bluuebunny/crossref_metadata_2025 huggingface dataset:
Neisseria gonorrhoeae utilizes and enhances the biosynthesis of the asialoglycoprotein receptor expressed on the surface of the hepatic HepG2 cell line
Authors: Porat N , Apicella M A , Blake M S
Published: January 2020

One of the lipooligosaccharide (LOS) structures of Neisseria gonorrhoeae contains a terminal Gal(beta 1-4)GlcNAc residue which is a good candidate to serve as a ligand for human asialoglycoprotein receptors (ASGP-R). These receptors have been shown to be present on macrophages, sperm cells, and hepatocytes. The human tissue culture cell line used most often to study this receptor, HepG2, was used in our investigations only as a model. We also chose N. gonorrhoeae 1291 for these studies because, unlike many other gonococcal strains, this strain expresses one main species of LOS. The LOS structure expressed by this strain has also been fully characterized. Using well-established assays for the utilization of the ASGP-R, we found that incubation of HepG2 cells with gonococci expressing the terminal Gal(beta 1-4)GlcNAc asialo-LOS carbohydrate structure competitively inhibited the ASGP-R from binding to one of its well-known ligands, asialo-alpha-acid-1-glycoprotein. The inhibition was specific to the ASGP-R, since binding of two other ligands to their specific receptors in the same model cell system was not affected. Immunoblot analysis for the ASGP-R suggested that gonococci seemed to stimulate the HepG2 cells to increase the expression of the major (46-kDa) receptor species. This observation was confirmed both by functional analysis, which showed that the concentration of total receptor molecules, as well as surface receptors, was about 60% higher after incubation with gonococci than in control cells and by Northern (RNA) blot analysis using a cDNA probe of the major human H1 subunit. Poly(A) RNA purified from control and HepG2 cells exposed to gonococci indicated the presence of increased amounts of mRNA coding for the ASGP-R after incubation with gonococci. This result supports the idea that the molecular mechanism controlling the receptor level after gonococcal exposure is under transcriptional regulation.
Borrelia burgdorferi mutant lacking Osp: biological and immunological characterization
Authors: Sadziene A , Thomas D D , Barbour A G
Published: January 2020

All Borrelia burgdorferi sensu lato isolates characterized to date have one or a combination of several major outer surface proteins (Osps). Mutants of B. burgdorferi lacking Osps were selected with polyclonal or monoclonal antibodies at a frequency of 10(-6) to 10(-5). One mutant that lacked OspA, -B, -C, and -D was further characterized. It was distinguished from the OspA+B+ cells by its (i) autoaggregation and slower growth rate, (ii) decreased plating efficiency on solid medium, (iii) serum and complement sensitivity, and (iv) diminished capacity to adhere to human umbilical vein endothelial cells. The Osp-less mutant was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant survived in mouse skin for the same duration as wild-type cells. Polyclonal mouse serum raised against Osp-less cells inhibited growth of the mutant but not of wild-type cells, an indication that other antigens are present on the surface of the Osp-less mutant. Two types of monoclonal antibodies (MAbs) with growth-inhibiting properties for mutant cells were identified. The first type bound to a 13-kDa surface protein of B. burgdorferi sensu stricto and of B. afzelii. The MIC of the Fab fragment of one MAb of this type was 0.2 micrograms/ml. The second type of MAb to the Osp-less mutant did not bind to B. burgdorferi components by Western blotting (immunoblotting) but did not bind to unfixed, viable cells in immunofluorescence and growth inhibition assays. These studies revealed possible functions Osp proteins in borrelias, specifically serum resistance, and indicated that in the absence of Osp proteins, other antigens are expressed or become accessible at the cell surface.
Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity
Authors: Dickinson B L , Clements J D
Published: January 2020

The heat-labile enterotoxin (LT) of Escherichia coli is immunologically and physiochemically related to cholera enterotoxin. A number of studies have been performed to determine the relationship of the ADP-ribosylating enzymatic activity of these enterotoxins to toxicity and adjuvanticity. These studies have generally examined the effect of abolishing the ADP-ribosyltransferase activity of A1 by a variety of chemical or genetic manipulations. In every case, loss of enzymatic activity was associated with loss of biological activity and also with the ability of the molecules to function as oral adjuvants. Consequently, we explored an alternate approach to detoxification of LT without altering its adjuvanticity. Specifically, we generated a novel mutant form of LT by genetic modification of the proteolytically sensitive residues that join the A1 and A2 components of the A subunit. This mutant contains a single amino acid substitution within the disulfide subtended region joining A1 and A2. This mutant toxin, designated LT(R192G), is not sensitive to proteolytic activation, has negligible activity on mouse Y-1 adrenal tumor cells, and is devoid of ADP-ribosyltransferase activity. Nonetheless, LT(R192G) retains the ability to function as a mucosal adjuvant, increasing the serum immunoglobulin G (IgG) and mucosal IgA responses to coadministered antigen (OVA) beyond that achieved with administration of that antigen alone. Further, LT(R192G) prevented the induction of tolerance to coadministered antigen and did not induce tolerance against itself, as demonstrated by the presence of significant serum anti-LT IgG and mucosal anti-LT IgA antibodies in immunized mice.
A plasmid-encoded regulatory region activates chromosomal eaeA expression in enteropathogenic Escherichia coli
Authors: Gómez-Duarte O G , Kaper J B
Published: January 2020

