulation of cell surface-associated mannoprotein antigen expression in experimental candidal vaginitis
Authors: De Bernardis F , Molinari A , Boccanera M , Stringaro A , Robert R , Senet J M , Arancia G , Cassone A
Published: January 2020

The monoclonal antibody (MAb) AF1 recognizes an oligosaccharide epitope present on highly immunogenic and immunomodulatory mannoproteins (MP) of Candida albicans. The expression of this epitope (AF1-MP) during experimental candidal vaginitis was studied in two strains of C. albicans (3153 and CA-2) which were equally vaginopathic but differed in the mode of hypha formation in the vagina. In both strains, immunofluorescence of vaginal samples, taken 1 h after challenge, revealed an intense, MAb AF1-specific labelling of the yeast cells. This labelling was very scarce in fungal cells taken at 24 h and on subsequent days during the development of filamentous forms. Electron-microscopic gold immunolabelling observations showed that molecules carrying AF1-MP spanned the entire cell wall in the initial yeast cells but were absent on the cell surface and in the outermost, capsular layer of the cell wall of the germ tubes and filamentous forms. In both strains, at any time and for any form of intravaginal growth, AF1-MP was clearly expressed in the cytoplasm and cytoplasmic vesicles, and was fully incorporated into the inner layers of the cell wall. As seen by immunofluorescence, the vaginal fluid from C. albicans-infected rats did not hinder the expression of AF1-MP on the yeast cells surface in vitro. In electron-microscopic gold immunolabelling, a hypha-specific MAb (3D9) labelled the surface of the hyphal but not of the yeast cells of C. albicans harvested from rat vagina. Overall, these data strongly suggest that cell surface expression of MP antigen is modulated during intravaginal growth and morphogenesis of C. albicans.
Conconavalin A-stimulated bovine T-cell supernatants inhibit growth of Cowdria ruminantium in bovine endothelial cells in vitro
Authors: Mahan S M , Smith G E , Byrom B
Published: January 2020

Conconavalin A-stimulated bovine T-cell supernatants inhibited the growth of Cowdria ruminantium in bovine endothelial cells in vitro but did not affect their entry. This finding represents one mechanism by which T cells may control C. ruminantium multiplication and hence affect the severity of disease.
Human secretory immunoglobulin A anti-Entamoeba histolytica antibodies inhibit adherence of amebae to MDCK cells
Authors: Carrero J C , Díaz M Y , Viveros M , Espinoza B , Acosta E , Ortiz-Ortiz L
Published: January 2020

The presence of secretory immunoglobulin A (IgA) anti-Entamoeba histolytica antibodies in the saliva of patients with intestinal amebiasis was demonstrated by immunoblot assay, and the capacity of these antibodies to inhibit amebic adherence to a monolayer of MDCK cells was analyzed. Inhibition was due to IgA antiamebic antibodies and in part to anti-Gal-binding-lectin antibodies, as demonstrated by absorption experiments with total amebic extract and with the fraction of Gal-binding lectin. These results emphasize the relevance of secretory IgA antibodies in the phenomenon of E. histolytica adherence to epithelial cells.
Identification and characterization of the Treponema pallidum tpn50 gene, an ompA homolog
Authors: Hardham J M , Stamm L V
Published: January 2020

Treponema pallidum is a pathogenic spirochete that has no known genetic exchange mechanisms. In order to identify treponemal genes encoding surface and secreted proteins, we carried out TnphoA mutagenesis of a T. pallidum genomic DNA library in Escherichia coli. Several of the resulting clones expressed enzymatically active T. pallidum-alkaline phosphatase fusion proteins. The DNA sequence of the 5' portion of a number of the treponemal genes was obtained and analyzed. A recombinant clone harboring plasmid p4A2 that encoded a treponemal protein with an approximate molecular mass of 50,000 Da was identified. Plasmid p4A2 contained an open reading frame of 1,251 nucleotides that resulted in a predicted protein of 417 amino acids with a calculated molecular mass of 47,582 Da. We have named this gene tpn50 in accordance with the current nomenclature for T. pallidum genes. A 1.9-kb HincII-ClaI fragment from p4A2 that contained the tpn50 gene was subcloned to produce p4A2HC2. Comparison of the predicted amino acid sequence of TpN50 with protein sequences in the National Center for Biotechnology Information data base indicated statistically significant homology to the Pseudomonas sp. OprF, E. coli OmpA, Bordetella avium OmpA, Neisseria meningitidis RmpM, Neisseria gonorrhoeae PIII, Haemophilus influenzae P6, E. coli PAL, and Legionella pneumophila PAL proteins. These proteins are all members of a family of outer membrane proteins that are present in gram-negative bacteria. The tpn50 gene complemented E. coli ompA mutations on the basis of two separate criteria. First, morphometry and electron microscopy data showed that E. coli C386 (ompA lpp) cells harboring plasmid vector pEBH21 were rounded while cells of the same strain harboring p4A2HC2 (TpN50+), pWW2200 (OprF+), or pRD87 (OmpA+) were rod shaped. Second, E. coli BRE51 (MC4100 delta sulA-ompA) cells harboring pEBH21 grew poorly at 42 degrees C in minimal medium, while the growth of BRE51 cells harboring p4A2HC2 was similar to that of the parental MC4100 cells. These results demonstrate that the TpN50 protein is functionally equivalent to the E. coli OmpA protein. If TpN50 functions in a similar fashion in T. pallidum, then it may be localized to the treponemal outer membrane.
Nitric oxide produced during murine listeriosis is protective
Authors: Boockvar K S , Granger D L , Poston R M , Maybodi M , Washington M K , Hibbs J B , Kurlander R L
Published: January 2020

