bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic control and properties of coli surface antigens of colonization factor antigen IV (PCF8775) of enterotoxigenic Escherichia coli
Authors: McConnell M M , Thomas L V , Willshaw G A , Smith H R , Rowe B
Published: January 2020

Enterotoxigenic Escherichia coli producing coli surface antigen 4 (CS4), CS5, and CS6 of colonization factor antigen IV were examined. This factor was originally called putative colonization factor 8775 (PCF8775). All of the coli surface antigens were plasmid coded and were usually carried on the same plasmid as the genes coding for heat-stable toxin (ST) or heat-labile toxin (LT); thus, CS5-CS6-ST, CS6-ST, and CS6-LT plasmids were found. In strains of serotype O25:H42, the genes coding for CS4 and CS6 were on a plasmid separate from that containing the genes coding for ST and LT. CS4 and CS5 were fimbrial antigens with a subunit molecular mass of about 17.0 and 21.0 kilodaltons (kDa), respectively. CS6 was found as a single polypeptide with a molecular mass of about 14.5 kDa in strains of serotypes O25:H42, O27:H7, and O27:H20 when heated extracts were run on sodium dodecyl sulfate-polyacrylamide gels. CS6-positive extracts of strains of serogroups O148, O159, and O167 showed two bands with molecular masses between 14.5 and 16.0 kDa.
Candida albicans C3d receptor, isolated by using a monoclonal antibody
Authors: Linehan L , Wadsworth E , Calderone R
Published: January 2020

Pseudohyphae of Candida albicans possess a receptor for C3d, a fragment of the complement component C3. This receptor was partially purified by using a monoclonal antibody (CA-A) that previously had been shown to inhibit the binding of C3d to C. albicans pseudohyphae. Purified immunoglobulin G from ascites fluid (CA-A) was coupled to a cyanogen bromide-activated Sepharose column, and an affinity-purified fraction (A2) from C. albicans pseudohyphae was obtained. This fraction inhibited rosetting of the EAC3d receptor by pseudohyphae and appeared to contain glycoprotein, since receptor activity could be removed when A2 was incubated with lectins specific for mannose and glucose. A2 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two polypeptides of approximately 60 and 70 kilodaltons (kDa) were consistently identified in reducing gels. The 60-kDa protein was identified as a glycoprotein by concanavalin A binding. A2 was further analyzed by high-pressure liquid chromatography (HPLC). Of three fractions obtained by HPLC, one containing the 60-kDa protein was found to have receptor activity. When analyzed by HPLC, this protein was found to contain mannose and glucose in approximately equal amounts. Both immunofluorescence and electron microscopy of pseudohyphae treated with CA-A identified A2 as a surface moiety. Thus, the C3d receptor of C. albicans, isolated with CA-A, is a glycoprotein of approximately 60 kDa.
Chemical and biological characterization of the lipopolysaccharide of the oral pathogen Wolinella recta ATCC 33238
Authors: Gillespie J , Weintraub S T , Wong G G , Holt S C
Published: January 2020

To investigate the potential pathogenic mechanisms of the oral periodontopathogen Wolinella recta ATCC 33238, we have isolated its lipopolysaccharide (LPS) and determined the chemical composition and selected in vitro biological activities of the molecule. Sodium desoxycholate-polyacrylamide gel electrophoresis revealed the W. recta LPS to be an atypical smooth LPS with short O-antigenic side chains. Chemically the LPS consisted of 47.2% lipid A, 19.6% polysaccharide, 9.0% heptose, 8.5% hexosamine, 3.2% phosphate, and 0.6% 2-keto-3-deoxyoctanoate. The major fatty acids were hexadecanoic acid (25.0%), 3-OH tetradecanoic acid (23.8%), tetradecanoic acid (15.4%), 3-OH hexadecanoic acid (11.6%), and octadecenoic acid (10.9%). Rhamnose constituted 87.8% of the carbohydrates generally associated with the O antigen, with smaller amounts of glucose (5.5%), mannose (4.9%), and an unidentified sugar (1.9%). CD-1 and C3H/HeN macrophages (M phi) exposed to 1 microgram of W. recta LPS per ml released 6.0 and 10.5 ng of prostaglandin E per ml of supernatant, representing 625% and 1,306% of prostaglandin E release by the control (without LPS). Maximum prostaglandin E release occurred in CD-1 M phi exposed to 100 micrograms of LPS per ml and was equivalent to 1,542% of release by the control. Interleukin-1 (IL-1) activities in CD-1 and C3H/HeN M phi exposed to 1 micrograms of LPS per ml were 257% and 1,941% of activities in the control, respectively. Maximum IL-1 release in CD-1 M phi occurred in response to 50 micrograms of LPS per ml and represented a 927% increase over release in the control, while 100 micrograms LPS per ml stimulated maximum IL-1 release in C3H/HeN M phi that was greater than 5,000% of release by the control.
Reduction of enterotoxic activity of Escherichia coli heat-stable enterotoxin by substitution for an asparagine residue
Authors: Okamoto K , Okamoto K , Yukitake J , Miyama A
Published: January 2020

