bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterium tuberculosis Des protein: an immunodominant target for the humoral response of tuberculous patients
Authors: Jackson M , Portnoï D , Catheline D , Dumail L , Rauzier J , Legrand P , Gicquel B
Published: January 2020

The phoA gene fusion methodology permitted the identification of a new Mycobacterium tuberculosis exported protein, Des. This protein has significant sequence similarities to plant acyl-acyl carrier protein desaturases, which are enzymes involved in general fatty acid biosynthesis as well as in mycolic acid biosynthesis in mycobacteria. As shown by Western blot and enzyme-linked immunosorbent assay experiments, the Des protein is a major B-cell antigen that was recognized by all the tuberculous M. tuberculosis- and M. bovis-infected human patients tested.
Protective immunity against Clostridium difficile toxin A induced by oral immunization with a live, attenuated Vibrio cholerae vector strain
Authors: Ryan E T , Butterton J R , Smith R N , Carroll P A , Crean T I , Calderwood S B
Published: January 2020

Clostridium difficile causes pseudomembranous colitis through the action of Rho-modifying proteins, toxins A and B. Antibodies directed against C. difficile toxin A prevent or limit C. difficile-induced colitis. We engineered plasmid pETR14, containing the hlyB and hlyD genes of the Escherichia coli hemolysin operon, to express a fusion protein containing 720 amino acid residues from the nontoxic, receptor-binding, carboxy terminus of C. difficile toxin A and the secretion signal of E. coli hemolysin A. We introduced pETR14 into Vibrio cholerae and found that the toxin A-HlyA fusion protein was secreted by a number of V. cholerae strains and recognized by both monoclonal and polyclonal anti-C. difficile toxin A antibodies. We introduced pETR14 into an attenuated V. cholerae strain, O395-NT, and inoculated rabbits orally with this construct. Colonization studies disclosed that the V. cholerae vector containing pETR14 was recoverable from rabbit ilea up to 5 days after oral inoculation. Vaccination produced significant systemic anti-C. difficile toxin A immunoglobulin G and anti-V. cholerae vibriocidal antibody responses. Vaccination also produced significant protection against toxin A in an ileal loop challenge assay, as assessed by determination of both fluid secretion and histological changes. These results suggest that the hemolysin system of E. coli can be used successfully in V. cholerae vector strains to effect secretion of large heterologous antigens and that a V. cholerae vector strain secreting a nontoxic, immunogenic portion of C. difficile toxin A fused to the secretion signal of E. coli HlyA induces protective systemic and mucosal immunity against this toxin.
FlaA, a putative flagellar outer sheath protein, is not an immunodominant antigen associated with Lyme disease
Authors: Ge Y , Charon N W
Published: January 2020

FlaA was recently found to be associated with flagellar filaments of Borrelia burgdorferi. We tested whether antibodies to this protein are a good indicator of infection, as antibodies to FlaA proteins in other spirochetal infections show an increase in titer. Although overproduction of intact FlaA was highly toxic to Escherichia coli, truncated proteins which lacked the N-terminal signal sequence could be successfully overexpressed. Immunoblotting with sera from mammalian hosts infected with B. burgdorferi indicated that FlaA is not an immunodominant antigen in Lyme disease. However, sera from two patients reacted with both recombinant and native FlaA protein, suggesting that B. burgdorferi FlaA was antigenic and expressed in vivo.
Dipeptidyl-peptidase IV secreted by Aspergillus fumigatus, a fungus pathogenic to humans
Authors: Beauvais A , Monod M , Wyniger J , Debeaupuis J P , Grouzmann E , Brakch N , Svab J , Hovanessian A G , Latgé J P
Published: January 2020

A dipeptidyl-peptidase IV was purified from the culture medium of the human-pathogenic fungus Aspergillus fumigatus. The enzyme has an apparent molecular mass of 95 kDa and contained approximately 10 kDa of N-linked carbohydrate. This glycoprotein is antigenic and has all characteristics of the class IV dipeptidyl-peptidases: removal of Xaa-Pro and to a lesser extent Xaa-Ala dipeptides from the N termini of peptides, including bioactive peptides such as neuropeptide Y, [des-Arg1] bradykinin, and glucagon-like peptide 1, activity at neutral pH, and presence in the amino acid sequence of the Gly-X-Ser-X-Gly consensus motif of the serine-hydrolases and the putative catalytic triad (Ser613, Asp690, His725) of the dipeptidyl-peptidases. Moreover, the last 200 amino acids displayed 60 to 65% similarity with the other dipeptidyl-peptidases IV from rat, mouse, human, and yeast. However, unlike the other dipeptidyl-peptidases, the dipeptidyl-peptidase IV of A. fumigatus is a secreted enzyme with a cleavable signal peptide. Expression of a recombinant dipeptidyl-peptidase IV of A. fumigatus has been attained in the yeast Pichia pastoris.
Attenuation and vaccine potential of aroQ mutants of Corynebacterium pseudotuberculosis
Authors: Simmons C P , Hodgson A L , Strugnell R A
Published: January 2020

