bluuebunny/crossref_metadata_2025 huggingface dataset:
Down regulation of intimin expression during attaching and effacing enteropathogenic Escherichia coli adhesion
Authors: Knutton S , Adu-Bobie J , Bain C , Phillips A D , Dougan G , Frankel G
Published: January 2020

Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions in the intestinal mucosa. The intimate bacterial adhesion associated with A/E lesion formation is promoted by intimin, a 94-kDa EPEC surface protein. Anti-intimin antisera raised in rabbits by using the purified 280-amino-acid cell binding domain of intimin as the immunogen were employed in immunofluorescence and immunoelectron microscopical studies to investigate the expression of intimin by classical EPEC strain E2348/69 (O127:H6) and defined E2348/69 derivatives during culture growth and A/E bacterium adhesion to cultured HEp-2 cells. In stationary-phase broth cultures, only a small fraction of E2348/69 bacteria expressed intimin, and of those that did, immunolabelling revealed a uniform distribution of intimin over the bacterial surface; increased numbers of bacteria expressing intimin were detected when E2348/69 was grown in tissue culture medium, an effect not seen with strain JPN15, a virulence plasmid-cured derivative of E2348/69. Strain CVD206, an eaeA mutant of E2348/69, did not stain with the anti-intimin antisera, but strain CVD206(pCVD438), containing a functional eaeA gene, stained uniformly. After a 3-h incubation of HEp-2 cells with strain E2348/69, double immunofluorescence labelling of intimin and cellular actin revealed strong intimin expression by all A/E bacteria, but after 6 h of incubation, intimin expression by most E2348/69 bacteria was greatly reduced or not detected. This effect on intimin expression was not observed with strain JPN15 but was restored for strain JPN15(pCVD450) harboring the virulence plasmid-encoded per genes. These results indicate that surface expression of intimin is regulated by environmental factors during bacterial growth and following A/E lesion formation and that virulence plasmid-encoded genes participate in these regulation processes.
Acute lethal toxicity following passive immunization for treatment of murine cryptococcosis
Authors: Savoy A C , Lupan D M , Manalo P B , Roberts J S , Schlageter A M , Weinhold L C , Kozel T R
Published: January 2020

Passive immunization with monoclonal antibodies (MAbs) specific for the major capsular polysaccharide of Cryptococcus neoformans alters the course of murine cryptococcosis. During studies of passive immunization for treatment of murine cryptococcosis, we noted the occurrence of an acute, lethal toxicity. Toxicity was characterized by scratching, lethargy, respiratory distress, collapse, and death within 20 to 60 min after injection of antibody. The toxic effect was observed only in mice with a cryptococcal infection and was reduced or absent in the early and late stages of disease. The clinical course and histopathology were consistent with those for shock. There was considerable variation between mouse strains in susceptibility to toxicity. Swiss Webster mice from the Charles River colony were most susceptible, followed by C3H/He, BALB/c, and C57BL/6 mice. DBA/2 mice and Swiss Webster mice from the Simonsen colony were resistant. Acute toxicity was mimicked by injection of preformed complexes of MAb and purified polysaccharide. The toxic effect was also produced by injection of MAbs into mice that were preloaded with polysaccharide. The toxic effect was not blocked by treatment of mice with chloropheniramine or anti-tumor necrosis factor alpha antibodies or by depletion of complement components via pretreatment with cobra venom factor. Toxicity was reduced by treatment of mice with high doses of epinephrine, dexamethasone, or chlorpromazine. Finally, the toxic effect was completely blocked by treatment of mice with the platelet-activating factor antagonist WEB 2170 BS or by pretreatment of mice with the liposome-encapsulated drug dichloromethylene diphosphonate, a procedure which depletes macrophages from the spleen and liver.
Oral vaccination against coccidiosis: responses in strains of mice that differ in susceptibility to infection with Eimeria vermiformis
Authors: Rose M E , Hesketh P , Wakelin D
Published: January 2020

Four strains of mice with different susceptibilities to Eimeria vermiformis were orally dosed with a crude antigen prepared from sporulated oocysts of the parasite, with or without cholera toxin as adjuvant. The effect on subsequent challenge infections depended on the resistance and susceptibility phenotypes of the host: oocyst production was reduced in susceptible C57BL/6 and NIH mice but increased in resistant BALB/c and C3H mice. Despite this contrast, no fundamental differences were detected between the immune responses of BALB/c and C57BL/6 mice, either to vaccination or after superimposed infection, but the suppressing and enhancing effects of vaccination were transmissible to naive recipients via suspensions of mesenteric lymph node cells. The results obtained are compared with those previously reported for parenterally immunized BALB/c and C57BL/6 mice.
Interleukin-12 secretion by Mycobacterium tuberculosis-infected macrophages
Authors: Ladel C H , Szalay G , Riedel D , Kaufmann S H
Published: January 2020

Infection with Mycobacterium tuberculosis or phagocytosis of large latex beads induced interleukin-12 (IL-12) production in macrophages. In contrast, tumor necrosis factor (TNF) was produced only in response to M. tuberculosis infection, not after phagocytosis of latex beads. Comparable results were obtained with cells from immunocompetent C57BL/6 and gamma interferon receptor-deficient mutant mice. Thus, phagocytosis by mechanisms not specific for M. tuberculosis was a sufficient trigger for IL-12 secretion, emphasizing the central role of this cytokine in the initiation of anti-infective immunity.
Nutritional requirements for growth of uropathogenic Escherichia coli in human urine
Authors: Hull R A , Hull S I
Published: January 2020