The Escherichia coli heat-stable enterotoxins (STs) are small peptide toxins consisting of 18 (STp) or 19 (STh) amino acids. STp and STh share biologically active sequences which reside in the C-terminal 13 amino acid residues, but the role of each amino acid in the active sequences is not clear. We substituted in vivo Asp, Tyr, His, Gln, Lys, and Arg for the Asn residue at position 11 of STp by oligonucleotide-directed site-specific mutagenesis and examined the biological activities of the resulting mutants. All mutant STs reacted with both monoclonal and polyclonal antibodies, demonstrating that the amino acid substitutions at position 11 did not cause a significant change in the conformation of STp. However, the substitutions invariably caused a significant decrease in enterotoxic activities. The most remarkable decrease was observed with Asn-11----Lys-11 and Asn-11----Arg-11 mutations; that is, enterotoxic activity could not be detected in the culture supernatant of either of these mutant strains. These results indicate that Asn-11 of STp plays an essential role in the enterotoxic activity. The amide group and the length of side chain of Asn-11 seem to be especially important for enterotoxic activity.
Differences in virulence for mice among strains of Mycoplasma pulmonis
Authors: Davidson M K , Lindsey J R , Parker R F , Tully J G , Cassell G H
Published: January 2020

The mouse model of acute murine respiratory mycoplasmosis was used to screen 18 strains of Mycoplasma pulmonis for their ability to establish respiratory infections and produce gross lung lesions in the susceptible C3H/HeN mouse strain. All experiments were designed to minimize host, environmental, and microbial differences to ensure that experimental results would reflect differences in mycoplasmal virulence. There were differences in the 50% infectious dose (range, 3 X 10(2) to greater than 10(7) CFU) and the 50% gross pneumonia dose (range, 10(3) to greater than 10(7) CFU) among the 18 mycoplasmal strains. Only 10 strains (UAB CT, M1, UAB 5782C, UAB 6510, 66, UAB T, UAB 8145D, Nelson C, Peter C, and Negroni) established respiratory infections, and only 2 of the 10 strains (UAB CT and M1) produced gross lung lesions. Strains UAB CT, UAB T, M1, UAB 5782C, and PG34(ASH) were chosen for qualitative and quantitative evaluation of lung lesions in C3H/HeN and C57BL/6N mice. Lesion incidence and severity was dependent on the mycoplasmal strain and the mouse strain. Microscopic lesions varied among mycoplasmal strains and mouse strains in the amount of lymphoid infiltrate, neutrophilic exudate, and consolidation, as well as overall severity. The most virulent strain, UAB CT, produced acute pneumonitis in the 10(7) CFU dosage group and required a threshold dose of 10(3) CFU to consistently produce microscopic lung lesions. These results suggest that M. pulmonis virulence is multifactorial and different strains of mycoplasmas yield disease expressions that differ both qualitatively and quantitatively.
Roles of motility, chemotaxis, and penetration through and growth in intestinal mucus in the ability of an avirulent strain of Salmonella typhimurium to colonize the large intestine of streptomycin-treated mice
Authors: McCormick B A , Stocker B A , Laux D C , Cohen P S
Published: January 2020

Previously, it had been shown that an avirulent strain of Salmonella typhimurium, SL5316, with wild-type lipopolysaccharide (LPS) was a far better colonizer of the streptomycin-treated CD-1 mouse large intestine, was far more motile, did not bind to mouse intestinal mucus nearly as well as, but penetrated through a layer of intestinal mucus in vitro far better than an almost isogenic LPS-deficient transductant, SL5325. In the present investigation, a nonflagellated transductant, SL5681, and a nonchemotactic transductant, SL5784, were isolated from SL5316 and tested for relative colonizing ability versus SL5316 (smooth) and SL5325 (rough) in streptomycin-treated mice. In addition, the Salmonella strains were tested for their ability to grow together in cecal intestinal mucus and in cecal luminal contents, for their tumbling and swimming activities after growth in cecal mucus, and for their ability to adhere to and travel through cecal mucus in vitro. The data show that the nonflagellated and nonchemotactic derivatives colonized large intestine nearly as well as their parent and were far better colonizers than the LPS-deficient mutant, that all the strains grew equally well in cecal mucus but did not grow in cecal luminal contents, and that cecal mucus-grown strains lost tumbling and swimming activities. Furthermore, the LPS-deficient strain adhered to cecal mucus far better but penetrated mucus far worse than did the nonflagellated transductant, the nonchemotactic transductant, and the parent. Thus, motility and chemotaxis do not appear to play a major role in the ability of the avirulent S. typhimurium strains to colonize the mouse large intestine, colonization may require growth in cecal mucus but does not depend on growth in cecal luminal contents, growth in cecal mucus inhibits S. typhimurium motility, and increased adhesion of the LPS-deficient mutant to cecal mucus and its poor ability to penetrate cecal mucus may play a role in its poor intestine-colonizing ability.
Purification and immunochemical studies of type b carbohydrate antigen of oral Streptococcus milleri
Authors: Yakushiji T , Inoue M , Koga T
Published: January 2020

The type-specific antigen of serotype b Streptococcus milleri was extracted with trichloroacetic acid from a purified cell wall preparation of the strain NCTC 10708 and then purified on a DEAE-Sephadex A-25 column, followed by a Sephadex G-100 column. The antigen was composed of rhamnose and glucose in a molar ratio of 1.7:1.0, with a trace of galactosamine (0.1). The quantitative precipitin inhibition test with various haptenic sugars showed that rhamnose gave the greatest inhibition, whereas glucose and its related carbohydrates were less effective. The major carbohydrate components of the Rantz-Randall extracts from cells of all four serotype b strains tested were also rhamnose and glucose. These results suggest that rhamnose is structurally involved in the immunodeterminant of the serotype b-specific antigen of oral S. milleri.
Synthesis and physicochemical and immunological characterization of pneumococcus type 12F polysaccharide-diphtheria toxoid conjugates
Authors: Fattom A , Vann W F , Szu S C , Sutton A , Li X , Bryla D , Schiffman G , Robbins J B , Schneerson R
Published: January 2020