Enteropathogenic Escherichia coli (EPEC) organisms produce a characteristic histopathology in intestinal epithelial cells called attaching and effacing lesions. The eaeA gene is associated with attaching and effacing lesions and encodes intimin, a 94-kDa outer membrane protein. A 60-MDa plasmid, pMAR2, is essential for full virulence of EPEC strain E2348/69 (O127:H6). We have cloned sequences from pMAR2 that increase expression of the chromosomal eaeA gene as shown by increased alkaline phosphatase activity of an eaeA::TnphoA gene fusion, increased expression of the intimin protein, and increased production of eaeA mRNA. These sequences are called per for plasmid-encoded regulator. pMAR2-cured JPN15 containing cloned per sequences adheres to HEp-2 cells in greater numbers than JPN15 carrying the plasmid vector only. The cloned per sequences contain four open reading frames (ORFs) which have been designated perA through perD. Only perC can by itself activate expression of eaeA::TnphoA, although the levels of alkaline phosphatase activity seen with this ORF alone are considerably lower than those seen when all four ORFs are present. The molecular sizes of polypeptides predicted from perA, perB, perC, and perD ORFs are 24, 14.8, 10.5, and 9.4 kDa, respectively. The PerA predicted protein shares homology with members of the AraC family of bacterial regulators, but PerB, PerC, and PerD have no striking homology with previously described prokaryotic proteins. Our studies indicate that plasmid-encoded factors regulate the expression of eaeA and possibly genes encoding other outer membrane proteins and may be important for virulence of EPEC.
Pseudomonas aeruginosa exoenzyme S induces proliferation of human T lymphocytes
Authors: Mody C H , Buser D E , Syme R M , Woods D E
Published: January 2020

Pseudomonas aeruginosa is a gram-negative bacterium that is responsible for devastating acute and chronic infections, which include bronchiectasis in cystic fibrosis, nosocomial pneumonia, and infection of burn wounds. Previous studies have demonstrated that these patients have impaired host responses, including cell-mediated immune responses, which are important in anti-Pseudomonas host defense. The P. aeruginosa exoproduct, exoenzyme S, has a number of characteristics which suggest that it might be important in cell-mediated immunity. To determine whether exoenzyme S activates lymphocytes to proliferate, peripheral blood mononuclear cells (PBMC) from normal volunteers were stimulated with purified exoenzyme S, and the lymphocyte response was assessed by measuring [3H]thymidine uptake and by counting the number of cells after various times in culture. Ninety-five percent of healthy adult donors had a lymphocyte response to exoenzyme S. The optimal lymphocyte response occurred on day 7, with 4 x 10(5) PBMC per microtiter well when cells were stimulated with 10 micrograms exoenzyme S per ml. [3H]thymidine uptake correlated with an increase in the number of mononuclear cells, indicating that proliferation occurred. In unseparated PBMC, T cells, and to a lesser extent B cells, proliferated. Purified T cells proliferated, while purified B cells proliferated only after the addition of irradiated T cells. Thus, T lymphocytes are necessary and sufficient for the proliferative response to exoenzyme S. We speculate that exoenzyme S from P. aeruginosa is important in T-lymphocyte-mediated host defense to P. aeruginosa. In strategies to enhance impaired cell-mediated immunity, exoenzyme S should be considered as a potential stimulant.
Human immunoglobulin M paraproteins cross-reactive with Neisseria meningitidis group B polysaccharide and fetal brain
Authors: Azmi F H , Lucas A H , Spiegelberg H L , Granoff D M
Published: January 2020

Three hundred fifty-nine serum samples from patients with immunoglobulin M (IgM) or IgG monoclonal gammopathies were tested for binding to the capsular polysaccharide (PS) of Neisseria meningitidis group B (MenB PS, poly-alpha[2-->8]-N-acetylneuraminic acid). Of 159 IgM paraproteins, 7 (4.4%) were positive, compared with 0 of 200 IgG paraproteins (P < 0.05). Since MenB PS reactivity was limited to the IgM paraproteins, the 159 IgM paraproteins were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with seven other bacterial PSs. None reacted with meningococcal A or C, Haemophilus influenzae type b, or Streptococcus pneumoniae type 3, 6, 14, or 23 PS. The specificity of the MenB PS-reactive antibodies was confirmed by demonstration of binding to N. meningitidis group B cells but not to a capsular PS-deficient mutant and by specific inhibition of binding to solid-phase MenB PS by soluble MenB PS in an ELISA. Five of five antibodies tested protected infant rats from bacteremia caused by Escherichia coli K1, an organism with a PS capsule that also is composed of poly-alpha[2-->8]-N-acetylneuraminic acid. Each of the seven MenB PS-reactive paraproteins had autoantibody activity as defined by binding to homogenates of calf brain in a radioimmunoassay. For six of the seven antibodies, binding to calf brain was inhibited by the addition of soluble MenB PS. Thus, approximately 4% of human IgM paraproteins have autoantibody activity to poly-alpha[2-->8]-N-acetylneuraminic acid, an antigen expressed in fetal brain and cross-reactive with the MenB capsular PS. The reason for this skewing of the IgM paraprotein repertoire toward reactivity with poly-alpha[2-->8]-N-acetylneuraminic acid antigenic determinants is unknown.
Cloning, expression, and nucleotide sequence of a Staphylococcus aureus gene (fbpA) encoding a fibrinogen-binding protein
Authors: Cheung A I , Projan S J , Edelstein R E , Fischetti V A
Published: January 2020

