bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection against endotoxic shock and lipopolysaccharide-induced local inflammation by tetracycline: correlation with inhibition of cytokine secretion
Authors: Shapira L , Soskolne W A , Houri Y , Barak V , Halabi A , Stabholz A
Published: January 2020

Septic shock results from excessive stimulation of host immune cells, particularly monocytes and macrophages, by lipopolysaccharide (LPS) released from gram-negative bacteria. Macrophage-derived cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1 beta), have been identified as central mediators in the pathogenesis of septic shock and the resultant mortality. Therefore, these cytokines were targets for experimental therapy for septic shock. Because of tetracycline's ability to intervene in cellular mechanisms involved in cytokine secretion, we tested the effect of tetracycline on LPS-induced septic shock and inflammatory lesions in mice. Tetracycline was found to protect mice against LPS-induced lethality and to abolish clinical signs of LPS-induced inflammatory lesions. This protection correlates with tetracycline's ability to reduce LPS-induced TNF-alpha levels in serum. Furthermore, tetracycline was found to inhibit LPS-induced TNF-alpha and IL-1 beta secretion, but not cytokine mRNA accumulation, in human monocytes in vitro. The results presented here suggest that tetracycline is a potent drug for LPS-induced pathology and that its mechanism of action involves blockage of posttranscriptional events of cytokine production.
Streptococcal cysteine protease augments lung injury induced by products of group A streptococci
Authors: Shanley T P , Schrier D , Kapur V , Kehoe M , Musser J M , Ward P A
Published: January 2020

Streptococcus pyogenes infections in humans may be associated with severe clinical manifestations, including adult respiratory distress syndrome and a toxic shock-like syndrome. These observations have led to the investigation of products of group A streptococci that may contribute to increased virulence. Streptococcal pyrogenic exotoxin B is a highly conserved precursor of an extracellular cysteine protease that is secreted by S. pyogenes. We investigated the ability of this streptococcal cysteine protease (SCP) to act synergistically with either streptococcal cell wall antigen (SCW) or streptolysin-O (SLO) to augment lung injury in rats. Intratracheal administration of either SCW or SLO alone caused lung injury, as measured by pulmonary vascular leak. Bronchoalveolar lavage (BAL) fluid analysis showed that SCW induced neutrophil accumulation and appearance of interleukin-1beta and tumor necrosis factor alpha. In contrast, SLO induced neither neutrophil influx nor significant cytokine elevations in BAL fluids. Intratracheal administration of SCP with either SCW or SLO resulted in synergistic augmentation of lung vascular permeability and accumulation of BAL neutrophils. The synergy was reduced when SCP was either heat inactivated or coinstilled with a peptide inhibitor of the protease. SCP in the presence of SCW resulted in a significant increase in BAL fluid tumor necrosis factor alpha content but not in immunoreactive interleukin-1beta. Moreover, the copresence of SAP with SAW resulted in increased BAL fluid nitrite-nitrate levels, indicative of nitric oxide production. These data demonstrate that SCP acts synergistically with other S. pyogenes products (SCW or SLO) to increase tissue injury and provide additional evidence that SCP may function as an important virulence factor in group A streptococcal infections.
Selection and phenotypic characterization of nonhemagglutinating mutants of Porphyromonas gingivalis
Authors: Chandad F , Mayrand D , Grenier D , Hinode D , Mouton C
Published: January 2020

To further investigate the relationship between fimbriae and the hemagglutinating adhesin HA-Ag2 of Porphyromonas gingivalis, three spontaneous mutants of the type strain ATCC 33277 were selected by a hemadsorption procedure. They were characterized for hemagglutination, trypsin-like and lectin-binding activities, and hydrophobicity and for the presence of fimbriae. The presence of the 42-kDa (the fimbrilin subunit) and the 43- and 49-kDa (the HA-Ag2 components) polypeptides was investigated by immunoblotting using polyclonal and monoclonal antibodies directed to fimbriae and to the hemagglutinating adhesin HA-Ag2. Cells from two of the three mutants (M1 and M2) exhibited no or little hemagglutination activity and very low trypsin-like activity and did not show the 43- and 49-kDa polypeptides. Abnormal fimbriation in M1 was deduced from the following observations of cells grown for 18 h: absence of the 42-kDa polypeptide and of a 14-kDa polypeptide and no fimbriae visible on electron micrographs. While the cells of mutant M2, irrespective of the age of the culture, were found to lack the 43- and 49-kDa polypeptides and hemagglutination activity, the supernatants of cultures grown for 72 h had high hemagglutination and trypsin-like activities and revealed the presence of the 42-, 43-, and 49-kDa polypeptides. This suggests that M2 may be missing some molecules which anchor the components to the cell surface. Mutant M3 showed levels of activities similar to those of the parental strain but lacked the 43-kDa polypeptide. Other pleiotropic effects observed for the mutants included loss of dark pigmentation and lower hydrophobicity. The data from this study fuel an emerging consensus whereby fimbriation, hemagglutination, and proteolytic activities, as well as other functions in P. gingivalis, are intricate.
Immune selection for antigenic drift of major outer membrane protein P2 of Haemophilus influenzae during persistence in subcutaneous tissue cages in rabbits
Authors: Vogel L , Duim B , Geluk F , Eijk P , Jansen H , Dankert J , vanAlphen L
Published: January 2020

