bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular studies of Ssa1, a serotype-specific antigen of Pasteurella haemolytica A1
Authors: Lo R Y , Strathdee C A , Shewen P E , Cooney B J
Published: January 2020

A serotype-specific antigen of Pasteurella haemolytica A1 encoded on the recombinant plasmid pSSA1 is characterized. Nucleotide sequence analysis of the insert DNA in pSSA1 identified the gene ssaI, which codes for a protein of approximately 100 kDa. In vivo labeling of pSSA1-encoded protein in Escherichia coli maxicells showed the expression of a 100-kDa protein from the insert DNA on the recombinant plasmid. Northern blot and primer extension analyses were used to identify the mRNA transcript in P. haemolytica A1 and the putative promoter of ssaI. The antigen (designated Ssa1) could be localized to the outer membrane of P. haemolytica A1 and E. coli clones carrying pSSA1. A rabbit serum against Ssa1 was produced by using whole cells of E. coli expressing Ssa1 on the surface as the immunogen, demonstrating that Ssa1 is immunogenic in rabbits. The results from colony immunoblot analysis with calf serum from animals that were resistant to P. haemolytica A1-induced pneumonia suggest indirectly that Ssa1 is also immunogenic in the animals.
Cloning of a Streptococcus sobrinus gtf gene that encodes a glucosyltransferase which produces a high-molecular-weight water-soluble glucan
Authors: Hanada N , Yamashita Y , Shibata Y , Sato S , Katayama T , Takehara T , Inoue M
Published: January 2020

The gtf gene coding for glucosyltransferase (GTF), which produces a water-soluble glucan, was cloned from Streptococcus sobrinus OMZ176 (serotype d) into plasmid vector pBR322. This gene was expressed in Escherichia coli, and the product was purified to near homogeneity. The antigenicity of recombinant GTF (rGTF) was examined with the antisera raised against purified GTF P1, P2, P3, and P4 obtained from S. sobrinus AHT (serotype g). The rGTF reacted only with anti-GTF P1 serum in a Western blot (immunoblot) analysis. The rGTF closely resembled GTF P1 in its molecular mass, Km value for sucrose, optimal pH, primer dependency, and immunological properties. The high-molecular-weight, water-soluble glucan produced by the rGTF also resembled that of GTF P1, which is the most efficient primer donor for primer-dependent, water-insoluble glucan synthesis. Properties of the rGTF were also compared with those of rGTFS, which was purified from E. coli carrying the gtfS gene isolated from Streptococcus downei (previously S. sobrinus serotype h) MFe28. Both rGTF and rGTFS synthesized water-soluble glucan from sucrose without primer dextran, but their characteristics in Km values for sucrose, optimal pHs, and polymer sizes of the glucan were different. Furthermore, the gtf gene did not hybridize with the gtfS gene in a Southern blot analysis. These results showed that rGTF is similar to S. sobrinus AHT GTF P1 but distinct from rGTFS that has been previously purified from E. coli carrying the gtfS gene.
Effect of carrier priming on immunogenicity of saccharide-protein conjugate vaccines
Authors: Peeters C C , Tenbergen-Meekes A M , Poolman J T , Beurret M , Zegers B J , Rijkers G T
Published: January 2020

Previous studies with saccharide-protein conjugates have demonstrated that antibody responses to the saccharide can be improved by the preexistence of carrier immunity. Here we report that prior exposure to the carrier protein can either enhance or suppress antibody response to polysaccharides administered in saccharide-protein conjugates. A dose-dependent role for carrier priming in the antisaccharide antibody response to three saccharide-protein conjugate vaccines, i.e., a Streptococcus pneumoniae type 4 polysaccharide-tetanus toxoid (TT) conjugate (PS4TT), a Neisseria meningitidis group C polysaccharide-TT conjugate (MenCTT), and a N. meningitidis group C oligosaccharide-diphtheria mutant toxin conjugate (MenCCRM), was investigated. The results showed that an increase in the antipolysaccharide antibody response could be obtained for both PS4TT and MenCTT but not for MenCCRM with low-dose carrier priming (0.025 to 0.25 microgram). However, suppression of the antipolysaccharide antibody response was observed with the PS4TT and MenCTT vaccines with high-dose (25-micrograms) carrier priming. There was no suppression effect with MenCCRM. The increase in the antipolysaccharide antibody response was shown to be restricted to the immunoglobulin G1 (IgG1) subclass, whereas suppression with high-dose carrier priming affected all antipolysaccharide subclass antibodies induced by PS4TT (IgG1, IgG2b, and IgG3) and only two of the four subclass antibodies induced by MenCTT (IgG2a and IgG2b). The increase in the antipolysaccharide antibody response was also present at the antipolysaccharide IgM antibody level but was not observed at the anti-carrier IgG antibody level.
Construction and expression of plasmids containing mutated diphtheria toxin A-chain-coding sequences
Authors: Fisher K S , Maxwell I H , Murphy J R , Collier J , Glode L M
Published: January 2020

