bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunogenicity of Outer Membrane Proteins in a Lipopolysaccharide-Deficient Mutant ofNeisseria meningitidis: Influence of Adjuvants on the Immune Response
Authors: Steeghs Liana , Kuipers Betsy , Hamstra Hendrik Jan , Kersten Gideon , van Alphen Loek , van der Ley Peter 
Published: December 2019

ABSTRACTThe immunogenicity of outer membrane complexes (OMCs) or heat-inactivated bacteria of a lipopolysaccharide (LPS)-deficient mutant derived from meningococcal strain H44/76 was studied. The immune response in BALB/c mice to the major outer membrane proteins was poor compared to the immune response elicited by wild-type immunogens. However, addition of external H44/76 LPS to mutant OMCs entirely restored the immune response. By using an LPS-deficient mutant, it may be possible to substitute a less toxic compound as adjuvant in meningococcal outer membrane vaccines. Therefore, a broad panel of adjuvants were tested for their potential to enhance the immunogenicity of LPS-deficient OMCs. AlPO4,Rhodobacter sphaeroidesLPS, monophosphoryl lipid A and alkali-hydrolyzed meningococcal LPS showed significantly lower adjuvant activity than did H44/76 LPS. Adjuvant activity similar to H44/76 LPS was found forEscherichia coliLPS, meningococcalicsBandrfaCLPS, QuilA, subfractions of QuilA, and MF59. Good adjuvant activity was also found with meningococcalhtrB1LPS, containing penta-acylated lipid A. Antisera elicited with the less active adjuvants showed relatively high immunoglobulin G1 (IgG1) titers, whereas strong adjuvants also induced high IgG2a and IgG2b responses in addition to IgG1. Antisera with the IgG2a and IgG2b isotypes showed high bactericidal activity, indicating that adjuvants promoting the IgG2a and IgG2b response contribute most to the protective mechanism. Thus, this study demonstrates that the immunogenicity of meningococcal LPS-deficient OMCs can be restored by using less toxic adjuvants, which opens up new avenues for development of vaccines against meningococcal disease.
Identification of theznuA-Encoded Periplasmic Zinc Transport Protein ofHaemophilus ducreyi
Authors: Lewis David A. , Klesney-Tait Julia , Lumbley Sheryl R. , Ward Christine K. , Latimer Jo L. , Ison Catherine A. , Hansen Eric J. 
Published: December 2019

ABSTRACTTheznuAgene ofHaemophilus ducreyiencodes a 32-kDa (mature) protein that has homology to both the ZnuA protein ofEscherichia coliand the Pzp1 protein ofH. influenzae; both of these latter proteins are members of a growing family of prokaryotic zinc transporters. Inactivation of theH. ducreyi35000znuAgene by insertional mutagenesis resulted in a mutant that grew more slowly than the wild-type parent strain in vitro unless ZnCl2was provided at a final concentration of 100 μM. Other cations tested did not restore growth of thisH. ducreyimutant to wild-type levels. TheH. ducreyiZnuA protein was localized to the periplasm, where it is believed to function as the binding component of a zinc transport system. Complementation of theznuAmutation with the wild-typeH. ducreyi znuAgene provided intransrestored the ability of thisH. ducreyimutant to grow normally in the absence of exogenously added ZnCl2. The wild-typeH. ducreyi znuAgene was also able to complement aH. influenzae pzp1mutation. TheH. ducreyi znuAisogenic mutant exhibited significantly decreased virulence (P= 0.0001) when tested in the temperature-dependent rabbit model for experimental chancroid. This decreased virulence was not observed when theznuAmutant was complemented with the wild-typeH. ducreyi znuAgene provided intrans.
Outer Membrane Proteins as a Carrier for Detoxified Lipooligosaccharide Conjugate Vaccines for NontypeableHaemophilus influenzae
Authors: Wu Ting-Huai , Gu Xin-Xing 
Published: December 2019