A scheme for the synthesis and purification of conjugates, composed of the type 12F capsular polysaccharide of Streptococcus pneumoniae (Pn12F) and diphtheria toxoid, is described. The scheme is a modification of that described previously for the Vi capsular polysaccharide of Salmonella typhi, a linear homopolymer of N-acetylgalactoseaminouronic acid (S. C. Szu, A. L. Stone, J. D. Robbins, R. Schneerson, and J. B. Robbins, J. Exp. Med. 166:1510-1524, 1986). Pn12F is a branched-chain copolymer composed of a hexasaccharide repeating unit containing an aminouronic acid, N-acetylmannoseaminouronic acid (K. Leontein, B. Lindberg, and J. Lonngren, Can. J. Chem. 59:2081-2085, 1981). Sulfhydryl groups were introduced into Pn12F by forming an amide bond between cystamine and carboxyl groups of N-acetylmannoseaminouronic acid in the presence of a carbodiimide. The disulfide moiety of cystamine was reduced to form the cysteamine derivative of Pn12F which was, in turn, covalently bound to diphtheria toxoid by using the heterobifunctional linker N-succinimidyl-3-(2-pyridylthio)propionate. Unbound, high-molecular-weight Pn12F was removed from the conjugate by hydrophobic interaction chromatography through octyl Sepharose by using n-octyl-beta-D-glucopyranoside as the eluent. In young outbred mice, Pn12F did not elicit detectable serum antibodies. Pn12F-diphtheria toxoid, in contrast, elicited antibodies after two injections and had T-cell-dependent properties as evidenced by a response to priming and by its ability to elicit booster responses. This scheme seems applicable to the synthesis of conjugates with other capsular polysaccharides containing aminouronic acids. Clinical evaluation of Pn12F-diphtheria toxoid conjugates in healthy and in immunocompromised hosts is planned.
Morphological differences in Neisseria meningitidis pili
Authors: Greenblatt J J , Floyd K , Philipps M E , Frasch C E
Published: January 2020

Disease and carrier isolates of Neisseria meningitidis were examined for their ability to adhere to human buccal epithelial cells and human cell lines and to hemagglutinate human erythrocytes, properties thought to be associated with the presence of pili. Seventy percent (7 of 10) of carrier isolates were found to be highly adherent to human buccal epithelial cells and to agglutinate human A, B, O, Rh-, and Rh+ erythrocytes. In contrast, 60% of the disease isolates adhered poorly to human buccal epithelial cells and 80% failed to agglutinate human erythrocytes. No adherence of either disease or carrier isolates was observed when several human cell lines were tested. When the meningococcal strains were examined by electron microscopy, 7 of 10 disease isolates were found to possess large bundles of aggregated pili (alpha-type pili), while 7 of 10 carrier isolates were found to have numerous unaggregated pili (beta-type pili). A monoclonal antibody against meningococcal pili and one against gonococcal pili reacted with 6 of 10 piliated carrier isolates and 4 of 10 piliated disease isolates. These results suggest that meningococci, like gonococci, possess different types of pili which differ in morphological, antigenic, and binding properties. In addition, antigenic and morphological differences between pili from carrier and disease isolates were observed as well as differences in adherence and hemagglutinating properties.
Resistance to Plasmodium chabaudi in B10 mice: influence of the H-2 complex and testosterone
Authors: Wunderlich F , Mossmann H , Helwig M , Schillinger G
Published: January 2020

Resistance to Plasmodium chabaudi has been examined in different inbred mouse strains bearing identical H-2 haplotypes on different genetic backgrounds as well as in H-2-congenic mouse strains on B10 background. Resistance is expressed in terms of percent survival after a challenge with 10(6) P. chabaudi-infected erythrocytes. We can show that murine resistance to P. chabaudi is under complex polygenic control involving a non-H-2 gene(s) as well as genes in both I-A and I-E subregions of the H-2 complex. Our data indicate in particular that malaria protective antigens can be presented in context with I-Ab molecules but not in context with I-Ak molecules. Resistance controlled by I-Ab does not become apparent when I-Ek molecules are coincidentally expressed. Moreover, testosterone abrogates I-Ab-controlled resistance to P. chabaudi.
Genetic relationships among pathogenic strains of avian Escherichia coli
Authors: Whittam T S , Wilson R A
Published: January 2020

Genetic relationships among 79 strains of Escherichia coli, isolated mostly from diseased chickens, were estimated on the basis of allelic variation at 15 enzyme-encoding loci, determined by multilocus enzyme electrophoresis. All 15 loci were polymorphic, with an average of 4.1 allelic states per locus. Comparisons of the observed combinations of alleles among strains revealed 37 distinct multilocus genotypes that were used to define naturally occurring cell lineages or clones. Two-thirds of the isolates were classified into 10 clones, including a single multilocus genotype that accounted for about a third of all isolates. For isolates of these clones, there was a high concordance (76%) between identity in multilocus genotype, O:K:H serotype, and pattern of resistance to five antibiotics. Cluster analysis disclosed two major complexes of closely related clones, in which more than 50% of the isolates were associated with localized infections (airsacculitis and pericarditis). Both complexes contained isolates with serotype O2:K1, indicating that this serotype can occur on diverse chromosomal backgrounds. The results suggest that colibacillosis within avian populations is caused by a relatively limited number of pathogenic clones representing at least two distinct clone complexes.
Changes in binding of staphylococcal leukocidin to HL-60 cells during differentiation induced by dimethyl sulfoxide
Authors: Morinaga N , Nagamori M , Kato I
Published: January 2020