We previously demonstrated that cells can be killed through transfection of an expression plasmid that encodes the diphtheria toxin A-chain fragment (DT-A). This report describes the construction of expression plasmids containing three mutant DT-A-coding sequences substituting glutamic acid 148 with aspartic acid, serine, or glutamine which are known to have 100- to 300-fold-reduced ADP-ribosylation activity measured in vitro. The toxicity of these constructs was determined in cotransfection experiments using HeLa and 293 cells with a luciferase expression plasmid as the reporter. Dose responses were compared for the three new DT-A mutant plasmids and for the corresponding plasmids containing wild-type DT-A and the previously characterized tox 176 mutant. The dose required to produce 50% inhibition of control luciferase expression in 293 embryonic kidney cells for the five plasmids ranged from 0.01 micrograms for wild-type DT-A to 1.2 micrograms for the least toxic plasmid, which replaces glutamic acid 148 with glutamine. In conclusion, a wide range of DT-A toxicity can be achieved by using plasmid expression vectors that encode different DT-A mutations.
Protective ribosomal preparation from Shigella sonnei as a parenteral candidate vaccine
Authors: Levenson V I , Egorova T P , Belkin Z P , Fedosova V G , Subbotina J L , Rukhadze E Z , Dzhikidze E K , Stassilevich Z K
Published: January 2020

A parenteral Shigella ribosomal vaccine (SRV) was investigated in animals for safety, antibody-inducing capacity, and protective activity. Ribosomal preparations from a Shigella sonnei phase I avirulent strain were obtained and shown to possess chemical, sedimentation, and other properties typical of bacterial ribosomes. No endotoxin contamination was revealed by a ketodeoxyoctonate assay, although the presence of some kind of O antigen was evidenced by serological findings and the high activity of SRV in inducing the O-antibody response and immunological memory in animals. SRV was nontoxic in mice, guinea pigs, and monkeys and induced no local reactions when injected subcutaneously in reasonable doses. Significant protection against a local Shigella infection (Sereny test) was seen in guinea pigs injected with SRV (efficiency index, about 60%) and the specificity of the protection was evident from cross-challenge experiments. The protective efficiency of SRV was especially high in rhesus monkeys challenged orally with virulent Shigella cells (89%, as calculated from the summarized data of several experiments in 71 animals). Protection in monkeys was long lasting and could be demonstrated several months after injection of SRV. An inexpensive technique can be used for the production of SRV on a large scale. The high immunogenicity of SRV is discussed in terms of the amplifying effect of the ribosome, which serves as a delivery system for polysaccharide O antigen. Further study of SRV as a candidate vaccine for humans seems justified by the data obtained.
Treponema phagedenis encodes and expresses homologs of the Treponema pallidum TmpA and TmpB proteins
Authors: Yelton D B , Limberger R J , Curci K , Malinosky-Rummell F , Slivienski L , Schouls L M , van Embden J D , Charon N W
Published: January 2020

We cloned and sequenced the genes from Treponema phagedenis Kazan 5 encoding proteins homologous to the TmpA and TmpB proteins of Treponema pallidum subsp. pallidum Nichols (hereafter referred to as T. pallidum). Although previous reports suggested that the TmpA and TmpB proteins were specific for T. pallidum, we found that homologs for both were expressed in T. phagedenis Kazan 5 and Reiter. The TmpA protein from T. phagedenis contained the consensus sequence that bacterial lipoproteins require for posttranslational modification and subsequent proteolytic cleavage by signal peptidase II and showed 42% amino acid sequence identity with the TmpA protein from T. pallidum. The TmpB proteins of T. phagedenis and T. pallidum had similar amino acid sequences at their amino- and carboxy-terminal ends. The central portions of both of these proteins contained four repeats of the amino acid sequence EAARKAAE. The TmpB protein from T. phagedenis had an additional amino acid sequence repeat (consensus sequence KAAKE/D) that was not found in the TmpB protein from T. pallidum; this repeat was most remarkable, as it occurred 17 times in succession. These repeated amino acid sequences probably created an extensive alpha-helix region within the TmpB proteins. As with T. pallidum, the stop codon of the T. phagedenis tmpA gene overlapped the start codon of its tmpB gene. Northern blot analysis showed that the T. phagedenis tmpA and tmpB genes were probably transcribed into a single 2.5-kb mRNA molecule. Western blot (immunoblot) analysis demonstrated that both proteins were expressed by T. phagedenis. The high degree of amino acid sequence conservation seen with the TmpA and TmpB proteins from two different Treponema species suggests that they may play crucial roles in the biology of these organisms.
Recombinant human tumor necrosis factor alpha promotes adherence of Staphylococcus aureus to cultured human endothelial cells
Authors: Cheung A L , Koomey J M , Lee S , Jaffe E A , Fischetti V A
Published: January 2020

Tumor necrosis factor (TNF), a potent inflammatory mediator secreted by monocytes during inflammation, was shown to significantly increase the adherence of Staphylococcus aureus to cultured human umbilical vein endothelial cells in vitro. The stimulatory effect of TNF was dose dependent and was bimodal with respect to time; bacterial adhesion peaked after 4 and 16 h of stimulation with recombinant human TNF-alpha. The ability of TNF-alpha to augment staphylococcal adherence to endothelial cells was contingent upon the presence of plasma factors. Thus, the complex interaction among cytokines (such as TNF), plasma factor(s), and the endothelium serves to modulate bacterial adherence to endothelial cells.
Variant surface antigens of Giardia lamblia are associated with the presence of a thick cell coat: thin section and label fracture immunocytochemistry survey
Authors: Pimenta P F , da Silva P P , Nash T
Published: January 2020