Septicemia due to Staphylococcus aureus often begins as a focal infection (e.g., colonized wounds or catheters) from which the organism gains access to the bloodstream. On the basis of recent data from this laboratory, it is likely that S. aureus colonizes catheters and endothelium by using a fibrinogen-binding protein to mediate adhesion to fibrinogen-coated surfaces. To characterize the fibrinogen-reactive protein, we screened a lambda Zap library of S. aureus DB, a clinical isolate, for clones that were reactive with fibrinogen. Of 100,000 plaques screened, 3 were found to react with fibrinogen on immunoblots. Plasmid DNA prepared from clones 14, 30, and 36, upon digestion with EcoR1, which released the insert, revealed fragments of 4.6, 3.6, and 3.2 kb, respectively. To identify the cloned protein expressed in E. coli, cells were fractionated into periplasmic, membrane, and cytoplasmic fractions. Expression studies of clone 14, which comprised approximately two-thirds of the mature molecule, including the C terminus, revealed a 34-kDa fibrinogen-reactive protein in both the periplasmic and membrane fractions. This protein, designated FbpA, could be partially purified on a fibrinogen column. By using both clones 14 and 36 as templates, the complete DNA sequence of the fibrinogen-binding protein was obtained, yielding a molecule with a predicted size of 69,991 Da. Although sequence analysis revealed a high degree of homology with coagulase, there is a unique sequence of 11 amino acids that is not found in three known coagulases as well as two recently cloned fibrinogen-binding proteins. This unique sequence shares homology with a cell wall anchor motif found in other gram-positive surface proteins.
Active release of bound antibody by Streptococcus mutans
Authors: Lee S F
Published: January 2020

Previous studies have shown that Streptococcus mutants is capable of releasing many surface protein antigens, particularly antigen P1. Antigen P1 is immunodominant and has been implicated in adherence of S. mutants to the acquired pellicles. The purpose of this study is to investigate the significance of release of this antigen by the cells. S. mutants NG8 (serotype c) was incubated with an anti-P1 rabbit immunoglobulin G (IgG) or a human colostral IgA which contains natural anti-P1 activity. Results indicated that the bound antibodies were released by the cells in a pH- and time-dependent manner. The optimal pH for release was between 6 and 8, and the release rate reached a plateau in 1 h at 37 degrees C. The release of bound antibodies was considered an active process, since heat-killed cells remained capable of antibody binding but failed to release the antibodies. The release was also dependent on the age of the culture, with early-exponential-phase cells releasing the maximum amount of bound IgG. The released IgG was isolated by polyethylene glycol precipitation and protein A-Sepharose column chromatography and found to be associated with antigen P1, indicating that the antibodies were released together with the antigen in the form of immune complexes. The binding of S. mutans by secretory IgA (SIgA) inhibited the adherence of the cells to salivary agglutinin-coated hydroxylapatite. However, when the SIgA-coated S. mutans was allowed to release the bound antibodies, the inhibitory effect of SIgA on adherence was abrogated. These results suggest that S. mutans is capable of shedding surface-bound antibodies in the form of antibody-antigen immune complexes. Such an action may be a strategy employed by the cells to counter the neutralizing effect of naturally occurring antibodies in the oral cavity.
Antibody and cytokine responses in a mouse pulmonary model of Shigella flexneri serotype 2a infection
Authors: van de Verg L L , Mallett C P , Collins H H , Larsen T , Hammack C , Hale T L
Published: January 2020

A murine pulmonary model was used to study the mucosal immune response to Shigella flexneri serotype 2a infection. Inoculation of BALB/cJ mice with shigellae via the intranasal route resulted in bacterial invasion of bronchial and alveolar epithelia with concomitant development of acute suppurative bronchiolitis and subsequent development of lethal pneumonia. The pathology of pulmonary lesions resembled the colitis that characterizes shigellosis in humans and primates. Significant protection against a lethal dose of S. flexneri 2a was observed in mice previously infected with two sublethal doses of the homologous strain. Immunity against lethal challenge was associated with decreased bacterial invasion of the mucosal epithelium. Over the course of two sublethal challenges, which constituted primary and secondary immunizations, mice developed pulmonary and serum immunoglobulin G and A antibody recognizing both lipopolysaccharide and invasion plasmid antigens IpaB and IpaC. Immune mice and naive control mice differed in lung lavage cytokine levels following lethal challenge. Immune mice developed significantly elevated levels of pulmonary gamma interferon within 6 h of challenge, while naive control mice developed elevated levels of this cytokine later during the initial 24-h period. Both groups had elevated levels of gamma interferon during the 24- to 48-h period of infection. Both groups also had elevated levels of tumor necrosis factor alpha within 6 h of challenge, but the control mice had significantly higher levels at the 48- and 72-h time points. Elevated levels of interleukin-4 were observed only in immunized mice. This cytokine appeared within 24 h and receded between 48 and 72 h. Fluorescence-activated cell sorter analysis of lung parenchymal cells showed that both groups experienced an initial influx of monocytes, but the proportion of this cell type began to recede in immunized mice after 48 h of infection, while peak levels were maintained in the control animals. These studies suggest that elements of local B lymphocyte activity, as well as Th1 and Th2 lymphocyte activity, may contribute to the survival of immune mice after intranasal challenge with shigellae.
Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sobrinus: dose response and induction of protective humoral responses in rats
Authors: Redman T K , Harmon C C , Lallone R L , Michalek S M
Published: January 2020