The susceptibility of HL-60 cells to the cytotoxic activity of leukocidin increased depending on the degree of differentiation induced by dimethyl sulfoxide (DMSO). To compare binding characteristics of two components (S and F) of leukocidin to HL-60 and DMSO-treated HL-60 cells, the S and F components were labeled with 125I. Scatchard analysis of the binding curve of the 125I-labeled S component to HL-60 cells showed two classes of binding sites. The binding sites with higher affinity had a dissociation constant of 3.39 nM, and the number of binding sites per cell was 730. The specific binding of the 125I-labeled S component to DMSO-treated cells increased depending on the period of DMSO treatment. Scatchard analysis of the binding curve of cells treated with DMSO for 7 days gave a straight line. The dissociation constant was 1.78 nM, and the number of binding sites per cell was 6,920. The total binding of the 125I-labeled F component to DMSO-treated cells increased about twofold over binding to HL-60 cells. However, in the presence of the unlabeled S component, the increase of binding of the F component to DMSO-treated cells was much greater. These data suggested that the increased susceptibility of DMSO-treated cells to leukocidin was dependent on the changes in the number of high-affinity binding sites of the S component and of the bound F component.
Antigenically related iron-regulated outer membrane proteins produced by different somatic serotypes of Pasteurella multocida
Authors: Ikeda J S , Hirsh D C
Published: January 2020

An 84-kilodalton outer membrane protein was expressed when Pasteurella multocida, somatic serotype 3, was grown in brain-heart infusion broth containing the iron chelator dipyridyl but not in brain-heart infusion broth alone. Antigenically related outer membrane proteins of various molecular masses were also expressed by P. multocida strains belonging to all of the other 15 somatic serotypes (somatic serotype 12 being the possible exception) as well as by isolates expressing somatic antigens representative of multiple somatic serotypes when grown under the same conditions of iron deprivation.
Immunogenicity of two formulations of oral cholera vaccine comprised of killed whole vibrios and the B subunit of cholera toxin
Authors: Migasena S , Desakorn V , Suntharasamai P , Pitisuttitham P , Prayurahong B , Supanaranond W , Black R E
Published: January 2020

Two formulations of oral cholera vaccine were evaluated for safety and immunogenicity in adult volunteers in Thailand, an area with sporadic cholera outbreaks. One formulation consisted of 2 x 10(11) killed vibrios and 5 mg of cholera toxin B subunit, as was previously evaluated in North American volunteers, and the other consisted of 1 x 10(11) killed vibrios and 1 mg of B subunit, as was recently evaluated in a field trial in Bangladesh. Three doses of each formulation were given with citrate-bicarbonate buffer. Neither formulation had adverse effects. The formulations stimulated similar serum immunoglobulin G (IgG) and IgA responses to Vibrio cholerae lipopolysaccharide and cholera toxin and intestinal secretory IgA responses to lipopolysaccharide and toxin after three doses. The formulation containing twice the quantity of killed vibrios stimulated better vibriocidal responses, especially to Ogawa serotype. A formulation of oral vaccine containing more killed vibrios than were included in the vaccine studied in the Bangladesh field trial may provide greater protection against cholera.
A protease of Bacteroides gingivalis degrades cell surface and matrix glycoproteins of cultured gingival fibroblasts and induces secretion of collagenase and plasminogen activator
Authors: Uitto V J , Larjava H , Heino J , Sorsa T
Published: January 2020

To assess the direct effects of Bacteroides gingivalis on periodontal cells, human gingival fibroblasts were cultured in the presence of B. gingivalis extracts or a trypsinlike enzyme partially purified from the bacteria by chromatography on benzamidine-Sepharose and Sephacryl S-200. Analysis of cell surface glycoproteins by the periodate-[3H]borohydride labeling technique combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-fluorography demonstrated that fibronectin and some other high-molecular-weight cell surface glycoproteins were degraded by a 35,000-Mr(35K) B. gingivalis protease. Immunostaining of the fibroblast cultures showed degradation of intercellular matrix fibronectin by the 35K protease. The pattern of fibronectin degradation was monitored by examining the reaction products with the SDS-PAGE-immunoblotting technique. The protease degraded fibronectin rapidly and more extensively than did corresponding amounts of pancreatic trypsin. Collagenase secretion by the fibroblasts was assayed by incubating cell culture medium with soluble type I [3H]collagen at 25 degrees C followed by SDS-PAGE-fluorography analysis of the reaction products. The medium was also assayed for plasminogen activator activity by using a casein-agarose diffusion plate assay. The fibroblasts cultured with the 35K protease secreted increased amounts of collagenase and plasminogen activator into the medium. The results suggest that periodontal infection by B. gingivalis causes proteolytic damage of the host cell surface structures. Concomitantly, B. gingivalis may induce the cells to degrade their pericellular matrix.
Pathogenicity of Mycobacterium avium for human monocytes: absence of macrophage-activating factor activity of gamma interferon
Authors: Toba H , Crawford J T , Ellner J J
Published: January 2020

