bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis vir-regulated genes
Authors: Weiss A A , Melton A R , Walker K E , Andraos-Selim C , Meidl J J
Published: January 2020

Mutants of Bordetella pertussis deficient in virulence-associated factors were identified by using the transposon Tn5 lac. Tn5 lac is a derivative of Tn5 which generates promoter fusions for beta-galactosidase. Tn5 lac insertions in the vir-regulated genes of B. pertussis were identified by selecting for kanamycin-resistant mutants that expressed beta-galactosidase when the vir-regulated genes were expressed but not when the vir-regulated genes were turned off. Fourteen different mutations in vir-regulated genes were identified. Two mutants were deficient in the production of the filamentous hemagglutinin, two mutants were deficient in the production of adenylate cyclase toxin and hemolysin, and one mutant was deficient in the production of dermonecrotic toxin. One insertion mapped adjacent to the pertussis toxin gene, but the mutant produced pertussis toxin. The phenotypes of the remaining eight mutants were not determined, but the mutants did not appear to be deficient in the production of the 69,000-dalton outer membrane protein (agglutinogen 3) or the capsule. Screening for mutations in either of the fimbrial genes proved to be problematic since the parental strain was found to switch from a fimbriated to a nonfimbriated state at a high frequency, which was suggestive of the metastable expression of pili in other bacteria. We used Southern blot analysis with a 30-mer specific for the fimbrial sequences. No bands with the predicted increase in size due to the 12 kilobases from Tn5 lac were observed, which suggests that none of these genes were mutated. Southern blot analysis also revealed that seven of the eight unidentified mutations mapped to different restriction fragments, which suggests that they could be deficient in as many as seven different genes.
Cross-reactivity of Pseudomonas aeruginosa antipilin monoclonal antibodies with heterogeneous strains of P. aeruginosa and Pseudomonas cepacia
Authors: Saiman L , Sadoff J , Prince A
Published: January 2020

Much of the morbidity and mortality in patients with cystic fibrosis (CF) is secondary to pulmonary infections with Pseudomonas aeruginosa and, more recently, with Pseudomonas cepacia. Prevention of colonization and subsequent infection would be a useful therapeutic strategy. The pili (fimbriae) of P. aeruginosa are a potential vaccine antigen, as they have been implicated in binding to respiratory epithelium and appear to have limited antigenic diversity. Monoclonal antibodies (MAbs) raised to P. aeruginosa pilin demonstrated significant cross-reactivity, as four of five P. aeruginosa strains with known pilin sequences and 10 of 15 P. aeruginosa clinical isolates hybridized by immunoblot with at least one of the three MAbs tested. The P. cepacia strains demonstrated minimal cross-reactivity with these MAbs, as only 2 of 16 strains hybridized immunologically. The three MAbs decreased the adherence of 35S-labeled P. aeruginosa PA1244 to bovine tracheal cells by 56, 45, and 31%. One of these MAbs decreased the adherence of strains P. aeruginosa PAO1 and P. cepacia 249 to CF epithelial cells by 46 and 25%, respectively. While antibodies to Pseudomonas pili must be shown to be protective in patients with CF, these studies give support for a multivalent vaccine strategy using P. aeruginosa pilin as the immunogen.
Definition of Brucella A and M epitopes by monoclonal typing reagents and synthetic oligosaccharides
Authors: Bundle D R , Cherwonogrodzky J W , Gidney M A , Meikle P J , Perry M B , Peters T
Published: January 2020

The paradigm that Brucella A and M epitopes are simultaneously expressed on single cells and within one antigen molecule was reinvestigated by using polysaccharide-specific murine monoclonal antibodies. Monoclonal antibodies were generated to the M antigen of Brucella melitensis 16M. Chemically defined lipopolysaccharides and O polysaccharides from Brucella abortus 1119-3, B. melitensis 16M, and Yersinia enterocolitica O:9 were used to dissect the binding profiles of the B. melitensis antibodies and an additional set of antibodies available from a B. abortus fusion experiment. Binding specificities were rationalized in terms of prototype A- and M-antigen structures, an interpretation supported by competitive binding studies with O polysaccharides and synthetic oligosaccharide analogs of the A and M antigens. Three binding patterns were characterized. Antibodies specific for the A antigen required five contiguous alpha 1,2-linked 4,6-dideoxy-4-formamido-D-mannopyranosyl residues, while antibodies with equal affinities for A or M epitopes were effectively inhibited by alpha 1,2-linked tri- or tetrasaccharides. Specificity for the M epitope correlated with binding of a critical disaccharide element alpha-D-Rha4NFo(1----3)alpha-D-Rha4NFo bracketed by alpha 1,2-linked residues. The binding profiles of Brucella monoclonal antibodies were consistent with the concept of simultaneous expression of A and M epitopes within a single molecule. A epitopes were present in the M antigen, and the discovery of isolated alpha 1,3 linkages in the A antigen suggests that M epitopes occur in all A antigens. Three monoclonal antibodies are proposed as standard reagents for the detection and identification of Brucella A and M antigens.
Salmonella typhimurium virulence in a burned-mouse model
Authors: Carsiotis M , Stocker B A , Holder I A
Published: January 2020