An attenuated, recombinant Salmonella typhimurium mutant, chi 4072(pYA2905), expressing the surface protein antigen A (SpaA) of Streptococcus sobrinus was investigated for its effectiveness in inducing protective immune responses against S. sobrinus-induced dental caries in an experimental caries model. Fischer rats were orally immunized with either 10(8) or 10(9) CFU of S. typhimurium chi 4072(pYA2905). Persistence of salmonellae in Peyer's patches and spleens and the induction of immune responses were determined. Maximum numbers of salmonellae were recovered from Peyer's patches of rats within the first week of immunization, with higher numbers recovered from rats given 10(9) CFU than from those given 10(8) CFU. Serum anti-Salmonella and anti-SpaA responses increased more rapidly in rats given 10(9) CFU than in rats given 10(8) CFU. The salivary antibody response to SpaA increased with time, but the response varied in the two groups. In a separate study, rats were orally immunized with the recombinant Salmonella mutant and then challenged with cariogenic S. sobrinus 6715. The levels of serum and salivary antibody and caries activity were assessed at the termination of the experiment. Higher levels of salivary immunoglobulin A antibody to SpaA and Salmonella carrier were detected in rats given 10(9) CFU than in those given 10(8) CFU, and these responses were higher than those in nonimmunized controls. Mandibular molars from immunized rats had lower numbers of recoverable streptococci and less extensive carious lesions than those from nonimmunized, control rats. These data indicate that oral immunization with an attenuated recombinant S. typhimurium expressing SpaA of S. sobrinus induces the production of antigen-specific mucosal antibody and confers protection against dental caries.
Inducible immunity with a pilus preparation booster vaccination in an animal model of Haemophilus ducreyi infection and disease
Authors: Desjardins M , Filion L G , Robertson S , Cameron D W
Published: January 2020

Using the temperature-dependent rabbit model of Haemophilus ducreyi infection as a quantitative virulence assay, we tested the abilities of two bacterial antigen preparations to induce protection against subsequent infection and disease. Lipooligosaccharide (LOS) and a pilus preparation were purified from H. ducreyi 35000 and were used in a booster immunization procedure. The serologic response to each immunogen was monitored by enzyme immunoassay. H. ducreyi virulence was assayed by intraepithelial inoculation and subsequent measurement of disease for homologous strain 35000 or clinical isolate RO-34. LOS and the pilus preparation induced humoral responses. The kinetics of the LOS antibody response suggest a type 1 T-independent response, whereas the pilus preparation induced an anamnestic response. An inoculum of 10(5) CFU of H. ducreyi 35000 or RO-34 consistently produced ulcerative chancroidal lesions in naive rabbit controls. Immunization with LOS did not modify the virulence of H. ducreyi 35000. Immunization with the strain 35000 pilus preparation significantly reduced the severity of disease and the duration of infection and disease compared with controls, with either homologous or heterologous strain infection. The histology of lesions from pilus preparation-vaccinated rabbits compared with that of lesions from controls revealed accelerated lymphoid cell recruitment, more prominent plasma cell infiltrate, and reduction in subsequent histiocytic infiltration. We conclude that both LOS and the pilus preparation are immunogenic and that the latter induces homologous and heterologous strain protection in this animal model of infection and disease.
Investigation of enterohemorrhagic Escherichia coli O157:H7 adherence characteristics and invasion potential reveals a new attachment pattern shared by intestinal E. coli
Authors: McKee M L , O'Brien A D
Published: January 2020

In this study, the interactions of enterohemorrhagic Escherichia coli (EHEC) O157 strains with human ileocecal (HCT-8) epithelial cells and HEp-2 cells were examined. EHEC adhered to, but did not invade, HCT-8 cells by the localized adherence mechanism and a heretofore unrecognized pattern which we called log jam. The log jam formation was (i) not observed on HEp-2 cells, (ii) independent of the EHEC eaeA gene required for localized adherence, and (iii) shared by pathogenic and nonpathogenic E. coli strains but not K-12 strains. The log jam phenotype may represent a basal means by which E. coli bacteria attach to the human intestine.
Circumvention of outer surface protein A immunity by host-adapted Borrelia burgdorferi
Authors: Barthold S W , Fikrig E , Bockenstedt L K , Persing D H
Published: January 2020

Outer surface protein A (OspA), which is abundantly expressed in cultured Borrelia burgdorferi, appears to be down-regulated or masked following low-dose infection, and OspA immunization did not prevent infection, dissemination, or disease development with host-adapted spirochetes. Seroconversion of mice to B. burgdorferi OspA depended on dose and viability of inoculated spirochetes. Mice inoculated with > 10(4) live spirochetes and > 10(7) heat-killed spirochetes seroconverted to OspA, but mice inoculated with fewer spirochetes did not seroconvert to OspA at 2 weeks after inoculation. Growth temperature of spirochetes was not a factor for infectious dose or seroconversion to OspA. Spirochetes grown at 30, 34, or 38 degrees C had the same median infectious dose. Growth temperature did not influence infectious dose when mice were inoculated intraperitoneally or intradermally and did not influence dose-related immunologic recognition of OspA. Mice hyperimmunized with recombinant OspA-glutathione S-transferase (GT) fusion protein or GT (controls) were challenged by syringe inoculation with 10(3) spirochetes or by transplantation of infected skin from syngenic mice infected for 2 or 8 weeks. OspA-GT-immunized mice resisted syringe challenge but developed disseminated infections following transplantation of infected skin. Identical results were obtained in mice passively immunized with hyperimmune serum to OspA-GT or GT and then challenged by syringe or infected skin transplant. The number of spirochetes in infected skin, determined by quantitative PCR directed toward both plasmid and genomic targets, was less than the syringe challenge dose.
Interleukin-6-deficient mice are highly susceptible to Listeria monocytogenes infection: correlation with inefficient neutrophilia
Authors: Dalrymple S A , Lucian L A , Slattery R , McNeil T , Aud D M , Fuchino S , Lee F , Murray R
Published: January 2020

