bluuebunny/crossref_metadata_2025 huggingface dataset:
Change in degree of type 1 piliation of Escherichia coli during experimental peritonitis in the mouse
Authors: Alkan M L , Wong L , Silverblatt F J
Published: January 2020

To determine whether expression of type 1 pili varies during the course of Escherichia coli infection in vivo, mice were injected intraperitoneally with 5 X 10(7) CFU of piliated or nonpiliated phase variants per ml, and the degree of piliation was measured in peritoneal exudate by an enzyme-linked immunosorbent assay inhibition method. In the animals challenged with the piliated bacteria, the numbers of organisms increased a log over 9 h and the amount of pilus antigen decreased from 3 to 0.075 micrograms/10 bacteria. After a 4-h delay, nonpiliated bacteria also increased by one log over 9 h; however, the amount of piliation remained virtually undetectable. Piliated E. coli were more virulent than nonpiliated variants in this model (50% lethal dose of 7.5 X 10(6) versus 3 X 10(7), respectively). The difference was significantly reduced by prior passive immunization with rabbit serum containing high titers of antipili antibody. Piliated bacteria adhered in significantly greater numbers to isolated mouse peritoneal membranes than did nonpiliated variants (15,400 +/- 2,700 versus 1,300 +/- 700 bacteria/mm2, respectively; P = 0.05). Adherence was inhibited by the presence of 0.1 M alpha methyl mannose (1,500 +/- 1,800 bacteria/mm2, P = 0.01). These results confirm the results of previous qualitative studies showing that phase variation of type 1 pili occurs in vivo and suggest that these pili may confer an initial advantage for growth of E. coli in the peritoneal cavity, presumably by fostering colonization of the peritoneal serosal surface.
Stage-specific and species-specific antigens of Plasmodium vivax and Plasmodium ovale defined by monoclonal antibodies
Authors: Andrysiak P M , Collins W E , Campbell G H
Published: January 2020

Monoclonal antibodies (MAbs) were produced against the asexual blood stages of Plasmodium vivax and Plasmodium ovale and used to define antigens of plasmodial parasites in an indirect fluorescent antibody assay. The anti-P. vivax MAbs produced two distinct patterns in the indirect fluorescent antibody assay. Four patterns were found with the anti-P. ovale MAbs. Species-specific epitopes were defined for P. vivax and P. ovale; epitopes shared among all four species of human malaria parasites were also defined. Some of the anti-P. vivax MAbs reacted only with mature stages, and others reacted with all asexual stages. No asexual blood-stage specificity could be found with the anti-P. ovale antibodies. Five of the anti-P. vivax MAbs and three of the anti-P. ovale MAbs also reacted with sporozoites.
Analysis of the genetic determinants coding for the S-fimbrial adhesin (sfa) in different Escherichia coli strains causing meningitis or urinary tract infections
Authors: Ott M , Hacker J , Schmoll T , Jarchau T , Korhonen T K , Goebel W
Published: January 2020

Recently we have described the molecular cloning of the genetic determinant coding for the S-fimbrial adhesin (Sfa), a sialic acid-recognizing pilus frequently found among extraintestinal Escherichia coli isolates. Fimbriae from the resulting Sfa+ E. coli K-12 clone were isolated, and an Sfa-specific antiserum was prepared. Western blots indicate that S fimbriae isolated from different uropathogenic and meningitis-associated E. coli strains, including O83:K1 isolates, were serologically related. The Sfa-specific antibodies did not cross-react with P fimbriae, but did cross-react with F1C fimbriae. Furthermore the sfa+ recombinant DNAs and some cloned sfa-flanking regions were used as probes in Southern experiments. Chromosomal DNAs isolated from O18:K1 and O83:K1 meningitis strains with and without S fimbriae and from uropathogenic O6:K+ strains were hybridized against these sfa-specific probes. Only one copy of the sfa determinant was identified on the chromosome of these strains. No sfa-specific sequences were observed on the chromosome of E. coli K-12 strains and an O7:K1 isolate. With the exception of small alterations in the sfa-coding region the genetic determinants for S fimbriae were identical in uropathogenic O6:K+ and meningitis O18:K1 and O83:K1 strains. The sfa determinant was also detected on the chromosome of K1 isolates with an Sfa-negative phenotype, and specific cross-hybridization signals were visible after blotting against F1C-specific DNA. In addition homology among the different strains was observed in the sfa-flanking regions.
Changes in isoenzyme patterns of a cloned culture of nonpathogenic Entamoeba histolytica during axenization
Authors: Mirelman D , Bracha R , Wexler A , Chayen A
Published: January 2020