Mycobacterium avium is a frequent opportunistic pathogen in the acquired immunodeficiency syndrome (AIDS). We compared 12 strains of M. avium in an in vitro model of pathogenicity. Peripheral blood-derived monocytes from healthy individuals were infected with M. avium in vitro. Bacterial uptake and intracellular replication were assessed by microscopic count of acid-fast bacilli and CFU of bacteria, respectively, in lysed monocytes. The CFU assay showed that among five AIDS-associated strains, only one replicated in monocytes. Two of seven non-AIDS-associated strains replicated intracellularly. In addition, we examined the effect of gamma interferon (IFN-gamma) on M. avium infection. IFN-gamma treatment of monocytes decreased phagocytosis and had no effect on the intracellular replication of M. avium. Thus, most strains of M. avium do not multiply within monocytes from healthy individuals and IFN-gamma does not have macrophage-activating factor activity for M. avium infection of human monocytes.
Lipid-modified surface protein antigens expressing size variation within the species Mycoplasma hyorhinis
Authors: Boyer M J , Wise K S
Published: January 2020

Monoclonal antibodies (MAbs) previously shown to recognize distinct epitopes selectively expressed on the surface of some Mycoplasma hyorhinis strains were used to define two discrete sets of lipid-modified membrane surface proteins showing marked size variation within this species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of Triton X-114 phase-fractionated proteins from six isolates of M. hyorhinis defined a set of amphiphilic integral membrane proteins of 23, 50, and 55 kilodaltons (kDa) recognized on respective isolates by one MAb and a second set of integral proteins of 88, 120, and 100 to 150 kDa recognized by another MAb. The first group of proteins all contained a common, amphiphilic 18-kDa limit tryptic polypeptide bearing the epitope. The size- and strain-variant surface antigens identified by the MAbs were shown to be lipid-modified proteins. Phase fractionation of [3H]palmitate-labeled organisms revealed numerous 3H-labeled proteins in all isolates, which partitioned exclusively into the hydrophobic phase. These proteins generally showed pronounced size variation among isolates and included the antigen variants recognized by the two MAbs, as demonstrated directly by immunoprecipitation of correspondingly sized 3H-labeled proteins from each isolate. A third MAb recognized an invariant, lipid-associated surface protein of 70 kDa on all M. hyorhinis isolates. Covalent modification of lipid-associated proteins was confirmed by identifying 3H-labeled methyl palmitate after acid methanolysis of Triton X-114 phase proteins derived from [3H]palmitate-labeled organisms. However, removal of covalently bound lipid from chloroform-methanol-extracted proteins by alkaline hydroxylamine was selective; complete removal was observed with only a few proteins, possibly including the 120-kDa form of one antigen variant. This suggested potential differences in the nature of covalent linkage among lipid-modified M. hyorhinis surface antigens. Intraspecies antigen variants described here in M. hyorhinis share some characteristics with size-variant antigens reported in phylogenetically related gram-positive eubacteria and may contribute to phenotypic diversification and differences in pathogenicity of mycoplasmas.
Fibrinogen binding and resistance to phagocytosis of Streptococcus sanguis expressing cloned M protein of Streptococcus pyogenes
Authors: Poirier T P , Kehoe M A , Whitnack E , Dockter M E , Beachey E H
Published: January 2020

The biological properties of Streptococcus pyogenes M protein cloned and expressed in S. sanguis were investigated. The spm-5 gene previously cloned into Escherichia coli was subcloned into the E. coli-S. sanguis shuttle plasmid pVA838 to produce a newly constructed plasmid, pBK100. Cells of S. sanguis transformed with pBK100 expressed 53-, 55-, and 58-kilodalton polypeptides reacting with type 5 M protein antiserum in immunoblots. The M protein was expressed on the surface of S. sanguis cells as shown by the capacity of the intact cells to (i) inhibit the reactivity of anti-type 5 antibodies with purified M protein as demonstrated by enzyme-linked immunosorbent assay; (ii) inhibit the opsonization by M5 antisera of type 5 S. pyogenes; (iii) express M-protein-like fibrils on the surface of the organisms that react with M5 antisera as revealed by immunoelectron microscopy; (iv) bind plasma fibrinogen and, as a consequence, resist phagocytosis by human blood neutrophils; and (v) be rendered susceptible to phagocytosis by opsonic M5 antisera. These results provide additional evidence that streptococcal M proteins bind host proteins as a ploy to evade host defense mechanisms.
Identification of Shigella invasion genes by isolation of temperature-regulated inv::lacZ operon fusions
Authors: Hromockyj A E , Maurelli A T
Published: January 2020

Penetration and multiplication within cells of the human colonic epithelium are hallmarks of Shigella spp. pathogenicity. Shigella spp. virulence is regulated by growth temperature. Strains phenotypically virulent when grown at 37 degrees C are phenotypically avirulent when grown at 30 degrees C. The number of genes involved in Shigella spp. pathogenicity and how many virulence genes are temperature regulated are unknown. To facilitate the study of temperature-regulated virulence in Shigella spp., we employed lacZ operon fusion technology to identify temperature-regulated invasion (inv) genes. Four inv::lacZ fusion mutants were identified and found to be unable to invade HeLa cells. The fusions were located in a region of the 220-kilobase invasion plasmid defined as the minimal amount of DNA required for invasion, and they were controlled by virR, the temperature-dependent virulence gene regulator. Western blot (immunoblot) and Southern hybridization analyses indicated that one of the fusions was located in a known inv gene, ipaB, which encodes one of the major immunogenic peptides of Shigella spp. This ipaB::lacZ operon fusion mutant synthesized a truncated IpaB protein recognized by IpaB-specific monoclonal antibodies. Three of the fusions were within novel genes mapping to regions previously identified as essential for a positive virulence phenotype. Analysis of bacterial surface proteins suggested that the genes marked by these fusions may play a role in the correct surface expression of the ipaB and ipaC gene products.
Treatment of mice with human recombinant interleukin-2 augments resistance to the facultative intracellular pathogen Listeria monocytogenes
Authors: Haak-Frendscho M , Young K M , Czuprynski C J
Published: January 2020