Corynebacterium pseudotuberculosis, a gram-positive intracellular bacterial pathogen, is the etiological agent of the disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel live veterinary vaccine vectors. We have cloned and sequenced the aroB and aroQ genes from C. pseudotuberculosis C231. By allelic exchange, aroQ mutants of both C231, designated CS100, and a pld mutant strain TB521, designated CS200, were constructed. Infection of BALB/c mice indicated that introduction of the aroQ mutation into C231 and TB521 attenuated both strains. In sublethally infected BALB/c mice, both CS100 and CS200 were cleared from spleens and livers by day 8 postinfection. The in vivo persistence of these strains was increased when the intact aroQ gene was supplied on a plasmid in trans. Mice infected with TB521 harbored bacteria in organs at least till day 8 postinfection without ill effect. When used as a vaccine, only the maximum tolerated dose of CS100 had the capacity to protect mice from homologous challenge. Vaccination with TB521 also elicited protective immunity, and this was associated with gamma interferon (IFN-gamma) production from splenocytes stimulated 7 days postvaccination. The role of IFN-gamma in controlling primary infections with C. pseudotuberculosis was examined in mice deficient for the IFN-gamma receptor (IFN-gammaR(-/-) mice). IFN-gammaR(-/-) mice cleared an infection with CS100 but were significantly more susceptible than control littermates to infection with C231 or TB521. These studies support an important role for IFN-gamma in control of primary C. pseudotuberculosis infections and indicate that aroQ mutants remain attenuated even in immunocompromised animals. This is the first report of an aroQ mutant of a bacterial pathogen, and the results may have implications for the construction of aromatic mutants of Mycobacterium tuberculosis for use as vaccines.
Contribution of proteases and LasR to the virulence of Pseudomonas aeruginosa during corneal infections
Authors: Preston M J , Seed P C , Toder D S , Iglewski B H , Ohman D E , Gustin J K , Goldberg J B , Pier G B
Published: January 2020

The roles of the Pseudomonas aeruginosa proteases LasB (elastase) and LasA and the transcriptional activator LasR, which regulates the expression of these proteases, were evaluated in a murine model of P. aeruginosa corneal infection. In scarified corneas, P. aeruginosa PAO-A1 (LasA negative) or PAO-B1A1 (LasB and LasA negative) at a dose of 10(8) CFU per eye caused very mild or no disease following infection; however, the defect in PAO-A1 could not be complemented by supplying a functional copy of lasA either on a plasmid or inserted into the chromosome. In contrast, PAO-B1 (LasB negative) colonized the cornea and caused disease equal in severity to disease caused by the parental strain, PAO1-I. Although LasR is a known regulator of lasA expression, PAO-R1, a lasR-negative derivative of PAO1-I, was as virulent as the parental strain during corneal infection. When transcriptional fusion plasmids were used to quantify the expression of the lasB and lasA genes in P. aeruginosa PAO1-I and PAO-R1, the lasB::lacZ fusion in PAO-R1 showed only 3.5% as much activity as it did in PAO1-I, while the activity of the lasA::lacZ fusion in PAO-R1 was 27.8% of that in PAO1-I. Coadministration of 5 microg of purified LasA protease with PAO-A1 did not reconstitute a wild-type infection. This treatment produced an acute toxic reaction leading to prolonged eyelid closure without inflammatory destruction of the cornea that was similar to that observed when LasA was administered alone. These results indicate that insertional inactivation of lasA renders P. aeruginosa avirulent in a murine model of keratitis and that neither LasR nor elastase production is required for the establishment and maintenance of corneal infection. However, the lack of virulence of the LasA-deficient strains cannot be ascribed with certainty to the deficiency of LasA from the available data.
Binding of pulmonary surfactant proteins A and D to Aspergillus fumigatus conidia enhances phagocytosis and killing by human neutrophils and alveolar macrophages
Authors: Madan T , Eggleton P , Kishore U , Strong P , Aggrawal S S , Sarma P U , Reid K B
Published: January 2020

To determine whether the lung surfactant proteins A (SP-A) and D (SP-D) are involved in the initial protective immunity against opportunistic pulmonary fungal infections caused by Aspergillus fumigatus, we performed a series of in vitro functional studies to see if SP-A and SP-D enhanced binding, phagocytosis, activation, and killing of A. fumigatus conidia by human alveolar macrophages and circulating neutrophils. Both SP-A and SP-D bound to carbohydrate structures on A. fumigatus conidia in a calcium-dependent manner. SP-A and SP-D were also chemoattractant and significantly enhanced agglutination and binding of conidia to alveolar macrophages and neutrophils. Furthermore, in the presence of SP-A and SP-D, the phagocytosis, oxidative burst, and killing of A. fumigatus conidia by neutrophils were significantly increased. These findings indicate that SP-A and SP-D may have an important immunological role in the early antifungal defense responses in the lung, through inhibiting infectivity of conidia by agglutination and by enhancing uptake and killing of A. fumigatus by phagocytic cells.
Neisserial porins may provide critical second signals to polysaccharide-activated murine B cells for induction of immunoglobulin secretion
Authors: Snapper C M , Rosas F R , Kehry M R , Mond J J , Wetzler L M
Published: January 2020