During persistence of nonencapsulated Haemophilus influenzae in the respiratory tracts of patients with chronic bronchitis, the major outer membrane proteins (MOMPs) P2 and P5 show antigenic drift. The hypothesis that appearance of antigenic variants is the consequence of antibody-dependent selection was tested in a rabbit model. Persistence of H. influenzae d1 was achieved in subcutaneous tissue cages for up to 948 days. During persistence in the rabbits, similar changes in MOMP P2 of H. influenzae occurred, as observed in isolates from chronic bronchitis patients. In rabbits vaccinated with strain d3 and in nonvaccinated rabbits, antigenic drift occurred later than in rabbits vaccinated with strain d1. High titers of antibodies against H. influenzae were measured in tissue cage fluid and serum. Vaccination of the rabbits with H. influenzae d1 or d3, an antigenic variant of strain d1, resulted neither in eradication of H. influenzae d1 nor in increased antibody titers in serum and tissue cage fluid. The sera of nonvaccinated rabbits during persistence had no strain d1-specific bactericidal activity in the presence of complement. Vaccination with H. influenzae d1 induced serum bactericidal activity against strain d1 in the presence of complement. However, a variant of strain d1 appearing in the tissue cages was not killed by this serum bactericidal activity. We conclude that immunological pressure leads to the selection of MOMP variants of H. influenzae and that these variants escape the antibody-mediated strain-specific bactericidal activity against H. influenzae.
Pregnancy failure following vaginal infection of sheep with Chlamydia psittaci prior to breeding
Authors: Papp J R , Shewen P E
Published: January 2020

Enzootic abortion in sheep, caused by Chlamydia psittaci, has been associated with pregnancy failure in most sheep-producing countries. Late-term abortions or the birth of weak low-birth-weight lambs occurred following primary C. psittaci infection in pregnant ewes. However, the mode by which C. psittaci can be transmitted among sheep has not been established. The present study was designed to determine whether the vaginal tracts of nonpregnant ewes were susceptible to C. psittaci infection and whether such infections had an impact during the next pregnancy. At day 0 of the estrus cycle, the vaginal tracts of 10 nonpregnant ewes were inoculated with C. psittaci and 10 ewes were exposed by subcutaneous injection. The ewes were bred 6 weeks postinfection. Five ewes from the vaginally infected group and four from the subcutaneously infected group were reinfected by subcutaneous injection at day 60 of gestation. Pregnancy outcomes and antibody responses to infection were compared with that of ewes that were infected with C. psittaci, either subcutaneously or intravaginally, for the first time during pregnancy and with that of noninfected control ewes. Subcutaneous infection of nonpregnant ewes did not cause subsequent pregnancy failure; rather, this provided protection against abortion following reinfection during pregnancy. As expected, abortions or the birth of weak lambs was observed in those ewes that received primary C. psittaci infection by either route during pregnancy. Similarly, abortion or the birth of weak lambs was a consequence of vaginal inoculation prior to breeding, thereby confirming the susceptibility of the vaginal mucosa to infection and demonstrating the potential for venereal transmission.
Toxoplasma gondii-induced immune suppression by human peripheral blood monocytes: role of gamma interferon
Authors: Channon J Y , Kasper L H
Published: January 2020

The ability of Toxoplasma gondii to evade the host immune response during primary infection in humans is poorly understood. In murine toxoplasmosis, infected spleen macrophages release soluble factors that mediate a transient immunosuppression, which may allow the parasite to become established. When an enriched population of human monocytes from seronegative individuals was incubated with toxoplasmas in vitro, soluble factors that mediated market suppression of mitogen-induced lymphocyte DNA synthesis were released. Irradiated tachyzoites that do not undergo replication were sufficient stimuli for near-maximal soluble factor release. Up to 50% of the soluble factor-mediated suppression is attributable to a gamma interferon (IFN-gamma)-dependent pathway, and the mediator of the remaining inhibition is neither interleukin-10, transforming growth factor beta, prostaglandin E2, lipoxygenase products, nitric oxide, nor tumor necrosis factor alpha-induced mitochondrial cell-derived reactive oxygen intermediates. IFN-gamma also mediates the up-regulation of an antigen-presenting cell phenotype by both infected and uninfected macrophages. However, IFN-gamma does not activate macrophages to become toxoplasmacidal; instead, intracellular toxoplasmas replicate and reinfect, eventually lysing the macrophage population. These results suggest that T. gondii is able to evade the naive host immune response by induction of soluble immunosuppressive factors that allow the parasite to become established during an acute infection.
Endogenous interleukin-4, but not interleukin-10, is involved in suppression of host resistance against Listeria monocytogenes infection in interferon-depleted mice
Authors: Nakane A , Nishikawa S , Sasaki S , Miura T , Asano M , Kohanawa M , Ishiwata K , Minagawa T
Published: January 2020