The axenization of an Entamoeba histolytica isolate with a nonpathogenic isoenzyme electrophoretic pattern (zymodeme) was recently achieved for the first time (15). Forty days after the cells were transferred to the medium used for axenic cultivation, the amebae developed virulence properties, and the zymodeme converted to a pathogenic pattern. To exclude the possibility that the original isolate consisted of two zymodeme populations and that conditions of growth selected for a particular population, the experiment was repeated with a cloned culture of a nonpathogenic (zymodeme III) strain, E. histolytica SAW 1734R clAR, isolated by and obtained from P. G. Sargeaunt. Axenization was accomplished, as before, by transferring trophozoites to TYI-S-33 medium containing a mixture of antibiotics to suppress the growth of the associated bacterial flora and a nutritional supplement consisting of gamma-irradiated bacteria. A change in the hexokinase and phosphoglucomutase isoenzyme pattern was observed 21 days after the amebae had been transferred to the axenic medium but before complete axenization of the amebae had occurred. The change in zymodeme was accompanied by an increase in virulence, as evidenced by the ability of fewer amebae to induce hepatic abscesses in hamsters. A reverse conversion to a nonpathogenic zymodeme was also accomplished by reassociating and subculturing the newly converted pathogenic trophozoites of strain SAW 1734R clAR with the bacterial flora that accompanied this ameba in the original xenic culture. The electromobilities of the hexokinase isoenzymes changed back to their original pattern 7 days after the amebae were returned to xenic growth conditions. Our in vitro results demonstrate that culture conditions and bacterial flora can cause changes in the zymodeme and virulence of a cloned ameba isolate and raise the concern that this could happen also in vivo. Thus, the finding of a particular zymodeme in a culture of E. histolytica isolated from a carrier should not be used to predict a clinical condition or serve as a basis for the recommendation of therapy.
Role of interleukin-1 in the depression of liver drug metabolism by endotoxin
Authors: Ghezzi P , Saccardo B , Villa P , Rossi V , Bianchi M , Dinarello C A
Published: January 2020

Endotoxin-resistant C3H/HeJ mice were used to test the hypothesis that a macrophage product, possibly interleukin-1, might mediate the depression of liver cytochrome P-450-dependent drug metabolism in endotoxin-treated mice. Depression of liver drug metabolism by endotoxin was observed in normal mice (C3H/HeN) but not in C3H/HeJ mice. Serum transfer experiments demonstrated that a serum factor was responsible for the depression of liver drug metabolism. Experiments of passive transfer of peritoneal macrophages showed that this endotoxin-induced factor might be a macrophage product. In vitro experiments showed that endotoxin-stimulated monocytes produced a factor that depressed cytochrome P-450-dependent metabolism in cultured hepatocytes. Homogeneous human monocyte and recombinant interleukin-1 also depressed liver drug metabolism both in vivo and in vitro, suggesting that this macrophage product might be involved in the regulation of liver function by the immune system.
Pneumococcal intracellular killing is abolished by polysaccharide despite serum complement activity
Authors: Schweinle J E
Published: January 2020

Normal human serum absorbed at 0 degrees C with pneumococcal serotype 1, 12, or 25 lost the ability to support polymorphonuclear leukocyte intracellular killing of some pneumococcal serotypes even if immunoglobulin was provided. The absorbed serum contained no organisms but had residual polysaccharide when measured by counterimmunoelectrophoresis against type-specific antisera. The influence of pneumococcal polysaccharide (PPS) on serum support of intracellular polymorphonuclear leukocyte killing was evaluated. Normal human serum was mixed with PPS serotype 1, 12, or 25 at 0 degrees C for up to 120 min and then used as the opsonic source in standard phagocytic killing assays with serotype 1, 12, or 25. Immediately after mixing, each serum combined with PPS serotype 1, 12, or 25 supported killing of all serotypes tested. With increasing time at 0 degrees C with serotype 1 PPS, serum exhibited a marked progressive decline in killing of serotype 25, a similar but less marked impairment in killing of serotype 12, and essentially no impairment for serotype 1. Serum treated with type 25 PPS did not support killing of type 25, but allowed normal killing of types 1 and 12. Incubation with type 12 PPS impaired opsonization of types 12 and 25, but not 1. Addition of PPS-specific antisera did not restore killing. Residual serum hemolytic activity of classic and alternative complement pathways was not reduced below opsonizing levels after 120 min at 0 degrees C. Furthermore, PPS treatment of normal human serum did not alter the attachment or ingestion of 14C-labeled pneumococci by polymorphonuclear leukocytes. Some serotypes of PPS reacted slowly with serum at 0 degrees C, diminishing its ability to support intracellular killing of pneumococci despite serum hemolytic complement activity. Phagocytosis was not inhibited. Specific antibody did not overcome inactivation. Different requirements for the inactivated factor among serotypes may be a characteristic related to organism virulence.
Presence and expression of aerobactin genes in virulent avian strains of Escherichia coli
Authors: Lafont J P , Dho M , D'Hauteville H M , Bree A , Sansonetti P J
Published: January 2020

Virulent and nonvirulent isolates of avian Escherichia coli were tested for the presence of aerobactin genes by colony hybridization with a specific gene probe constructed from plasmid pABN1 (A. Bindereif and J. B. Neilands, J. Bacteriol. 153:1111-1113, 1983). Positive hybridization with the gene probe was highly correlated with virulence, as measured by the 50% lethal dose of the strains for chicks. Evidence for the expression of aerobactin genes in the virulent strains was obtained by demonstrating their susceptibility to cloacin DF13, which binds to the same receptor that binds aerobactin, and their ability to produce aerobactin, as revealed by cross-feeding the E. coli mutant WO987 (aroB fepA iuc iut+), which is unable to synthesize but capable of taking up aerobactin. We suggest that the production of aerobactin is involved in the virulence of avian septicemic E. coli.
Histoplasma capsulatum fails to trigger release of superoxide from macrophages
Authors: Eissenberg L G , Goldman W E
Published: January 2020