Resting B cells stimulated with dextran-conjugated anti-immunoglobulin D (anti-IgD) antibodies (anti-Ig-dex), a model for B-cell activation in response to polysaccharide antigens, proliferate but secrete little if any Ig, unless additional stimuli are present. In order to elucidate the parameters which costimulate T-cell-independent antipolysaccharide antibody responses during bacterial infections, we tested the capacities of highly purified porin proteins from Neisseria meningitidis and Neisseria gonorrhoeae to augment in vitro proliferation and induce Ig secretion by anti-Ig-dex-activated B cells. Resting B cells, from lipopolysaccharide (LPS)-nonresponsive C3H/HeJ mice, proliferated and secreted IgM in response to each of three distinct porins acting alone. Further, porins, even at concentrations that were minimally inductive when acting alone, were strongly synergistic with anti-Ig-dex for proliferation and Ig secretion. Similar synergistic effects of porins with CD40-ligand were also observed. These effects of porins were shown to occur directly at the level of the B cell. The predominant Ig isotype elicited in response to porins plus anti-Ig-dex or CD40-ligand was IgM (>97%), with the remainder comprising IgG. Surprisingly, picogram-per-milliliter amounts of neisserial LPS were also found to be highly synergistic with anti-Ig-dex for induction of IgM secretion by LPS-responsive C3H/HeN, but not C3H/HeJ, B cells. Thus, these data suggest that porins, as well as LPS, may provide critical second signals for T-cell-independent induction of polysaccharide-specific Ig in response to neisserial and other gram-negative porin-expressing bacterial pathogens, without a requirement for the participation of non-B cell types. These data may also help to explain the potent immunopotentiating effects of porins for polysaccharide-specific, as well as protein-specific, humoral responses in vivo.
The baboon as a nonhuman primate model for assessing the effects of maternal immunization with Haemophilus influenzae type b polysaccharide vaccines
Authors: Shearer M H , Lucas A H , Anderson P W , Carey K D , Jenson H B , Chanh T C , Stanley J R , Kennedy R C
Published: January 2020

These studies were performed to assess the utility of the baboon as a nonhuman primate model to evaluate vaccines for use in humans. Specifically, we examined the antibody response of baboons immunized during the third trimester of pregnancy with Haemophilus influenzae type b (Hib) polyribosylribitol phosphate (PRP) conjugate and unconjugated polysaccharide vaccines. Some of the vaccinated mothers failed to respond to a single immunization with unconjugated Hib PRP. Specific Hib PRP immunoglobulin G (IgG) but not IgM antibodies cross the baboon placenta and are detected in the offspring. Higher-titer baboon anti-Hib PRP did not express two previously defined cross-reactive human anti-Hib PRP idiotypes and was biased towards lambda light-chain expression. Spectrotype analysis indicated that baboon anti-Hib PRP was restricted in heterogeneity and oligoclonal.
Activation of host cell protein kinase C by enteropathogenic Escherichia coli
Authors: Crane J K , Oh J S
Published: January 2020

Enteropathogenic Escherichia coli (EPEC) consists of a group of diarrhea-producing E. coli strains, common in developing countries, which do not produce classical toxins and are not truly invasive. EPEC strains adhere to mammalian cells in an intimate fashion, trigger a localized increase in intracellular calcium levels, and elevate inositol phosphate production. We hypothesized that these mediators could activate host cell protein kinase C (PKC) and tested this idea in vitro with two cultured human cell lines, HeLa cells and T84 cells. Using a recently described subculturing protocol to "induce" or accelerate EPEC adherence, we infected the cells with EPEC at a multiplicity of infection of approximately 100:1 for 30 to 60 min. Under these conditions, EPEC E2348 increased membrane-bound PKC activity 1.5- to 2.3-fold in HeLa cells and T84 cells, respectively. The increase in membrane-bound PKC activity was accompanied by a decrease in cytosolic PKC activity in EPEC-infected HeLa cells. Nonadherent laboratory E. coli strains such as HB101 and H.S. failed to trigger any consistent change in PKC production, similar to the nonadherent mutant strains derived from E2348, JPN15 (plasmid cured) and CVD206 (eaeA). In addition, immunoblots performed on extracts of T84 cells with a monoclonal antibody against PKC-alpha showed an increased PKC content in membranes of EPEC-infected cells. Finally, EPEC-infected T84 cells showed a 60% increase in responsiveness to the E. coli heat-stable toxin. We conclude that mediators produced in response to EPEC adherence activate PKC in intestinal and nonintestinal cells.
Identification of a Coccidioides immitis antigen 2 domain that expresses B-cell-reactive epitopes
Authors: Zhu Y , Tryon V , Magee D M , Cox R A
Published: January 2020

Antigen 2 (Ag2), a major immunoreactive component of Coccidioides immitis mycelium- and spherule-phase cell walls, was recently cloned in our laboratory and was shown to elicit T-cell responses in Coccidioides-immune mice. In this investigation, we evaluated recombinant Ag2 (rAg2) and PCR-generated Ag2 truncations for expression of B-cell-reactive epitopes in enzyme-linked immunosorbent and immunoblot assays with sera from patients with active coccidioidomycosis, a hyperimmune goat anti-Ag2 serum, and a murine anti-Ag2 monoclonal antibody that recognizes a conformational epitope. The results established that rAg2 expresses both linear and conformational B-cell-reactive epitopes which are localized to a domain comprised of amino acids 19 through 96 (designated A19-96). Truncations designed to identify epitopes within the A19-96 domain yielded fragments that either were nonreactive (A62-194, A19-61, and A49-79) or showed reduced reactivity (A19-79). Hence, A19-96 was the shortest domain expressing epitopes recognized by the panel of antibodies. The prevalence of antibodies to the A19-96 domain was evaluated in enzyme-linked immunosorbent assays of sera from 28 coccidioidomycosis patients. Antibody reactivity was detected in 79% of the patients' sera, and the level of antibody reactivity was directly correlated with disease severity. Whereas patients with pulmonary disease showed a mean response (A405) of 0.16 +/- 0.04, patients with disseminated coccidioidomycosis showed a mean response of 0.69 +/- 0.17 (P < 0.05). No reactivity was detected with sera from histoplasmosis or blastomycosis patients. The production of a recombinant peptide that expresses C. immitis-specific Ag2 epitopes provides a useful reagent for examining the role of anti-Ag2 antibodies in coccidioidomycosis.
Monophosphoryl lipid A enhances both humoral and cell-mediated immune responses to DNA vaccination against human immunodeficiency virus type 1
Authors: Sasaki S , Tsuji T , Hamajima K , Fukushima J , Ishii N , Kaneko T , Xin K Q , Mohri H , Aoki I , Okubo T , Nishioka K , Okuda K
Published: January 2020