The production and roles of endogenous interleukin-4 (IL-4) and IL-10 in a sublethal infection with Listeria monocytogenes were studies in normal mice and anti-gamma interferon (IFN-gamma) monoclonal antibody (MAb)-pretreated mice. In normal mice, the expression of mRNAs for IL-4 and IL-10, which was amplified by reverse transcription-PCR, was induced in the spleens and livers either early or late in infection, although the serum IL-4 and IL-10 were not detectable by enzyme-linked immunosorbent assays. In vivo administration of anti-IL-4 MAb showed no effect on antilisterial resistance, whereas anti-IL-10 MAb partially diminished the defense. In anti-IFN-gamma MAb-pretreated mice, a delay in the bacterial elimination from the spleens and livers was observed and high titers of serum IL-4 and IL-10 were induced late in infection. Production of endogenous IL-4 and IL-10 was suppressed in both CD4+ cell-and CD8+ cell depleted mice. The suppression of antilisterial resistance in anti-IFN-gamma MAb-pretreated mice was canceled when anti-IL-4 MAb was injected late in infection, whereas anti-IL-10 MAb showed no effect. These results suggest that the host immune responses were polarized into the T-helper 2 phenotype in anti-IFN-gamma MAb-pretreated mice and that inhibition of host resistance against L. monocytogenes by depletion of IFN-gamma might be attributable to IL-4 produced by T cells polarized into the T-helper 2 phenotype as well as the inhibition of the IFN-gamma effects.
Nitric oxide induction by pertussis toxin in mouse spleen cells via gamma interferon
Authors: Sakurai S , Kamachi K , Konda T , Miyajima N , Kohase M , Yamamoto S
Published: January 2020

We examined the major pathogenic substances of Bordetella pertussis for the ability to induce nitric oxide, and important biological function of macrophages, via gamma interferon in spleen cells. B. pertussis, which produces a variety of pathogenic substances, including pertussis toxin and filamentous hemagglutinin, causes a severe respiratory disease. Nitric oxide was detected in the culture fluid of spleen cells stimulated with pertussis toxin or its B oligomer but not in the culture fluid of spleen cells stimulated with the A protomer of pertussis toxin or with filamentous hemagglutinin. Incubation of the peritoneal exudate macrophages with pertussis toxin, B oligomer, A protomer, or filamentous hemagglutinin induced little nitric oxide, whereas incubation with gamma interferon induced a significant amount of nitric oxide. The induction of nitric oxide in spleen cells stimulated with pertussis toxin was completely inhibited by anti-gamma interferon antibody. The treatment of spleen cells with anti-Thy-1.2 antibody plus complement followed by stimulation with pertussis toxin decreased the secretion of gamma interferon and nitric oxide. These results suggest that gamma interferon from T lymphocytes stimulated with pertussis toxin induces nitric oxide.
Functional chemotactic factor CP-10 and MRP-14 are abundant in murine abscesses
Authors: Kocher M , Kenny P A , Farram E , Abdul Majid K B , Finlay-Jones J J , Geczy C L
Published: January 2020

Murine abscesses induced by intraperitoneal injection of a mixture of Escherichia coli, Bacteroides fragilis, and bran are established models for the study of localized infectious and inflammatory lesions. Chemotactic factors are though to mediate the directed migration of large numbers of leukocytes into the abscess. Microorganisms located within the encapsulated lesion are not readily eliminated by the leukocytes, but their numbers are controlled over many weeks. We report the presence of large amounts of two murine S100 proteins, CP-10 and migration inhibition factor-related protein 14 (MRP-14), in abscesses as demonstrated by immunohistochemistry and measured by enzyme-linked immunosorbent assay and Western blotting (immunoblotting). High levels of CP-10 (7.7 +/- 1 mg/ml) and MRP-14 (5.5 +/- 1 mg/ml) were found throughout the time course of abscess development from early acute-phase lesions, which are predominantly neutrophilic, to late chronic-phase lesions, which contained more mononuclear cells. Approximately one-third of these amounts occurred as monomers (2.0 mg/ml for MRP 14 and 2.2 mg/ml for CP-10). Abscess fluid was strongly chemotactic, and a portion of the activity was due to CP-10, indicating its important role in leukocyte recruitment. CP-10-MRP-14 complexes were present in abscess fluid, and the proteins were immunoabsorbed together. In analogy with the related human MRP-8-MRP-14 complex, these proteins could be involved in the inhibition of microbial growth. No growth inhibition occurred with 20 microgram of CP-10 or MRP-14 per ml or with mixtures of both, but these concentrations may have been insufficient and were not representative of the high concentrations found within abscesses. CP-10 may contribute indirectly to the antimicrobial response in abscesses by virtue of its strong chemotactic properties and its capacity to modulate the activation state of recruited leukocytes.
Serological analysis of antigenic heterogeneity of Giardia lamblia variant surface proteins
Authors: Müller N , Stäger S , Gottstein B
Published: January 2020

Neonatal ZU.ICR mice were infected with trophozoites of Giardia lamblia clone GS/M-83-H7 expressing the variant surface protein (VSP) H7 and were subsequently investigated for their serum antibody response directed against VSPH7. Recombinant polypeptides, representing overlapping segments of VSPH7, and native Giardia proteins were used as antigenic reagents to examine the antigenic substructure of VSPH7 and the extent of antigenic variation in vivo. VSPH7 proved to be the predominant antigen of the parasite with respect to serum antibody reactivity. The data indicated that VSPH7 basically consists of two antigenically distinct parts: (i) a unique, variant-specific 314-amino-acid N-terminal region which elicits a low antibody response preferentially detectable during the early phase of the infection and (ii) a 171-amino-acid C-terminal region which elicits a high antibody response during the later phase or after resolution of the infection. The epitopes of the C-terminal region appear to be shared by other, as yet uncharacterized, variant antigens. The highly conserved 34-amino-acid stretch at the extreme C terminus of VSPH7 exhibited no immunoreactivity to any of the sera tested. Further investigations indicated that antigenic variation of the intestinal parasite population was associated with a diversification into at least six to nine new antigen types. These variant antigens were extremely heterogeneous in size ranging from approximately 50 to 115 kDa. None of these proteins shared antigenic epitopes with the 314-amino-acid N-terminal portion, but several of them cross-reacted with antibodies specific to the 171-amino-acid C-terminal portion of the VSP from the original inoculum.
Endogenous and exogenous interleukin-12 augment the protective immune response in mice orally challenged with Salmonella dublin
Authors: Kincy-Cain T , Clements J D , Bost K L
Published: January 2020