Enrichment with D-cycloserine was used to identify Escherichia coli auxotrophic mutants that exhibited limited growth in human urine. Bacterial synthesis of guanine, arginine, and glutamine was found to be required for optimal growth in urine. Mutants that required leucine, methionine, serine, phenylalanine, or proline also exhibited reduced growth in urine. Several other nutritional mutants, including nicotinamide auxotrophs, which are found frequently among cystitis isolates, exhibited normal growth in urine.
Adherence of Mycoplasma gallisepticum involves variable surface membrane proteins
Authors: Athamna A , Rosengarten R , Levisohn S , Kahane I , Yogev D
Published: January 2020

Adherence of Mycoplasma gallisepticum to erythrocytes was examined by colony immunoblotting, detergent phase fractionation, trypsin treatment, comparison of protein profiles, and comparison of erythrocyte-bound mycoplasma protein fractions of hemadsorption-positive and -negative mutants. The binding of M. gallisepticum to chicken or human erythrocytes was found to be mediated via surface-exposed membrane proteins undergoing high-frequency phase variation.
Expression of the Streptococcus mutans fructosyltransferase gene within a mammalian host
Authors: Grey W T , Curtiss R , Hudson M C
Published: January 2020

In vivo expression of the virulence-associated fructosyltransferase gene (ftf) of Streptococcus mutans has been examined. S. mutans ftf expression is affected by both the specific carbohydrate consumed and the age of the host animal.
Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a
Authors: Ward C K , Inzana T J
Published: January 2020

Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens.
Enteropathogenic Escherichia coli O103 from rabbit elicits actin stress fibers and focal adhesions in HeLa epithelial cells, cytopathic effects that are linked to an analog of the locus of enterocyte effacement
Authors: De Rycke J , Comtet E , Chalareng C , Boury M , Tasca C , Milon A
Published: January 2020

Escherichia coli O103, a major agent of weaned-rabbit diarrhea in Western Europe, was previously shown to produce diarrhea and attaching-and-effacing intestinal lesions in experimentally infected rabbits and to possess a homolog of the eaeA gene of enteropathogenic E. coli (EPEC). In the present study, we have shown that although negative in the fluorescent-actin staining test on HeLa cells, prototype rabbit E. coli O103 strain B10 was able to induce an original cytopathic effect (CPE) in the same interaction model. This CPE was characterized by a generalized reorganization of the actin cytoskeleton and the formation of focal adhesions on the entire surface of the target cells. These effects amplified with time, leading to cell death about 5 days after the interaction. They were produced by all rabbit E. coli O103 strains tested, by rabbit-infecting E. coli RDEC-1, and also by two human EPEC isolates. We localized genes associated with CPE by using TnphoA insertion mutagenesis in strain B10. In all five independent CPE-negative mutants that we were able to generate, the insertion was located in a region of the genome homologous to the 35-kb locus of enterocyte effacement (LEE locus) of EPEC E2348/69. The mutants concurrently lost the ability to secrete four major supernatant proteins of 25, 37, 39, and 40 kDa, which were shown by immunoprecipitation to share antigenic determinants with secreted proteins of human EPEC E2348/69. The virulence of one of these mutants (strain B10/CA1) was compared with that of the parental strain in the weaned-rabbit diarrhea model. The mutant was totally deprived of virulence, although it colonized the intestine as efficiently as the parental strain did. This study points to a new pathogenic trait of EPEC strains, which is associated with the LEE locus and, possibly, with in vivo virulence.
Characterization of a class II pilin expression locus from Neisseria meningitidis: evidence for increased diversity among pilin genes in pathogenic Neisseria species
Authors: Aho E L , Botten J W , Hall R J , Larson M K , Ness J K
Published: January 2020

Strains of Neisseria meningitidis elaborate one of two classes of pili. Meningococcal class I pili have many features in common with pili produced by N. gonorrhoeae, including the ability to bind monoclonal antibody SM1 and a common gene and protein structure consisting of conserved, semivariable, and hypervariable regions. Class II pili are SM1 nonreactive and display smaller subunit molecular weights than do gonococcal or meningococcal class I pili. In this study, we have determined the N-terminal amino acid sequence for class II pilin and isolated the expression locus encoding class II pilin from N. meningitidis FAM18. Meningococcal class II pilin displays features typical of type IV pili and shares extensive amino acid identity with the N-terminal conserved regions of other neisserial pilin proteins. However, the deduced class II pilin sequence displays several unique features compared with previously reported meningococcal class I and gonococcal pilin sequences. Class II pilin lacks several conserved peptide regions found within the semivariable and hypervariable regions of other neisserial pilins and displays a large deletion in a hypervariable region of the protein believed to be exposed on the pilus face in gonococcal pili. DNA sequence comparisons within all three regions of the coding sequence also suggest that the meningococcal class II pilin gene is the most dissimilar of the three types of neisserial pilE loci. Additionally, the class II locus fails to display flanking-sequence homology to class I and gonococcal genes and lacks a downstream Sma/Cla repeat sequence, a feature present in all other neisserial pilin genes examined to date. These data indicate meningococcal class II pili represent a structurally distinct class of pili and suggest that relationships among pilin genes in pathogenic Neisseria do not necessarily follow species boundaries.
Intranasal vaccination of humans with recombinant cholera toxin B subunit induces systemic and local antibody responses in the upper respiratory tract and the vagina
Authors: Bergquist C , Johansson E L , Lagergård T , Holmgren J , Rudin A
Published: January 2020