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a common cause of otitis media and respiratory tract infections. Outer membrane proteins (OMP) and lipooligosaccharide (LOS) are major surface antigens of NTHi and potential vaccine candidates. De-O-acylated LOS (dLOS) or oligosaccharide (OS) was coupled to total OMP to form dLOS-OMP and OS-OMP conjugates, while a dLOS-tetanus toxoid (TT) was synthesized for comparison. These conjugates were evaluated in mice and rabbits for immunogenicity. dLOS-OMP elicited a better boostable antibody response against LOS than did dLOS-TT, while OS-OMP was not immunogenic. Formulation of the conjugates with Ribi adjuvant significantly enhanced the immunogenicity of dLOS-OMP and dLOS-TT but not that of OS-OMP. In addition, rabbit antisera elicited by dLOS-OMP but not dLOS-TT (or OMP alone) demonstrated bactericidal activity against 40% of the NTHi strains tested. These results indicate that dLOS is a better derivative of LOS than OS and that OMP is a good carrier for NTHi LOS-based conjugate vaccines.
Differential T-Cell Recognition of Native and RecombinantMycobacterium tuberculosisGroES
Authors: Rosenkrands Ida , Weldingh Karin , Ravn Pernille , Brandt Lise , Højrup Peter , Rasmussen Peter Birk , Coates Anthony R. , Singh Mahavir , Mascagni Paolo , Andersen Peter 
Published: December 2019

ABSTRACTMycobacterium tuberculosisGroES was purified from culture filtrate, and its identity was confirmed by immunoblot analysis and N-terminal sequencing. Comparing the immunological recognition of native and recombinant GroES, we found that whereas native GroES elicited a strong proliferative response and release of gamma interferon-γ by peripheral blood mononuclear cells from healthy tuberculin reactors, the recombinant protein failed to do so. The same difference in immunological recognition was observed in a mouse model of TB infection. Both the native and recombinant preparations were recognized by mice immunized with the recombinant protein. Biochemical characterization including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional electrophoresis, and mass spectrometry analysis of both proteins demonstrated no differences between the native and recombinant forms of GroES except for the eight additional N-terminal amino acids derived from the fusion partner in recombinant GroES. The recombinant fusion protein, still tagged with the maltose binding protein, was recognized by T cells isolated from TB-infected mice if mixed with culture filtrate before affinity purification on an amylose column. The maltose binding protein treated in the same manner as a control preparation was not recognized. Based on the data presented, we suggest that the association of biologically active molecules from culture filtrate with the chaperone GroES may be responsible for the observed T-cell recognition of the native preparation.
Identification of Regions of the Chromosome of
Neisseria meningitidis
and
Neisseria gonorrhoeae
Which Are Specific to the Pathogenic
Neisseria
Species
Authors: Perrin Agnes , Nassif Xavier , Tinsley Colin 
Published: December 2019

ABSTRACT

Neisseria meningitidis
and
Neisseria gonorrhoeae
give rise to dramatically different diseases. Their interactions with the host, however, do share common characteristics: they are both human pathogens which do not survive in the environment and which colonize and invade mucosa at their port of entry. It is therefore likely that they have common properties that might not be found in nonpathogenic bacteria belonging to the same genetically related group, such as
Neisseria lactamica
. Their common properties may be determined by chromosomal regions found only in the pathogenic
Neisseria
species. To address this issue, we used a previously described technique (C. R. Tinsley and X. Nassif, Proc. Natl. Acad. Sci. USA 93:11109–11114, 1996) to identify sequences of DNA specific for pathogenic neisseriae and not found in
N. lactamica
. Sequences present in
N. lactamica
were physically subtracted from the
N. meningitidis
Z2491 sequence and also from the
N. gonorrhoeae
FA1090 sequence. The clones obtained from each subtraction were tested by Southern blotting for their reactivity with the three species, and only those which reacted with both
N. meningitidis
and
N. gonorrhoeae
(i.e., not specific to either one of the pathogens) were further investigated. In a first step, these clones were mapped onto the chromosomes of both
N. meningitidis
and
N. gonorrhoeae
. The majority of the clones were arranged in clusters extending up to 10 kb, suggesting the presence of chromosomal regions common to
N. meningitidis
and
N. gonorrhoeae
which distinguish these pathogens from the commensal
N. lactamica
. The sequences surrounding these clones were determined from the
N. meningitidis
genome-sequencing project. Several clones corresponded to previously described factors required for colonization and survival at the port of entry, such as immunoglobulin A protease and PilC. Others were homologous to virulence-associated proteins in other bacteria, demonstrating that the subtractive clones are capable of pinpointing chromosomal regions shared by
N. meningitidis
and
N. gonorrhoeae
which are involved in common aspects of the host interaction of both pathogens.
Inhibition of Neutrophil Apoptosis by Verotoxin 2 Derived from
Escherichia coli
O157:H7
Authors: Liu Jiajia , Akahoshi Tohru , Sasahana Takeshi , Kitasato Hidero , Namai Rie , Sasaki Takeji , Inoue Matsuhisa , Kondo Hirobumi 
Published: December 2019