Various features of salmonellosis were examined in a burned-mouse model. In this model, which uses an outbred mouse strain, a challenge dose of ca. 100 CFU with any of several strains of Salmonella typhimurium caused a fatal infection. A variety of mutated strains attenuated for virulence in Salmonella-susceptible parenterally infected mice were also attenuated in the burned-mouse model. When administered as live vaccines injected intraperitoneally the same attenuated strains provided between slight and complete protection against subsequent lethal challenge subcutaneously at the site of a burn. The correspondence of results obtained in the burned-mouse model with those seen in other mouse models coupled with the unique advantages of the burned-mouse model argue for the usefulness of the model in studies of salmonellosis and in testing of strains constructed for use as live vaccines.
Identification and characterization of a serotype b-specific segment of the Haemophilus influenzae genome
Authors: Ely S , Tippett J , Moxon E R
Published: January 2020

Within the region of the Haemophilus influenzae serotype b genome required for capsule expression, a 4.6-kilobase segment was cloned, used as a hybridization probe, and found to be specific for serotype b strains. Deletion mutagenesis within this segment defined a locus necessary for serotype b capsule production and another involved in maintenance of wild-type colony appearance.
Immunosuppressive properties of the Mycoplasma arthritidis T-cell mitogen in vivo: inhibition of proliferative responses to T-cell mitogens
Authors: Cole B C , Wells D J
Published: January 2020

We have previously shown that Mycoplasma arthritidis produces a soluble T-cell mitogen (MAM) which is active for most mouse strains that express the alpha chain of the I-E molecule (E alpha) encoded within the murine major histocompatibility complex. The lymphocytes from mice injected intravenously with the MAM exhibited a marked decrease in their ability to respond in vitro to MAM, to phytohemagglutinin, or to concanavalin A T-cell mitogens. Suppression could only be induced in MAM-responsive mouse strains and was most marked 1 to 4 days postinjection. Splenic and node cells and, to a lesser extent, thymic cells from MAM-injected mice could inhibit the ability of lymphocytes from normal mice to respond to MAM and lectin mitogens. A minimum of 2.5 x 10(4) viable cells was required for significant transfer of suppression, and no major histocompatibility complex restrictions were seen. Unlike concanavalin A-induced suppressor cells, which consist of a CD4-, CD8+ T-cell subset, suppressor cells induced by MAM were due to a CD4+ CD8- subset. We hypothesize that MAM may play a role in M. arthritidis-mediated disease by both its inflammatory and immunosuppressive properties.
Evidence for three separate genes encoding the proteins of the mycobacterial antigen 85 complex
Authors: Wiker H G , Sletten K , Nagai S , Harboe M
Published: January 2020

The secreted Mycobacterium bovis BCG antigen 85 complex, which is known to bind to human fibronectin, consists of three closely related cross-reacting antigens. Amino-terminal sequence analysis of the purified proteins showed distinct differences. Data are presented to show that the three components are produced by individual cells, which indicates that three separate genes are involved.
Genomic variation of Trypanosoma cruzi: involvement of multicopy genes
Authors: Wagner W , So M
Published: January 2020

By using improved pulsed field gel conditions, the karyotypes of several strains of the protozoan parasite Trypanosoma cruzi were analyzed and compared with those of Leishmania major and two other members of the genus Trypanosoma. There was no difference in chromosome migration patterns between different life cycle stages of the T. cruzi strains analyzed. However, the sizes and numbers of chromosomal bands varied considerably among T. cruzi strains. This karyotype variation among T. cruzi strains was analyzed further at the chromosomal level by using multicopy genes as probes in Southern hybridizations. The chromosomal location of the genes encoding alpha- and beta-tubulin, ubiquitin, rRNA, spliced leader RNA, and an 85-kilodalton protein remained stable during developmental conversion of the parasite. The sizes and numbers of chromosomes containing these sequences varied among the different strains analyzed, implying multiple rearrangements of these genes during evolution of the parasites. During continuous in vitro cultivation of T. cruzi Y, the chromosomal location of the spliced leader gene shifted spontaneously. The spliced leader gene encodes a 35-nucleotide RNA that is spliced in trans from a 105-nucleotide donor RNA onto all mRNAs in T. cruzi. The spliced leader sequences changed in their physical location in both the cloned and uncloned Y strains. Associated with the complex changes was an increase in the infectivity of the rearranged variant for tissue culture cells. Our results indicate that the spliced leader gene clusters in T. cruzi undergo high-frequency genomic rearrangements.
Role of granulocytes in increased host resistance to Candida albicans induced by recombinant interleukin-1
Authors: Kullberg B J , van 't Wout J W , van Furth R
Published: January 2020

The effect of human recombinant interleukin-1 alpha (IL-1 alpha) on a systemic candidal infection in mice under various conditions of immunosuppression was investigated. In normal mice and in mice pretreated with cyclophosphamide, hydrocortisone acetate, or sublethal total body irradiation, the outgrowth of Candida albicans in the kidney was significantly reduced by the administration of a single intraperitoneal dose of 80 ng of IL-1 (P less than 0.05). In mice treated with either cyclophosphamide or irradiation, IL-1 also significantly reduced the outgrowth of C. albicans in the spleen. The protective effect of IL-1 was present when given 24 h before injection of C. albicans but also when IL-1 was given simultaneously with or 6 h after injection of C. albicans in cyclophosphamide-treated mice. The effect of IL-1 was independent of the presence or recruitment of granulocytes, since IL-1 inhibited the outgrowth of C. albicans in the kidneys and spleens of mice that were rendered severely granulocytopenic (less than 50 granulocytes per mm3) throughout the duration of the infection by either repeated injections of cyclophosphamide or sublethal total body irradiation. These results indicate that the enhancement of host resistance by IL-1 is not due solely to increased granulocytopoiesis or chemotaxis of granulocytes but strongly suggest that other mechanisms play a role in the protective effect of IL-1 against systemic infections.
Protective activities in mice of monoclonal antibodies against pertussis toxin
Authors: Sato H , Sato Y
Published: January 2020