Forty-five volunteers were vaccinated twice intranasally with 10, 100, or 1,000 microg of cholera toxin B subunit (CTB). Blood and nasal and vaginal secretions were collected before and 1 week after the second vaccination from all volunteers, and the specific and total immunoglobulin A (IgA) and IgG titers were determined by enzyme-linked immunosorbent assay. Samples were also taken 6 months (n = 16) and 1 year (n = 14) after the vaccination. The 10- and 100-microg doses were well tolerated by the volunteers, but the 1,000-microg dose induced increased secretions from the nose and repetitive sneezings for several hours. The CTB-specific serum IgA and IgG increased 21- and 7-fold, respectively, 1 week after vaccination with the medium dose and increased 61- and 37-fold, respectively, after the high dose. In nasal secretions the specific IgA and IgG increased 2- and 6-fold after the medium dose and 2- and 20-fold after the high dose, respectively. In vaginal secretions the specific IgA and IgG increased 3- and 5-fold after the medium dose and 56- and 74-fold after the high dose, respectively. The lowest dose did not induce any significant antibody titer increases in serum or in secretions. The specific IgA and IgG levels in secretions were still elevated after 6 months but were decreasing 1 year after the vaccination. These results show that intranasal vaccination of humans with CTB induces strong systemic and mucosal antibody responses and suggest that CTB may be used as a carrier for antigens that induce protective immunity against systemic as well as respiratory and genital infections.
Cytostatic and cytotoxic effects of activated macrophages and nitric oxide donors on Brugia malayi
Authors: Thomas G R , McCrossan M , Selkirk M E
Published: January 2020

The susceptibility of Brugia malayi microfilariae and adults to injury by the murine macrophage cell line J774 activated with gamma interferon and bacterial lipopolysaccharide has been examined in vitro. Parasites of both stages showed a decline in viability over 48 h of coculture with activated macrophages, assessed by their capacity to reduce the tetrazolium salt 3-[4,5-diethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), although adult parasites were more resistant than microfilariae. Removal of parasites to cell-free medium following exposure to activated macrophages for up to 48 h resulted in partial recovery of their capacity to reduce MTT, suggesting that the effects were primarily cytostatic. However, prolonged exposure to activated J774 cells for 72 h resulted in parasite death. Addition of the nitric oxide synthase inhibitor L-NMMA (N(G)-monomethyl-L-arginine monoacetate) indicated that nitric oxide derivatives were responsible for cytostasis and ultimate toxicity. The toxicity of nitric oxide derivatives was confirmed by coincubation of parasites with chemical donors, although far higher concentrations were required than those generated by activated J774 cells, implying additional complexity in macrophage-mediated cytotoxicity. These experiments further suggested that peroxynitrite or its by-products were more potently damaging to filariae than nitric oxide per se. Examination of ultrastructural changes on exposure of parasites to activated macrophages or donors of nitric oxide indicated that hypodermal mitochondria were highly vacuolated, with less prominent cristae. The data are discussed with reference to immunity to lymphatic filariae and their mechanisms of energy generation.
Pseudomonas aeruginosa lipopolysaccharide binds galectin-3 and other human corneal epithelial proteins
Authors: Gupta S K , Masinick S , Garrett M , Hazlett L D
Published: January 2020

The aim of this study was to test whether galectin-3 is present in human corneal epithelium and whether lipopolysaccharide (LPS) purified from Pseudomonas aeruginosa ATCC 19660 binds to this animal lectin and/or to another human corneal epithelial protein(s) (HCEP) and to confirm which component of LPS (inner or outer core or lipid A) is important in bacterial binding by using the eye in organ culture. LPS isolated and purified from P. aeruginosa ATCC 19660 and a commercial LPS (serotype 10) differed in polyacrylamide gel analysis but bound similarly to blotted HCEP. Binding was determined to be a receptor-ligand type of interaction by the solid-phase assay, because it was both specific and saturable. Several LPS binding proteins in HCEP were identified by an overlay method. Western blotting with antibody against galectin-3 revealed the presence of this protein in both freshly isolated and cultured transformed human corneal epithelium. Binding inhibition assays showed that antibody specific for the outer core region of LPS and an anti-galectin antibody significantly inhibited bacterial binding in vitro. These data provide further evidence that LPS is an important adhesin of P. aeruginosa, that it binds to protein receptor molecules in HCEP, that one of the LPS binding proteins is galectin-3, and that the outer core portion of the molecule appears to be critical for LPS binding to the eye.
A synthetic lipopolysaccharide-binding peptide based on the neutrophil-derived protein CAP37 prevents endotoxin-induced responses in conscious rats
Authors: Brackett D J , Lerner M R , Lacquement M A , He R , Pereira H A
Published: January 2020