We have produced interleukin-6 (IL-6)-deficient mice to examine, in vivo, the wide variety of biological activities attributed to this multifunctional cytokine. To investigate the role of IL-6 during infectious disease, IL-6-deficient mice were challenged with sublethal doses of Listeria monocytogenes, a facultative intracellular bacterium. While normal control animals were able to clear the infection, mutant animals exhibited a high mortality rate and showed uncontrolled replication of the bacteria in the spleen and liver at 2 and 3 days postinfection. Sections of infected tissues showed an increase in the number and severity of inflammatory foci. All aspects of this phenotype in the mutant animals were completely reverted upon administration of recombinant murine IL-6 (rIL-6). Various parameters of natural killer (NK) cell and macrophage function were unaffected in the challenge of the mutant animals. However, IL-6-deficient animals failed to mount peripheral blood neutrophilia in response to listeriosis, whereas control animals displayed a prominent neutrophilia in the blood at 24 and 48 h postinfection. Additionally, we analyzed the efficacy of rIL-6 in protecting animals devoid of lymphocytes or devoid of neutrophils during listeriosis. Administration of rIL-6 was protective to animals devoid of lymphocytes, suggesting that the rIL-6 protective effect was not mediated through lymphocytes. In contrast, control and mutant animals depleted of neutrophils were refractory to the rIL-6 protective effect. These data suggest that IL-6 is critical early during listeriosis, perhaps acting by stimulating neutrophils either directly or indirectly. Additionally, these data show a promising therapeutic potential for rIL-6 administration during opportunistic infection.
Human T-cell recognition of Listeria monocytogenes: recognition of listeriolysin O by TcR alpha beta + and TcR gamma delta + T cells
Authors: Guo Y , Ziegler H K , Safley S A , Niesel D W , Vaidya S , Klimpel G R
Published: January 2020

The cell-mediated immune response to Listeria monocytogenes has been well characterized in the mouse. Listeriolysin O (LLO) is a major antigen in murine T-cell recognition of L. monocytogenes. In this study, we show that LLO is also recognized by human TcR alpha beta T cells and TcR gamma delta T cells. Human peripheral blood mononuclear cells (PBMC) cultured in vitro with live listeriae and then expanded with interleukin 2 were shown to respond to purified LLO. The generation of LLO-responsive T cells was dependent on the use of live bacteria during the initial in vitro challenge. LLO-induced proliferation of T cells expanded by exposure of PBMC to live listeriae was major histocompatibility complex restricted. PBMC cultured with formalin-fixed listeriae and subsequently expanded by interleukin 2 gave high proliferative responses to fixed bacteria but failed to respond to LLO. PBMC stimulated in vitro with fixed listeriae contained predominantly TcR alpha beta + T cells. In contrast, PBMC obtained from 85% of the donors studied generated high numbers of TcR gamma delta + T cells following in vitro culture with live listeriae. Using a panel of synthetic amphipathic LLO peptides, we found that LLO-specific T cells from different individuals recognized both common and unique peptides. LLO 470-508 was recognized by three of five individuals, while LLO 203-226 and LLO 107-126 were recognized by two of six individuals. A TcR gamma delta + T-cell line was established from PBMC stimulated with live listeriae and was shown to recognize LLO 470-508. Proliferative responses could be induced in this cell line by peptide-pulsed autologous PBMC but not by peptide-pulsed allogeneic PBMC. Our results establish the importance of LLO in human T-cell recognition of listeriae and show that both TcR alpha beta + T cells and TcR gamma delta + T cells recognize this antigen. Finally, since LLO 470-508 has a high degree of homology with other gram-positive bacterial toxins, the recognition of this peptide by TcR gamma delta + T cells suggests that an important role of these T cells in host defense is the recognition of bacterium-derived toxins.
Construction of nontoxic derivatives of cholera toxin and characterization of the immunological response against the A subunit
Authors: Fontana M R , Manetti R , Giannelli V , Magagnoli C , Marchini A , Olivieri R , Domenighini M , Rappuoli R , Pizza M
Published: January 2020