Pertussis toxin (PT) protein, which is the most important protective antigen of Bordetella pertussis, has a hexameric structure composed of five subunits, designated S1 through S5. Immunoprotective activity of 20 different mouse monoclonal antibodies (MAbs) against pertussis toxin, 10 anti-S1, 1 anti-S2, 2 anti-S3, 4 anti-S23, and 3 anti-S4 antibodies, were investigated by aerosol and intracerebral challenges with virulent B. pertussis organisms in mice. Four anti-S1, named 1B7, 1D7, 3F11, and 10D6, and three anti-S23 antibodies, named 11E6, 10B5, and 10C9, showed the highest, and almost complete, protectivity against the aerosol challenge. Mouse protectivity against the intracerebral challenge was significant for these four anti-S1 MAbs but not for any of the three anti-S23 MAbs. Four anti-S1 and two anti-S4 MAbs did not protect the mice against either challenge. The other seven MAbs also showed dose-dependent moderate but significant protection against the aerosol challenge. In the aerosol challenge system, bacterial numbers and amounts of PT detected in the lung and the number of peripheral leukocytes were lower in the mice given the protective MAbs. All mice surviving 5 weeks after the infection produced high titers of antibodies against PT, filamentous hemagglutinin (FHA), and agglutinogens from the challenge organisms. A combination of the protective MAbs 1B7 and 11E6 strongly suppressed the disease and mortality of the mice at smaller amounts than with the anti-PT polyclonal antibody. Although combinations of one of the protective MAb and anti-FHA or anti-agglutinogen 2 also showed extremely high mouse protection without development of symptoms of the disease, antibody titers of the survivors against PT, FHA, and agglutinogens were significantly low. The foregoing results suggest that some important protective epitopes should be in S1 and S2 and/or S3, although there are both differences and similarities in the protective roles between anti-S1 and anti-S23 antibodies and also in the pathogenic mechanisms between aerosol and intracerebral infections. Furthermore, it was suggested that although not only FHA and agglutinogen 2 but also PT have roles as attachment factors, the processes of infection and protection are different between mice immunized with antibody against FHA or agglutinogen 2 and that against PT because the latter mice are also able to neutralize toxicity of PT diffused into the mice.
Genetically engineered S and F1C fimbriae differ in their contribution to adherence of Escherichia coli to cultured renal tubular cells
Authors: Marre R , Kreft B , Hacker J
Published: January 2020

Escherichia coli K-12 strains producing S-fimbrial adhesins, F1C fimbriae, and mutagenized fimbriae were tested in a binding assay with a renal tubular cell line. S-fimbrial adhesins and F1C fimbriae mediated binding to tubular cells. The SfaA, SfaG, and SfaS subunits of S fimbriae contributed to attachment. Site-specific mutations in the sfaS gene reduced binding. The inhibition profile of F1C fimbriae resembled that of S fimbriae.
Construction of a bifunctional Escherichia coli heat-stable enterotoxin (STb)-alkaline phosphatase fusion protein
Authors: Urban R G , Dreyfus L A , Whipp S C
Published: January 2020

A fusion between the genes encoding the Escherichia coli STb heat-stable enterotoxin (estB) and alkaline phosphatase (phoA) was constructed, and the expressed protein product was characterized. The STb-alkaline phosphatase protein (STb-PhoA) had an apparent molecular mass of 50,000 daltons and was detected with both monoclonal anti-alkaline phosphatase and polyclonal anti-STb antibodies. Expression of the gene fusion resulted in high-level production of alkaline phosphatase activity, indicating that STb-PhoA was processed and exported into the periplasm of the E. coli host strain. Amino acid sequence analysis of the hybrid protein yielded the sequence Ser-Thr-Gln-Ser-Asn-Lys-Lys, indicating that STb-PhoA was processed during export in a fashion identical to that of native STb (Y. M. Kupersztoch, K. Tachias, C. R. Moomaw, L. A. Dreyfus, R. G. Urban, C. Slaughter, and S. Whipp, J. Bacteriol. 172: 2427-2432, 1990). STb-PhoA was purified from an expressed bacterial lysate by preparative isoelectric focusing. In a rat ligated intestinal loop model, purified STb-PhoA induced highly significant (P less than 0.002) fluid secretion. In addition, the specific activity of STb-PhoA was nearly identical to that of purified STb. Thus, the STb-PhoA hybrid protein represents a readily obtainable source of biologically active (STb) enterotoxin that may prove useful in studies to determine the mode of toxin action.
Resistance of Congenitally Immunodeficient Gnotobiotic Mice to Vaginal Candidiasis
Authors: Cantorna Margherita , Mook Dianna , Balish Edward
Published: January 2020

Congenitally immunodeficient beige, athymic, and beige athymic mice whose orogastric mucosal tissues were chronically colonized and infected with a pure culture of
Candida albicans
were found to be resistant to naturally occurring vulvovaginal candidiasis.
Metalloproteases of infective Ancylostoma hookworm larvae and their possible functions in tissue invasion and ecdysis
Authors: Hotez P , Haggerty J , Hawdon J , Milstone L , Gamble H R , Schad G , Richards F
Published: January 2020