Nitric oxide (NO) has been shown to be important for intracellular microbiostasis in vitro. To determine the role of NO in immune function in vivo, groups of C57BL/6 mice were given a sublethal intravenous inoculum of Listeria monocytogenes EGD, and their urine was monitored daily for nitrate, the mammalian end product of NO metabolism. Urinary nitrate levels peaked at 5 to 10 times the basal level on days 5 to 6, when spleen and liver Listeria counts declined most steeply, and decreased thereafter, when spleens and livers were nearly sterile. Peritoneal macrophages explanted from Listeria-infected mice produced nitrite spontaneously, whereas macrophages from uninfected mice did not. The inducible NO synthase mRNA was detectable in the spleens of infected mice on days 1 to 4 of infection. When Listeria-infected mice were treated orally throughout the infection with NG-monomethyl-L-arginine (NMMA), a specific NO synthase inhibitor they showed no detectable rise in urinary nitrate excretion. Mean Listeria counts in the livers and spleens NMMA-treated mice were 1 to 3 orders of magnitude greater than counts in control mice on days 4 through 8 of infection. Compared with control mice, NMMA-treated mice also showed worse clinical signs of infection, namely, weight loss, hypothermia, decreased food and water intake, and decreased urine output. Histologically NMMA-treated mice had many more inflammatory foci in their livers and spleens than control mice. The histologic observation that mononuclear cells are present at sites of infection suggests that inhibiting NO production did not block the flux of macrophages into infected viscera. As controls for possible drug toxicity, a group of uninfected mice given NMMA orally showed no detrimental effects on weight, temperature, and food and water intake. These experiments demonstrate that inhibition of NO production in Listeria-infected mice results in an exacerbated infection and thus that NO synthesis is important for immune defense against Listeria infection in mice.
Heparin-inhibitable lectin activity of the filamentous hemagglutinin adhesin of Bordetella pertussis
Authors: Menozzi F D , Mutombo R , Renauld G , Gantiez C , Hannah J H , Leininger E , Brennan M J , Locht C
Published: January 2020

Bordetella pertussis, the etiologic agent of whooping cough, produces an outer membrane-associated filamentous hemagglutinin (FHA) which is the major adhesin of this organism. FHA exhibits a lectin-like activity for heparin and dextran sulfate. By using in vitro adherence assays to cultured epithelial cells, the attachment of B. pertussis was reduced in the presence of sulfated polysaccharides such as heparin and dextran sulfate but not in the presence of dextran, indicating the crucial role of polysaccharide sulfation. In addition, inhibition of cellular sulfation by chlorate treatment of the cells resulted in a reduction of B. pertussis adherence, suggesting that epithelial cell surface-exposed sulfated glycoconjugates may serve as receptors for the microorganism. B. pertussis mutant strains deficient in FHA production expressed residual adherence that was no longer inhibited by sulfated polysaccharides. In addition, purified FHA displayed heparin-inhibitable binding to epithelial cells. Mapping experiments of the heparin-binding site of FHA indicated that this site is different from the RGD site and the recently proposed carbohydrate-binding site involved in the interaction of FHA with lactosylceramide. This result demonstrates that FHA contains at least three different binding sites, a feature unusual for bacterial adhesions but similar to features of eukaryotic adhesins and extracellular matrix proteins.
Isolation and characterization of a gene encoding a Chlamydia pneumoniae 76-kilodalton protein containing a species-specific epitope
Authors: Perez Melgosa M , Kuo C C , Campbell L A
Published: January 2020