To enhance immunity induced by DNA vaccination against human immunodeficiency virus type 1 (HIV-1), we evaluated the efficacy of monophosphoryl lipid A (MPL), an adjuvant of bacterial origin. BALB/c mice were intramuscularly injected with immunogenic DNA, encoding the env and rev genes of the HIV-1(IIIB) strain, formulated with MPL dissolved in different vehicles (MPL in stable emulsion and MPL in aqueous formulation). The sera from mice immunized with the two preparations of MPL revealed 2(6) to 2(9) times higher HIV-1-specific immunoglobulin G (IgG) titers than the sera from mice immunized without MPL. In virus neutralization tests for HIV-1(IIIB), by p24 assay and antifusion assay of infected MOLT-4 cells, MPL tends to elicit antibody more protective than antibody elicited without adjuvant. MPL also elicited stronger delayed-type hypersensitivity and cytotoxic-T-lymphocyte activity against HIV-1(IIIB) compared to DNA alone. HIV-1-specific IgG subclass analysis showed that MPL tends to facilitate IgG2a production, suggesting enhancement of a predominant T-helper-type-1 response, and this enhancement may help to facilitate protective-antibody induction. Furthermore, a chloramphenicol acetyltransferase (CAT) assay was employed to determine whether MPL affected the gene expression process. Interestingly, both MPL preparations reduced CAT activity in the muscle injected with CAT expression vector but increased anti-CAT antibody production. These results indicate that MPL acts as an effective adjuvant for immunogenic DNA injection despite reduced expression of encoding protein in muscle. We conclude that MPL has a strong adjuvant effect on DNA vaccination against HIV-1.
Necrosis versus apoptosis as the mechanism of target cell death induced by Entamoeba histolytica
Authors: Berninghausen O , Leippe M
Published: January 2020

The human pathogen Entamoeba histolytica is known to kill a variety of host cells, including leukocytes. Using human myeloid cells as targets, we studied whether cytotoxicity of amoebic trophozoites in vitro is equivalent to the induction of apoptosis or whether these target cells die via necrosis. Based upon morphological criteria, incubation of target cells with amoebae resulted in necrosis, with cell swelling, rupture of plasma membrane, and release of cell contents including nucleic acids being detected by light and transmission electron microscopy. On the other hand, the characteristic features of apoptosis such as cell shrinking, surface blebbing, and chromatin condensation were not observed. Moreover, internucleosomal fragmentation of genomic DNA within target cells as a characteristic feature of apoptotic cell death did not occur as judged by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique in combination with flow cytometry. Consistently, cleavage of DNA was detectable upon agarose gel electrophoresis only after a substantial part of the target cell population had already been lysed. We also analyzed the mechanism of cell death induced by amoebapores, pore-forming peptides and primary candidate molecules for mediating the cytolytic activity of E. histolytica. At a time point at which the majority of target cells showed membrane injury upon incubation with purified amoebapores, no DNA degradation was detectable in the victim cells. The data suggest that the target cells used in our study undergo necrosis rather than apoptosis when they are killed by viable trophozoites as well as by isolated amoebapores.
Role of adherence in interleukin-8 induction in Helicobacter pylori-associated gastritis
Authors: Rieder G , Hatz R A , Moran A P , Walz A , Stolte M , Enders G
Published: January 2020

Active Helicobacter pylori-associated gastritis is characterized by a dense mucosal infiltration with granulocytes. Since H. pylori is noninvasive, secondary signals must induce the accumulation of granulocytes. Interleukin-8 (IL-8) has been shown to play a key role in this event. Using competitive reverse transcriptase-PCR on mRNA from gastric biopsies, we could show a clear correlation between the amount of IL-8 transcripts and the activity of H. pylori gastritis. Due to the inability of the bacterium to invade host cells, the epithelial layer is a potential candidate as an IL-8 source. To study the mechanism of IL-8 induction, established gastric carcinoma epithelial cell lines (AGS and Kato III) and well-defined H. pylori strains were used in a modified in vitro system. The experimental design enabled us to prevent direct contact of bacteria with epithelial cells by use of a filter membrane which did not block secreted bacterial products crossing the membrane. The data clearly showed that the direct contact of the bacterial cell with the epithelial cell is necessary for optimal IL-8 production because not only live bacteria, but also metabolically inactive bacteria, increased IL-8 secretion. Neither purified lipopolysaccharide nor water-soluble protein fractions of H. pylori NCTC 11637 and Tx30a nor the cytotoxin of H. pylori was able to increase IL-8 production significantly by the epithelial cells used. Furthermore, preparations of total membrane and outer membrane proteins of H. pylori were not able to stimulate IL-8 release in vitro. Accumulatively, these results imply that active metabolism is not necessary for stimulation as long as there is an intact membrane aiding the presentation of a stimulating membrane complex or aggregate on the surface of the bacteria. From these results, we conclude that whole bacteria and their direct contact with epithelial cells may be critical for IL-8 induction in vivo.
Baculovirus-mediated expression of Plasmodium falciparum erythrocyte binding antigen 175 polypeptides and their recognition by human antibodies
Authors: Daugherty J R , Murphy C I , Doros-Richert L A , Barbosa A , Kashala L O , Ballou W R , Snellings N J , Ockenhouse C F , Lanar D E
Published: January 2020