To infect their hosts, hookworm larvae must exsheath and migrate through connective tissue. A modified in vitro skin chamber was used to show that the human hookworm Ancylostoma duodenale and the zoonotic canine hookworm Ancylostoma caninum penetrate epidermis, basement membrane, and dermis in similar ways. These similarities in tissue invasion properties reflect the observed biochemical similarities in parasite protease composition. The larvae of both species contain protease activity that is inhibited by o-phenanthroline; this identifies the proteases as metalloproteases. The enzyme activities exhibit an alkaline pH optimum between pH 9 and 10. During modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which a protein substrate (either casein or gelatin) was used, the protease activities resolved into a major band at an Mr of 68,000 and a minor band at an Mr of 38,000. Proteases were released by living A. caninum larvae in vitro and degraded purified and radiolabeled casein to smaller peptides. Motile hookworm larvae were also incubated with purified and radiolabeled connective tissue macromolecules in vitro. Both Ancylostoma species degraded human fibronectin to a 60,000-Mr polypeptide intermediate, but could not degrade solubilized bovine elastin or human laminin. In contrast, the obligate skin-penetrating nematode Strongyloides stercoralis degraded all three substrates. This biochemical difference may explain some observed differences in invasiveness.
Experimental infection of pig-tailed macaques (Macaca nemestrina) with Campylobacter cinaedi and Campylobacter fennelliae
Authors: Flores B M , Fennell C L , Kuller L , Bronsdon M A , Morton W R , Stamm W E
Published: January 2020

Campylobacter cinaedi and C. fennelliae have been associated with proctocolitis, bacteremia, and asymptomatic rectal infection, primarily in homosexual men. To more directly assess the pathogenic role of these organisms, we studied their disease-producing potential in 12- to 25-day-old pig-tailed macaques (Macaca nemestrina). Four infant monkeys were challenged with 10(8) to 10(9) C. cinaedi, three were challenged with C. fennelliae, two were challenged with C. jejuni, and one received no microorganisms. Watery or loose stools without associated fever or fecal leukocytes developed 3 to 7 days postinoculation in all of the animals given C. cinaedi, C. fennelliae, and C. jejuni, but not in the control animal. Stool cultures were simultaneously positive and remained so in the animals challenged with C. cinaedi or C. fennelliae for 3 weeks after inoculation despite the resolution of clinical illness. All of the animals challenged with C. cinaedi and C. fennelliae became bacteremic, and three had clinical evidence of septicemia. Histopathologic evaluation of rectal biopsies (five animals) and necropsy (one animal) showed no evidence of mucosal disruption. Specific immunoglobulin M and immunoglobulin G antibody responses occurred in all of the animals challenged with C. cinaedi and C. fennelliae, as determined by enzyme-linked immunosorbent assay and immunoblotting. We conclude that C. cinaedi and C. fennelliae consistently produce a diarrheal illness accompanied by bacteremia and followed by prolonged gastrointestinal colonization in M. nemestrina.
Iron-regulated outer membrane protein of Bacteroides fragilis involved in heme uptake
Authors: Otto B R , Sparrius M , Verweij-van Vught A M , MacLaren D M
Published: January 2020

Under iron starvation, Bacteroides fragilis expresses various iron-regulated outer membrane proteins. In this study, a deferrated minimal medium was used in growth experiments, and the role of one of these iron-regulated outer membrane proteins (a 44-kDa protein) in an iron uptake mechanism which acquires iron from heme compounds was elucidated. When a specific 44-kDa protein antiserum was used in a medium with heme as the only iron source, growth inhibition was observed. These results demonstrate that the 44-kDa outer membrane protein plays an important role in the uptake of heme in B. fragilis.
Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin type II
Authors: Wadolkowski E A , Sung L M , Burris J A , Samuel J E , O'Brien A D
Published: January 2020

Escherichia coli O157:H7 strains have been implicated as etiologic agents in food-borne outbreaks of hemorrhagic colitis and the hemolytic-uremic syndrome. A prototype E. coli O157:H7 strain, designated 933, produces Shiga-like toxin I (SLT-I) and SLT-II and harbors a 60-MDa plasmid. In a previous study, streptomycin-treated mice were fed 933 together with a derivative cured of the 60-MDa plasmid (designated 933cu). Strain 933cu colonized poorly, but in approximately one-third of the animals, an isolate of 933cu was obtained from the feces that had regained the ability to colonize well. This isolate, designated 933cu-rev, killed all of the animals when fed alone to mice. In this investigation, two types of experiments were done to assess whether SLT-I, SLT-II, or both contributed to the death of mice fed 933cu-rev. (i) Mice were pretreated with monoclonal antibodies to SLT-I, SLT-II, SLT-I and SLT-II, or cholera toxin (as a control) before infection with 933cu-rev. (ii) Mice were fed either an E. coli K-12 strain carrying cloned SLT-I genes or the same K-12 strain carrying cloned SLT-II genes. The results of both types of experiments indicated that the deaths of the orally infected mice were due solely to SLT-II. Extensive histological and selected electron microscopic examinations of various tissues from the infected animals suggested that death was due to acute renal cortical tubular necrosis consistent with toxic renal damage. These data indicate a critical role for SLT-II, but not SLT-I, in renal damage associated with E. coli O157:H7 infection of streptomycin-treated mice.
Genetic characterization and virulence of enterotoxigenic Escherichia coli mutants which have lost virulence genes in vivo
Authors: Casey T A , Moon H W
Published: January 2020

Loss of K99 and STaP genes from enterotoxigenic Escherichia coli 431 during infection occurred by either plasmid curing or plasmid deletion. These mutants expressed the F41 adhesion and colonized neonatal pigs, but only those mutants that retained STaP caused diarrhea with significant weight loss.
Identification of a 68-kilodalton outer membrane protein as the major protective antigen of Bordetella bronchiseptica by using specific-pathogen-free piglets
Authors: Kobisch M , Novotny P
Published: January 2020