The yeast form of the dimorphic fungus Histoplasma capsulatum survives within macrophages after phagocytosis. To do so, it must avoid, inhibit, or resist a variety of toxic oxygen metabolites. Using ferricytochrome c reduction to assay superoxide release, we examined the response of mouse macrophages to the yeast form of various H. capsulatum strains. Doses of zymosan as low as 20 particles per macrophage elicited superoxide, whereas H. capsulatum failed to induce superoxide even at 160 yeast cells per macrophage. This phenomenon was observed with two virulent strains of H. capsulatum (G217B and G186A) and with an avirulent variant of G186A. Over a 15- to 150-min observation period, zymosan stimulated increasing reduction of ferricytochrome c, but H. capsulatum did not. When added concurrently with zymosan, H. capsulatum had no effect on superoxide production. Therefore, H. capsulatum was unable either to inactivate the oxygen radical or inhibit host cell superoxide response to other competent stimuli. Enzymatically generated superoxide reduced ferricytochrome c even in the presence of H. capsulatum, again implying that the organism does not readily inactivate superoxide. This experiment also demonstrated that the yeast did not interfere with the assay used. Thus, rather than inhibiting superoxide generation or inactivating the anion, H. capsulatum yeast cells appear to avoid the toxic effects of superoxide by failing to trigger its release.
Oxidative response of human neutrophils, monocytes, and alveolar macrophages induced by unopsonized surface-adherent Staphylococcus aureus
Authors: Devalon M L , Elliott G R , Regelmann W E
Published: January 2020

In contrast to results with bacterial suspensions, phagocytosis of unopsonized bacteria readily occurs when bacteria are adhered to glass or plastic surfaces. However, in contrast to neutrophils, alveolar macrophages produced much less DNA denaturation as measured by acridine orange metachromasia of phagocytized Staphylococcus aureus. We have studied the phagocytosis of unopsonized surface-adherent S. aureus and the subsequent production of reactive oxygen species by peripheral blood neutrophils, monocytes, and alveolar macrophages. Phagocyte-free systems were then used to show the relationship of the reactive oxygen species produced by neutrophils and alveolar macrophages and the denaturation of unopsonized S. aureus DNA with acridine orange. Peripheral blood neutrophils, monocytes, and alveolar macrophages from normal human volunteers were added to vials with adherent S. aureus without opsonin. Bacterial uptake and luminol- and lucigenin-dependent chemiluminescence were measured. Neutrophils developed much greater luminol-dependent chemiluminescence than monocytes or alveolar macrophages. Compared with neutrophils and monocytes, alveolar macrophages developed significantly greater concentrations of superoxide, as measured by lucigenin-dependent chemiluminescence and ferricytochrome c reduction. These findings suggested that products of the myeloperoxidase-hydrogen peroxide-halide pathway were generated when peripheral blood neutrophils were stimulated and that alveolar macrophages primarily produced superoxide. When these reactive oxygen species were generated in phagocyte-free systems containing S. aureus, products of the myeloperoxidase-hydrogen peroxide-halide pathway produced denaturation of S. aureus DNA, whereas superoxide did not. Thus, differences in reactive oxygen species produced during phagocytosis may be related to the different capacities of neutrophils and alveolar macrophages to denature unopsonized adherent S. aureus DNA.
Effect of immunization on experimental Bacteroides gingivalis infection in a murine model
Authors: Chen P B , Neiders M E , Millar S J , Reynolds H S , Zambon J J
Published: January 2020

BALB/c mice were immunized with an invasive (A7A1-28) or noninvasive (381) Bacteroides gingivalis strain, Bacteroides intermedius, or Ringer solution. All immunized mice were subsequently challenged with the invasive B. gingivalis strain and examined for septicemia or secondary spread of the infection or both. Mice immunized with the invasive B. gingivalis strain localized the infection to the challenge site. Mice immunized with the noninvasive B. gingivalis strain, B. intermedius, or Ringer solution developed spreading infections. These data suggest that immunization with an invasive B. gingivalis strain can alter the course of subsequent infections.
Oral immunization of humans with Streptococcus sobrinus glucosyltransferase
Authors: Smith D J , Taubman M A
Published: January 2020

The effect of oral administration of glucosyltransferase (GTF) from Streptococcus sobrinus 6715 on levels of immunoglobulin A (IgA) antibody to GTF in parotid saliva and on the number of indigenous Streptococcus mutans in the whole saliva was studied in young adult males. GTF combined with aluminum phosphate (AP) was administered in capsules to 14 subjects, while sodium phosphate buffer combined with AP was administered in the same way to 11 control subjects. Thirteen administrations were given during the first immunization regimen, and five administrations, approximately 3 months later, constituted the second immunization regimen. All subjects were given professional dental prophylaxis immediately prior to each immunization. Each subject served as his own control by using antibody and bacterial data collected prior to antigen administration for comparison. After the first immunization regimen, the GTF vaccine group exhibited a significantly higher distribution (P less than 0.05) of normalized parotid saliva IgA antibody elevations than observed in the placebo group. Between the first and second immunization regimens a significant increase (P less than 0.05) in parotid salivary anti-GTF activity also occurred in the GTF vaccine but not the placebo group. No significant differences between these two groups were observed on any occasion when serum IgG or IgA antibody to GTF was analyzed. Comparison of the group mean log ratios (post- to prevaccine administration) of S. mutans to total streptococci in whole saliva revealed that the GTF vaccine group values were always lower than those of the placebo group. These differences reached significance (P less than 0.01) on three of the last four sampling occasions (days 21, 35, and 42) following initiation of the first immunization regimen. The mean log ratios of the GTF vaccine group were also lower than those of the placebo group after the second immunization regimen but did not reach significance. These data indicate that oral administration of GTF from the mutans streptococci has the potential to elicit a salivary IgA antibody response when combined with an aluminum-based adjuvant and that this response can interfere with the reaccumulation of indigenous S. mutans following dental prophylaxis.
The low-molecular-mass, cysteine-rich outer membrane protein of Chlamydia trachomatis possesses both biovar- and species-specific epitopes
Authors: Zhang Y X , Watkins N G , Stewart S , Caldwell H D
Published: January 2020