The lipid A component of lipopolysaccharide (LPS) derived from Escherichia coli has been implicated as a significant mediator in the development of circulatory and metabolic dysfunction and lethality associated with sepsis. A synthetic peptide corresponding to amino acid residues 20 through 44 of the neutrophil-derived 37-kDa cationic antimicrobial protein (CAP37 P(20-44)) possesses lipid A binding characteristics which may be useful in attenuating in vivo responses induced during circumstances of endotoxemia, including sepsis. The E. coli LPS to be used in the in vivo study was shown to be attenuated by CAP37 P(20-44) in a dose-dependent manner in the in vitro reaction with Limulus amoebocyte lysate. Intravenous infusion of CAP37 P(20-44) (1.5 or 3.0 mg/kg of body weight) with E. coli LPS (250 microg/kg over 30 min) into conscious, unrestrained rats prevented LPS-induced hyperdynamic and hypodynamic circulatory shock, hyperlactacidemia, and leukopenia in a dose-related fashion. CAP37 P(20-44) (0.2, 1.0, and 5.0 mg/kg) administered intravenously to conscious, actinomycin D-sensitized rats following a lethal dose of LPS neutralized LPS toxicity, resulting in dose-dependent 7-day survival rates of 30, 50, and 80%, respectively. CAP37 P(20-44) (5.0 mg/kg) significantly inhibited the endotoxin-induced increase in circulating tumor necrosis factor alpha in sensitized rats. These data demonstrate that CAP37 P(20-44) has the capacity to abolish in vivo biological responses to LPS that are relevant to human sepsis and to significantly neutralize the toxicity of circulating E. coli LPS.
T-helper-cell cytokines in the early evolution of murine Lyme arthritis
Authors: Kang I , Barthold S W , Persing D H , Bockenstedt L K
Published: January 2020

Genetic susceptibility to murine Lyme arthritis has been correlated with the dominance of T-helper (Th1)- or Th2-cell-associated cytokines. To determine when commitment of the Th cell phenotype occurs, we examined the kinetics of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production by lymph node T cells of disease-susceptible C3H/HeN and disease-resistant BALB/c mice from days 2 through 30 of infection, a period encompassing the evolution of disease and early regression. BALB/c mice produced more IFN-gamma on day 2 of infection than did C3H/HeN mice, whereas IL-4 was first detected on day 14. In contrast, only IFN-gamma could be detected in C3H/HeN mice, and the levels steadily increased from day 2 to surpass those seen in BALB/c mice by day 14 of infection. Despite the difference in cytokine profiles, both BALB/c and C3H/HeN mice developed comparable arthritis assessed at 14 days of infection. Arthritis regressed by day 30 in BALB/c mice but persisted in C3H/HeN mice. These studies are the first to demonstrate that the Th2 response to Borrelia burgdorferi infection of BALB/c mice is preceded by a Th1 cytokine response. Moreover, the timing of the appearance of IL-4 suggests that its primary effect is not in preventing disease, as suggested by others, but, rather, in hastening the resolution of inflammation. The implications of these findings for the orchestration of host defense against B. burgdorferi infection are discussed.
Identification and characterization of a metalloprotease activity from Helicobacter pylori
Authors: Windle H J , Kelleher D
Published: January 2020

Helicobacter pylori produces a metalloprotease with a native molecular size of approximately 200 kDa, as determined by size-exclusion chromatography. Subcellular distribution studies demonstrated that the activity was associated with the outer membrane fraction of the bacterium. In addition, the protease was secreted by the bacterium when grown in liquid culture. The enzyme activity was measured by hydrolysis of azocasein and biotinylated casein and exhibited optimal caseinolytic activity at pH 8.0 (37 degrees C). The activity was inhibited by EDTA, 1,10-phenanthroline, phosphoramidon, pyridine-2,6-dicarboxylic acid, and 8-hydroxyquinoline-5-sulfonic acid (HQSA). Inhibition by HQSA was reversed by zinc, whereas inhibition due to EDTA was reversed by excess calcium, thus indicating that the enzyme was a zinc-dependent, calcium-stabilized endoproteinase. Furthermore, titration with Zn2+ of a desalted, active-site zinc-chelated preparation of the protease demonstrated that Zn2+ was essential for activity. Leupeptin, phenylmethylsulfonyl fluoride, E-64, pepstatin A, dithiothreitol, and 2-mercaptoethanol had no effect on enzymatic activity. Addition of Ca2+ or Mg2+ to the incubation medium resulted in approximately a twofold stimulation of the azocaseinolytic activity of the enzyme. The protease was stably expressed since it was active even after repeated subculture of the bacterium. Bovine serum albumin, hide powder azure, and elastin-Congo red remained intact even after prolonged exposure to the enzyme. The surface expression of this metalloprotease activity raises the possibility that this enzyme may be involved in the proteolysis of a variety of host proteins in vivo and thereby contributes to gastric pathology.
Structurally defined epitopes of Haemophilus ducreyi lipooligosaccharides recognized by monoclonal antibodies
Authors: Ahmed H J , Frisk A , Månsson J E , Schweda E K , Lagergård T
Published: January 2020