Maternal antibody to an outer membrane 68-kilodalton (kDa) protein of Bordetella bronchiseptica was shown to be protective in experiments on specific-pathogen-free piglets. After challenge with B. bronchiseptica, 100% (n = 19) control piglets from nonimmunized sows developed pneumonia, coughing, and sneezing, and 74% of the animals developed severe atrophic rhinitis. In 12 piglets from a sow immunized with 68-kDa protein, pneumonia occurred only in 34% of offspring, coughing was reduced, the duration of coughing bouts was shortened, and severe atrophic rhinitis occurred in one animal only (8%). The difference in the occurrence of atrophic rhinitis and of pneumonia in immunized and nonimmunized offspring was statistically significant (P less than 0.05). Sera of protected piglets had high titers (enzyme-linked immunosorbent assay) of antibodies that showed a high specificity for the 68-kDa protein isolated from B. bronchiseptica, whereas their reactivity with an analogous 69-kDa protein isolated from Bordetella pertussis was low or absent. The 68-kDa protein of B. bronchiseptica appeared to be the major protective antigen in B. bronchiseptica infection; however, isolated protein alone did not induce such a solid protection, as observed in a previous study after the application of an effective whole cell vaccine.
Invasion and replication of Salmonella typhimurium in animal cells
Authors: Gahring L C , Heffron F , Finlay B B , Falkow S
Published: January 2020

A total of 81 avirulent Tn10 insertion mutants of Salmonella typhimurium have previously been described. These mutants were selected for the inability to survive in murine macrophages. We have characterized the abilities of the most avirulent of these mutants to adhere to, invade, and replicate in both macrophages and nonphagocytic epithelial cells. The results suggest that most mutants contain a defect that is specific to survival within professional phagocytes. These mutants invaded and replicated normally within nonphagocytic human colon adenocarcinoma cells (Caco-2) but did not survive in the macrophage cell line J774. One mutant invaded both macrophages and epithelial cells much less efficiently than the parental strain. The defect associated with this mutant appears to be a result of decreased adherence to animal cells.
Macrophages are stimulated by muramyl dipeptide to induce polymorphonuclear leukocyte accumulation in the peritoneal cavities of guinea pigs
Authors: Nagao S , Nakanishi M , Kutsukake H , Yagawa K , Kusumoto S , Shiba T , Tanaka A , Kotani S
Published: January 2020

N-Acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]) injected intraperitoneally significantly increased the number of cells entering the peritoneal cavity of guinea pigs primed with liquid paraffin or thioglycollate. There was a close relationship between peritoneal polymorphonuclear leukocyte (PMN) accumulation and the uptake of glucosamine by macrophages in guinea pigs treated with a variety of bacterial cell surface components such as cell wall peptidoglycan subunits and bacterial or synthetic lipid A. The PMN accumulation was also facilitated by the intraperitoneal transfer of the peritoneal macrophages that had been stimulated by MDP in vitro. Furthermore, cell-free lavage fluids taken from the peritoneum of MDP-treated guinea pigs also initiated the influx of PMNs when introduced into the peritoneal cavities of liquid paraffin-pretreated guinea pigs. These results suggest that a soluble factor which attracts neutrophils is produced by MDP-treated macrophages. Partial characterization of the factor is described.
Variation in expression of class 1 and class 5 outer membrane proteins during nasopharyngeal carriage of Neisseria meningitidis
Authors: Woods J P , Cannon J G
Published: January 2020

Accidental nasopharyngeal colonization of a laboratory worker by a human disease isolate of Neisseria meningitidis allowed us to examine the variation in expression of outer membrane components and the host humoral response over time. There were quantitative differences in class 1 outer membrane protein expression in nasopharyngeal isolates obtained at different times. Isolates also showed phase variation of all four class 5 outer membrane proteins produced by the colonizing strain. After colonization, we detected new host serum immunoglobulin G antibodies directed against class 1, class 5, and H.8 outer membrane proteins.
A recombinant Rickettsia conorii vaccine protects guinea pigs from experimental boutonneuse fever and Rocky Mountain spotted fever
Authors: Vishwanath S , McDonald G A , Watkins N G
Published: January 2020

There are no vaccines against boutonneuse fever and Rocky Mountain spotted fever. Previous studies have identified a Rickettsia rickettsii surface protein as a vaccine candidate and shown that an antigenically related protein is present in R. conorii, which causes boutonneuse fever. The gene encoding the R. rickettsii protein has been cloned and expressed in Escherichia coli. We confirmed by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rickettsial lysates followed by immunoblotting with a monoclonal antibody raised against the R. rickettsii protein that an analogous protein exists in R. conorii. Although these proteins were previously called 155-kilodalton (kDa) proteins, we found that their apparent molecular masses were 198 kDa for R. conorii Kenya tick typhus and 190 kDa for R. rickettsii R. Using the R. rickettsii gene probe, we cloned and expressed a 5.5-kilobase HindIII fragment from R. conorii Kenya tick typhus genomic DNA in E. coli JM107. The expressed recombinant product was recognized by a monospecific polyclonal rabbit antiserum prepared against the 198-kDa protein. Guinea pigs immunized with sonic lysates of the E. coli strain expressing the recombinant gene product developed antibodies recognizing R. conorii when tested by a microimmunofluorescence antibody assay. Upon immunoblotting of rickettsial lysates, those antisera specifically recognized the 198-kDa R. conorii protein and its 190-kDa analog in R. rickettsii. Guinea pigs immunized with sonic lysates of the recombinant E. coli expressing the 198-kDa protein were protected from experimental infections with the homologous R. conorii strain and partially protected from experimental infections with a strain of the heterologous species R. rickettsii. These findings show that the 198-kDa R. conorii protein is a candidate for a vaccine against boutonneuse fever.
Characterization of monoclonal antibodies against the Escherichia coli hemolysin
Authors: Pellett S , Boehm D F , Snyder I S , Rowe G , Welch R A
Published: January 2020