We isolated, by hydroxylapatite high-performance liquid chromatography, 14- and 15-kilodalton (kDa), cysteine-rich outer membrane proteins from Chlamydia trachomatis TW-5/OT (serovar B) and LGV-434 (serovar L2), respectively. Monoclonal antibodies (MAbs) were generated against the purified proteins, and their specificities were determined by immunoblotting. MAb B-14k recognized an epitope located on the 14-kDa cysteine-rich protein of the TW-5/OT strain and was immunoreactive with a comigrating 14-kDa protein that was common to all trachoma biovar strains, but it did not react with the 15-kDa, cysteine-rich protein of LGV biovar strains. In contrast, MAb L2-15k, which recognized an epitope located on the 15-kDa protein of the LGV-434 strain, reacted with the 15- and 14-kDa, cysteine-rich proteins of both LGV and trachoma biovar strains, but did not react with related proteins of two Chlamydia psittaci strains. Thus, the low-molecular-mass, cysteine-rich outer membrane proteins of C. trachomatis possess antigenic determinants that are both biovar and species specific. Neither MAbB-14k nor MAb L2-15k was reactive by dot-blot assay when viable chlamydiae were used as test antigens, indicating that the cysteine-rich proteins are not accessible to antibody on the native chlamydial cell surface.
Proteinase production by the parasitic cycle of the pathogenic fungus Coccidioides immitis
Authors: Resnick S , Pappagianis D , McKerrow J H
Published: January 2020

Coccidioides immitis is the causative agent of coccidioidomycosis (valley fever), a potentially disseminated fungal disease. We hypothesized that proteinases are expressed by the parasitic life cycle of C. immitis and that they might play an important role in the pathogenesis of coccidioidomycosis by facilitating spherule rupture, endospore dissemination, and tissue invasion and destruction. Filtrate from cultures of the parasitic life cycle of C. immitis was therefore assayed for proteolytic activity at neutral pH. The filtrate degraded 68% of a radiolabeled model of an elastin-rich extracellular matrix. The principal activity was against elastin and glycoprotein in the matrix. Degradation of purified elastin by filtrate was 222 micrograms/h per mg of filtrate protein at 37 degrees C. Denatured type I collagen (Azocoll) degradation was 13.5 mg/h per mg of filtrate protein at 37 degrees C. Proteinase activity peaked at 60 h of culture, correlating with release of endospores from mature spherules in the in vitro culture system. Elastase activity was attributed to a serine proteinase which exhibited an active-site preference for phenylalanine at the P1 site. The subunit molecular mass of the elastase determined by [3H]diisopropylfluorophosphate labeling was approximately 25 kilodaltons. Inhibition of the azocollytic activity of crude filtrate by 2 mM 1,10-phenanthroline and 10 mM EDTA, and stimulation by 2 mM CaCl2, suggested that a metalloproteinase was also present. Gelatin substrate gel electrophoresis with and without inhibitors confirmed that two proteinases were expressed, and they were separated by fast protein liquid chromatography.
Expression of a cloned lipopolysaccharide antigen from Neisseria gonorrhoeae on the surface of Escherichia coli K-12
Authors: Palermo D A , Evans T M , Clark V L
Published: January 2020

A gonococcal gene bank maintained in Escherichia coli K-12 was screened by colony immunoblotting, and a transformant expressing a surface antigen reactive to anti-gonococcal outer membrane antiserum was isolated. The isolate carried a recombinant plasmid, pTME6, consisting of approximately 9 kilobases of Neisseria gonorrhoeae DNA inserted into the BamHI site of pBR322. Surface labeling of E. coli HB101(pTME6) confirmed that the antigen was expressed on the E. coli cell surface. The antigenic material was resistant to proteinase K digestion and sensitive to periodate oxidation, indicating that the material was carbohydrate. Purified lipopolysaccharide (LPS) from HB101(pTME6) produced a unique band on silver-stained polyacrylamide gels that contained immunoreactive material as seen on Western blots of LPS samples. Only two of three E. coli LPS mutant strains carrying pTME6 reacted with the antigonococcal antiserum, suggesting that a certain E. coli core structure is necessary for antigen expression. We conclude that pTME6 contains one or more gonococcal genes encoding an LPS core biosynthetic enzyme(s) which can modify E. coli core LPS to produce a gonococcuslike epitope(s).
A species-specific DNA probe for the detection of Mycoplasma gallisepticum
Authors: Santha M , Burg K , Rasko I , Stipkovits L
Published: January 2020