The erythrocyte binding antigen EBA-175 is a 175-kDa Plasmodium falciparum protein which mediates merozoite invasion of erythrocytes in a sialic acid-dependent manner. The purpose of this study was to produce recombinant EBA-175 polypeptide domains which have previously been identified as being involved in the interaction of EBA-175 with erythrocytes and to determine whether these polypeptides are recognized by malaria-specific antibodies. The eba-175 gene was cloned by PCR from genomic DNA isolated from the 3D7 strain of P. falciparum. The predicted protein sequence was highly conserved with that predicted from the published eba-175 gene sequences from the Camp and FCR-3 strains of P. falciparum and contained the F segment divergent region. Purified recombinant EBA-175 polypeptide fragments, expressed as glutathione S-transferase fusion proteins in insect cells by using the baculovirus system, were recognized by antibodies present in serum from a drug-cured, malaria-immune Aotus nancymai monkey. The fusion proteins were also recognized by antibodies present in sera from individuals residing in areas where malaria is endemic. In both cases the antibodies specifically recognized the EBA-175 polypeptide portion of the fusion proteins. Antibodies raised in rabbits immunized with the recombinant fusion proteins recognized parasite proteins present in schizont-infected erythrocytes. Our results suggest that these regions of the EBA-175 protein are targets for the immune response against malaria and support their further study as possible vaccine components.
Binding of Pasteurella haemolytica leukotoxin to bovine leukocytes
Authors: Brown J F , Leite F , Czuprynski C J
Published: January 2020

Pasteurella haemolytica is the principal bacterial pathogen in the bovine respiratory disease complex. This organism produces an exotoxin (referred to as leukotoxin) during logarithmic-phase growth that is a potent leukocyte-modulating agent. At low concentrations, it activates neutrophils and mononuclear phagocytes to release inflammatory mediators, while at the same time making these cells destined to undergo apoptotic cell death. At higher concentrations, the toxin causes rapid swelling and loss of cell viability. In this study, we demonstrated that toxin binding can be directly evaluated by flow cytometry with biologically active biotinylated leukotoxin. Leukotoxin binding was blocked by the addition of a neutralizing anti-leukotoxin monoclonal antibody and was not detected when bovine leukocytes were incubated with culture filtrates from a mutant strain of P. haemolytica that does not produce biologically active leukotoxin. In addition, treatment of bovine leukocytes with protease K eliminated subsequent binding of leukotoxin, suggesting that there is a protein on the leukocyte surface that is either a leukotoxin binding site or is required for stabilization of leukotoxin binding. We did not detect binding of biotinylated leukotoxin to porcine or human leukocytes, which have been reported previously to be resistant to the lytic effects of the leukotoxin. These findings suggest that there may be a specific binding site for P. haemolytica leukotoxin on bovine but not on porcine or human leukocytes and that it might be involved in the activation and lytic activities of the leukotoxin.
A proposed role for the lutropin receptor in contact-inducible gonococcal invasion of Hec1B cells
Authors: Spence J M , Chen J C , Clark V L
Published: January 2020

We previously reported the existence of a contact-inducible, enhanced invasion phenotype in the obligate human pathogen Neisseria gonorrhoeae. Our present studies showed that the ability of glutaraldehyde-fixed eucaryotic cells to convert gonococci (GC) to this invasive phenotype (Inv+) is limited to cells derived from reproductive tissues. We present evidence that GC recognize the lutropin receptor (LHr), which recognizes both luteinizing hormone and human chorionic gonadotropin (hCG), as the tissue-specific environmental signal that induces the conversion of GC to the Inv+ phenotype. By competitive binding studies, we showed that Inv+ GC bind to Hec1B cells, a human endometrial cell line, by a unique adhesin not present on noninduced GC and that this Inv+ GC-specific binding is completely blocked by the addition of hCG. We demonstrated that limiting the access of GC to LHr decreases the ability of the host cell to both convert GC to the Inv+ phenotype and serve as a target for Inv+ GC invasion. We propose a model of GC invasion of Hec1B cells in which the LHr plays a dual role both as an induction signal and as part of the internalization mechanism. This utilization of LHr could account for both the preponderance of complicated GC disease in women and the observed correlation of the disease with the onset of menses.
Identification of a Streptococcus gordonii SspB domain that mediates adhesion to Porphyromonas gingivalis
Authors: Brooks W , Demuth D R , Gil S , Lamont R J
Published: January 2020

Porphyromonas gingivalis, a primary pathogen in adult periodontitis, may establish itself in the oral cavity by adhering to early plaque bacteria such as Streptococcus gordonii. Our previous studies (R. J. Lamont et al., Microbiology 140:867-872, 1994) suggested that this interaction is mediated by the SspB polypeptide, a member of the antigen I/II family of streptococcal surface proteins. S. gordonii was recently shown to express a second Ssp polypeptide (SspA) that resembles SspB and the structurally homologous antigen I/II polypeptide (Pac) of Streptococcus mutans. To determine if all of these related antigen I/II proteins interacted with P. gingivalis, SspA, SspB, and Pac were tested for adhesion to P. gingivalis cells. Both of the S. gordonii Ssp proteins bound labeled target cells, whereas the S. mutans Pac polypeptide did not, suggesting that antigen I/II-mediated binding of P. gingivalis by streptococci may be species specific. To investigate the molecular basis for this functional difference, the P. gingivalis binding domain of SspB was mapped. The binding properties of a family of truncated SspB polypeptides lacking C-terminal sequences were determined. In addition, the lack of binding activity exhibited by the Pac protein was exploited to construct and analyze chimeric SspB-Pac polypeptides. Both approaches revealed that the region defined by residues 1167 to 1250 of SspB was essential for P. gingivalis binding. This region of SspA and SspB is entirely conserved, consistent with the binding properties determined for these proteins. However, the corresponding region of Pac differs in both the primary sequence and predicted secondary structure, suggesting that the overall structure of this domain may define its functional activity.
Shiga toxin-producing Escherichia coli isolates from cases of human disease show enhanced adherence to intestinal epithelial (Henle 407) cells
Authors: Paton A W , Voss E , Manning P A , Paton J C
Published: January 2020