Giardia lamblia undergoes surface antigenic variation. The ultrastructural location of antigens on four different variants was studied by label fracture and immunocytochemistry with four monoclonal antibodies (MAbs), each of which recognized the predominant variant in a particular clone. Each Giardia clone and its reacting MAb showed similar findings. The entire surface of the organism was covered by a surface coat which contained the variant surface protein. The surface coat was densely and uniformly labeled. Unreacting MAbs failed to label the surface. Label-fractured Giardia trophozoites exposed to reactive MAb revealed a planar distribution of reactivity with no discernible relationship to visualized intramembranous particles. Unexpectedly, some Giardia trophozoites lacked a surface coat and consequently failed to react with the appropriate MAb. The biological relevance of coatless Giardia trophozoites is unknown. These findings localize the variant antigens to the surface coat of the parasite and identify a minority of the population which lacks a surface coat.
Analysis of egg granuloma formation in Schistosoma japonicum-infected mice treated with antibodies to interleukin-5 and gamma interferon
Authors: Cheever A W , Xu Y H , Sher A , Macedonia J G
Published: January 2020

Schistosoma japonicum-infected mice were treated with antibodies to interleukin-5 (IL-5) or gamma interferon (IFN-gamma) from week 3 or 4 to week 10 of infection. Neither antibody affected egg production by the parasite, and neither had a consistent effect on the secretion of IFN-gamma or IL-5 cell-related cytokines by spleen cells from infected mice. Mice treated with antibody to murine IL-5 had only rare eosinophils in hepatic circumoval granulomas. Granulomas around single eggs were reduced in volume by a third, but hepatic fibrosis was unaffected. Treatment with antibody to murine IFN-gamma also reduced the size of granulomas and also did not affect hepatic fibrosis, which was measured as hydroxyproline. Our results, taken together with the studies of others, indicate that a complex interaction of cytokines affects granuloma size and that the size and fibrosis of granulomas are to some extent regulated independently.
Functional and structural mapping of Chlamydia trachomatis species-specific major outer membrane protein epitopes by use of neutralizing monoclonal antibodies
Authors: Peterson E M , Cheng X , Markoff B A , Fielder T J , de la Maza L M
Published: January 2020

Three monoclonal antibodies (MAbs), E4, L1-4, and L1-24, to the major outer membrane protein (MOMP) of Chlamydia trachomatis were identified that neutralized in vitro the infectivity of members of the B- and C-related complex as well as the mouse pneumonitis strain. MAbs L1-4, L1-24, and E4 gave a strong signal in an indirect immunofluorescence assay and/or Western immunoblot with all serovars of the lymphogranuloma venereum and trachoma biovars and a weak signal with the mouse biovar. In addition, C. psittaci and C. pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by the technique of pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by by the technique of overlapping peptides, all three MAbs showed reactivity to variable domain (VD) IV of MOMP. While all three MAbs had different recognition patterns, all strongly bound to the peptides TLNPTI and LNPTIA within the species-conserved region of VD IV. MAb E4 also recognized the peptide SATAIF in the subspecies region of VD IV. Peptides corresponding to VD IV of MOMP were synthesized and used in competitive inhibition experiments to determine the functional location of the epitope recognized by these three MAbs. Both the serological and neutralizing activities of MAb E4 were inhibited by the peptides ATAIFDTTTLNPTIAG and FDTTTLNPTIAG; however, none of the peptides made to the VD IV region blocked the neutralizing activity of MAbs L1-4 and L1-24. Therefore, the neutralizable domain of the epitope recognized by MAb E4 is contiguous and may be an important candidate for inclusion in a subunit vaccine.
Sulfhydryl-dependent attachment of Treponema denticola to laminin and other proteins
Authors: Haapasalo M , Singh U , McBride B C , Uitto V J
Published: January 2020

Attachment of Treponema denticola ATCC 35405 to laminin, a major basement membrane protein, and to other proteins was studied. Microdilution plates were coated with the proteins, and the attachment of T. denticola was measured by the enzyme-linked immunosorbent assay technique. Compared with bovine serum albumin (BSA), T. denticola had a high affinity to laminin, fibronectin, fibrinogen, and gelatin, as well as to type I and type IV collagens. Attachment to RGD peptide (Gly-Arg-Gly-Asp-Ser, the integrin recognition sequence) was only about 30% of that to laminin and was comparable to attachment to BSA. Tests with laminin fragments obtained through elastase digestion showed that the spirochetes attached well to an A-chain 140-kDa fragment involved in eukaryote cell attachment but did not attach to a 50-kDa fragment that includes the heparin binding site. Pretreatment of T. denticola with soluble laminin, fibronectin, gelatin, BSA, or fibrinogen had no effect on the attachment of the bacteria to laminin or fibronectin. A wide variety of compounds were tested for their possible inhibitory actions on the attachment. While most treatments of T. denticola ATCC 35405 had little or no effect on the attachment to proteins, sulfhydryl reagents p-chloromercuribenzoic acid (pCMBA) and oxidized glutathione inhibited the attachment by 70 to 99%, depending on the protein. When T. denticola was first allowed to attach to proteins, addition of pCMBA or oxidized glutathione could no longer reverse the attachment. Heat treatment of the spirochetes also markedly reduced the attachment to laminin, gelatin, and fibrinogen but not to BSA. Mixed glycosidase treatment of the spirochetes inhibited the attachment by 20 to 80%. None of the above treatments of the substrate proteins had any marked effect on the spirochete attachment. The results indicate that T. denticola has the capacity to bind to many different kinds of proteins by utilizing specific attachment mechanisms. The binding appears to involve protein SH groups and/or carbohydrate residues on the surface of T. denticola.
Mapping T-cell epitopes in group A streptococcal type 5 M protein
Authors: Robinson J H , Atherton M C , Goodacre J A , Pinkney M , Weightman H , Kehoe M A
Published: January 2020