An 800-base-pair DNA fragment from a partial genomic library of Mycoplasma gallisepticum was selected and used as a probe for the selective detection of this avian pathogen. The specificity and sensitivity of this probe were demonstrated by using dot blot and Southern hybridizations.
Most Mycobacterium leprae carbohydrate-reactive monoclonal antibodies are directed to lipoarabinomannan
Authors: Gaylord H , Brennan P J , Young D B , Buchanan T M
Published: January 2020

Each of more than 30 monoclonal antibodies that had been raised against Mycobacterium leprae and previously classified as reactive with carbohydrate was shown to be directed against lipoarabinomannan, a prominent, highly pervasive, myo-inositol-phosphate-containing, cross-reactive antigen within the leprosy bacillus. Some of the antibodies preferentially bound to the lipopolysaccharide of M. leprae rather than to that of Mycobacterium tuberculosis, suggesting the presence of distinguishing structural features. The presence of alkali-labile inositol 1-phosphate in the lipopolysaccharide from M. tuberculosis and its apparent absence from the M. leprae product may account for the difference.
Phenotypic variation of Staphylococcus epidermidis slime production in vitro and in vivo
Authors: Christensen G D , Baddour L M , Simpson W A
Published: January 2020

Clinical studies performed by us and others have found an association between slime production and strains of coagulase-negative staphylococci that infect indwelling medical devices. By serial low-speed centrifugation of broth cultures we have isolated a stable, weakly adherent strain (RP62A-NA) from a strongly adherent, slime-producing, pathogenic strain of Staphylococcus epidermidis sensu stricto (RP62A, ATCC 35984). We obtained a second strain from RP62A-NA (RP62A-NAR) by serial subculture of glass-adherent cells of RP62A-NA. All three strains had the same pattern of biochemical reactions, antimicrobial susceptibilities, and plasmid analysis. Transmission electron micrograph sections stained with the mucopolysaccharide-specific stain alcian blue demonstrated that the adherent strains RP62A and RP62A-NAR were covered with an extracellular coat of polysaccharide-rich material. In contrast, the nonadherent RP62A-NA strain lacked this external coat. All three strains were used in a mouse model of foreign body infection and a rat model of catheter-induced infective endocarditis. The adherence characteristics of isolates of RP62A and RP62A-NA recovered from experimental animals were relatively stable, although we noted a slight but a significant increase in the adherence of RP62A-NA isolates recovered from the foreign body model. The adherence characteristics of RP62A-NAR isolates recovered from infected animals were variable; in general these isolates were less adherent than the laboratory strain of RP62A-NAR. In both models the 50% infective dose (calculated by the Reed and Muench method) was three times greater for the RP62A-NA strain than for the RP62A strain. The phenotypic expression of slime production is subject to both in vitro and in vivo variation and could play a role in the pathogenesis of foreign body infection.
Cloning and expression in Escherichia coli of the gene encoding the heat-modifiable major outer membrane protein of Haemophilus influenzae type b
Authors: Gonzales F R , Leachman S , Norgard M V , Radolf J D , McCracken G H , Evans C , Hansen E J
Published: January 2020

One major outer membrane protein (P1) of Haemophilus influenzae type b (Hib), with an apparent molecular weight of 34,000 (34K) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), has been shown to be heat modifiable. After heating at 100 degrees C for 5 min in 2% SDS, the P1 protein exhibits an apparent molecular weight of 49,000 (49K) in SDS-PAGE. Monoclonal antibodies (MAbs) reactive with P1 bound to the surface of Hib, and one of these MAbs had a protective effect against the development of Hib bacteremia in an animal model for invasive Hib disease. A 6-kilobase Hib DNA insert containing the gene encoding this P1 protein was cloned into Escherichia coli by using the gamma gt11 expression vector. Recombinant phage expressing P1 were identified by screening phage plaques with a MAb directed against the P1 protein. Expression of the P1 protein by an E. coli lysogen carrying the recombinant phage was independent of both vegetative phage growth and induction of lacZ gene-directed transcription of the Hib DNA insert. The Hib DNA insert encoding the P1 protein was subcloned into the plasmid vector pBR322, and a transformant containing the recombinant plasmid pFRG100 was identified with the P1 protein-directed MAb in a colony blot-radioimmunoassay. Western blot (immunoblot) analysis determined that the recombinant P1 protein possessed heat-modifiability characteristics identical to those of the native Hib protein. The P1 protein was expressed on the surface of both the E. coli lysogen containing the recombinant phage and the E. coli transformant containing pFRG100. Western blot analysis of acute- and convalescent-phase sera from infants with Hib meningitis showed that antibodies in the convalescent-phase sera recognized the P1 protein expressed by the E. coli transformant containing pFRG100. The availability of this cloned Hib DNA insert encoding the Hib P1 protein and the expression of this protein on the surface of recombinant E. coli should facilitate the investigation of P1 for both its vaccinogenic potential and its functional role in the outer membrane of Hib.
In vivo emergence of enterotoxigenic Escherichia coli variants lacking genes for K99 fimbriae and heat-stable enterotoxin
Authors: Mainil J G , Sadowski P L , Tarsio M , Moon H W
Published: January 2020