Chlamydia pneumoniae is a human respiratory pathogen. Unlike the other two Chlamydia species, no species-specific antigen has been defined for C. pneumoniae. An immunoreactive clone containing a 0.8-kb fragment was isolated from a C. pneumoniae (AR-39) genomic library by using anti-C. pneumoniae rabbit immune serum. By Southern hybridization analysis of chromosomal digests of the different Chlamydia spp., the 0.8-kb fragment was shown to react specifically with C. pneumoniae. Subcloning of this fragment into the pGEX-1 lambda T expression vector resulted in the expression of a 62-kDa fusion protein. This fusion protein as well as the cleaved C. pneumoniae peptide were recognized by anti-C. pneumoniae rabbit immune serum, while the glutathione S-transferase moiety was not recognized. The fusion protein was used to produce monospecific rabbit antiserum. This antiserum was shown to react with a 76-kDa protein in all C. pneumoniae isolates tested, specifically recognize C. pneumoniae inclusions in tissue culture, and neutralize infectivity of C. pneumoniae in cell culture. No reactivity was observed with Chlamydia trachomatis or Chlamydia psittaci. To isolate the entire coding sequence of the 76-kDa protein, two partially overlapping fragments of C. pneumoniae DNA, a 3.2-kb HindIII fragment and a 1.2-kb PvuII fragment, were isolated, cloned, and sequenced. No significant sequence similarity was found with any previously reported nucleotide or amino acid sequence of the other Chlamydia species. This C. pneumoniae protein containing a species-specific epitope could play a role in pathogenesis and may be useful as a diagnostic tool.
Pentoxifylline and CD14 antibody additively inhibit priming of polymorphonuclear leukocytes for enhanced release of superoxide by lipopolysaccharide: possible mechanism of these actions
Authors: Yasui K , Komiyama A , Molski T F , Sha'afi R I
Published: January 2020

Lipopolysaccharide (LPS) primes human polymorphonuclear leukocytes (PMN) for enhanced O2- production in response to stimulation by N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Serum factor is essential for priming at lower concentrations of LPS. Complexes of LPS and LPS-binding protein are recognized by CD14 on PMN. We investigated the effects of a monoclonal antibody against CD14 (MY4) and of pentoxifylline (POF), a membrane fluidizer, alone and in combination, on LPS-LPS-binding protein activation of phospholipase D evidenced by increased phosphatidic acid formation. Phosphatidic acid formation and O2- production were inhibited by MY4 and POF. Our results suggest that the actions of these agents occur at an early step in the excitation-response sequence. In the absence of a second stimulus, LPS plus serum caused an increase in the amount of Gi alpha 2 associated with the membrane via CD14. POF, however, had no effect on Gi alpha 2 in the membrane. POF alone significantly changed the affinity (KD) of the fMet-Leu-Phe receptor of PMN (from 25.2 +/- 4.5 nM to 15.2 +/- 2.4 nM [P < 0.01; n = 4]) at 37 degrees C. The differences between the sites of action of MY4 and POF may lead to cooperation by these agents for inhibition of priming by LPS plus serum for enhanced O2- production. Clinical use of the antibody and POF may diminish tissue damage caused by PMN in clinical endotoxic shock.
Alterations of neutrophil responses to tumor necrosis factor alpha and interleukin-8 following human endotoxemia
Authors: Solomkin J S , Bass R C , Bjornson H S , Tindal C J , Babcock G F
Published: January 2020

Interleukin-8 (IL-8), a neutrophil chemoattractant and activating cytokine, has been implicated as a proinflammatory mediator in gram-negative sepsis. In vitro data support the notion of IL-8 as an endothelial adherence inhibitor. To evaluate this issue, we infused six volunteers with reference endotoxin and measured plasma levels of IL-8, neutrophil tumor necrosis factor alpha (TNF-alpha) receptors, TNF-alpha-induced adherence to fibronectin, and neutrophil chemotaxis to IL-8 and other attractants. We found that, at 3 h postinfusion, IL-8 but not TNF-alpha plasma levels were elevated. Neutrophils had shed L-selectin (mean channel fluorescence decrease, 79 +/- 9 to 49 +/- 7; P = 0.0625) and TNF-alpha receptors (decrease in number of receptors per cell, 1,596 +/- 340 to 574 +/- 93; P = 0.004). Cells were chemotactically desensitized to IL-8. TNF-alpha-induced adherence to fibronectin was suppressed from 69% +/- 5% of the phorbol myristate acetate response to 38% +/- 7% (P = 0.0154). These findings support the notion that release of IL-8 into the vascular space may be an in vivo mechanism for suppression of neutrophil accumulation at extravascular sites. L-Selectin loss would reduce the ability of neutrophils to adhere to activated endothelial cells. The specific loss of migratory response to IL-8 would impair neutrophil delivery to areas where IL-8 was the predominant chemoattractant. Loss of TNF-alpha-induced adherence to fibronectin would blunt those responses, including production of oxidants, capacitated by adherence.
Binding of Yersinia enterocolitica to purified, native small intestinal mucins from rabbits and humans involves interactions with the mucin carbohydrate moiety
Authors: Mantle M , Husar S D
Published: January 2020