Group A streptococcal cell surface M proteins elicit highly protective, serotype-specific opsonic antibodies and many serotypes also elicit host cross-reactive antibodies, which may contribute to the pathogenesis of poststreptococcal autoimmune disease. To date, studies aimed at designing safe (non-host-cross-reactive, defined-epitope) M vaccines have focused almost exclusively on antibody epitopes. Here we identify T-cell epitopes recognized by T cells from BALB/c, C57BL/6, and CBA/Ca mice immunized with purified, recombinant serotype 5 M protein (rM5). The responses of rM5-specific, major histocompatibility complex class II-restricted, T-cell clones to synthetic peptides representing most of the M5 sequence identified at least 13 distinct T-cell recognition sites, including sites recognized by more than one major histocompatibility complex haplotype of mice. Although none of these sites appeared to be strongly immunodominant, an N-terminal peptide, sM5[1-35], was recognized by lymph node T cells of rM5-immunized mice and by a larger proportion of rM5-specific T-cell clones than any other individual peptide. The fine specificity of these clones was mapped with subpeptides to a single site at or overlapping the sequence ELENHDL at residues 21 to 27, which is in close proximity to previously mapped protective antibody epitopes. Other T-cell recognition sites are distributed throughout the M protein and include several in the highly conserved C-terminal region of the molecule. One of these C-terminal sites, located within residues 300 to 319, was recognized by a significant proportion of T-cell clones from two strains of mice. Helper T-cell epitopes located in the C-terminal region of M5 are likely to be widely conserved between different M serotypes and could be particularly useful in designing multivalent, defined-epitope M vaccines.
Analysis of Aspergillus nidulans conidial antigens and their prevalence in other Aspergillus species
Authors: Puente P , Ovejero M C , Fernández N , Leal F
Published: January 2020

Aspergillus nidulans is an ascomycetous fungus that reproduces asexually by forming multicellular conidiophores and uninucleate spores called conidia. These elements constitute the main vehicle for the transmission of this and other pathogenic Aspergillus species and are the starting point of the different forms of aspergillosis. In order to use A. nidulans as a potential source of useful antigens for the immunodiagnosis of these diseases, we have examined the total protein composition of conidial extracts of this fungus by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in gels of different percent T. Injection of SDS-extracted conidial proteins into rabbits allowed us to raise a battery of polyclonal antibodies which have defined some important immunogenic polypeptides. Several of these immunogens were both present in mycelial extracts and recognized by antimycelium antibodies. Four of them, designated cdA, cdB, cdC, and cdE, were also found in conidial extracts of other pathogenic Aspergillus species. Only cdE was undetectable in cell extracts of the nonrelated species Fusarium culmorum and Phycomyces blakesleeanus.
Intracellular targeting of the Yersinia YopE cytotoxin in mammalian cells induces actin microfilament disruption
Authors: Rosqvist R , Forsberg A , Wolf-Watz H
Published: January 2020

Pathogenic Yersinia spp., including the etiological agent of plague, Y. pestis, all carry a common plasmid that encodes a number of essential virulence determinants, the Yop proteins. One of these, YopE, has been shown to be involved in the obstruction of the primary host defense by a molecular mechanism leading to inhibition of phagocytosis (R. Rosqvist, A. Forsberg, M. Rimpiläinen, T. Bergman, and H. Wolf-Watz, Mol. Microbiol. 4:657-667, 1990). Although the Yop proteins are secreted into the culture supernatant in vast amounts, in vitro studies of the function of the Yop proteins have so far been unsuccessful. We show that isolated Yop proteins indeed can cause cytotoxic effects in vitro if the proteins are introduced intracellularly into the eukaryotic cell. Isolated Yop proteins of Yersinia pseudotuberculosis were found to disrupt the microfilament structure when microinjected intracellularly into the host cell. In particular, YopE was demonstrated to be directly involved in the cytotoxic action, whereas YopD seems to have a critical role in translocating the YopE protein through the host cell membrane. These results elucidate the requirement for at least some of the Yop proteins to leave the pathogen during infection.
Production and characterization of neutralizing and nonneutralizing monoclonal antibodies against listeriolysin O
Authors: Nato F , Reich K , Lhopital S , Rouyre S , Geoffroy C , Mazie J C , Cossart P
Published: January 2020

Listeriolysin O (LLO) is a thiol-activated toxin secreted by the facultative intracellular pathogen Listeria monocytogenes. LLO is essential for the survival of the bacterium in the infected cell because it promotes lysis of the phagosome membrane and escape of the bacterium into the cytosol. LLO was used as an antigen for the production of nine monoclonal antibodies (MAbs) in mice. Three of these could inhibit the hemolytic activity of LLO. One of them inhibited binding of LLO to erythrocyte membranes. The two other antibodies blocked the activity of LLO at a step subsequent to membrane binding. Only two of the nine MAbs recognized three other purified SH-activated toxins, streptolysin O, alveolysin, and pneumolysin. Western blot (immunoblot) analysis of culture supernatants of Listeria ivanovii and Listeria seeligeri, two hemolytic species of the genus Listeria, revealed that two MAbs recognized ivanolysin and seeligerolysin. The latter was also recognized by two other MAbs, including one of the neutralizing antibodies. MAbs raised against a peptide, ECTG LAWEWWR, present in all thiol-activated toxins sequenced to date, recognized all toxins and were not neutralizing. Taken together, these results demonstrate the existence of regions important for hemolytic activity that are unique to hemolysins of the genus Listeria and show that regions outside the conserved peptide are important for activity of LLO.
Detection of Bordetella pertussis associated with the alveolar macrophages of children with human immunodeficiency virus infection
Authors: Bromberg K , Tannis G , Steiner P
Published: January 2020