Neonatal pigs were inoculated with porcine enterotoxigenic Escherichia coli 431, which carries genes for K99 fimbriae and STaP enterotoxin. Colonies of strain 431 were recovered from feces of pigs for up to 17 days after inoculation and tested for hybridization with gene probes for K99 and STaP. Variants of strain 431 that did not hybridize with the probes were considered to have lost the genes. Variants were recovered from 10 of 13 suckling pigs that survived the infection. Only 0.4% of the isolates recovered during the first 2 days after inoculation were variants. Of the isolates recovered 3 to 5 days after inoculation, 20 to 36% were variants. Variant colonies were detected more frequently among pigs in some litters than in others. The litter with the highest number of variant-shedding pigs had the dam with the highest titer of K99 antibody in her colostrum. Variants also occurred in colostrum-deprived, artificially reared pigs. However, the number of variants detected was lower and they occurred later in the course of the infection in colostrum-deprived pigs than in suckling pigs. More variants were detected and they were detected earlier in colostrum-deprived pigs fed anti-K99 monoclonal antibody than in controls fed anti-K88 monoclonal antibody. Loss of STaP appeared to be secondary to loss of K99 in that some variants lacked only K99 (K99- STaP+) and some lacked both genes (K99- STaP-), but none was of the K99+ STaP- type. Our results confirmed reports of gene loss from enterotoxigenic E. coli during infection. They are consistent with the hypothesis that variants emerge under in vivo selection pressure of K99 antibody and with the speculation that gene loss may be an important component of protection in vaccinated populations. However, the emergence of variants did not appear to play a major role in the recovery of individual pigs from clinical disease.
Role of a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic Escherichia coli O157:H7 in gnotobiotic piglets
Authors: Tzipori S , Karch H , Wachsmuth K I , Robins-Browne R M , O'Brien A D , Lior H , Cohen M L , Smithers J , Levine M M
Published: January 2020

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has two putative virulence factors: (i) a fimbrial adhesin, specified by a 60-megadalton (MDa) plasmid, and (ii) bacteriophage-specified cytotoxin(s), known as Shiga-like toxin (SLT) or verotoxin. The contribution of these factors to the pathogenesis of EHEC-induced disease in gnotobiotic piglets was examined. The bacterial strains included the following: two EHEC strains and their corresponding plasmid-cured derivatives; another EHEC isolate and its derivative which had spontaneously lost the ability to produce SLT; one E. coli K-12 transconjugatant containing a 60-MDa plasmid from an EHEC strain; two K-12 strains into which an SLT-producing phage had been transduced (one of these strains also carried a 60-MDa EHEC-derived plasmid); and the parent K-12 strain. Each strain was fed to four piglets, which were observed for diarrhea and examined for development of characteristic mucosal lesions 3 or 5 days after inoculation. All 24 piglets inoculated with the three EHEC strains and their respective derivatives (two plasmid cured and one SLT negative) showed the typical mucosal lesions of bacterial attachment: effacement of microvillous border and cell membrane dissolution culminating in destruction of surface and glandular epithelium in the cecum and colon. No such lesions were observed in 12 piglets inoculated with three strains of E. coli K-12, including the strain which carried both the 60-MDa plasmid and a phage which specified production of SLT. Moderate to severe diarrhea was observed in 16 piglets inoculated with two EHEC strains and their derivatives (one plasmid cured and one SLT negative). The third EHEC strain and its plasmid-cured derivative produced fewer typical mucosal lesions and no diarrhea. The reason for the reduced virulence of this strain was not clear. These results demonstrate that neither the 60-MDa plasmid nor the capacity to produce SLT is essential for expression of virulence by E. coli O157:H7 in gnotobiotic piglets.
Hemagglutinating activity of lipopolysaccharides from subgingival plaque bacteria
Authors: Okuda K , Kato T
Published: January 2020

Lipopolysaccharide (LPS) preparations from the Bacteroides species B. endodontalis, B. intermedius, B. denticola, and B. melaninogenicus and from Fusobacterium nucleatum, Haemophilus actinomycetemcomitans, Capnocytophaga gingivalis, and Eikenella corrodens directly agglutinated erythrocytes of some kinds of animals. LPSs from the Bacteroides species B. gingivalis, B. asaccharolyticus, B. corporis, and B. loescheii and from Capnocytophaga ochracea did not possess any hemagglutinating activity. Pretreatment of LPSs with lipase and phospholipase C completely eliminated the hemagglutinating activity. The hemagglutinating activity was also affected by human serum, saliva, colistin, and polymyxin B but was not affected by sugars, amino acids, EDTA, or proteolytic enzymes. The role of this hemagglutinating activity in colonization by these microorganisms of the periodontal region is discussed.
Phospholipase D activity of Vibrio damsela cytolysin and its interaction with sheep erythrocytes
Authors: Kreger A S , Bernheimer A W , Etkin L A , Daniel L W
Published: January 2020

Exposure of sheep erythrocytes to sublytic amounts of Vibrio damsela cytolysin markedly reduced their membrane sphingomyelin content and their sensitivity to lysis by the sphingomyelin-dependent cytolysins staphylococcal sphingomyelinase C (beta-toxin) and helianthin. The toxin was found to be a phospholipase D active against sphingomyelin.
Differentiation of F38 mycoplasmas causing contagious caprine pleuropneumonia with a growth-inhibiting monoclonal antibody
Authors: Rurangirwa F R , McGuire T C , Musoke A J , Kibor A
Published: January 2020