Following oral challenge with Salmonella dublin, we observed significant increases in interleukin-12 (IL-12) protein expression in the mesenteric lymph nodes. The importance of this endogenous cytokine production in the immune response against S. dublin was demonstrated by in vivo depletion of IL-12 with an anti-IL-12 monoclonal antibody prior to oral S. dublin challenge. Mice pretreated with anti-IL-12 antibody had increased salmonellosis and reduced survival times compared with mice receiving control antibody. Furthermore, administration of exogenous murine recombinant IL-12 dramatically increased survival times of mice challenged orally with S. dublin. Together, these results demonstrate that endogenous and exogenous IL-12 significantly augment the mucosal immune response against the intracellular pathogen S. dublin.
Cloning and characterization of a putative cytadhesin gene (mgc1) from Mycoplasma gallisepticum
Authors: Keeler C L , Hnatow L L , Whetzel P L , Dohms J E
Published: January 2020

A 150-kDa cytadhesin-like protein from Mycoplasma gallisepticum has been identified. A previously described 583-bp fragment (J.E. Dohms, L.L. Hnatow, P. Whetzel, R. Morgan and C.L. Keeler, Jr., Avian Dis. 37:380-388, 1993) was used to probe a genomic library of M. gallisepticum DNA. An 8.0-kb SacI fragment was identified, cloned, and partially sequenced. Analysis of the resulting 3,750-bp sequence revealed the presence of a 3,366-nucleotide open reading frame, mgc1. The 1,122-amino-acid protein encoded by this open reading frame, MGC1, has characteristics of a class I membrane protein and has homology with the MgPa cytadhesin of Mycoplasma genitalium (26.3%) and the P1 cytadhesin of Mycoplasma pneumoniae (28.7%). A portion of MGC1 was expressed as a glutathione S-transferase fusion protein and used to produce antiserum in rabbits. The antiserum recognizes a 150-kDa protein from M. gallisepticum. The protein is sensitive to trypsin, confirming that it is surface exposed. Primer extension analysis indicates that the mgc1 RNA starts within an upstream open reading frame, suggesting complex control of its expression. This is the first description of a functional gene from M. gallisepticum showing homology to cytadhesin genes from human mycoplasmas.
Activation of Shiga-like toxins by mouse and human intestinal mucus correlates with virulence of enterohemorrhagic Escherichia coli O91:H21 isolates in orally infected, streptomycin-treated mice
Authors: Melton-Celsa A R , Darnell S C , O'Brien A D
Published: January 2020

The enterohemorrhagic Escherichia coli (EHEC) O91:H21 isolates B2F1 and H414-36/89 are virulent in an orally infected streptomycin-treated mouse model. Previous studies demonstrated that B2F1 and H414-36/89 grow to high levels in mucus isolated from mouse small intestine and colon and that growth in small-intestine mucus is related to virulence. We measured the levels of Shiga-like toxins (SLTs) SLT-IIvha and SLT-IIvhb produced by B2F1 after growth in Luria-Bertani (LB) broth supplemented with mouse intestinal mucus by assaying the cytotoxicity of culture supernatants on Vero cells. Culture supernatants from B2F1 grown in mouse intestinal mucus, but not EHEC strains that produce SLT-II or SLT-IIc, were approximately 35- to 350-fold more toxic for Vero cells than supernatants from B2F1 grown in LB broth. This increased toxicity was not reflected by a concomitant increase in SLT antigen content. Furthermore, when culture supernatants from B2F1 or K-12 strains carrying plasmids encoding SLTs cloned from H414-36/89 or purified SLT-IIvhb from B2F1 were incubated with mouse intestinal mucus, the samples exhibited greater cytotoxicity than when they were incubated with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer alone. These toxin preparations also showed increased cytotoxicity after incubation with human colonic mucus. In contrast, culture supernatants from LB-grown EHEC isolates that produced SLT-I, SLT-II, SLT-IIc or SLT-IIe did not show increased cytotoxicity after incubation with mouse or human intestinal mucus. The A subunits of purified SLT-II and SLT-IIvhb that had been treated with mouse intestinal mucus or trypsin were cleaved to A1 fragments by the mucus, but trypsin-mediated cleavage, unlike treatment with mouse intestinal mucus, did not result in increased Vero cell cytotoxicity activity. This finding implies that the increased cytotoxicity of SLT-IIvhb detected after incubation with mucus is probably not due to cleavage of the A subunit into the A1 and A2 fragments. Taken together, these results indicate that mouse or human intestinal mucus directly activates SLT-II-related toxins from B2F1 and H414-36/89 and suggest that toxin activation may explain the low 50% lethal doses of B2F1 and H414-36/89 in streptomycin-treated mice.
Antibody-mediated shift in the profile of glycoprotein A phenotypes observed in a mouse model of Pneumocystis carinii pneumonia
Authors: Gigliotti F , Garvy B A , Harmsen A G
Published: January 2020