ABSTRACT
In order to evaluate the pathological role of verotoxin 2 (VT2), we investigated the effects of VT2 on neutrophil apoptosis in vitro. The results showed that VT2 caused a significant delay in spontaneous neutrophil apoptosis and that the effect was abrogated by a protein kinase C inhibitor. These data indicate that longer survival of neutrophils may aggravate neutrophil-mediated tissue damage in VT2-associated diseases.
Induction of Protective T Cells againstListeria monocytogenesin Mice by Immunization with a Listeriolysin O-Negative Avirulent Strain of Bacteria and Liposome-Encapsulated Listeriolysin O
Authors: Tanabe Yoshinari , Xiong Huabao , Nomura Takamasa , Arakawa Masaaki , Mitsuyama Masao 
Published: December 2019

ABSTRACTOnly listeriolysin O (LLO)-producing strains ofListeria monocytogenesgenerate protective immunity in mice. Based on the findings that endogenous gamma interferon (IFN-γ) production was induced only by such strains and that purified LLO could induce IFN-γ from NK cells, we have postulated that LLO may play a pivotal role in the induction of Th1-type protective T cells, which are highly dependent on IFN-γ. In this study, mice were immunized withL. monocytogenesATCC 15313, an LLO-nonproducing avirulent strain, along with LLO encapsulated in liposome (LLO-liposome). LLO-liposome was highly potent in the induction of various cytokines, including IFN-γ. Immunization of mice with either LLO-liposome or the viable strain ATCC 15313 alone did not induce protection against challenge infection. In contrast, the combination of LLO-nonproducing bacteria plus LLO-liposome induced a significant level of protective immunity mediated mainly by Th1-type cells capable of producing a large amount of IFN-γ in an antigen-specific manner. The protection afforded by the combination was not dependent on LLO-specific cytotoxic T cells. These results support the idea that the inability of an LLO-nonproducing avirulent strain or killed bacteria to induce the generation of protective T cells is due not to the lack of a central T-cell epitope(s) but to the lack of ability to induce the production of endogenous cytokine during the early stage of immunization; the results also suggest that an appropriate use of LLO at least in an animal model may be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria.
Urokinase-Type Plasminogen Activator in Inflammatory Cell Recruitment and Host Defense againstPneumocystis cariniiin Mice
Authors: Beck James M. , Preston Angela M. , Gyetko Margaret R. 
Published: December 2019

ABSTRACTEffective host defense againstPneumocystis cariniidepends upon the integrated actions of inflammatory cells and mediators in the lungs. Using immunocompetent and immunosuppressed mice, our laboratory has defined inflammatory changes in the lungs in response toP. carinii. However, the essential molecules and mechanisms required for cellular recruitment and for host defense againstP. cariniiare undefined. We hypothesized that urokinase-type plasminogen activator (uPA), a protease intimately involved in inflammatory cell migration and activation, is required for clearance ofP. carinii. To test this hypothesis in vivo, we compared the intensity ofP. cariniiinfection and inflammation in the lungs of mice lacking the uPA gene (uPA knockout mice) and in the lungs of wild-type mice. After intratracheal inoculation withP. cariniiorganisms, uPA knockout mice developed uniformly heavyP. cariniipneumonia while wild-type mice cleared theP. cariniiinoculum. Bronchoalveolar lavage fluid from uPA knockout mice contained significantly smaller numbers of cells than did lavage fluid from wild-type mice. We conclude that deletion of the uPA gene prevents the clearance ofP. cariniiand reduces inflammatory cell recruitment. Therefore, uPA is an important participant in the network of inflammatory events required for the clearance ofP. carinii, confirming an important role for this molecule in pulmonary host defense against opportunistic pathogens.
Alpha-Toxin and Gamma-Toxin Jointly PromoteStaphylococcus aureusVirulence in Murine Septic Arthritis
Authors: Nilsson Ing-Marie , Hartford Orla , Foster Timothy , Tarkowski Andrzej 
Published: December 2019