Monoclonal antibody WM-25 inhibited the in vitro growth of 13 F38 isolates from goats with contagious caprine pleuropneumonia but not 7 heterologous mycoplasma isolates representing four different species. In contrast to results with polyclonal antisera, growth inhibition by monoclonal antibody WM-25 was specific for F38 mycoplasma isolates and constituted a reliable means of distinguishing F38 from other mycoplasmas.
Ionic requirements for entry of Shiga toxin from Shigella dysenteriae 1 into cells
Authors: Sandvig K , Brown J E
Published: January 2020

The ionic requirements for entry of Shiga toxin into cells were examined by measuring inhibition of protein synthesis after short-term incubations with toxin. The sensitivity of Vero cells and HeLa cells to Shiga toxin was strongly dependent on the divalent cation present. Vero cells were most sensitive in the presence of CaCl2 and SrCl2, whereas HeLa cells were equally sensitive in the presence of MgCl2, SrCl2, and CaCl2. Both cell lines were protected by BaCl2, CoCl2, and MnCl2. Inhibitors of Ca2+ transport, like verapamil, D600, and Co2+ as well as the calcium-ionophores A23187 and ionomycin, protected both cell lines. HEp-2 cells were protected against Shiga toxin by a high concentration of potassium in the medium as well as by potassium depletion of the cells. Substitution of chloride in the medium with slowly permeable anions, like SO42- and SCN-, protected the cells against Shiga toxin. High concentrations of the ionophore nigericin that increase pH of acidic intracellular vesicles did not protect Vero cells against Shiga toxin. Shiga Toxin X-114 at pH values below 4.5. This binding was shifted to higher pH values after pretreatment of the toxin with dithiothreitol. The results indicate that Ca2+ transport through physiologically occurring Ca2+ channels is required for entry of Shiga toxin into cells. Furthermore, the sensitivity of cells of Shiga toxin is strongly dependent on the anions present.
Oxygen sensitivity of sugar metabolism and interconversion of pyruvate formate-lyase in intact cells of Streptococcus mutans and Streptococcus sanguis
Authors: Takahashi N , Abbe K , Takahashi-Abbe S , Yamada T
Published: January 2020

Pyruvate formate-lyase (PFL) (formate acetyltransferase; EC 2.3.1.54) of oral streptococci is essential for metabolizing sugar into volatile compounds (formate, acetate, and ethanol). This enzyme is extremely sensitive to oxygen, and its activity is irreversibly inactivated by oxygen. When Streptococcus sanguis was anaerobically starved, a part of the active form of PFL was converted into a reversible inactive form that was tolerant of oxygen. This reversible inactive enzyme could be reactivated to the active enzyme by anaerobic sugar metabolism, with the recovery of volatile compound production. The PFL in Streptococcus mutans was not converted into an oxygen-tolerant inactive form by anaerobic starvation, and after exposure of the cells to oxygen the PFL could not be reactivated. These findings suggest that S. mutans can produce acids rapidly under anaerobic conditions because of its capacity to keep PFL active and that S. sanguis can protect its sugar metabolism from oxygen impairment because of its interconversion of PFL.
Purification and biochemical properties of a bacteriocin from Actinobacillus actinomycetemcomitans
Authors: Stevens R H , Lillard S E , Hammond B F
Published: January 2020

Extracts of certain strains of Actinobacillus actinomycetemcomitans are inhibitory to strains of Streptococcus sanguis such as S. sanguis ATCC 10556. The isolation of a protein from an A. actinomycetemcomitans sonic extract which copurified with the inhibitory activity was accomplished by preparative isoelectric focusing, Sephadex G-100 gel filtration chromatography, and preparative polyacrylamide gel electrophoresis (PAGE). The resulting isolated protein, which focused at a pH of 6.1 to 6.3, appeared as a single band in anionic nondissociating PAGE analysis. This protein could be dissociated into two subunits with molecular weights of 50,000 and 70,000, which were resolvable by PAGE analysis. A 1,758-fold increase in specific activity was seen in the purified inhibitory protein compared with the crude sonic extract starting material. The properties of the inhibitory activity in the A. actinomycetemcomitans extract are characteristic of a bacteriocin. Accordingly, we propose the name actinobacillicin for the inhibitory protein.
M proteins of group G streptococci isolated from bacteremic human infections
Authors: Bisno A L , Craven D E , McCabe W R
Published: January 2020

We studied seven strains of group G streptococci isolated from clinically severe bacteremic infections in six intravenous drug abusers. These group G strains multiplied luxuriantly in fresh human blood. On electron microscopy, they exhibited surface fibrillae similar to those observed in M-protein-rich group A streptococci, but they were not serologically M typable with a battery of 39 M antisera. Rabbit antisera raised against two of the group G strains (1618 and 1750) opsonized the homologous but not the heterologous isolates and exhibited type-specific Ouchterlony immunoprecipitin reactions. Moreover, antisera raised against peptic extracts of strain 1750 also promoted phagocytic killing of that strain. Anti-1750 reacted in high titer in an enzyme-linked immunosorbent assay against peptic extracts of the homologous strain; these antibodies were removed by absorption with 1750 cells but not by absorption with heterologous strains. These studies represent, to our knowledge, the first analysis of virulence factors of group G streptococci isolated from invasive human disease. The seven epidemiologically related blood isolates of group G streptococci possess distinct type-specific, antiphagocytic surface virulence factors analogous to the M proteins of group A streptococci.
Mediation of Staphylococcus saprophyticus adherence to uroepithelial cells by lipoteichoic acid
Authors: Teti G , Chiofalo M S , Tomasello F , Fava C , Mastroeni P
Published: January 2020