Using computer modelling, we have identified some of the residues of the A subunit of cholera toxin (CT) and heat-labile toxin that are involved in NAD binding, catalysis, and toxicity. Here we describe the site-directed mutagenesis of the CT gene and the construction of CT mutants. Nine mutations of the A subunit gene were generated. Six of them encoded proteins that were fully assembled in the AB5 structure and were nontoxic; these proteins were CT-D53 (Val-53-->Asp), CT-K63 (Ser-63-->Lys), CT-K97 (Val-97-->Lys), CT-K104 (Tyr-104-->Lys), CT-S106 (Pro-106-->Ser), and the double mutant CT-D53/K63 (Val-53-->Asp, Ser-63-->Lys). Two of the mutations encoded proteins that were assembled into the AB5 structure but were still toxic; these proteins were CT-H54 (Arg-54-->His) and CT-N107 (His-107-->Asn). Finally, one of the mutant proteins, CT-E114 (Ser-114-->Glu), was unable to assemble the A and the B subunits and produced only the B oligomer. The six nontoxic mutants were purified from the culture supernatants of recombinant Vibrio cholerae strains and further characterized. The CT-K63 mutant, which was the most efficient in assembly of the AB5 structure, was used to immunize rabbits and was shown to be able to induce neutralizing antibodies against both the A and B subunits. This molecule may be useful for the construction of improved vaccines against cholera.
Intransal or intragastric immunization with proteosome-Shigella lipopolysaccharide vaccines protects against lethal pneumonia in a murine model of Shigella infection
Authors: Mallett C P , Hale T L , Kaminski R W , Larsen T , Orr N , Cohen D , Lowell G H
Published: January 2020

Mice immunized intransally or intragastrically with proteosome vaccines containing either Shigella sonnei or S. flexneri 2a lipopolysaccharide were protected against lethal pneumonia caused by homologous organisms in an experimental murine intranasal challenge model of Shigella infection. Histopathological analysis demonstrated that immunization also protected against the progressive lesions resulting from invasion of the pulmonary mucosa by S. sonnei. These data show that mucosal proteosome-lipopolysaccharide vaccines can protect against lethal bacterial pneumonia and indicate that such vaccines are promising candidates for protection against intestinal shigellosis.
Salmonella enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens
Authors: Keller L H , Benson C E , Krotec K , Eckroade R J
Published: January 2020

Salmonella enteritidis colonizes the tissues of the chicken ovary and oviduct, presumably contaminating eggs and thereby contributing to human outbreaks of salmonellosis. In this study, commercial adult laying hens were given an oral inoculation of 10(8) S. enteritidis organisms. Tissues from various organs, the intestines, and the reproductive tract, including freshly laid eggs, were collected daily for up to 40 days postinoculation (p.i.). Within 2 days p.i. S. enteritidis was detected by culture in pools of the spleen, liver, heart, and gallbladder tissues, in intestinal tissues of all infected birds, and in various sections of the ovary and oviduct. Detection of organisms by immunohistochemical staining was rare for most tissues in spite of their culture-positive status, suggesting a low level of tissue colonization. However, S. enteritidis could be detected by immunohistochemical staining in oviduct tissues associated with four forming eggs, indicating the possibility of a heavier colonization in the egg during its development. In two subsequent experiments, forming eggs taken from the oviduct with their associated tissue, were found to be culture positive for S. enteritidis at a rate of 27.1 and 31.4%, while freshly laid eggs in these experiments were culture positive at the rate of 0 and 0.6%. These observations suggest that while forming eggs are significantly colonized in the reproductive tract, factors within the eggs may control the pathogen before the eggs are laid. The data show that prior to egg deposition, forming eggs are subject to descending infections from colonized ovarian tissue, ascending infections from colonized vaginal and cloacal tissues, and lateral infections from colonized upper oviduct tissues. The data are consistent with an ascending infection of freshly laid eggs from the cloaca, as the incidence of positive eggs in experiments 1 and 3 coincided with heavily contaminated cloacal tissues (50.7 and 80%, respectively), while no positive eggs were detected in experiment 2 when cloacal colonization was low (8.3%). The data do not support the possibility of egg invasion by bacterial translocation from the peritoneal cavity.
Putative glycoprotein and glycolipid polymorphonuclear leukocyte receptors for the Actinomyces naeslundii WVU45 fimbrial lectin
Authors: Sandberg A L , Ruhl S , Joralmon R A , Brennan M J , Sutphin M J , Cisar J O
Published: January 2020

Recognition of receptors on sialidase-treated polymorphonuclear leukocytes (PMNs) by the Gal/GalNAc lectin associated with the type 2 fimbriae of certain strains of actinomyces results in activation of the PMNs, phagocytosis, and destruction of the bacteria. In the present study, plant lectins were utilized as probes to identify putative PMN receptors for the actinomyces lectin. The Gal-reactive lectin from Ricinus communis (RCAI), the Gal/GalNAc-reactive lectins from R. communis (RCAII) and Bauhinia purpurea (BPA), as well as the Gal beta 1-3GalNAc-specific lectins from Arachis hypogaea (PNA) and Agaricus bisporus (ABA) inhibited killing of Actinomyces naeslundii WVU45 by sialidase-treated PMNs. These five lectins detected a 130-kDa surface-labeled glycoprotein on nitrocellulose transfers of PMN extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This glycoprotein was revealed only after treatment of the transfers with sialidase, a condition analogous to the sialidase dependence of the lectin-mediated biological responses of the PMNs to the actinomyces. The mannose-reactive lectin concanavalin A did not inhibit killing of the actinomyces and failed to detect the 130-kDa glycoprotein but did block PMN-dependent killing of Escherichia coli B, a bacterium that possesses mannose-sensitive fimbriae. Therefore, the PMN glycoprotein receptor for A. naeslundii is clearly distinct from those recognized by E. coli. Two major putative glycolipid receptors were also identified by actinomyces and RCAI overlays on sialidase-treated thin-layer chromatograms of PMN gangliosides. Thus, both a 130-kDa glycoprotein and certain gangliosides are implicated in the attachment of the actinomyces to PMNs.
Enhancement of uptake of lipopolysaccharide in macrophages by the major outer membrane protein OmpA of gram-negative bacteria
Authors: Korn A , Rajabi Z , Wassum B , Ruiner W , Nixdorff K
Published: January 2020