The effects of exogenously administered human recombinant IL-2 (hrIL-2) on resistance to Listeria monocytogenes infection were examined. Intravenous injection of hrIL-2 significantly enhanced antibacterial resistance in both BDF1 and C3H/HeJ mice. The beneficial effect of hrIL-2 was observed with as little as 0.6 micrograms per mouse, whereas optimum protection occurred at 6 micrograms per mouse, hrIL-2 was equally protective when administered concomitant with the listeriae or up to 24 h prior to infection; it had little effect if given after the bacterial challenge. Kinetic experiments indicated that both the peak bacterial burden and the time lag before L. monocytogenes began to be cleared from the spleen and liver were reduced in hrIL-2-treated mice as compared with control mice. Histopathological examination of spleens and livers confirmed that hrIL-2-treated Listeria-infected mice experienced considerably less damage to these organs than did control mice. Spleen cells from Listeria-infected mice exhibited depressed levels of mitogen-induced proliferation coincident with the peak bacterial burden in the spleen and liver and during the subsequent recovery from the infection. Administration of hrIL-2 to uninfected mice had no effect on spleen cell proliferation in response to mitogens in vitro, nor did hrIL-2 treatment restore normal levels of splenocyte proliferative responses to Listeria-infected mice. In addition, hrIL-2 treatment resulted in attenuated levels of serum colony-stimulating activity in infected mice as compared with control infected mice. Coadministration of both hrIL-2 and human recombinant interleukin-1 alpha at various dose and time combinations had no detectable additive or synergistic effect. Although these data do not suggest an obvious mechanism of action, they clearly demonstrate that hrIL-2 can augment host defense against the facultative intracellular pathogen L. monocytogenes.
Molecular analysis of a region of the group B streptococcus chromosome involved in type III capsule expression
Authors: Kuypers J M , Heggen L M , Rubens C E
Published: January 2020

Type III group B streptococci (GBS) are the most common cause of neonatal sepsis and meningitis in the United States. The important role of the type III polysaccharide capsule and of the terminal sialic acid moiety of the capsule in the virulence of GBS has been demonstrated by using Tn916 mutagenesis. Several of the transposon insertion sites that resulted in defective type III capsule synthesis were located in a 30-kilobase (kb) region of the chromosome. Hybridization analysis of two other type III strains that differed in their relative virulence and of GBS serotypes Ia, Ib, Ic, and II showed that this region of the chromosome was highly conserved. A repetitive 1.4-kb sequence was found only in the 30-kb region of the more virulent type III strain, COH 1. The Escherichia coli maxicell in vivo expression system and an in vitro coupled transcription-translation system successfully identified the proteins expressed from the 30-kb region. Comparison of the proteins expressed from the same DNA fragments in these two assays indicated that some of these proteins may contain leader sequences that would ultimately result in their secretion to the cell surface. Identification and further characterization of the genes and their products will provide the foundation for understanding the genetic and biochemical events in GBS capsular polysaccharide production.
Antigenic variation within serotypes of Cryptococcus neoformans detected by monoclonal antibodies specific for the capsular polysaccharide
Authors: Spiropulu C , Eppard R A , Otteson E , Kozel T R
Published: January 2020

The serotype-specific polysaccharide was isolated from four to seven strains of each of the four major serotypes of Cryptococcus neoformans. Reactivities of polysaccharides with seven monoclonal antibodies raised against polysaccharides of serotypes A, B, C, and D were assessed by an enzyme-linked immunosorbent assay and double immunodiffusion. The results indicated that there is heterogeneity within all four serotypes in the expression of epitopes reactive with monoclonal antibodies.
Relationship of Haemophilus influenzae type b pilus structure and adherence to human erythrocytes
Authors: Gilsdorf J R , Judd W J , Cinat M
Published: January 2020

Six strains of Haemophilus influenzae type b, some expressing immunologically different pili, showed identical patterns of binding to erythrocytes that were characterized for 38 blood group antigens. All six strains appeared to bind to the Anton antigen, as they agglutinated all erythrocytes tested except cord erythrocytes and those characterized as Lu(a-b-), dominant type, including Anton-negative cells.
Binding of Yersinia enterocolitica to rabbit intestinal brush border membranes, mucus, and mucin
Authors: Mantle M , Basaraba L , Peacock S C , Gall D G
Published: January 2020