Treatment of uroepithelial cells with lipoteichoic acid from Staphylococcus saprophyticus resulted in a decrease in the adherence of this organism. Similar effects were observed when bacteria were pretreated with the lipoteichoic acid ligands albumin and anti-polyglycerophosphate monoclonal antibodies. Lipoteichoic acid might behave as an adhesin of S. saprophyticus.
Expression and immunological properties of the five subunits of pertussis toxin
Authors: Nicosia A , Bartoloni A , Perugini M , Rappuoli R
Published: January 2020

Pertussis toxin, a protein composed of five different subunits, is responsible for the pathogenicity of Bordetella pertussis and is the main component of a new vaccine against whooping cough. The genes coding for the five subunits, recently cloned and sequenced, are organized as an operon. We approached the problem of expression of the five genes in Escherichia coli and, although we obtained high levels of transcription of the native pertussis toxin genes, the amount of proteins produced was very low or undetectable. To obtain suitable expression of each of the five subunits, we fused their genes to the gene coding for the DNA polymerase of MS2 in the expression vector pEx31. A total of 5 to 30 mg of purified fusion proteins could be obtained from 1 liter of culture. The purified fusion proteins were used to immunize rabbits to obtain sera against each of the five subunits. These sera, although able to recognize the toxin in an enzyme-linked immunosorbent assay and the corresponding subunits in Western blots, were not able to protect CHO cells from the action of pertussis toxin. Mice immunized with the five subunits were not protected from an intracerebral challenge with B. pertussis. Subunits S2 and S3, which are 67% homologous, were shown to cross-react immunologically. The fused subunit S1 was able to ADP-ribosylate transducin as efficiently as the native pertussis toxin.
Production and characterization of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa
Authors: Lam J S , MacDonald L A , Lam M Y , Duchesne L G , Southam G G
Published: January 2020

Monoclonal antibodies against 12 of the 17 IATS serotype strains of Pseudomonas aeruginosa were produced. Eighty-seven hybridoma clones were isolated, and the antibodies secreted were found to be reactive with both Formalin-fixed whole cells and purified lipopolysaccharide of homologous strains in enzyme-linked immunosorbent assays. Among these monoclonal antibodies, the predominant antibody class was immunoglobulin M (IgM) (76%), although antibodies of the IgG2a and IgG3 isotypes were also produced. The monoclonal antibodies could further be divided into two groups based on their ability to agglutinate whole cells of homologous strains. The agglutinating monoclonal antibodies were found to immunoblot with the O side chains of homologous lipopolysaccharide, while the nonagglutinating monoclonal antibodies were found to be reactive with outer membrane protein-associated lipopolysaccharide. The applicability of monoclonal antibodies for serotyping was examined, and several antibodies were found to agglutinate whole cells and immunoblot with the O antigen of corresponding serotypes of clinical isolates from cystic fibrosis patients. In conclusion, a set of monoclonal antibodies against the IATS serotype strains of P. aeruginosa have been produced. These antibodies represent a bank of invaluable immunological reagents which may have application in serotyping, epitope mapping, lipopolysaccharide structural determination, and studies of protection against P. aeruginosa.
Activation of botulinum C2 toxin by trypsin
Authors: Ohishi I
Published: January 2020

C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two dissimilar protein components, designated components I and II. The biological activity of C2T is enhanced by treating the toxin with trypsin. This activation of C2T is observed as a result of mixing untrypsinized component I and trypsinized component II but not as a result of mixing trypsinized component I and untrypsinized component II. The data presented here show that the maximum lethality of C2T, determined by mixing untrypsinized component I and trypsinized component II, was attained by treating component II with trypsin at a ratio of 10:1 on a protein basis for 30 min at 35 degrees C at pH 7.5. The activation of component II was always accompanied by a change in the molecular weight of the component from 101,000 to 88,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the gel filtration of trypsinized component II resulted in the separation of two active components, with apparent molecular weights, estimated from the elution volume by gel filtration, of 365,000 and 74,000. The high-molecular-weight component II had hemagglutination and hemolytic activities, whereas the low-molecular-weight component II has only hemagglutination activity. These two molecular species of active component II had approximately the same lethality, when mixed with component I, and gave a single band in SDS-PAGE, with a molecular weight of 88,000, the same as that of trypsin-activated component II under different reaction conditions. The results indicate that the activation of C2T by trypsin is due to the molecular conversion of component II from molecular weight 101,000 to 88,000 as determined by SDS-PAGE and that the trypsin-activated component II tends to form an oligomer of the active component II.
Adherence of Pseudomonas aeruginosa to cilia of human tracheal epithelial cells
Authors: Franklin A L , Todd T , Gurman G , Black D , Mankinen-Irvin P M , Irvin R T
Published: January 2020

Pseudomonas aeruginosa was found to adhere selectively to cilia of human ciliated tracheal epithelial cells (TECs). P. aeruginosa bound in equal numbers to TECs of smokers and nonsmokers, with the mean adhesion index for binding of P. aeruginosa 492c to TECs of healthy individuals (+/- standard deviation) being 6.83 +/- 6.00 bacteria per TEC.