By use of enzyme-linked immunosorbent assay and immunoblotting techniques, the migration patterns and binding epitopes of lipooligosaccharides (LOS) from 10 Haemophilus ducreyi strains were investigated with two monoclonal antibodies (MAbs), MAHD6 and MAHD7, raised against LOS from H. ducreyi ITM 2665. Closely related LOS, with defined structures, from Haemophilus influenzae, Bordetella pertussis, Aeromonas spp., and synthetic glycoproteins were also included in the analyses. The MAbs bound to conserved epitopes of LOS exposed on the surface of H. ducreyi. The MAb MAHD6 reacted with 8 of the 10 LOS from H. ducreyi but with none of the other Haemophilus or Bordetella spp. with structurally defined LOS. It is suggested that MAb MAHD6 binds to a LOS epitope (-DD-Hepp-1-->6-beta-D-Glcp-). This LOS epitope is not present in the hexasaccharide structure of LOS from H. ducreyi ITM 4747 (E. K. H. Schweda, A. C. Sundström, L. M. Eriksson, J. A. Jonasson, and A. A. Lindberg, J. Biol. Chem. 269:12040-12048, 1994). Because MAb MAHD6 reacts with the epitope mentioned above, it also discriminates between the two LOS structures, the hexasaccharide group and the nonasaccharide group, of H. ducreyi strains. MAb MAHD7 recognizes the common conserved inner core region of the LOS because it reacts with all H. ducreyi strains and with LOS with minor components in the inner core epitope structure. Rabbit polyclonal sera raised against the LOS from strains CCUG 4438 and CCUG 7470 were tested with the 10 LOS from the H. ducreyi strains. The antiserum to CCUG 7470 reacted with all H. ducreyi strains as did MAb MAHD7, whereas the antiserum to CCUG 4438 reacted with only its homologous strain and strain ITM 4747. Also, the LOSs of our reference strains CCUG 4438 and CCUG 7470 were structurally analyzed by use of sugar analyses and electrospray ionization-mass spectrometry. The hexasaccharide and nonasaccharide structures obtained from LOS of strains CCUG 4438 and CCUG 7470 were identical to the described LOS structures from H. ducreyi ITM 4747 and ITM 2665, respectively. In conclusion, the MAb MAHD6 recognizes an epitope present in the nonasaccharide LOS group, whereas the MAb MAHD7 recognizes a conserved epitope on LOS of H. ducreyi, which is present in all strains of H. ducreyi tested. Two major groups of oligosaccharides were distinguished by their LOS structures and the reactivity of monoclonal as well as polyclonal antibodies. The majority of H. ducreyi strains possess a nonasaccharide structure of LOS.
Ecto-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exoenzyme S
Authors: Knight D A , Barbieri J T
Published: January 2020

Pseudomonas aeruginosa produces two ADP-ribosyltransferases, exotoxin A and exoenzyme S (ExoS). Although the physiological target protein remains to be defined, ExoS has been shown to ADP-ribosylate several eukaryotic proteins in vitro, including vimentin and members of the family of low-molecular-weight GTP-binding proteins. Recently, ExoS ADP-ribosyltransferase activity has been detected in the pleural fluid of rabbits infected with P. aeruginosa. This observation prompted an examination of the potential for ExoS to function as an ecto-ADP-ribosyltransferase. We have observed that ExoS preferentially ADP-ribosylated two extracellular serum proteins with molecular masses of 150 and 27 kDa. The ADP-ribosylation of these serum proteins by ExoS was stimulated by, but not dependent upon, exogenous FAS (for factor activating exoenzyme S), which indicated that serum contained endogenous FAS activity. Biochemical analysis showed that the 150-kDa ADP-ribosylated protein was immunoglobulin of the immunoglobulin G (IgG) and IgA classes. Subtyping showed that ExoS preferentially ADP-ribosylated human IgG3 and that ADP-ribosylation occurred within its Fc region. The 27-kDa protein ADP-ribosylated by ExoS was determined to be apolipoprotein A1. These data demonstrate ecto-ADP-ribosyltransferase activity by ExoS. This may extend the potential physiological consequences of ExoS during infection by P. aeruginosa beyond the implicated type III secretion-mediated intracellular delivery of ExoS into sensitive eukaryotic cells.
Immunologic and genetic analyses of VmpA of a neurotropic strain of Borrelia turicatae
Authors: Cadavid D , Pennington P M , Kerentseva T A , Bergström S , Barbour A G
Published: January 2020

In mice infected with serotype A but not serotype B of the relapsing fever spirochete Borrelia turicatae, early invasion of the brain occurs. Serotypes A and B are further distinguished by the abundant surface protein they produce: VmpA and VmpB, respectively. Western blotting with monoclonal antibodies, one-dimensional peptide mapping, and partial amino acid sequencing demonstrated regions of the VmpA protein that differed from VmpB. Oligonucleotide primers based on the partial amino acid sequences of unique regions were used to amplify a portion of the VmpA gene (vmpA) by PCR, and the product was used as a probe in Southern blot and Northern blot analyses. These experiments showed that (i) expression of the vmpA sequence was determined at the level of transcription and (ii) the vmpA sequence was in two locations in serotype A and one location in serotype B. The vmpA gene at the expression-linked locus of serotype A was cloned and sequenced. An open reading frame would encode a polypeptide of 214 amino acids. The polypeptide expressed by Escherichia coli was bound by VmA-specific but not VmpB-specific antibody. Primer extension analysis identified a consensus sigma70-type promoter for vmpA at the expression locus. Phylogenetic analysis revealed that VmpA is homologous to small Vmp (Vsp) proteins of B. hermsii and to OspC proteins of B. burgdorferi. These findings indicate that a function of the Vsp-OspC family of proteins of Borrelia spp. may be differential localization in organs, including the brain, during infection.
Protective role of antimannan and anti-aspartyl proteinase antibodies in an experimental model of Candida albicans vaginitis in rats
Authors: De Bernardis F , Boccanera M , Adriani D , Spreghini E , Santoni G , Cassone A
Published: January 2020