It is well established that Pneumocystis carinii has the molecular capability for variation of a major surface antigen, glycoprotein A (gpA). However, the extent of expression of gpA variation among P. carinii organisms infecting a single host and whether this variation has any impact on host-parasite immunological interactions is unknown. Using a mouse model of P. carinii pneumonia, we were able to demonstrate the expression of more than one gpA phenotype in a closed population of infected mice. Administration of monoclonal antibody (MAb) 2B5, which is specific for one of the gpA phenotypes, resulted in a marked diminution in the frequency of this particular gpA phenotype in the population of organisms. This effect was due to a loss of trophozoites bearing the specific epitope recognized by MAb 2B5; cysts bearing the same epitope appeared unaffected. Interestingly, P. carinii was unable to introduce a new phenotype into the population to compensate for the loss of trophozoites bearing the epitope recognized by MAb 2B5. Discontinuing administration of MAb 2B5 allowed the MAb 2B5-binding phenotype to reemerge. This finding suggests that the phenotype recognized by MAb 2B5 was continually produced even when MAb 2B5 was present. Thus, although P. carinii exhibited a form of antigenic variation, it did not appear able to rapidly introduce new phenotypes into the population in response to destruction by antibodies.
Cytotoxic-T-lymphocyte-mediated cytolysis of L cells persistently infected with Chlamydia spp
Authors: Rasmussen S J , Timms P , Beatty P R , Stephens R S
Published: January 2020

Persistent chlamydial infections have been proposed as a means whereby chlamydiae evade immune resolution of infection. Such a mechanism would require evasion not only of the humoral immune responses but also of cell-mediated immune responses. We hypothesized that if such a mechanism is important, persistently infected cells should not be recognized by cytotoxic T cells. Persistent infections were simulated in vitro by treatment of Chlamydia trachomatis- or Chlamydia psittaci-infected cells with gamma interferon (IFN-gamma), penicillin, or tryptophan depletion. Cultures were examined for induction of a chlamydial stress response (measured by transcription of groesl RNA) and for the effects on viability, infectivity, morphology, and immune recognition. Although both IFN-gamma and penicillin induced aberrant chlamydial morphology and growth, we did not find evidence that these treatments elicited a classical stress response. In addition, T-cell-mediated lysis of Chlamydia-infected target cells treated with IFN-gamma or penicillin or grown in tryptophan-deficient media was examined. The immune cell-mediated lysis of these treated infected cells demonstrated that despite the effects of these compounds on chlamydial growth and development, the infected cells continued to be efficiently recognized and killed by cytotoxic T cells. Thus, it seems unlikely that these in vitro models of persistence represent functional mechanisms to evade immune clearance.
The conserved 18,000-molecular-weight outer membrane protein of Haemophilus ducreyi has homology to PAL
Authors: Spinola S M , Hiltke T J , Fortney K , Shanks K L
Published: January 2020

Haemophilus ducreyi expresses an 18,000-molecular-weight outer membrane protein that contains a conserved surface-exposed epitope recognized by monoclonal antibody 3B9. Monoclonal antibody 3B9 cross-reacts with proteins of similar molecular weight found in many Haemophilus sp. strains, including P6, a candidate vaccine for Haemophilus influenzae. The gene encoding the 18,000-molecular-weight outer membrane protein was identified by screening a lambdagt11 genomic library with 3B9. The coding sequence of the gene was localized to a 471-bp open reading frame, designated pal (peptidoglycan-associated lipoprotein). Translation of pal predicted a mature polypeptide with a molecular weight of 15,000 that had extensive homology with P6 and Escherichia coli PAL. The predicted signal peptide had features characteristic of a prokaryotic lipoprotein, and processing of PAL was sensitive to globomycin in H. ducreyi. The sequences encoding mature H. ducreyi PAL were subcloned into the vector pRSET B and expressed as a polyhistidine-containing fusion protein that bound 3B9. In Western blot (immunoblot) analysis, serum samples obtained from healthy subjects and patients with chancroid or other genital ulcer diseases contained antibodies to purified PAL. Antibodies that bound to PAL were removed by absorption with a lysate of Haemophilus sp. antigens, suggesting that patients with chancroid do not develop an H. ducreyi-specific antibody response to PAL.
Conservation of the sporozoite p67 vaccine antigen in cattle-derived Theileria parva stocks with different cross-immunity profiles
Authors: Nene V , Musoke A , Gobright E , Morzaria S
Published: January 2020

Immunity to Theileria parva infection in cattle is often parasite stock specific. The antigenic diversity which is expressed at the schizont stage of the parasite together with a wild reservoir of the organism in buffalo has complicated the development of effective disease control by immunization. We have previously shown that about 70% of cattle inoculated with recombinant forms of p67, a sporozoite stage-specific surface antigen from the cattle-derived Muguga stock of the parasite, are immune to a homologous challenge. Thus, immune responses to p67 can play a role in immunity. The genes encoding this protein in five other parasite stocks have been sequenced. Here, we report that the p67 molecule encoded by four cattle-derived parasite stocks (Boleni, Uganda, Mariakani, and Marikebuni) that fall into different cross-immunity groups is identical in sequence to Muguga p67. The protein encoded by a buffalo-derived parasite exhibits 95% sequence identity with Muguga p67, the major difference being the presence of a 43-residue peptide insert. As predicted by these data, cattle inoculated with recombinant p67 can resist a heterologous cattle-derived parasite challenge. Seven of 12 cattle receiving a homologous Muguga challenge and 6 of 11 cattle receiving a heterologous Marikebuni challenge were immune to East Coast fever. These results extend earlier data suggesting that p67 is a conserved molecule and confirm its potential as a broad-spectrum vaccine antigen for the control of T. parva infection.
Contribution of YopB to virulence of Yersinia enterocolitica
Authors: Hartland E L , Bordun A M , Robins-Browne R M
Published: January 2020