Shiga toxin-producing Escherichia coli (STEC) strains are a diverse group of organisms which are known to cause diarrhea and hemorrhagic colitis in humans. We have recently described a large food-borne outbreak of STEC disease caused by contaminated semidry fermented sausage (A. W. Paton, R. Ratcliff, R. M. Doyle, J. Seymour-Murray, D. Davos, J. A. Lanser, and J. C. Paton, J. Clin. Microbiol. 34:1622-1627, 1996). STEC strains belonging to several O serotypes were isolated from the contaminated food source, but of these, only a subset were isolated from patients with diarrhea or hemolytic-uremic syndrome (HUS). In the present study, we characterized these STEC isolates with respect to the presence of putative virulence-associated genes and the capacity to adhere to a human intestinal epithelial cell line (Henle 407). The O111:H- STEC strain 95NR1 (isolated from one of the outbreak HUS patients) was shown to adhere to Henle 407 cells in a dose-dependent, mannose-resistant fashion. Microscopic examination revealed a diffuse pattern of adherence for this as well as several other STEC strains. Interestingly, the adherence of STEC strains from HUS cases (both outbreak related and sporadic) was significantly greater than that of STEC strains found in the contaminated food source but not found in any patients. These studies support the hypothesis that an enhanced capacity to adhere to intestinal cells is one of the factors which distinguishes human-virulent STEC strains from those of lesser clinical significance.
Streptococcal mitogenic exotoxin Z, a novel acidic superantigenic toxin produced by a T1 strain of Streptococcus pyogenes
Authors: Kamezawa Y , Nakahara T , Nakano S , Abe Y , Nozaki-Renard J , Isono T
Published: January 2020

Streptococcus pyogenes T1 was previously found to produce an acidic mitogenic exotoxin, designated A beta, antigenically distinct from erythrogenic toxin type A (ETA) of strains T1 and NY5. Following chemical analysis and biological characterization, we have renamed this toxin streptococcal mitogenic exotoxin Z (SMEZ). Physicochemical separation of SMEZ from ETA was successfully performed on a hydrophobic chromatograph. The isoelectric point was pH 5.3, and the molecular size was estimated to be 28 kDa. These values were similar to those of ETA, but the amino acid composition and the NH2-terminal sequence of SMEZ were distinct from those of any mitogenic exotoxins hitherto described. Its mitogenic activity was found to be more potent than that of ETA in rabbit lymphocyte cultures. A specific antiserum raised against SMEZ did not cross-react with ETA, ETB, or ETC in the neutralization tests of mitogenic and erythrogenic activities. Its superantigenic nature was evident from the reverse transcriptase PCR findings of the T-cell receptor Vbeta profiles of rabbit lymphocytes stimulated in vitro. The Vbeta 8 subfamily was unique to SMEZ, while the Vbeta 2 and 6 subfamilies were found to be common among lymphocytes stimulated with ETA, ETB, ETC, or SMEZ. The results from this study provide an additional example of the diversity that exists among mitogenic or superantigenic exotoxins of streptococcal origin.
Expression of a Schistosoma mansoni 28-kilodalton glutathione S-transferase in the livers of transgenic mice and its effect on parasite infection
Authors: Xu X , Lemaire C , Grzych J M , Pierce R J , Raccurt M , Mullier F , Zerimech F , Decavel J P , Peyrol S , Liu J , Fontaine J , Lafitte S , Capron A , Cesbron J Y
Published: January 2020

Schistosomiasis is a debilitating tropical disease for which an effective vaccine is needed. A 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) has been shown to induce protective immunity. Sm28GST possesses significant sequence identity to mammalian GST isoforms. In order to study self-reactivity in mice immunized with Sm28GST and the concomitant phenomena of immune tolerance and epitope suppression, as well as their consequences for the protective immunity induced by this vaccination, we developed transgenic (Tg) mice that express Sm28GST under the control of a part of the mouse transferrin gene promoter. A study of (P28)Tg mice showed that the expression of Sm28GST was strictly localized in pericentrolobular hepatocytes. No histological change, inflammatory infiltrates, or modification of seric L-aspartate: 2-oxoglutarate aminotransferase concentration was observed over an 18-month period, despite a cross-reactivity between Sm28GST and a mouse molecule of 30 kDa. The immunoglobulin G anti-Sm28GST response of (P28)Tg mice immunized with recombinant Sm28GST was lower (P < 0.001) than that observed in non-(P28)Tg littermates and inversely proportional of Sm28GST liver expression. The response of non-(P28)Tg mouse spleen cells to Sm28GST stimulation was greater (P < 0.01) than that observed with (P28)Tg mouse spleen cells. (P28)Tg mice infected with 40 S. mansoni furcocercariae harbored more worms (P < 0.05) than did non-(P28)Tg control mice. The increase in the level of infection in (P28)Tg mice was reflected in concomitant increases in the numbers of adult worms and schistosome eggs found in livers and intestines after whole-body perfusion at 56 days postinfection, but no relative increase in the fertility of individual female worms was observed. The results obtained argue for the involvement of Sm28GST in reducing levels of infection and support the view that this enzyme has a central role in the maintenance of parasite viability, at least during its migration through host tissues.
Circulating promyelocytes and low levels of CD16 expression on polymorphonuclear leukocytes accompany early-onset periodontitis
Authors: Nemoto E , Nakamura M , Shoji S , Horiuchi H
Published: January 2020