In humans, infection with Bordetella pertussis is considered to be localized to an epithelial surface. However, an intracellular state in cultured cells and in the macrophages of infected animals has been shown. By using indirect immunofluorescence with a monoclonal antibody, it was found that 3 of 20 bronchoalveolar lavage specimens from children with human immunodeficiency virus infection had B. pertussis associated with pulmonary alveolar macrophages. None of the cultures from the patients grew B. pertussis. The B. pertussis appeared to be intracellular.
Properties of the B oligomer of pertussis toxin
Authors: Nencioni L , Pizza M G , Volpini G , De Magistris M T , Giovannoni F , Rappuoli R
Published: January 2020

The B oligomer of pertussis toxin was purified from culture supernatants of Bordetella pertussis strains which do not secrete the S1 subunit. The purified B oligomer is devoid of toxicity for CHO cells and other in vivo properties of the toxin, such as leukocytosis and histamine sensitization, but it retains the abilities to agglutinate erythrocytes and to induce the proliferation of T lymphocytes. The B oligomer is also able to induce protective immunity in mice but is less potent than molecules containing the S1 subunit also.
Endotoxin shedding by enterobacteria: free and cell-bound endotoxin differ in Limulus activity
Authors: Mattsby-Baltzer I , Lindgren K , Lindholm B , Edebo L
Published: January 2020

The endotoxin activities of gram-negative bacteria and their lipopolysaccharides (LPS) have been quantitated by a chromogenic Limulus amocbocyte lysate (CLAL) assay. When bacterial cell exposing various cell surface structures were compared, the highest Limulus activities were found in R strains of Escherichia coli and Salmonella typhimurium mutants. E. coli with K antigens did not differ from K-negative strains. By measuring beta-hydroxymyristic acid (3-OH tetradecanoic acid, beta-OHC14:0), it was possible to compare the CLAL activities of LPS bound to bacterial cells, LPS shed into the culture medium, and purified LPS. After 16 h of growth, the cell-free culture supernatants of three E. coli O1K1 strains and S. typhimurium showed CLAL activities 14.3 to 20.3 times higher than did the corresponding bacterial cell suspensions in relation to their beta-OHC14:0 contents. Four other E. coli strains (O serotypes O14, O24, and O75) and the S. typhimurium 395 R mutants MR5 and MR6 showed CLAL values 2.8 to 7.9 times higher in their culture supernatants. LPS of E. coli O1K1 and S. typhimurium had lower CLAL activities than the culture supernatants (1/10 and 1/4, respectively). Although the beta-OHC14:0 concentrations of the culture supernatants were approximately half those of the corresponding bacterial cells, all had CLAL values that were 2 to 21 times higher. The bacterial cell suspension, culture supernatant, and purified LPS of S. typhimurium MS were compared by CLAL assay and a quantitative enzyme-linked immunosorbent assay based on monoclonal antibodies to the O5 antigen. Endotoxin shed into the culture medium was the most CLAL-active form of LPS, while purified LPS was the most antigen-active form. The results emphasize the importance of appropriate standards when quantifying endotoxin in various states. In conclusion, E. coli and S. typhimurium bacteria shed significant amounts of endotoxin into the surrounding medium during growth. This form of LPS is more CLAL active than the cell-bound or purified LPS.
Effect of sulfide ions on complement factor C3
Authors: Granlund-Edstedt M , Johansson E , Claesson R , Carlsson J
Published: January 2020

In infected sites such as the gingival pockets of patients with periodontal disease, sulfide levels up to 1 mmol/liter may be reached. There is little information, however, on how sulfide may interact with the host defense. In a previous study (R. Claesson, M. Granlund-Edstedt, S. Persson, and J. Carlsson, Infect. Immun. 57:2776-2781, 1989), it was shown that polymorphonuclear leukocytes were able to kill bacteria in the presence of 1 mM sulfide. However, sulfide seemed to interfere with the opsonization of the bacteria. It has been claimed that sulfide may be toxic by splitting disulfide bonds of proteins. In the present study, serum was exposed to 2 mM sulfide under anaerobic conditions, and the capacity of sulfide to split disulfide bonds of 10 serum proteins involved in opsonization was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection of the proteins after blotting. Sulfide had a low capacity to split the disulfide bonds of most proteins. Sulfide had, however, a pronounced effect on the complement component C3 in the form of C3bi. Sulfide released the C-terminal region of the alpha chain from C3bi. When C3 opsonizes bacteria, it is this region of C3bi which binds to complement receptor 3 (CR3) of the polymorphonuclear leukocytes. If sulfide has the same effect on C3bi deposited on the bacterial surface as it has on C3bi in solution, it will annihilate the very important contribution of C3bi to opsonization.
Isolation and characterization of a transposon-induced cytotoxin-deficient mutant of Pseudomonas aeruginosa
Authors: Bopp L H , Baltch A L , Hammer M C , Franke M A , Smith R P , Lutz F
Published: January 2020

In order to provide a better system for investigating the role of cytotoxin in pathogenesis, we mutated wild-type Pseudomonas aeruginosa PA158 by introducing a transposon. The resulting pool of mutants was screened for cytotoxin-deficient strains. One mutant strain, PA114F5, was compared with PA158. Except for cytotoxin production and antibiotic resistance (specified by the transposon), the two strains appear isogenic. This mutant strain should be useful in further clarifying the role of cytotoxin in pathogenesis.
Protective immunity to heartwater (Cowdria ruminantium infection) is acquired after vaccination with in vitro-attenuated rickettsiae
Authors: Jongejan F
Published: January 2020