Twelve monoclonal antibodies (MAbs) produced against the Escherichia coli hemolysin (HlyA) encoded by the hemolysin recombinant plasmid pWAM04 were studied. HlyA derivatives from recombinant strains with different plasmids encoding HlyA amino-terminal and carboxy-terminal truncates, HlyA in-frame deletions, and HlyA frameshift mutations were used in immunoblots to localize the antigenic determinants for the anti-HlyA MAbs. The mapping of the MAb epitopes was also facilitated by immunoblotting analysis of HlyA polypeptide fragments derived by cyanogen bromide cleavage. The HlyA epitopes for 11 of the MAbs were mapped to relatively small linear regions of the cytolysin ranging from 28 to 160 amino acids. Five of the MAbs (C10, G8, E2, B7, and D12) neutralized HlyA hemolytic activity to varying degrees. The epitopes for these neutralizing MAbs were found to reside within the following HlyA regions: C10 and G8, amino acids 2 to 160; E2, amino acids 161 to 194; B7, amino acids 518 to 598; and D12, amino acids 626 to 726. Hemolytically active HlyA was dependent on the action of the hlyC gene product. The D12 MAb recognized only HlyA produced by strains with an intact hlyC function. MAb A10 recognized an epitope within the HlyA region from amino acids 728 to 829 where a glycine-rich repeat domain exists; however, this MAb did not neutralize HlyA hemolytic activity. A HlyA domain map showing the anti-HlyA epitope location was constructed.
Nonreciprocal complementation of the hlyC and lktC genes of the Escherichia coli hemolysin and Pasteurella haemolytica leukotoxin determinants
Authors: Forestier C , Welch R A
Published: January 2020

The genetic organization of the Pasteurella haemolytica leukotoxin operon (lktCABD) is similar to that of the Escherichia coli hemolysin (hlyCABD). Their gene products share a sequence similarity of 66, 62, 90.5, and 75.6%, respectively. We investigated the role of the C proteins (LktC and HlyC) by performing reciprocal transcomplementation analyses in an E. coli recombinant background. In the absence of the C genes, neither LktA nor HlyA had their respective cytotoxic activities. When hlyC was provided in trans to lktA, the toxin that was produced had the same activity and target cell specificity as the wild-type leukotoxin; it was leukotoxic for bovine lymphoid cells but not human lymphoblast cells when it was evaluated by a 51Cr-release assay. We also detected a weak hemolytic activity for the active form of LktA against sheep erythrocytes. In contrast, an E. coli strain containing lktC with hlyA produced a form of HlyA which was neither hemolytic nor cytotoxic. A monoclonal antibody (D12) against HlyA which recognized an epitope specific to the active form of HlyA did not cross-react in immunoblots with LktA that was activated by either LktC or HlyC. We conclude that the mechanism for activation of leukotoxin and hemolysin by their respective C proteins (LktC and HlyC) is mechanistically similar but that the exact structural requirements involved in the process are different.
Protein and antigenic heterogeneity among isolates of Bacillus piliformis
Authors: Riley L K , Besch-Williford C , Waggie K S
Published: January 2020

Protein and antigenic heterogeneity among isolates of Bacillus piliformis, the etiologic agent of Tyzzer's disease, were investigated. The seven isolates utilized in this study were originally isolated from naturally infected animals of different animal species and diverse geographical locations. Isolates were propagated in mammalian cell lines, and bacterial extracts were prepared. Protein and antigenic profiles were compared among isolates, using Coomassie blue-stained polyacrylamide gels and Western blot (immunoblot) analyses, respectively. Results showed differences in protein and antigen banding patterns, indicating diversity among isolates. Western blots probed with serum preabsorbed with a heterologous bacterial extract revealed that numerous antigens have different electrophoretic mobilities among isolates but apparently share common epitopes. Immunodominant cross-reactive antigens may be candidate proteins useful for development of improved serologic diagnostic tests, allowing identification of animals infected with a wide range of B. piliformis isolates.
Identification of plasmid-encoded mannose-resistant hemagglutinin and HEp-2 and HeLa cell adherence factors of two diarrheagenic Escherichia coli strains belonging to an enteropathogenic serogroup
Authors: Pal R , Ghose A C
Published: January 2020