Early-onset periodontitis (EOP) is characterized by rapidly progressive alveolar bone loss, chemotactic defects of neutrophils, and significant familial aggregation. We found immature myeloid lineage cells, defined as promyelocytes, in the peripheral blood in patients with EOP. A hematological examination of peripheral blood cells showed normal reference values regarding cell proportions. Flow cytometry revealed significantly lower expression of CD16, a glycosylphosphatidylinositol (GPI)-anchored protein, on peripheral neutrophils in patients compared with those in age- and sex-matched healthy controls, whereas the levels of CD11a and CD11b expression were similar. The chemotactic response of neutrophils was lower toward not only formyl-methionyl-leucyl-phenylalanine but also complement fragment C5a than that of healthy controls. The expression of another GPI-anchored protein, CD14, was equally expressed by controls and patients. Therefore, the low level of CD16 expression was not due to the incomplete synthesis of the GPI anchor. GPI anchors of CD16 on neutrophils from controls and patients were both partially resistant to phosphatidylinositol-specific phospholipase C. The presence of promyelocytes in peripheral blood, low expression of CD16, and low chemotactic response of neutrophils suggest that patients with EOP have an abnormal maturation system in myeloid lineage cells in the bone marrow, which may be associated with the onset and course of EOP.
Giardia lamblia infections in B-cell-deficient transgenic mice
Authors: Stager S , Muller N
Published: January 2020

In the present study, we infected B-cell (and antibody-)-deficient transgenic mice with the Giardia lamblia clone GS/M-83-H7. These animals were inhibited in intestinal anti-Giardia immunoglobulin A (IgA) production and could not resolve the parasite infection, and antigenic diversification within the respective parasite populations occurred in an unusually slow manner. These findings indicate an important immunological function of local IgA antibodies which promotes antigenic variation of the parasite and is involved in control of the parasite infection.
A human monoclonal antibody which inhibits the coaggregation activity of Porphyromonas gingivalis
Authors: Abiko Y , Ogura N , Matsuda U , Yanagi K , Takiguchi H
Published: January 2020

A B-cell line producing a human monoclonal antibody (HuMAb) against a recombinant 40-kDa outer membrane protein (OMP) of Porphyromonas gingivalis was constructed by in vivo immunization of a severe combined immunodeficiency C.B.-17/Icr mouse, which had been injected with human peripheral blood lymphocytes, with recombinant 40-kDa OMP and subsequent Epstein-Barr virus immortalization of B cells isolated from the spleen of the mouse. This HuMAb inhibited coaggregation between P. gingivalis vesicles and Actinomyces naeslundii cells.
Characterization of Neisseria meningitidis by polymerase chain reaction and restriction endonuclease digestion of the porA gene
Authors: Kertesz D A , Byrne S K , Chow A W
Published: January 2020

Subtype classification based on the use of monoclonal antibodies to the class 1 outer membrane protein combined with techniques such as multilocus enzyme electrophoresis remains the standard method of characterizing isolates during outbreaks of invasive meningococcal disease. We developed a rapid typing method based on the restriction fragment length polymorphisms (RFLPs) within the polymerase chain reaction (PCR) product of the porA gene, which encodes the class 1 outer membrane protein, reflecting genotypic rather than phenotypic variability between strains. Forty-five isolates of invasive Neisseria meningitidis obtained from October 1990 to April 1992 were studied after randomization and coding. Included among these were isolates from a local outbreak that resulted in a mass vaccination program. PCR amplification for each isolate was followed by restriction digestion with the following enzymes in the indicated sequence: HaeIII, RsaI, HinfI, HpaII, and AluI. Eighteen different patterns were demonstrated on the basis of RFLPs, whereas only seven groups were identified after standard subtyping. The most common isolate identified by serosubtyping was serogroup C, serotype 2a, subtype P1.2 (C:2a:P1.2) (38%). Thirteen (76%) of these group C isolates shared a common RFLP pattern after digestion with the five restriction enzymes. We were able to further differentiate strains of C:2a:P1.2 with electrophoretic type 5 from electrophoretic types 1, 9, and 15 that occurred during an apparent outbreak. We were also able to characterize 15 isolates (33%) which could not be subtyped with monoclonal antibodies. Our method offers a convenient alternative to standard subtyping procedures and is particularly useful in outbreak situations in which rapid characterization of N. meningitidis is essential so that rational public health policy regarding preventative measures can be formulated.
Isolation and characterization of "Flexispira rappini" from laboratory mice
Authors: Schauer D B , Ghori N , Falkow S
Published: January 2020