A Senegalese (S) stock of Cowdria ruminantium was passaged on bovine umbilical endothelial cells with an average interval of 13.9 days (range, 8 to 34 days) between passages. The virulence of infected bovine umbilical endothelial cultures was tested in susceptible goats and sheep by intravenous inoculation of culture supernatant from passages 2 (51 days in vitro), 3 (69 days), 11 (229 days), 14 (264 days), and 16 (291 days). Both animals inoculated with passages 2 and 3 died of heartwater. However, clinical reactions were completely absent in goats and sheep that were inoculated with C. ruminantium from passages 11, 14, and 16. High antibody titers were detected, with immunofluorescence in all vaccinated animals, and a strong signal was found against a 32-kDa Cowdria protein in Western blots (immunoblots). Moreover, the vaccinated animals proved solidly immune when challenged with virulent Cowdria sp.-infected blood stabilate (S strain), whereas all control goats died. No attenuation of a second Cowdria stock (W) was achieved after 226 days in culture, at which time passage 17 was tested in a recipient goat which died of typical heartwater. This is the first report of vaccination with live attenuated C. ruminantium. These attenuated organisms may replace vaccination with virulent blood currently in use in areas where heartwater is endemic.
Inhibition of intracellular growth of Histoplasma capsulatum yeast cells by cytokine-activated human monocytes and macrophages
Authors: Newman S L , Gootee L , Bucher C , Bullock W E
Published: January 2020

Human monocytes/macrophages (M psi) were infected with Histoplasma capsulatum yeast cells, and intracellular growth was quantified after 24 h of incubation in medium alone or in medium containing cytokines. Yeast cells multiplied within freshly isolated monocytes, cultured M psi, and alveolar M psi with intracellular generation times of 14.2 +/- 1.4, 18.5 +/- 2.1, and 19.9 +/- 1.9 h (mean +/- standard error of the mean), respectively. Monocytes and M psi inhibited the intracellular growth of yeast cells in response to cytokine supernatant; maximum inhibition was obtained when cytokines were added to cell monolayers immediately after infection. Opsonization of yeast cells in normal serum or in H. capsulatum-immune serum did not affect the intracellular generation time of yeast cells in either control M psi or cytokine-activated M psi.
Two copies of Shiga-like toxin II-related genes common in enterohemorrhagic Escherichia coli strains are responsible for the antigenic heterogeneity of the O157:H- strain E32511
Authors: Schmitt C K , McKee M L , O'Brien A D
Published: January 2020

Thirty-two clinical isolates of Shiga-like toxin (SLT)-producing Escherichia coli associated with single cases or outbreaks of bloody diarrhea, hemorrhagic colitis, the hemolytic uremic syndrome, or edema disease of swine were examined for multiple copies of genes belonging to the slt-I or slt-II toxin families. Five of 19 strains that were known to produce SLT-II or to hybridize to slt-II-specific probes by colony blot were found by Southern hybridization to contain two copies of toxin genes related to slt-II. The genes for two toxins closely related to slt-II were cloned from one of the isolates, Escherichia coli O157:H- strain E32511. One copy of the operon was found to be essentially identical to slt-II; it differed from slt-II by only one nucleotide base. This single nucleotide difference did not affect the predicted amino acid sequence. The predicted amino acid sequence of the A subunit of the second operon was identical to that of SLT-II, but the predicted amino acid sequence of the B subunit was identical to that of the B2F1 toxin VT2ha. We designated this second operon slt-IIc. Neutralization assays using several monoclonal antibodies and polyclonal antiserum prepared against SLT-II showed that SLT-IIc was antigenically related to but distinct from SLT-II.
Helicobacter felis gastritis in gnotobiotic rats: an animal model of Helicobacter pylori gastritis
Authors: Fox J G , Lee A , Otto G , Taylor N S , Murphy J C
Published: January 2020

The gastric spirillum Helicobacter felis, originally isolated from the cat stomach, colonizes the stomachs of germfree rats. Studies were designed to examine the pathological and serological responses of germfree rats inoculated orally with H. felis. At 2 weeks postinoculation, the gastric mucosa of germfree rats had lymphocytes and eosinophils scattered in small foci throughout the subglandular region of the antrum. Small numbers of lymphocytes were present in the subglandular portion of the antral mucosa that focally extended through the lamina propria towards the luminal surface. Eight weeks postinoculation, the inflammation was confined to the antrum. It was characterized by increased numbers of lymphocytes and eosinophils in the subglandular areas, with focal aggregates of lymphocytes in the submucosa. Some lymphoid aggregates extended from the submucosa through the muscularis mucosa and lamina propria to the luminal surface. H. felis was demonstrated with the Warthin-Starry stain, bacterial culture, and urease assay, particularly in the antrum. H. felis also produced a significant immunoglobulin G antibody titer at 2, 4, and 8 weeks postinoculation as well as a transitory immunoglobulin M response at 2 to 4 weeks postinoculation. Contact control rats were not infected, inferring that fecal-oral spread of H. felis did not occur.
The msp1 beta multigene family of Anaplasma marginale: nucleotide sequence analysis of an expressed copy
Authors: Barbet A F , Allred D R
Published: January 2020

A gene for the beta subunit of the immunoprotective surface antigen MSP-1 of Anaplasma marginale was previously cloned and expressed in Escherichia coli. A nucleic acid probe based on this gene detects A. marginale infection in carrier cattle and in the tick vector. We report here the sequence and structural features of the cloned msp1 beta gene and expressed polypeptide. The gene codes for a polypeptide of 756 amino acids that contains domains of tandemly repeated sequence and glutamine-rich regions at the N and C termini. The cloned copy is a member of a multigene family with multiple restriction fragment length polymorphisms in isolates of this rickettsia from different geographical regions. The availability of the sequence will allow use of the polymerase chain reaction in diagnostic assays and the preparation and testing of different vaccine constructs in cattle.
A 43-kilodalton pneumococcal surface protein, PspA: isolation, protective abilities, and structural analysis of the amino-terminal sequence
Authors: Talkington D F , Crimmins D L , Voellinger D C , Yother J , Briles D E
Published: January 2020