Monoclonal antibodies (MAb) to lipopolysaccharide (LPS) and to the major outer membrane protein OmpA from Proteus mirabilis were generated and used to monitor the kinetics of uptake in macrophages of LPS as well as LPS bound to OmpA. Uptake was measured by a modified enzyme-linked immunosorbent assay (ELISA) in a microtiter culture system. The MAb were of various immunoglobulin G subclasses and showed strong reactivities with their antigens. Four hybridoma clones recognizing LPS and three recognizing OmpA from P. mirabilis 19 were selected for the present study on the basis of reactions in ELISA and Western blot (immunoblot) analyses. In the uptake assay, it was possible to differentiate between antigen on the cell surface and antigen which had been internalized. Uptake of LPS by macrophages was relatively rapid during the first 4 h of culture and then progressed more slowly over the remaining 24-h observation period. The level of detection of LPS in this assay system was in the nanogram range. When macrophages were pulsed with LPS for 30 min and subsequently washed to remove antigen not bound to the cells, the amount of LPS detectable on the macrophage surface decreased progressively for 3 h after the pulse, which indicated internalization of the antigen. Thereafter, LPS rose to an increased level on the cell surface. The rate of uptake of LPS was more rapid when it was in complex with OmpA. When the fate of OmpA was monitored in the same LPS-protein complexes by use of MAb to OmpA in a pulse experiment, the level of protein measured on the cell surface decreased after an initial rise, which again indicated internalization, but the protein did not reappear on the cell surface in a form detectable with the MAb. Compared with the LPS monitoring system, detection of OmpA associated with macrophages was weak, although the MAb to OmpA reacted strongly with the protein in the ELISA and Western blot analyses.
Enhanced respiratory clearance of nontypeable Haemophilus influenzae following mucosal immunization with P6 in a rat model
Authors: Kyd J M , Dunkley M L , Cripps A W
Published: January 2020

Nontypeable Haemophilus influenzae (NTHi) is a common cause of infection of the respiratory tract in children and adults. The search for an effective vaccine against this pathogen has focused on components of the outer membrane, and peptidoglycan-associated lipoprotein P6 is among the proposed candidates. This study investigated the immunogenicity of P6 in a rat respiratory model. P6 was purified from two strains of NTHi, one capsule-deficient strain and an H. influenzae type b strain, and assessed for clearance of both homologous and heterologous bacterial strains following mucosal immunization. A protective immune response was determined by enhancement of pulmonary clearance of live bacteria and an increased rate of recruitment of phagocytic cells to the lungs. This was most effective when Peyer's patch immunization was accompanied by an intratracheal (IT) boost. However, the rate of bacterial clearance varied between strains, which suggests some differences in anti-P6 immunological defenses recognizing the expression of the highly conserved P6 lipoprotein on the bacterial surface in some strains. P6-specific antibodies in both serum and bronchoalveolar lavage fluid were cross-reactive and did not differ significantly in strain specificity, demonstrating that difference in clearance was unlikely due to differences in P6-specific antibody levels. Serum homologous and heterologous P6-antibody was bactericidal against NTHi even when enhanced clearance had not been observed. Peyer's patch immunization induced P6-specific CD4+ T-helper cell proliferation in lymphocytes isolated from the mesenteric lymph nodes. An IT boost increased the level of P6-specific antibodies in serum and bronchoalveolar lavage fluid, and P6-specific mesenteric node lymphocyte proliferation. Cells from rats immunized with P6 demonstrated proliferation following stimulation with P6 from nonhomologous strains; however, there was some variation in proliferative responses to P6 from different strains in lymphocytes isolated from animals immunized with killed bacteria. The increase in P6-specific antibodies and T-helper cell responses following an IT boost correlated with an increased rate of recruitment of phagocytic cells and enhanced bacterial clearance of both homologous and heterologous bacteria in the lungs. The data suggests that P6 has the potential to afford protection against pulmonary infection by NTHi following the induction of effective antigen-specific B- and T-cell responses in mucosal tissues.
Purification and characterization of a Shiga toxin A subunit-CD4 fusion protein cytotoxic to human immunodeficiency virus-infected cells
Authors: al-Jaufy A Y , King S R , Jackson M P
Published: January 2020

In a previous paper, we reported that a chimeric toxin composed of the enzymatic domain of the Shiga toxin A polypeptide (StxA1) genetically fused to the human CD4 (hCD4) molecule selectively kills cells infected with human immunodeficiency virus type 1 (HIV-1). Although other hCD4-containing chimeras cytotoxic to HIV-infected cells have been developed, there is limited information regarding their receptor binding and internalization. Therefore, the goals of this study were to purify the StxA1-hCD4 fusion protein, identify the receptor(s), and investigate the cytosolic trafficking route used by the chimeric toxin. Sufficient quantities of the StxA1-hCD4 hybrid were isolated for this investigation by using the pET expression and purification system. Cos-1 cells were rendered sensitive to the StxA1-hCD4 chimera by transfection with the env gene, which encodes HIV-1 envelope glycoproteins. The entry and translocation pathway used by the StxA1-hCD4 hybrid toxin was investigated by assessing the protective capacities of chemical reagents which interfere with microfilament movement, acidification of endosomes, and the integrity of the Golgi apparatus. Our findings indicated that the chimera uses HIV-1 glycoprotein gp120, and perhaps gp41, as a receptor which directs its entry through receptor cycling. Uptake is pH independent, and the StxA1-hCD4 hybrid is apparently translocated to the Golgi complex as with other bipartite toxins.
Immunobiological activity of recombinant H antigen from Histoplasma capsulatum
Authors: Deepe G S , Durose G G
Published: January 2020