A bacterium with an unusual ultrastructure and possessing a fusiform protoplasmic cylinder, spiral periplasmic fibers, and bipolar tufts of sheathed flagella was identified in the intestinal mucosae of laboratory mice. The organism was cultured under microaerophilic conditions and was found to rapidly hydrolyze urea. On the basis of 16S rRNA gene sequence analysis, the organism was shown to be "Flexispira rappini." "F. rappini" is closely related to members of the genus Helicobacter and has been reported to be associated with human gastroenteritis and ovine abortion. "F. rappini" has not previously been observed in the gastrointestinal tracts of mice.
Cultivation of cilia-associated respiratory bacillus in artificial medium and determination of the 16S rRNA gene sequence
Authors: Schoeb T R , Dybvig K , Davidson M K , Davis J K
Published: January 2020

Cilia-associated respiratory (CAR) bacillus, an unclassified gliding bacterium associated with respiratory disease in rats, mice, and rabbits, has previously been cultivated only in embryonated chicken eggs, cell culture, or cell culture medium supplemented with conditioned medium from cultured tracheas. A reference strain of CAR bacillus, originally isolated in eggs, grew in cell culture flasks as adherent individual bacilli and ropy, whorled fascicles in cell culture media supplemented only with fetal calf serum. Using Dulbecco's minimal essential medium, we isolated CAR bacillus from naturally infected rats and a naturally infected rabbit and from experimentally inoculated mice and rats. Isolates were maintained for up to 20 passages. Isolates from rats were similar in morphology to the reference strain, but most were more actively motile and formed pincushion-like aggregates. The rabbit bacilli were smaller and formed fewer aggregates. DNAs of rat isolates differed only slightly in restriction fragment patterns from that of the reference strain, whereas that of the rabbit isolate was distinctly different. Cultures of CAR bacilli of all strains from rats contained Mycoplasma fermentans, Mycoplasma pulmonis, or both, and cultures of the CAR bacillus from the rabbit contained an unidentified arginine-utilizing mycoplasma. The sequence of the 16S rRNA gene of the reference strain was determined by amplification by polymerase chain reaction, cloning of the product, and sequencing by the dideoxynucleotide chain termination method. Comparison of the sequence with sequences in the GenBank data base indicated that CAR bacillus is a unique organism most closely related to Flavobacterium ferrugineum and Flexibacter sancti.
Multilocus enzyme typing of Cryptococcus neoformans
Authors: Brandt M E , Bragg S L , Pinner R W
Published: January 2020

Multilocus enzyme electrophoresis was adapted for subtyping Cryptococcus neoformans. The two cryptococcal varieties were clearly distinguishable. Isolates of the C. neoformans var. neoformans were sorted according to serotype and were sorted into four to five subtypes within each serotype. Nearly no two isolates of the C. neoformans var. gattii displayed the same enzyme electrophoretic type. This method may be a useful adjunct to current methods for classification and epidemiologic studies of cryptococci.
Interpretive accuracy of the disk diffusion method for testing newer orally administered cephalosporins against Morganella morganii
Authors: Biedenbach D J , Jones R N , Erwin M E
Published: January 2020

Eight newer orally administered cephems (cefdinir, cefetamet, cefixime, cefpodoxime, cefprozil, ceftibuten, cefuroxime, and loracarbef) were tested against 100 clinical strains of Morganella morganii to determine the extent of serious interpretive very major (false-susceptible) errors when current criteria for the disk diffusion test are applied. Agar dilution MICs and disk diffusion tests were performed as recommended by the National Committee for Clinical Laboratory Standards (Villanova, Pa.) (NCCLS), and the methods were compared by regression analysis using the method of least squares and by error rate bounding. The following results are listed in the order of increasing error rates: cefdinir, loracarbef, and cefprozil, < or = 1% very major error; ceftibuten, 8% minor errors; cefuroxime, 21% minor errors; cefixime, cefpodoxime, and cefetamet, very major errors of 15, 24, and 36%, respectively. M. morganii produces unacceptable rates of test error with cefuroxime, cefixime, cefpodoxime, and cefetamet. The latter two cephalosporins currently have NCCLS table footnote warnings covering the problem observed with this organism. The inclusion of cefuroxime and cefixime in the NCCLS table footnote is strongly recommended.
Reliable confirmation of antibodies to bovine respiratory syncytial virus (BRSV) by enzyme-linked immunosorbent assay using BRSV nucleocapsid protein expressed in insect cells
Authors: Samal S K , Pastey M K , McPhillips T , Carmel D K , Mohanty S B
Published: January 2020

The nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) in the baculovirus expression system was evaluated as a source of antigen in an enzyme-linked immunosorbent assay (ELISA) for the detection of respiratory syncytial virus (RSV) antibodies. The recombinant N protein was purified from infected-cell extracts by sucrose gradient centrifugation and used in the ELISA for the detection of antibodies to various RSV strains. The ELISA was compared with the virus neutralization (VN) test for determining BRSV antibodies in 10 consecutive serum samples from four calves vaccinated with a live modified BRSV vaccine and from two nonvaccinated control calves. The ELISA compared favorably with the VN test for detecting serological responses. All serum samples which were positive in the VN test were also positive in the ELISA. None of the serum samples collected from the two nonvaccinated calves reacted in the ELISA. To determine the usefulness of the ELISA for epidemiological studies, 58 cattle serum samples were tested in the ELISA and the VN test. Approximately 94% (42 of 45) of field serum samples which were positive in the ELISA were also positive in the VN test. No case was found in which the ELISA result was negative and the VN test result was positive. Thirteen of the serum samples were negative in both methods. Our results indicate that the ELISA with the baculovirus-expressed N protein as an antigen is an efficient, sensitive, and specific method for detecting serum antibodies to RSV.