PspA is an antigenically variable surface protein of Streptococcus pneumoniae that appears to be essential for full pneumococcal virulence. In addition, monoclonal antibodies to PspA protect mice against infection with specific strains of pneumococci virulent for mice. In this study, we have isolated the 43-kDa N-terminal half of the native 84-kDa PspA and determined the sequence of the first 45 amino acids. This sequence, the first obtained for a pneumococcal surface protein, is consistent with that of an amphiphatic coiled-coil alpha helix with a 7-residue periodicity common to fibrous proteins such as tropomyosin and streptococcal M protein. The 7-residue periodicity begins with residue 8 and extends throughout the remaining sequence for nearly 11 turns of the helix. Mice immunized with this purified PspA segment were protected from fatal pneumococcal challenge, thus demonstrating that those PspA epitopes eliciting protection were present in the N-terminal half of the molecule.
New adhesive factor (antigen 8786) on a human enterotoxigenic Escherichia coli O117:H4 strain isolated in Africa
Authors: Aubel D , Darfeuille-Michaud A , Joly B
Published: January 2020

An enterotoxigenic Escherichia coli strain, E. coli 8786, of serotype O117:H4 produced only heat-stable enterotoxin and gave mannose-resistant hemagglutination with human and bovine erythrocytes. The strain adhered to the brush border of human enterocytes and to enterocytelike cell line Caco-2. Adhesion inhibition assays using Caco-2 cells with different adhesive factor extracts showed that the adhesive factor of E. coli 8786 is different from colonization factor antigen I (CFA/I). CFA/II, CFA/III of Darfeuille et al. (A. Darfeuille, B. Lafeuille, B. Joly, and R. Cluzel, Ann. Microbiol. Inst. Pasteur 134A:53-64, 1983), CS6, and antigen 2230. A bacterial surface protein, designated antigen 8786, with a molecular mass of 16,300 Da was responsible for the adhesion to intestinal cells. It was immunologically different from previously described adhesive factors as determined by immunoblotting. Antigen 8786 was detected on the bacterial cell surface and appeared to be nonfimbrial. NH2-terminal analysis of antigen 8786 showed no homology with the previously described adhesive factors. Nevertheless, antigen 8786 is closely related to the NH2-terminal sequence of Salmonella enteritidis fimbrin. A hybridization experiment using a synthetic oligonucleotide probe based on the NH2-terminal amino acid sequence of antigen 8786 revealed that the coding region was located on a 70-MDa plasmid.
Cell walls of normal and lysozyme-damaged blastoconidia of Candida albicans: localization of surface factor 4 antigen and vicinal-glycol staining
Authors: Marquis G , Garzon S , Strykowski H , Auger P
Published: January 2020

The fungicidal effect of lysozyme on Candida albicans involves ultrastructural modifications previously described (G. Marquis, S. Montplaisir, S. Garzon, H. Strykowski, and P. Auger, Lab. Invest. 46:627-636, 1982). To further define the action of lysozyme on the yeast cell wall, we used the following: (i) the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method to highlight vicinal-glycol-reactive sites of complex carbohydrates; (ii) a monospecific antiserum and a protein A-gold complex to study the expression of surface factor 4, a major Candida antigen; and (iii) the periodic acid-silver methenamine method to stain cell wall glycoproteins. All Candida cells were found to express surface factor 4 antigen. In normal blastoconidia, surface factor 4 was located in a glycoprotein-rich cell wall layer, underneath radially oriented bundles of filaments which form the outermost wall layer. In lysozyme-treated blastoconidia, this glycoprotein-rich layer was lost and the regular brushlike organization of the outer fibrillogranular layer was disrupted. PA-TCH-SP staining and localization of surface factor 4 antigen demonstrated an altered arrangement of bundles of filaments in the outer wall layers of blastoconidia which were morphologically intact but had abnormal cell wall appearance. Next, there was a reduction in thickness of the outer layer and the expression of surface factor 4 antigen was limited to the cytoplasmic membrane area. Later on, the cell wall was almost uniformly highlighted by PA-TCH-SP staining. These data evinced a highly plastic architecture of the cell wall in C. albicans.
Regulation of leukotoxin in leukotoxic and nonleukotoxic strains of Actinobacillus actinomycetemcomitans
Authors: Spitznagel J , Kraig E , Kolodrubetz D
Published: January 2020