Mucus and its gel-forming glycoprotein component, mucin, are thought to protect the gastrointestinal tract from enteric pathogens by inhibiting their attachment to enterocytes. In this study, we investigated interactions between Yersinia enterocolitica (isogenic strains of virulent and nonvirulent organisms) and crude mucus, highly purified mucin, and brush border membranes (BBMs) isolated from the upper mid-, and distal small intestine and the proximal colon of the rabbit. Adherence of radiolabeled bacteria was assessed to BBMs, mucus, and mucin immobilized in polystyrene microtiter plate wells. Virulent Y. enterocolitica showed saturable binding to mucus, mucin, and BBMs from all four regions of the intestinal tract, although adherence to BBMs was appreciably greater than that to mucus or mucin. Maximal binding of bacteria was higher to BBMs from the distal small intestine and the proximal colon than to those from the upper and mid-small intestine, which may in part explain why the organism localizes to the ileo-caecal regions of the gut. Adherence of virulent Y. enterocolitica to BBMs was significantly reduced in the presence of homologous mucus or mucin preparations. Binding of virulent bacteria appears to depend on plasmid-encoded proteins located on the outer surface membrane, since (i) the isogenic strain lacking the virulence plasmid showed markedly less binding to all BBM, mucus, and mucin preparations; (ii) growth of the virulent strain at 25 degrees C, which inactivates its plasmid, significantly diminished binding to BBMs, mucus, and mucin; and (iii) mild proteolysis substantially decreased adherence of virulent bacteria to BBMs. Compared with rabbit intestinal and colonic mucins, binding of virulent Y. enterocolitica was significantly greater to purified human intestinal mucin and significantly less to rat intestinal mucin. These findings provide support for the role of mucus and mucin in host defense by preventing adherence of virulent Y. enterocolitica to epithelial cell membranes.
High-resolution 31P nuclear magnetic resonance study of Chlamydia trachomatis: induction of ATPase activity in elementary bodies
Authors: Peeling R W , Peeling J , Brunham R C
Published: January 2020

ATPase activity of elementary bodies (EBs) of Chlamydia trachomatis was investigated by using high-resolution 31P nuclear magnetic resonance spectroscopy. ATPase activity was detected in EBs of C. trachomatis serovars A, B, and L2 after treatment with the reducing agents 2-mercaptoethanol and glutathione. ATPase activity was oligomycin sensitive and magnesium ion dependent. EBs heated at 60 degrees C for 10 min or pretreated with Triton X-100 before exposure to 2-mercaptoethanol did not exhibit ATPase activity. Monoclonal antibody to the major outer membrane protein abrogated ATPase activity of EBs, whereas monoclonal antibody to chlamydial lipopolysaccharide only marginally reduced the level of ATPase activity. These findings suggest that EBs possess intrinsic ATPase activity and that cysteine-rich outer membrane proteins of EBs are important in the regulation of ATPase activity. The major outer membrane protein may be the major route through which ATP accesses ATPase.
Activation of C3 and binding to Aspergillus fumigatus conidia and hyphae
Authors: Kozel T R , Wilson M A , Farrell T P , Levitz S M
Published: January 2020

Complement activation by Aspergillus fumigatus may play a crucial role in stimulating binding and killing of this organism by phagocytes. We examined the amount and type of C3 deposited on resting conidia, swollen conidia, and hyphae of A. fumigatus after incubation in pooled human serum. All three life forms of A. fumigatus were potent activators of the complement cascade, with deposition on the organisms of similar amounts of C3 per unit of surface area. The rate of deposition was slowest for resting conidia, although maximal deposition was still achieved within 40 min. The roles of the alternative and classical pathways were assessed by use of serum chelated with magnesium EGTA [magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and with an alternative pathway reconstituted from the six purified alternative-pathway proteins. Complement activation by resting conidia was mediated by the alternative pathway. In contrast, there was a progressive dependence on the classical pathway as the fungal particles matured into swollen conidia and then hyphae. Treatment with hydroxylamine, which disrupts ester linkages, removed 89 to 95% of the C3 bound to all three forms of A. fumigatus. This released C3 contained a mixture of C3b and iC3b, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. These data demonstrate that although all three forms of A. fumigatus are potent activators of the complement system, the transition from resting conidia to swollen conidia to hyphae results in progressive changes in the manner in which the fungal particles interact with the complement system. The lack of participation of the classical pathway in complement activation by resting conidia may have important implications regarding their ability to effectively stimulate phagocytes.
Production and characterization of monoclonal antibodies to a pilus colonization factor (colonization factor antigen III) of human enterotoxigenic Escherichia coli
Authors: Honda T , Wetprasit N , Arita M , Miwatani T
Published: January 2020

Three monoclonal antibodies (MAbs) to a pilus colonization factor (colonization factor antigen III [CFA/III]) of human enterotoxigenic Escherichia coli (ETEC) were developed and characterized. All of the MAbs isolated belonged to the immunoglobulin G2a subclass. The specificity of these MAbs for CFA/III pili was demonstrated by the immunogold-labeling technique. The presence of more than one epitope in CFA/III pili was suggested. One of the three MAbs appears to recognize a polymeric conformational epitope(s) of CFA/III. CFA/III antigenicity distinct from that of other pilus colonization factors of ETEC was demonstrated by both a bacterial agglutination test and a sandwich enzyme-linked immunosorbent assay using the MAbs. Of the 100 strains of ETEC isolated from persons with traveler's diarrhea, 8% were found to carry CFA/III pili. Two enzyme-linked immunosorbent assay systems which could detect as little as several or 50 ng of CFA/III per ml were developed.
Purified P fimbriae from two cloned gene clusters of a single pyelonephritogenic strain adhere to unique structures in the human kidney
Authors: Karr J F , Nowicki B , Truong L D , Hull R A , Hull S I
Published: January 2020