The H antigen from Histoplasma capsulatum is one of the known principal antigens of this fungus, but information regarding its identity and its participation in cellular immunity is largely unavailable. Therefore, we sought to determine both the nature of this antigen and the nature of its involvement in the cell-mediated immune response. The antigen was isolated from histoplasmin and digested with selected proteinases, and the cleavage products were subjected to reverse-phase high-performance liquid chromatography. Amino acid sequences of protein fragments were obtained by Edman degradation. A fragment of the gene encoding the H antigen was isolated by using degenerate primers in the PCR. This fragment was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence revealed homology to extracellular beta-glucosidases. A cDNA was generated by reverse transcription PCR and cloned into the expression vector pET 19b. Recombinant H antigen was isolated from inclusion bodies of Escherichia coli and tested for its ability to elicit and induce an in vitro cell-mediated immune response in BALB/c mice. Recombinant antigen stimulated splenocytes from mice immunized with viable yeast cells or with antigen suspended in adjuvant. Mice inoculated with H antigen were not protected against either a sublethal or a lethal inoculum of yeast cells. Thus, H antigen stimulates a cell-mediated immune response but does not induce a protective response to H. capsulatum.
T-cell cytokine responses in human infection with Mycobacterium tuberculosis
Authors: Zhang M , Lin Y , Iyer D V , Gong J , Abrams J S , Barnes P F
Published: January 2020

Compared with healthy tuberculin reactors, Mycobacterium tuberculosis-stimulated peripheral blood mononuclear cells from tuberculosis patients had diminished production and mRNA expression of the Th1 cytokines gamma interferon and interleukin 2 (IL-2), with no change in production and mRNA expression for the Th2 cytokines IL-4, IL-10, and IL-13. These results were confirmed by evaluation of T cells and CD4+ cells. At the level of systemic T cells, development of tuberculosis is associated with diminished Th1 but not enhanced Th2 responses.
Variable efficacy of passive antibody administration against diverse Cryptococcus neoformans strains
Authors: Mukherjee J , Scharff M D , Casadevall A
Published: January 2020

The efficacy of monoclonal antibody (MAb 2H1) against diverse strains of Cryptococcus neoformans was studied in a murine model of intravenous infection. For six of eight strains, administration of MAb prior to infection prolonged survival of mice. For two strains, 371 and SB4A, administration of MAb prior to infection did not prolong survival in multiple experiments with inocula ranging from 10(2) to 10(6) yeast cells per mouse. Mice infected with strains 371 and SB4A had fewer CFU than non-MAb-treated controls, but the CFU reduction was not sufficient to affect survival. Serum glucuronoxylomannan (GXM) levels varied for the different C. neoformans strains. For mice that did not receive MAb 2H1, there was a positive correlation between lung fungal burden and serum GXM levels. MAb 2H1-treated mice had significantly reduced serum GXM levels. The results indicate that the efficacy of MAb 2H1 administration in prolonging survival and/or reducing organ CFU varies with the C. neoformans strain.
Cytopathic effects of Treponema denticola chymotrypsin-like proteinase on migrating and stratified epithelial cells
Authors: Uitto V J , Pan Y M , Leung W K , Larjava H , Ellen R P , Finlay B B , McBride B C
Published: January 2020

The effects of Treponema denticola and its outer membrane-bound chymotrypsin-like proteinase on periodontal ligament epithelial cell cultures at different stages of maturity were studied. In sparse cultures with migrating epithelial cells, large intracellular vacuoles were formed rapidly following exposure to live T. denticola. Treponemes showing structural damage were seen occasionally inside membrane-bound vesicles. Intensive membrane blebbing occurred in infected cells and continued for up to 48 h before the cell died. Blebbing could also be induced by a purified chymotrypsin-like proteinase of T. denticola. Cortical actin and alpha-actinin of the bacterium-treated cells showed disorganization, and pericellular fibronectin was degraded by both whole T. denticola and the isolated proteinase. Epithelial cells with well-formed lateral cell contacts appeared to be more resistant to the effects of T. denticola than migrating isolated cells. In multilayer epithelial cultures, adhesion of T. denticola and membrane blebbing were observed infrequently. There was no evidence of invasion of T. denticola into epithelial multilayers. However, immunogold electron microscopy showed rapid transport of T. denticola chymotrypsin-like proteinase into newly formed large intracellular vacuoles within the epithelial layers. These vacuoles were lined by membranes studded with ribosomes. T. denticola-treated epithelial multilayers had loose cell contacts, collapsed intercellular spaces, and increased permeability. Through its capacity to cause these unique cytopathic effects, the chymotrypsin-like proteinase of T. denticola has the potential to contribute to the initiation of periodontal disease.