Actinobacillus actinomycetemcomitans is a gram-negative bacterium that has been implicated in the etiology of several forms of periodontitis, especially localized juvenile periodontitis. A potent leukotoxin (Lkt) is produced by most A. actinomycetemcomitans isolates from patients with periodontal disease, but some isolates are leukotoxin nonproducing (Lkt-). The molecular bases for the differences in leukotoxin expression are being explored to clarify the role of leukotoxin in pathogenesis. We have previously cloned the leukotoxin structural gene, lktA, from the leukotoxin-producing (Lkt+) strain JP2 and have shown that it is linked to three other genes, lktB, lktC, and lktD, whose gene products are thought to be required for activation and localization of the leukotoxin. These genes have now been used in Southern blot analysis to demonstrate that Lkt- strains, like Lkt+ strains, contain all four genes of the lkt gene cluster. While restriction fragment length polymorphisms were detected, they did not correlate with toxin phenotype. RNA blot analysis demonstrated that Lkt+ strains produced two transcripts, one 9.3 kb in length and the other 4.3 kb. They encode lktCABD and lktCA. respectively. Lkt- strains contained significantly lower levels of the 4.3-kb transcript with no discernible 9.3-kb message. The leukotoxic activity of the A. actinomycetemcomitans strains, measured by chromium release assays, correlated with the lkt RNA content. Therefore, a major component of leukotoxin regulation is at the level of RNA transcription or stability. Interestingly, the lkt RNAs in JP2 are regulated during growth phase, being greatly reduced in cells approaching stationary phase. Thus, the regulation of lkt RNA can be affected by both genotype and environment.
Hafnia alvei, a probable cause of diarrhea in humans
Authors: Albert M J , Alam K , Islam M , Montanaro J , Rahaman A S , Haider K , Hossain M A , Kibriya A K , Tzipori S
Published: January 2020

Hafnia alvei, a member of the family Enterobacteriaceae, was the only species of bacteria cultured from the stool of a 9-month-old child who was admitted with a 3-day history of watery diarrhea. The isolated strain of H. alvei failed to produce heat-labile or heat-stable enterotoxins or Shiga-like toxin I or II and did not invade HeLa cells, nor did it cause keratoconjunctivitis (determined by the Sereny test) in a guinea pig's eye. The strain, however, induced diarrhea in 8 of 12 adult rabbits with removable intestinal ties (removable intestinal tie-adult rabbit diarrhea [RITARD] assay) and in 1 of 2 orally fed animals. No diarrhea could be induced with Escherichia coli K-12 in eight RITARD assay rabbits and three orally fed rabbits, respectively. Microscopic examination of affected animals revealed moderate inflammatory cellular infiltration of the intestinal mucosa, in which bacterial attachment to the surface epithelium and loss of the microvillus border were evident in the ileum and colon. Electron microscopy demonstrated cellular modifications of the apical surface, with cupping or pedestal formation and increased terminal web density at sites of bacterial "attachment-effacement," a well-known characteristic and mechanism of diarrhea of enteropathogenic E. coli. Identical lesions were also induced by H. alvei in rabbit ileal loops, which ruled out naturally occurring rabbit enteropathogenic E. coli strains, which are known to produce similar lesions. It is concluded that at least some strains of H. alvei have the potential to cause diarrhea and that attachment-effacement is a virulence characteristic shared by bacteria other than E. coli.
Effect of challenge with Candida albicans strains with different levels of virulence on plasma proteins in burned mice
Authors: Neely A N , Childress C M , Holder I A
Published: January 2020

Immunoglobulin G, immunoglobulin A, transferrin, and fibrinogen were measured by radial immunodiffusion in plasma samples from burned versus unburned mice challenged with high-virulence Candida albicans MY 1044 or its low-virulence mutant, MY 1049. Early decreases in these proteins were found after burn and/or MY 1044 but not MY 1049 challenge. These decreases may contribute to increased susceptibility of mice that were burned and challenged by MY 1044 to lethal candidiasis.
Direct sequence evaluation of the major outer membrane protein gene variant regions of Chlamydia trachomatis subtypes D', I', and L2'
Authors: Dean D , Patton M , Stephens R S
Published: January 2020

The nucleotide sequences of variable segments (VS) 1, 2, and 4 for the major outer membrane protein gene (omp1) of Chlamydia trachomatis were determined for serologically defined subtypes D', I', and L2'. Asymmetric DNA amplification was used to produce single-stranded DNA for direct sequencing. Amino acid substitutions were detected in VS1, VS2, and VS4 for I', in VS2 for L2', and in VS4 for D'. DNA sequencing of omp1 variant regions may be an important method for evaluating the molecular epidemiology of Chlamydia spp.
Monoclonal antibodies to surface antigens of a pathogenic Mycoplasma hominis strain
Authors: Olson L D , Shane S W , Karpas A A , Cunningham T M , Probst P S , Barile M F
Published: January 2020

Three monoclonal antibodies (MAbs) were prepared against an arthritogenic strain of Mycoplasma hominis isolated from the joint aspirates of a patient with chronic septic arthritis. Immunoblots of polyacrylamide gel-electrophoresed proteins before and after surface proteolysis showed that the predominant antigenic determinants were on surface-exposed polypeptides. These polypeptides have extensive hydrophobic characteristics, as demonstrated by Triton X-114 phase partitioning. The electrophoresed proteins from cells grown in medium containing [14C]palmitate were blotted onto nitrocellulose which was both reacted with the MAbs and exposed to X-ray film. Superimposable bands on both the immunoblots and the exposed film suggested that the proteins might be acylated. The MAbs were further tested for reactivity with 16 other strains of M. hominis isolated from patients with septic arthritis (1 strain), septicemia (10 strains), or nongonococcal urethritis (1 strain); from the cervix (1 strain), rectum (1 strain), or surgical wound (1 strain) of patients; and from a contaminated cell culture. No single protein was consistently recognized from strain to strain, although a 94-kDa protein from 16 of the 17 strains tested was bound by at least one of the MAbs. The apparent antigenic heterogeneity among strains of M. hominis, including those isolated from the same tissue source and/or from patients with the same type of clinical disease, might be misleading in that all strains express epitopes associated with a discrete number of proteins to which one, two, or all three MAbs bind. The expression of the epitopes on multiple proteins from the same or different strains may reflect a mechanism for generating antigenic diversity.