Two Escherichia coli strains (B/M 369 and C-35) belonging to enteropathogenic serogroup O86 were isolated from patients with infantile diarrhea and studied with respect to their cellular adherence properties. Both strains exhibited adherence (Ad+) to HEp-2 and HeLa cell monolayers in vitro and expressed mannose-resistant hemagglutinating (MRHA+) activity towards human, chicken, and sheep (but not mouse, rabbit, or guinea pig) erythrocytes. Cellular adherence properties of both strains could be substantially reduced by pronase treatment and by heat treatment (100 degrees C for 5 min) of bacteria. Electron microscopic examination failed to reveal fimbria- or pilus-like structures on the bacterial cell surface. Conjugation experiments conducted with these strains suggested that both MRHA and HEp-2 and HeLa cell adherence factors were encoded by the same plasmid, with a size of 55 to 57 megadaltons (MDa). Further biochemical studies indicated that the cellular adherence factors were associated with cell surface structures of bacteria that were proteinaceous in nature. An antiserum, rendered specific for the 57-MDa plasmid (pRP201) products of B/M 369 by adsorption, reacted with both MRHA+ Ad+ strains, B/M 369 and C-35, but not with their 57- or 55-MDa plasmidless MRHA- Ad- transconjugants or with other MRHA- Ad- E. coli strains. Immunological studies showed that the absorbed antiserum recognized two proteins with subunit molecular sizes of 18 and 14.5 kDa that were present on the cell surfaces of both strains. Furthermore, the absorbed antiserum at subagglutinating dilutions did inhibit, although only partially, the MRHA and HEp-2 and HeLa cell adherence activities of both E. coli strains. All these results would indicate that some of the E. coli strains belonging to enteropathogenic serogroups express their adherence potential through factors that were hitherto unrecognized.
Cloning and expression in Escherichia coli of LKP pilus genes from a nontypeable Haemophilus influenzae strain
Authors: Kar S , To S C , Brinton C C
Published: January 2020

Nontypeable Haemophilus influenzae HF0295, isolated by aspiration from the middle ear of a patient with otitis media, expresses long, thick, and hemagglutinating pili of a single serotype (LKP1) on its surface. An intact pilus vaccine consisting of the LKP1 serotype protected chinchillas against experimental otitis media (C. C. Brinton, Jr., M. J. Carter, D. B. Derber, S. Kar, J. A. Kramarik, A. C. C. To, S. C. M. To, and S. W. Wood, Pediatr. Infect. Dis. J. 8:554-561, 1989; R. B. Karasic, D. J. Beste, S. C. M. To, W. J. Doyle, S. W. Wood, M. J. Carter, A. C. C. To, K. Tanpowpong, C. D. Bluestone, and C. C. Brinton, Jr., Pediatr. Infect. Dis. J. 8:562-565, 1989). The genes encoding LKP1 pili were cloned from a genomic library of the clinical strain as a 12.5-kilobase insert on a plasmid vector and inserted into Escherichia coli K-12. Transposon mutagenesis and deletion constructs mapped the pilus-coding region within a 7-kilobase region of insert DNA. The recombinant bacteria were found by electron microscopy to express pili morphologically similar to LKP1 pili. Purified pilus rods from the recombinant and its parental strain were composed of a single detectable protein with an apparent molecular weight of 27,500. Antibodies raised against LKP1 pili purified from H. influenzae immunologically reacted with pili from the recombinant bacteria. Pili from both strains also adhered to human erythrocytes and buccal cells with the same specificity.
Nucleotide sequence of the leukotoxin gene from Actinobacillus actinomycetemcomitans: homology to the alpha-hemolysin/leukotoxin gene family
Authors: Kraig E , Dailey T , Kolodrubetz D
Published: January 2020

The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of localized juvenile periodontitis. To initiate a genetic analysis into the role of this protein in disease, we have cloned its gene, lktA. We now present the complete nucleotide sequence of the lktA gene from A. actinomycetemcomitans. When the deduced amino acid sequence of the leukotoxin protein was compared with those of other proteins, it was found to be homologous to the leukotoxin from Pasteurella haemolytica and to the alpha-hemolysins from Escherichia coli and Actinobacillus pleuropneumoniae. Each alignment showed at least 42% identity. As in the other organisms, the lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be involved in the activation of the leukotoxin. The predicted LktC protein was related to the leukotoxin/hemolysin C proteins from the other bacteria, since they shared a minimum of 49% amino acid identity. Surprisingly, although actinobacillus species are more closely related to pasteurellae than to members of the family Enterobacteriaciae, LktA and LktC from A. actinomycetemcomitans shared significantly greater sequence identity with the E. coli alpha-hemolysin proteins than with the P. haemolytica leukotoxin proteins. Despite the overall homology to the other leukotoxin/hemolysin proteins, the LktA protein from A. actinomycetemcomitans has several unique properties. Most strikingly, it is a very basic protein with a calculated pI of 9.7; the other toxins have estimated pIs around 6.2. The unusual features of the A. actinomycetemcomitans protein are discussed in light of the different species and target-cell specificities of the hemolysins and the leukotoxins.
Growth inhibition of Cryptococcus neoformans by cultured human monocytes: role of the capsule, opsonins, the culture surface, and cytokines
Authors: Levitz S M , Farrell T P
Published: January 2020

Despite a presumed critical role of macrophages in the host response to cryptococcal infections, previous studies have failed to show growth inhibition of encapsulated Cryptococcus neoformans by human peripheral blood cultured monocyte-derived macrophages (MO-M phi). Here, we examined whether MO-M phi could be induced to inhibit growth of an encapsulated strain and an isogenic acapsular mutant strain of C. neoformans. MO-M phi were cultured in microwells, and inhibition was measured by comparing CFU at 0 and 24 h after fungal challenge. MO-M phi cultured on plastic surfaces failed to inhibit growth of the encapsulated strain, even in the presence of pooled human serum and/or anticapsular antibody. Moreover, the presence of anticapsular antibody significantly enhanced fungal growth. However, if MO-M phi were cultured on surfaces coated with fibronectin or poly-L-lysine (but not laminin or collagen) and yeast cells were opsonized with pooled human serum, then complete growth inhibition occurred. Preincubation with various concentrations of tumor necrosis factor, granulocyte macrophage colony-stimulating factor, 1,25-dihydroxycholecalciferol, or supernatants from C. neoformans-stimulated lymphocytes failed to activate macrophages for enhanced antifungal activity. The addition of gamma interferon resulted in a significant loss of growth inhibition. For the acapsular strain, complete growth inhibition was observed regardless of the choice of culture surface, opsonins, or cytokines. Fungicidal activity, as measured by a significant decrement in CFU compared with the initial inoculum, was not observed under any conditions tested. These data demonstrate that macrophages are capable of inhibiting cryptococcal growth but that this capacity is markedly influenced by the culture surface, opsonins, cytokines, and the fungal capsule.
Antibody responses to Toxoplasma gondii in sera, intestinal secretions, and milk from orally infected mice and characterization of target antigens
Authors: Chardès T , Bourguin I , Mevelec M N , Dubremetz J F , Bout D
Published: January 2020