Plasmid-bearing (but not plasmid-cured) Yersinia enterocolitica is known to bind to purified small intestinal mucins from rabbits and humans. This study examined which region(s) of the mucin molecule is important for bacterial adherence. Pronase digestion of mucin and removal of nonglycosylated or poorly glycosylated peptide regions had no effect on bacterial binding, suggesting that plasmid-bearing Y. enterocolitica interacts with mucin carbohydrate. Periodate oxidation also did not alter bacterial adherence, indicating that vicinal hydroxyl groups in the mucin sugars are not important for binding. Boiling of mucin, depolymerization by reduction of disulfide bonds, or removal of noncovalently associated lipid actually enhanced bacterial adherence, suggesting that plasmid-bearing Y. enterocolitica can interact with additional domains in the mucin molecule revealed by these treatments. These domains were destroyed by pronase digestion. In delipidated mucin (but not in reduced or boiled mucin), binding to these domains appeared to be hydrophobic since it could be prevented by treatment of bacteria with tetramethyl urea. Oligosaccharides obtained from both human and rabbit small intestinal mucins were capable of inhibiting attachment of plasmid-bearing (but not plasmid-cured) Y. enterocolitica to mucin. After removal of terminal and backbone sugar residues by treatment of mucin with trifluoromethanesulfonic acid, binding of plasmid-bearing bacteria increased significantly when N-acetylgalactosamine, either alone or with galactose attached, was revealed, indicating that core regions of the sugar side chains are involved in bacterial binding. Adherence of plasmid-cured organisms was unaffected by trifluoromethanesulfonic acid treatment of mucin. We concluded that virulent Y. enterocolitica interacts with the carbohydrate moiety of native small intestinal mucin through a plasmid-mediated process. When mucin becomes denatured, binding of the organism can increase through hydrophobic and nonhydrophobic interactions with (most likely) the mucin protein.
Differential interaction of Escherichia coli heat-labile toxin and cholera toxin with pig intestinal brush border glycoproteins depending on their ABH and related blood group antigenic determinants
Authors: Balanzino L E , Barra J L , Monferran C G , Cumar F A
Published: January 2020

The ability of glycoproteins from pig intestinal brush border membranes (BBM) to bind cholera toxin (CT) or heat-labile toxins from strains of Escherichia coli isolated from human (LTh) or pig (LTp) intestines was studied. Glycoproteins capable of binding the toxins are also recognized by antibodies or lectins specific for ABO(H) blood group and related antigens. Pigs expressing A, H, or I antigenic determinants were used for comparison. The toxin-binding capacity of a glycoprotein depends on the toxin type and the blood group epitope borne by the glycoprotein. LTh and LTp preferably bound to several blood group A-active glycoproteins rather than H-active glycoproteins. By contrast, CT practically did not recognize either blood group A- or blood group H-active glycoproteins, while glycoproteins from pigs expressing I antigenic determinants were able to interact with LTh, LTp, and CT. LTh, LTp, or CT glycoprotein binding was selectively inhibited by specific lectins or monosaccharides. Affinity purification of the toxin binding brush border glycoproteins on the basis of their blood group reactivity suggests that such glycoproteins are hydrolytic enzymes. BBM from A+ pigs contain about 27 times more LTh binding sites, in addition to those recognized by CT, than an equivalent membrane preparation from H+ pigs. The present findings may help clarify some previous unclear results on LTh binding to intestinal BBM glycoproteins obtained by use of animals not typed by their ABO(H) blood group phenotype.
Pneumolysin stimulates production of tumor necrosis factor alpha and interleukin-1 beta by human mononuclear phagocytes
Authors: Houldsworth S , Andrew P W , Mitchell T J
Published: January 2020

Human peripheral blood monocytes and a human monocyte cell line were exposed to the toxin pneumolysin. Pneumolysin-exposed cells produced significantly larger amounts of tumor necrosis factor alpha and interleukin-1 beta than cells not exposed to the toxin. The viability of cells was not affected by the concentrations of pneumolysin used in the experiments.