The 70-kb virulence plasmid, pYV, of Yersinia enterocolitica encodes a number of secreted proteins (Yops) which are essential for virulence. YopD, the 33-kDa product of the lcrGVHyopBD operon, appears to be involved in delivering YopE and YopH (the Yersinia protein tyrosine phosphatase) into target cells. These proteins then act in concert to cause cytotoxicity in host cells. Previously, we reported that bacteria carrying transposon insertions in yopD are not cytotoxic for macrophages, show impaired tyrosine phosphatase activity in host cells, and are avirulent for mice (E. L. Hartland, S. P. Green, W. A. Phillips, and R. M. Robins-Browne, Infect. Immun. 62:4445-4453, 1994). trans complementation of yopD mutants of Y. enterocolitica with the yopD gene restores all these properties. In this study, we show that polar mutations in proximal genes of the lcrGVHyopBD operon also abrogated bacterial virulence and the capacity to induce cytotoxicity in mouse bone marrow-derived macrophages and HEp-2 epithelial cells. Moreover, trans complementation of a yopBD mutant with the yopD gene alone was not sufficient to restore the ability of the bacteria to cause cytotoxicity. Further work showed that YopB was required for cytotoxicity, dephosphorylation of host proteins, and virulence for mice. These findings indicate that YopB and YopD may serve a related function in Y. enterocolitica and that they may act together to deliver intracellularly acting Yops to their respective targets in host cells.
Comparison of haemophilus influenzae type b interaction with respiratory mucosa organ cultures maintained with an air interface or immersed in medium
Authors: Jackson A D , Cole P J , Wilson R
Published: January 2020

Haemophilus influenzae type b infection of immersed and air interface organ cultures for 24 h caused significant epithelial damage. Bacterial association with mucus, damaged epithelium, and unciliated cells was significantly higher in air interface than immersed organ cultures, and total bacterial association was 55.8 times greater. Bacteria exhibited tropism for mucus only on explants maintained with an air interface. We conclude that immersion of nasopharyngeal tissue in medium may influence bacterial interaction with the mucosal surface.
Melanin, melanin "ghosts," and melanin composition in Cryptococcus neoformans
Authors: Wang Y , Aisen P , Casadevall A
Published: January 2020

Melanin synthesis is associated with virulence for the pathogenic fungus Cryptococcus neoformans. Exposure of nonmelanized C. neoformans 4067 cells to 4 M guanidinium isothiocyanate followed by 6 M HCl at 100 degrees C resulted in complete solubilization of cellular structures. However, exposure of melanized C. neoformans 24067 to the same conditions produced a suspension of black particles. Analysis of black particles with transmission and scanning electron microscopy revealed cell "ghosts" with electron-dense walls. Electron spin resonance (ESR) spectroscopy of suspensions of black particles revealed a signal indicative of a stable free radical population. The magnitude of the ESR signal was increased by illumination, Zn2+ ions, and basic pH. Boiling the black particles produced only a small reduction in the magnitude of the ESR signal. These ESR changes are characteristic of melanin. Exposure of melanized black cells to 0.1 or 0.5% NaOCl bleached the cells white and abolished the ESR signal, consistent with melanin disruption. ESR spectroscopy was used to study the rate of stable free radical formation, taken as an index of melanization, of C. neoformans 24067 cells growing in medium with 1.0 mM L-dopa at 30 and 37 degrees C over the course of 14 days. Melanization was slower at 37 degrees C. but by day 14, cells grown at 30 and 37 degrees C. had comparable levels or melanin. ESR spectroscopy of seven melanized C. neoformans strains revealed that some strains differed by up to eightfold in melanin content. The ESR spectra of C. neoformans 24067 grown in minimal medium containing either 1.0 mM catechol, dopamine, or L-dopa revealed large differences in signal amplitude. The relative melanin contents were 0.05, 0.45, and 1.0 for C. neoformans grown in media with catechol, dopamine. and L-dopa, respectively. Quantitative analysis revealed that melanin comprised 15.4% of the dry mass of the cell after 10 days of growth in medium containing 1.0 mM L-dopa. The results suggest (i) a useful protocol for isolating C. neoformans melanin, (ii) a role for melanin in maintaining cell wall integrity, (iii) disruption of melanin by NaOCl, (iv) strain differences in melanin content after growth in L-dopa, and (v) quantitative and/or qualitative differences in the melanin produced after growth in various substrates.
Comparison of in vitro models for the study of Mycobacterium tuberculosis invasion and intracellular replication
Authors: Mehta P K , King C H , White E H , Murtagh J J , Quinn F D
Published: January 2020