The role of antibodies (Abs) in the resistance to vaginal infection by Candida albicans was investigated by using a rat vaginitis model. Animals receiving antimannoprotein (anti-MP) and anti-aspartyl proteinase (Sap) Ab-containing vaginal fluids from rats clearing a primary C. albicans infection showed a highly significant level of protection against vaginitis compared to animals given Ab-free vaginal fluid from noninfected rats. Preabsorption of the Ab-containing fluids with either one or both proteins MP and Sap sequentially reduced or abolished, respectively, the level of protection. A degree of protection against vaginitis was also conferred by postinfectious administration of anti-Sap and anti-MP monoclonal antibodies (provided the latter were directed against mannan rather than protein epitopes of MP) and by intravaginal immunization with a highly purified, polysaccharide-free Sap preparation. Postinfectious administration of pepstatin A, a potent Sap inhibitor, greatly accelerated the clearance of C. albicans from rat vagina. No anti-MP or anti-Sap Abs were elicited during a C. albicans vaginal infection of congenitally athymic nude rats. Although they were as able as their euthymic counterparts to clear the primary infection, these animals did not show increased resistance to a rechallenge, demonstrating that induction of anticandidal protection in normal rats was a thymus-dependent Ab response. Overall, our data strengthen the concept that Abs against some defined Candida antigens are relevant in the mechanism of acquired anticandidal protection in vaginitis. The T-cell dependence of this protection may also provide a link between cell-mediated and humoral immunity in vaginal infection.
Effects of granulocyte and granulocyte-macrophage colony-stimulating factors in a neutropenic murine model of trichosporonosis
Authors: Muranaka H , Suga M , Nakagawa K , Sato K , Gushima Y , Ando M
Published: January 2020

We produced disseminated trichosporonosis in a neutropenic murine model with Trichosporon asahii, which was identified by DNA relatedness analysis. We then assessed the efficacy of granulocyte colony-stimulating factor (G-CSF) (30 to 100 microg/kg of body weight per day) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.8 to 2 microg/kg x day). The administration of G-CSF either before or after infection improved the survival rate from less than 25% up to 100% (P < 0.05). The effects of G-CSF on organ clearance and histological examinations were most remarkable in the lungs. The levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage fluid (BALF) of neutropenic and G-CSF-pretreated mice were 60 +/- 6 ng/ml and 18 +/- 6 pg/ml, respectively, at 24 h after infection. Immunohistologically, alveolar macrophages proved to be the main source of TNF-alpha in BALF. GM-CSF increased neutrophil counts less significantly than did G-CSF and increased the lethality (P < 0.05) with a high level of TNF-alpha in BALF. Expecting to inhibit TNF-alpha, we administered anti-TNF-alpha intraperitoneally at the dose completely inhibiting TNF-alpha in plasma (2 x 10(4) U), but the TNF-alpha level in BALF and the lethality increased. Though the number of neutrophils at the early stage of infection appeared to be the most critical, the results suggest that other host defense mechanisms, such as TNF-alpha overproduction in the lungs, have an important role in the prognosis of trichosporonosis.
Relevance of immunoglobulin G Fc receptor polymorphism to recurrence of adult periodontitis in Japanese patients
Authors: Kobayashi T , Westerdaal N A , Miyazaki A , van der Pol W L , Suzuki T , Yoshie H , van de Winkel J G , Hara K
Published: January 2020

Polymorphonuclear neutrophil (PMN) phagocytic function has been shown to be impaired in some patients with periodontitis. PMN constitutively express members of two immunoglobulin G receptor (Fc gammaR) classes: Fc gammaRIIa (CD32) and Fc gammaRIIIb (CD16). Both receptors exhibit genetically determined structural and functional biallelic polymorphisms, which have been shown to influence PMN phagocytic function. In this study, we assessed the relevance of these Fc gammaR polymorphisms to susceptibility to adult periodontitis and recurrence rate. The distribution of Fc gammaRIIa and Fc gammaRIIIb genotypes of 100 Japanese patients with adult periodontitis during follow-up was compared to the distribution of genotypes in 105 race-matched healthy controls. No significant skewing of distributions of Fc gammaRIIa and Fc gammaRIIIb genotypes was observed between patients and controls. Notably, however, a significant overrepresentation of the Fc gammaRIIIb-NA2 allotype was found in patients with disease recurrence (P < 0.05; odds ratio, 4.29; 95% confidence interval, 1.19 to 16.24). Moreover, the annual rate of recurrence was significantly higher in patients with the Fc gammaRIIIb-NA2/NA2 and Fc gammaRIIIb-NA1/NA2 genotypes than in Fc gammaRIIIb-NA1/NA1 individuals (P < 0.05). Fc gammaRIIa-R/R131 individuals also exhibited higher recurrence rates, though the difference was not statistically significant (P = 0.06). These results suggest that the Fc gammaRIIIb-NA2 allotype represents a risk factor for recurrence of adult periodontitis.
Bacteroides fragilis toxin exhibits polar activity on monolayers of human intestinal epithelial cells (T84 cells) in vitro
Authors: Chambers F G , Koshy S S , Saidi R F , Clark D P , Moore R D , Sears C L
Published: January 2020