We have completed immunofluorescence binding studies of purified fimbriae from two clones which express Pap or Pap-2 fimbriae. Although the two fimbrial types exhibited common binding to the uroepithelia of the bladder and renal pelvis and to occasional cells located within the glomeruli which we have termed glomerular elements, only Pap-2 fimbriae adhered to Bowman's capsule. Previous studies have demonstrated that the Gal alpha 1----4Gal disaccharide moiety is capable of inhibiting Pap hemagglutination and adherence to uroepithelial cells. Results of our experiments demonstrate that this disaccharide is not sufficient for blocking binding of Pap-2 fimbriae to Bowman's capsule but that GalNAc beta 1----3Gal completely blocks Pap-2 adherence to Bowman's capsule. These results indicate that the different hemagglutination capacities of the two clones reflect different receptor specificities and differential tissue tropisms in the urinary tract. These unique receptor specificities may provide uropathogenic strains of Escherichia coli carrying multiple chromosomal copies of pap-like gene clusters with the advantage of increased numbers of binding sites within the urinary tract. This, in turn, might improve the chances of colonization and the establishment of infection.
Biochemical and immunological characterization of the surface proteins of Borrelia burgdorferi
Authors: Luft B J , Jiang W , Munoz P , Dattwyler R J , Gorevic P D
Published: January 2020

The immunodominant proteins and glycoproteins of Borrelia burgdorferi were analyzed by one-dimensional (1D) and 2D gel electrophoresis. More than 100 polypeptide species could be detected on silver-stained 2D gels. Separation of sonic extracts of the organism by differential centrifugation (100,000 X g) revealed several of the major proteins to reside predominantly within the pellet fraction. The antigenicity of the individual polypeptides was determined by Western (immuno-) blot analysis with sera from humans with chronic Lyme disease and from rabbits immunized with B. burgdorferi. Surface proteins of viable B. burgdorferi labeled with 125I or long-arm hydroxysuccinimide biotin were identified by gel analyses. Thirteen major surface proteins were apparent, including the highly immunogenic 41-kilodalton (kDa) endoflagellar antigen. Two of these proteins, with molecular masses of 22 and 41 kDa, were further characterized by electroblotting and microsequencing their amino termini. Significant (35%) homology between the first 20 amino acids of the 22-kDa protein and the deduced amino acid sequence of the 31-kDa (outer surface protein A) protein of B. burgdorferi may indicate that these proteins are processed similarly or are part of a gene family expressed at the surface of the organism. In addition, highly significant (88%) homology was found between the first nine amino acids of the 41-kDa protein of B. burgdorferi and the 33-kDa endoflagellar protein of Treponema pallidum, after which the sequences diverge. This observation provides in part a structural basis for the observed cross-reactivity between the two organisms and suggests alternative approaches to the development of specific immunodiagnostics.
Fusion of genes encoding Escherichia coli heat-stable enterotoxin and outer membrane protein OmpC
Authors: Saarilahti H T , Palva E T , Holmgren J , Sanchez J
Published: January 2020

The OmpC outer membrane protein of Escherichia coli was used as a carrier molecule for the nonimmunogenic heat-stable enterotoxin STa. Two fragments of different lengths of the gene encoding STa were fused in vitro to the 3' terminus of the truncated ompC gene. The resulting OmpC-STa hybrid proteins could be detected by L-[35S]cysteine labeling, and they were processed and thus exported. All synthesized hybrid protein remained cell bound and was found by fractionation mainly in the periplasm. Immunoblot analysis showed that the hybrid proteins reacted in vitro both with anti-OmpC and anti-STa antibodies, and immunization of rabbits evoked an antibody response to either of these proteins.
Adhesion of colonization factor antigen II-positive enterotoxigenic Escherichia coli strains to human enterocytelike differentiated HT-29 cells: a basis for host-pathogen interactions in the gut
Authors: Neeser J R , Chambaz A , Golliard M , Link-Amster H , Fryder V , Kolodziejczyk E
Published: January 2020

Enterotoxigenic Escherichia coli are the most common cause of travelers' and infant diarrhea in less-developed countries. In the present work, among several metabolically labeled human diarrheagenic E. coli strains, enterotoxigenic strains expressing colonization factor antigen II were shown to bind to HT-29 intestinal cell monolayers when these cells were grown in conditions promoting their enterocytic differentiation. Indirect immunofluorescence with fimbrial antisera revealed that pathogen attachment was associated with the production of a specific bacterial adhesin, the E. coli surface antigen CS3. Scanning and transmission electron micrographs showed an apical pattern of colonization, characteristic of enterotoxigenic E. coli infections. The above data were consistent with all observations previously made with human enterocytes obtained from intestinal biopsies. The lectin-carbohydrate nature of this cell-cell recognition mechanism was also established. Bacterial binding to differentiated HT-29 cells was inhibited by a mixture of newborn meconium glycopeptides. By coating the cell layers with the plant agglutinin from Evonymus europaea, pathogen attachment was also prevented. Binding of 125I-labeled CS3 adhesin and E. europaea agglutinin to brush border membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose revealed three bands of about 30, 20, and 13 kilodaltons, which acted as receptors for both bacterial and plant lectins. These data suggest that the sugar units to which the bacterial colonization factor CS3 binds are synthesized as carbohydrate chains of three brush border membrane glycoproteins in HT-29 cells by a differentiation-specific pathway.