We recently evaluated several tissue culture model systems for the study of invasion and intracellular multiplication of Mycobacterium tuberculosis. These model systems include a human alveolar pneumocyte epithelial cell line, a murine macrophage cell line (J774), and fresh human peripheral blood-derived macrophages. Our data indicated that the initial level of association of M. tuberculosis with human alveolar pneumocyte cells (2%) was less than that observed with fresh human peripheral blood macrophages (9%) or J774 murine macrophages (13%) within 6 h of the addition of the bacteria. M. tuberculosis replicated in association with the pneumocyte cells by more than 55-fold by day 7 postinfection. In contrast, total bacteria] growth in the J774 cells and human macrophages was considerably less, with increases of only fourfold and threefold, respectively, over the same 7-day period. Amikacin, an aminoglycoside antimicrobial agent, was added to inhibit the growth of extracellular bacteria after the initial 6-h infection period. Decreases in viable counts were observed in all three cell cultures within the first 3 days after infection. However, unlike the case with either macrophage culture, intracellular bacterial CFU obtained from the infected pneumocytes increased by fourfold by day 7 after the addition of amikacin. These data indicate that M. tuberculosis infects and multiplies intracellularly in human lung epithelial cells and that these cells may be an alternative in vitro model for the study of intracellular multiplication of M. tuberculosis in the human lung.
Studies of immunity and bacterial invasiveness in mice given a recombinant salmonella vector encoding murine interleukin-6
Authors: Dunstan S J , Ramsay A J , Strugnell R A
Published: January 2020

Interleukin-6 (IL-6) was expressed in Salmonella typhimurium in an attempt to increase the mucosal immune response against the bacterium. Murine IL-6 was PCR amplified from cDNA, cloned, sequenced, and found to be functionally active when expressed in S. typhimurium BRD509, the (delta)aroA (delta)aroD vaccine strain. Expression of murine IL-6 did not appear to adversely affect the growth of salmonellae, as the construct was retained in the absence of antibiotic selection and the growth rate was unaffected compared with that of the parent strain in vitro. However, IL-6 expression led to a significant reduction in bacterial invasiveness in vitro and in vivo. Splenocytes and small intestinal lamina propria lymphocytes were isolated from mice orally immunized with BRD509 expressing IL-6 (pKK233-2/IL-6), and the number of antibody-secreting cells was determined by the ELISPOT technique. No differences were observed between mice immunized with BRD509(pKK.233-2/IL-6) and those immunized with BRD509(pKK233-2) with respect to the antibody subclass-specific responses elicited despite the markedly reduced invasiveness of the former. Serum antibody responses were also examined by a kinetic enzyme-linked immunosorbent assay (ELISA), and equivalent levels of antibody response were detected in mice given BRD509(pKK233-2/IL-6) and those given BRD509(pKK233-2). The humoral immune response against bacterial lipopolysaccharides was also examined in transgenic IL-6-deficient mice given oral inocula of BRD509. Equivalent numbers of antibody-secreting cells (ELISPOTs) were observed in the spleens and laminae propriae of both IL-6-deficient (-/-) mice and control (+/+) mice harboring an intact IL-6 gene, whereas small, yet significant differences in the serum immunoglobulin A ELISA titers were observed. These data suggest that the immunoglobulin A response against Salmonella lipopolysaccharides is largely IL-6 independent.
Tumor necrosis factor alpha and interleukin-12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms
Authors: Zhan Y , Liu Z , Cheers C
Published: January 2020

Both interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) are produced early in intracellular bacterial infection. Depletion of either IL-12 or TNF-alpha by a single injection of specific antibody 4 h before the injection of Brucella abortus 19 led to the exacerbation of infection 2 weeks later. Whereas the effect of IL-12 depletion on resistance was persistent and exacerbation was still significant 6 weeks later, the bacterial numbers in mice depleted of TNF-alpha were similar to the bacterial numbers in control infected mice by 6 weeks postinfection. Massive splenomegaly, which is often seen in 2-week Brucella-infected mice, was not observed in IL-12- or TNF-alpha-depleted mice. Both IL-12- and TNF-alpha-depleted mice showed reduced cell accumulation in the spleen compared with the massive cell accumulation in control infected mice. Granuloma formation in livers was much reduced in IL-12-depleted mice but not in TNF-alpha-depleted mice. Gamma interferon (IFN-gamma) production by cells from TNF-alpha-depleted mice was not significantly different from that of cells from control infected mice. In contrast, the production of IFN-gamma by both CD4+ and CD8+ T cells from IL-12-depleted mice was greatly reduced, compared with that from control infected mice. This effect was still observed when the antibody injection was delayed for up to 7 days postinfection, but injections of anti-IL-12 antibody into mice with established Brucella infection had no significant effect on IFN-gamma production by T cells. Taken together, these results suggested that IL-12 contributed to resistance mainly via an IFN-gamma-dependent pathway and had a profound effect on the induction of acquired cellular resistance. In contrast, TNF-alpha was involved in resistance possibly via direct action on effector cells and may not be essential for the induction of acquired cellular resistance.
Identification of an immunoglobulin A binding motif located in the beta-antigen of the c protein complex of group B streptococci
Authors: Jerlström P G , Talay S R , Valentin-Weigand P , Timmis K N , Chhatwal G S
Published: January 2020

The beta-antigen of the c protein complex of group B streptococci contains two immunoglobulin A (IgA)-binding domains called A and B. A 73-amino-acid segment in domain A is responsible for most of the IgA-binding activity. To identify the IgA binding motif, the 73-amino-acid domain was divided into 60 14-amino-acid overlapping peptides spot synthesized onto a cellulose membrane. A 20-residue putative antigenic epitope was identified and expressed as a fusion protein. The fusion protein was purified by fast protein liquid chromatography and used to raise rabbit antiserum. By use of a membrane with spot-synthesized peptide amino acids of decreasing length (from 14 to 6 amino acids), the major antigenic epitope recognized by the anti-fusion protein antibodies was mapped to motif MLKKIE. Anti-fusion protein antibodies inhibited the binding of IgA to group B streptococci. This inhibition could be blocked by the peptide containing the motif MLKKIE. These results indicate that the motif MLKKIE is located in the IgA-binding site. The IgA-binding domain of beta-antigen from three group B streptococcal strains reacted with the anti-fusion protein antibodies, and their coding sequences gave positive signals in Southern hybridization. The sequences of beta-antigen from these strains were amplified by PCR, and sequence analysis showed them to be identical. The results indicate that the motif MLKKIE is required for IgA binding and is present in different group B streptococcal strains.
Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity
Authors: Bauerfeind P , Garner R M , Mobley L T
Published: January 2020