Strains of Bacteroides fragilis associated with diarrhea in children (termed enterotoxigenic B. fragilis, or ETBF) produce a heat-labile ca. 20-kDa protein toxin (BFT). The purpose of this study was to examine the activity of BFT on polarized monolayers of human intestinal epithelial cells (T84 cells). In Ussing chambers, BFT had two effects. First, BFT applied to either the apical or basolateral surfaces of T84 monolayers diminished monolayer resistance. However, the time course, magnitude, and concentration dependency differed when BFT was applied to the apical versus basolateral membranes. Second, only basolateral BFT stimulated a concentration-dependent and short-lived increase in short circuit current (Isc; indicative of C1- secretion). Time course experiments indicated that Isc returned to baseline as resistance continued to decrease, indicating that these two electrophysiologic responses to BFT are distinct. Light microscopic studies of BFT-treated monolayers revealed only localized cellular changes after apical BFT, whereas basolateral BFT rapidly altered the morphology of nearly every cell in the monolayer. Transmission and scanning electron microscopy after basolateral BFT confirmed a striking loss of cellular microvilli and complete dissolution of some tight junctions (zonula occludens) and zonula adherens without loss of desmosomes. The F-actin structure of BFT-treated monolayers (stained with rhodamine-phalloidin) revealed diminished and flocculated staining at the apical tight junctional ring and thickening of F-actin microfilaments in focal contacts at the basolateral monolayer surface compared to those in similarly stained control monolayers. BFT did not injure T84 monolayers, as assessed by lactic dehydrogenase release and protein synthesis assays. These studies indicate that BFT is a nonlethal toxin which acts in a polar manner on T84 monolayers to stimulate C1- secretion and to diminish monolayer resistance by altering the apical F-actin structure of these cells. BFT may contribute to diarrheal disease associated with ETBF infection by altering epithelial barrier function and stimulating C1- secretion.
Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of gamma interferon by natural killer cells
Authors: Zhang T , Kawakami K , Qureshi M H , Okamura H , Kurimoto M , Saito A
Published: January 2020

We examined the ability of interleukin-12 (IL-12) and IL-18 to induce the production of gamma interferon (IFN-gamma) and nitric oxide (NO) by murine peritoneal exudate cells (PEC) and to stimulate the growth-inhibitory activity of these cells against Cryptococcus neoformans. PEC produced IFN-gamma and NO when stimulated with a combination of IL-12 and IL-18 but little or no IFN-gamma or NO when either cytokine was used alone. PEC anticryptococcal activity was mediated by IFN-gamma and NO production, since it was completely inhibited by a neutralizing anti-IFN-gamma monoclonal antibody (MAb) and N(G)-monomethyl-L-arginine, a competitive inhibitor of NO synthesis, respectively. To identify the IFN-gamma-producing cells among PEC stimulated with IL-12 and IL-18, we depleted NK cells, gammadelta T cells, or CD4+ T cells by treating PEC with specific Abs and complement. NK cell depletion strongly suppressed IFN-gamma production and almost completely inhibited NO production and anticryptococcal activity, while depletion of other cells had no such influence. Alternatively, purified NK cells by two cycles of glass adherence and magnetic separation with anti-CD3, -CD4, -CD8, and -B220 MAbs produced a greater amount of IFN-gamma by stimulation with IL-12 and IL-18 than unseparated non-glass-adherent PEC. Our results demonstrated that IL-12 and IL-18 synergistically induced NO-dependent anticryptococcal activity of PEC by stimulating NK cells to produce IFN-gamma.
Bordetella pertussis binds the human complement regulator C4BP: role of filamentous hemagglutinin
Authors: Berggård K , Johnsson E , Mooi F R , Lindahl G
Published: January 2020

C4BP (C4b-binding protein) is a high-molecular-weight plasma protein that inhibits the classical pathway of complement activation. Recent experiments have demonstrated that C4BP binds to many strains of the gram-positive bacterium Streptococcus pyogenes, a major respiratory tract pathogen. Binding to S. pyogenes was shown to be due to members of the M protein family, a group of surface proteins important for virulence. Here we report that human C4BP also binds to all clinical isolates of the gram-negative bacterium Bordetella pertussis, the etiologic agent of whooping cough. In addition, binding of C4BP was demonstrated for other Bordetella species that can cause disease in humans. Characterization of different B. pertussis mutants showed that the binding of C4BP is strongly dependent on the expression of the cell surface protein filamentous hemagglutinin, a well-known virulence factor. Inhibition experiments suggested that B. pertussis and S. pyogenes bind to the same region in C4BP. The finding that B. pertussis and S. pyogenes both have the ability to bind human C4BP suggests that these two unrelated respiratory tract pathogens may use a common mechanism during the establishment of an infection.
Vibrio mimicus attaches to the intestinal mucosa by outer membrane hemagglutinins specific to polypeptide moieties of glycoproteins
Authors: Alam M , Miyoshi S , Tomochika K , Shinoda S
Published: January 2020