Toxoplasma gondii-specific antibody responses in serum, intestinal secretions, and milk were identified with an enzyme-linked immunosorbent assay following a single oral infection of mice with strain 76K cysts of T. gondii. Immunoglobulin A (IgA) production began during week 2 of infection in serum and milk and during week 3 of infection in intestinal secretions and persisted in all three throughout the experiment (17 weeks). IgG but not IgM antibodies were detected in intestinal secretions later in the infection. Serum and milk IgG and IgM production began at the same time after infection as did the IgA response. In Western blotting (immunoblotting), intestinal IgA antibodies were shown to react with antigens comigrating with the T. gondii proteins p22, p23, p30, and p43, the 28-kilodalton antigen, and the 55- and 60-kilodalton rhoptry proteins, as recognized by specific monoclonal antibodies. Milk IgA antibodies reacted with antigens comigrating with p30 and p43. Most of the antigens recognized by IgA antibodies were also detected by IgG antibodies. IgA antibodies from all three biological samples detected the same major T. gondii antigens; thus, there was apparently no specific antibody production unique to one locality.
In vivo activation of macrophage oxidative burst activity by cytokines and amphotericin B
Authors: Wolf J E , Massof S E
Published: January 2020

Alterations in macrophage oxidative burst activity following in vivo administration of recombinant murine gamma interferon (IFN-gamma), recombinant murine tumor necrosis factor alpha, and the antifungal antibiotic amphotericin B were investigated. Mice were given intraperitoneal injections of these agents alone and in combination, and the oxidative responses of their resident peritoneal macrophages to challenge with Histoplasma capsulatum or zymosan particles were measured 1 to 5 days later. Various degrees of enhanced oxidative burst activity were achieved following treatment with each agent. However, a synergistic response was observed only when mice were treated with the combination of recombinant murine IFN-gamma and amphotericin B. These results not only confirm the dual role of amphotericin as an antifungal agent and as an immunomodulator but also suggest that IFN-gamma may serve as a useful adjunct in the treatment of intracellular fungal infections.
Isolation of Neisseria meningitidis mutants deficient in class 1 (porA) and class 3 (porB) outer membrane proteins
Authors: Tommassen J , Vermeij P , Struyvé M , Benz R , Poolman J T
Published: January 2020

The class 1 major outer membrane protein of Neisseria meningitidis is a serious candidate for a meningococcal vaccine. To facilitate studies on the function of this protein, mutants were isolated that lacked this protein or the structurally related class 3 protein. These mutants were obtained by using the antibody-dependent bactericidal action of the complement system. The class 1 protein-deficient strain grew normally in vitro, whereas growth of the class 3 protein-deficient strain was slightly retarded. The class 3 protein-deficient strain displayed increased resistance to the antibiotics tetracycline and cefsulodin, which is consistent with the proposed role of the protein as a pore-forming protein. The class 1 protein was purified to homogeneity from the class 3 protein-deficient strain. Lipid bilayer experiments revealed that this protein also formed pores. The class 1 protein pores were cation selective.
Specific labeling of cysteine proteinases in pathogenic and nonpathogenic Entamoeba histolytica
Authors: de Meester F , Shaw E , Scholze H , Stolarsky T , Mirelman D
Published: January 2020

Growth of Entamoeba histolytica trophozoites was inhibited by 50% at low concentrations (2.0 micrograms/ml) of the diazopeptidyl inhibitor benzyloxycarbonyl-leucyl-L-tyrosyldiazomethane (Z-L-Leu-L-Tyr-CHN2). Iodination of the tyrosine residue lowered the growth inhibitory efficacy of the diazopeptidyl inhibitor (50% inhibition, approximately 10 micrograms/ml). However, even at this concentration, practically all of the cysteine proteinase activity of the cells was irreversibly inactivated as shown by fluorescence microscopy with the dipeptide substrate L-Arg-L-Arg-4-methoxy-beta-napthylamide or colorimetrically with azocasein as the substrate. Growth of trophozoites of E. histolytica from various strains, including both pathogenic and nonpathogenic zymodemes, was similarly inhibited. The concentration of inhibitor required to inactivate the proteinase activity of nonpathogenic cells was lower. Lysates from trophozoites grown in the presence of sublethal concentrations of 125I-labeled protease inhibitor (10 micrograms/ml) showed as many as eight radioactive bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular sizes, 73, 68, 56, 40, 39, 35, 29, and 27 kilodaltons). Two of these bands (molecular sizes, 29 and 27 kilodaltons) could be seen in gels of the cytoplasmic fraction, whereas the high-molecular-size bands were mostly associated with the membrane fraction. The radioactive bands in pathogenic and nonpathogenic strains were very similar with only minor differences. The results obtained show that E. histolytica cells, irrespective of their pathogenicity, possess a number of cysteine proteinases of similar molecular sizes which are vital for cell growth.