Helicobacter pylori, an etiologic agent of gastritis and peptic ulceration in humans, synthesizes urease, a nickel metalloenzyme, as its most abundant protein. NixA, a high-affinity nickel transport protein, allows synthesis of catalytically active urease when coexpressed with H. pylori urease in an Escherichia coli host. To determine whether NixA is essential for the production of active urease in H. pylori, nixA was insertionally inactivated with a kanamycin resistance cassette (aphA) and this construct was electroporated into H. pylori ATCC 43504; allelic exchange mutants were selected on kanamycin-containing medium. The nixA mutation, confirmed by PCR, reduced urease activity by 42% (140 +/- 70 micromol of NH3/min/mg of protein in the mutant versus 240 +/- 100 micromol of NH3/min/mg of protein in the parent (P = 0.037). Rates of nickel transport were dramatically reduced (P = 0.0002) in the nixA mutant (9.3 +/- 3.7 pmol of Ni2+/min/10(8) bacteria) of H. pylori as compared with the parent strain (30.2 +/- 8.1 pmol of Ni2+/min/10(8) bacteria). We conclude that NixA is an important mediator of nickel transport in H. pylori. That residual nickel transport and urease activity remain in the nixA mutant, however, provides evidence for the presence of a redundant transport system in this species.
Epidermal growth factor-binding protein in Mycobacterium avium and Mycobacterium tuberculosis: a possible role in the mechanism of infection
Authors: Bermudez L E , Petrofsky M , Shelton K
Published: January 2020

Epidermal growth factor (EGF) is a potent mitogen for a variety of eukaryotic cells. EGF is found in a number of tissues and is prevalent in necrotic tissues and granulomata. The biological effect of EGF on mammalian cells is initiated by the binding to a specific receptor. Both Mycobacterium avium and Mycobacterium tuberculosis cause lung infections and localized or disseminated disease in both patients without AIDS and those with AIDS. Histopathologic studies show necrosis in the lung, liver, and splenic tissues of patients with disseminated mycobacterial infection. In the course of experiments to examine the effect of growth factors on macrophages, it was observed that M. avium and M. tuberculosis but not Mycobacterium smegmatis cultured in the presence of 5, 50, or 500 ng of EGF per ml grew significantly faster than mycobacteria cultured in the absence of EGF. 125I-EGF was found to bind to M. avium and M. tuberculosis, and the binding was competitively inhibited by unlabeled EGF. A receptor for EGF was identified on mycobacteria. Incubation of mycobacteria with EGF prior to infection of macrophage monolayers resulted in faster bacterial growth within macrophages compared with that of mycobacteria not incubated with EGF. EGF-binding protein was cloned and expressed in Escherichia coli, and subsequently the protein was purified and the N-terminal amino acids were sequenced. These results suggest that EGF is a growth factor for pathogenic mycobacteria in granulomatous tissues and within macrophages and might enhance growth rates of both intracellular and extracellular mycobacteria in the site of infection.
Invasion of epithelial cells by Actinobacillus actinomycetemcomitans: a dynamic, multistep process
Authors: Meyer D H , Lippmann J E , Fives-Taylor P M
Published: January 2020

The invasion process of Actinobacillus actinomycetemcomitans, a periodontopathogen, was studied with microscopy and viable quantitative assays using both KB and Madin-Darby canine kidney (MDCK) epithelial cells. Microscopy revealed that the events associated with the A. actinomycetemcomitans invasion process occurred rapidly. Scanning electron micrographs revealed A. actinomycetemcomitans associated with craters on the KB cell surface and others entering the KB cells through apertures with lip-like rims within 30 min of infection. Both transmission electron and immunofluorescence micrographs demonstrated that by this time some bacteria had, in fact, already entered, replicated, and exited host cells. Scanning electron micrographs revealed that infected KB cells exhibited fibrillar protrusions which contained bulges with the conformation of bacteria. Some protrusions formed intercellular connections between KB cells. Immunofluorescence micrographs revealed protrusions which harbored A. actinomycetemcomitans. The spread of internalized A. actinomycetemcomitans from one MDCK epithelial cell monolayer to another was demonstrated using a sandwich assay developed in our laboratory. Transcytosis of A. actinomycetemcomitans through polarized MDCK cells was also demonstrated. This study indicates that soon after entry of A. actinomycetemcomitans bacteria into epithelial cells, they undergo rapid multiplication and may subsequently be found in protrusions which sometimes extend between neighboring epithelial cells. The protrusions are thought to mediate the cell-to-cell spread of A. actinomycetemcomitans. Cell-to-cell spread may also occur by the endocytosis of A. actinomycetemcomitans bacteria which have been released into the medium via rudimentary protrusions which do not interconnect epithelial cells. The finding that the A. actinomycetemcomitans invasion process is so dynamic sheds significant new light on the interaction of this periodontopathogen with mammalian cells.