Vibrio mimicus is the closest organism to Vibrio cholerae. V. mimicus E-33, which is a highly adhesive and enteropathogenic strain, is known to produce three types of hemagglutinins (HAs), i.e., a 31-kDa exocellular metalloprotease (Vm-HA/protease), lipopolysaccharide (Vm-LPSHA), and a 39-kDa major outer membrane protein (Vm-OMPHA). Hemagglutination induced by Vm-LPSHA and Vm-OMPHA was inhibited by glycoproteins, including mucin, fetuin, and asialofetuin, but not by monosaccharides, disaccharides, or N-acetylated saccharides. The inhibitory potential of each glycoprotein for Vm-OMPHA was greatly augmented by treatment with a glycolytic enzyme such as beta-D-galactosidase or beta-D-glucosidase, while pronase treatment achieved complete abolition of the inhibitory potential. The inhibitory ability of the glycoproteins for Vm-LPSHA was also abolished by pronase treatment; however, glycolytic enzyme treatment showed no effect. Hence, the polypeptide portion of glycoproteins may directly associate with Vm-OMPHA and Vm-LPSHA, but the sugar moiety may act as a barrier to interaction with Vm-OMPHA. The glycoproteins as well as Fab antibodies against Vm-OMPHA and Vm-LPSHA eliminated the ability of E-33 cells to agglutinate rabbit erythrocytes and to attach to rabbit intestinal mucosa. Additionally, expression of the hemagglutinating ability by the bacterial cells was accompanied by efficient bacterial adherence to the intestinal mucosa. Finally, the hemagglutinating activity of Vm-OMPHA was markedly increased by incubation with Vm-HA/protease. These results indicate that all three HAs may have significant roles in the glycoprotein-mediated intestinal adherence of V. mimicus E-33.
Pathogenicity of three strains of Serpulina pilosicoli in pigs with a naturally acquired intestinal flora
Authors: Thomson J R , Smith W J , Murray B P , McOrist S
Published: January 2020

Serpulina pilosicoli is an anaerobic spirochete which has been isolated from the colons of pigs with enteric disease. The clinical and pathologic features of experimental infections of conventional pigs (born by normal farrowing with a naturally acquired intestinal flora) with three strains of S. pilosicoli were determined in order to confirm the enteropathogenicity of this species. Strains were derived from the colons of British pigs with colitis and passaged 8 to 10 times during expansion and purification in vitro. Eighteen ten-week-old Large White-Landrace cross pigs were each inoculated once orally with 0.7 x 10(9) to 1.6 x 10(9) of one of three strains of S. pilosicoli. Six pigs were challenged with each strain. Control pigs were dosed with uninfected broth medium or with 1.8 x 10(7) cells of the nonpathogenic Serpulina innocens. Eight pigs (two to four per S. pilosicoli challenge group) developed soft or diarrheic feces (fecal dry matter < 24%) between 3 and 8 days after challenge, which persisted for 7 to 8 days or until necropsy at 14 days after challenge. Average weight gains in two of the three groups challenged with S. pilosicoli were significantly less than controls. The feed conversion ratios of all the groups challenged with S. pilosicoli were impaired compared to controls. The mean values for daily liveweight gain (and feed conversion ratio) for the three groups challenged with S. pilosicoli were 0.799 (2.13), 0.783 (2.05), and 0.844 kg (2.10), respectively, while that of the uninoculated controls was 0.944 kg (1.70). Gross lesions with slight mucosal thickening, congestion, and multifocal erosions were evident in seven of eight diarrheic pigs. The relative weights of the large intestines of pigs challenged with S. pilosicoli were significantly less than controls. Histologic lesions with an increase in mucosal height, infiltration of the lamina propria with mononuclear cells, mucosal erosion with mixed inflammatory cell infiltration, and goblet cell hyperplasia in colonic glands were evident in 15 of the 18 challenged pigs. S. pilosicoli was recovered on bacterial culture of the colon from all except one of the pigs with these histologic lesions. Serpulina sp. was clearly visible within the colonic glands of these affected pigs in silver-stained sections of the gut. Clinical and pathologic findings in control pigs were unremarkable, with no diarrhea or colonic lesions evident. The results provide further evidence that S. pilosicoli is a specific enteric pathogen for conventional pigs. It is capable of colonizing the large intestine and causing mucosal damage, which although mild is sufficient to result in significant adverse effects on growth.
Mannan-specific immunoglobulin G antibodies in normal human serum mediate classical pathway initiation of C3 binding to Candida albicans
Authors: Zhang M X , Lupan D M , Kozel T R
Published: January 2020

Candida albicans activates both the classical and alternative complement pathways. Previous studies found that immunoglobulin G (IgG) in normal human serum (NHS) mediates classical pathway initiation. The goal of this study was to determine the role of candidal mannan-specific human IgG antibodies in complement activation. Mannan was purified from the yeast cells, and naturally occurring antimannan IgG was isolated from pooled NHS or plasma samples by immunoaffinity chromatography. Early activation and binding of C3, characteristics of classical pathway activity, were abolished in yeast- or mannan-absorbed serum but could be restored to absorbed serum with added purified antimannan IgG in a dose-dependent manner. Microscopically, the immunofluorescence pattern of initial C3 binding was diffuse over the entire cell surface for yeast cells incubated in NHS or in mannan-absorbed NHS supplemented with antimannan IgG but was asynchronous and focal for yeast cells incubated in EGTA-treated or mannan-absorbed NHS. The antimannan IgG level in serum samples from 21 donors varied from 17 to 570 microg/ml of serum compared to 220 microg in pooled NHS samples. The rate of initial C3 binding to yeast cells correlated with the level of antimannan IgG in sera from different individuals (r2 = 0.94) and could be accelerated in sera containing lower amounts of antimannan IgG with exogenous antimannan IgG. These observations identify antimannan IgG as the initiator of classical pathway C3 deposition on C. albicans. Given the variability in the levels of antimannan antibodies in sera from different individuals, the presence or absence of these antibodies may be an important determinant of host resistance to disseminated candidiasis.