ABSTRACTSeptic arthritis is a common and feared complication of staphylococcal infections.Staphylococcus aureusproduces a number of potential virulence factors including certain adhesins and enterotoxins. In this study we have assessed the roles of cytolytic toxins in the development of septic arthritis by inoculating mice withS. aureuswild-type strain 8325-4 or isogenic mutants differing in the expression of alpha-, beta-, and gamma-toxin production patterns. Mice inoculated with either an alpha- or beta-toxin mutant showed degrees of inflammation, joint damage, and weight decrease similar to wild-type-inoculated mice. In contrast, mice inoculated with either double (alpha- and gamma-toxin-deficient)- or triple (alpha-, beta-, and gamma-toxin-deficient)-mutantS. aureusstrains showed lower frequency and severity of arthritis, measured both clinically and histologically, than mice inoculated with the wild-type strain. We conclude that simultaneous production of alpha- and gamma-toxin is a virulence factor inS. aureusarthritis.
Protective Efficacy of Recombinant
Yersinia
Outer Proteins against Bubonic Plague Caused by Encapsulated and Nonencapsulated
Yersinia pestis
Authors: Andrews G. P. , Strachan S. T. , Benner G. E. , Sample A. K. , Anderson G. W. , Adamovicz J. J. , Welkos S. L. , Pullen J. K. , Friedlander A. M. 
Published: December 2019

ABSTRACT

To evaluate the role of
Yersinia
outer proteins (Yops) in conferring protective immunity against plague, six
yop
loci from
Yersinia pestis
were individually amplified by PCR, cloned, and expressed in
Escherichia coli
. The recombinant proteins were purified and injected into mice. Most Yop-vaccinated animals succumbed to infection with either wild-type encapsulated
Y. pestis
or a virulent, nonencapsulated isogenic variant. Vaccination with YpkA significantly prolonged mean survival time but did not increase overall survival of mice infected with the nonencapsulated strain. The only significant protection against death was observed in YopD-vaccinated mice challenged with the nonencapsulated strain.
Epitope Mapping of Monoclonal Antibodies againstBordetella pertussisAdenylate Cyclase Toxin
Authors: Lee S.-J. , Gray M. C. , Guo L. , Sebo P. , Hewlett E. L. 
Published: December 2019

ABSTRACTAdenylate cyclase (AC) toxin fromBordetella pertussisis a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity withEscherichia colihemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.
Yersinia pseudotuberculosis-Induced Calcium Signaling in Neutrophils Is Blocked by the Virulence Effector YopH
Authors: Andersson Kerstin , Magnusson Karl-Eric , Majeed Meytham , Stendahl Olle , Fällman Maria 
Published: December 2019

ABSTRACTPathogenic species of the genusYersiniaevade the bactericidal functions of phagocytes. This evasion is mediated through their virulence effectors, Yops, which act within target cells. In this study we investigated the effect ofYersinia pseudotuberculosison Ca2+signaling in polymorphonuclear neutrophils. The intracellular free calcium concentration in single adherent human neutrophils was monitored during bacterial infection and, in parallel, the encounter between the bacteria and cells was observed. When a plasmid-cured strain was used for infection, adherence of a single bacterium to the cellular surface induced a β1integrin-dependent transient increase in the intracellular concentration of free calcium. This was, however, not seen with Yop-expressing wild-type bacteria, which adhered to the cell surface without generating any Ca2+signal. Importantly, the overall Ca2+homeostasis was not affected by the wild-type strain; the Ca2+signal mediated by the G-protein-coupled formyl-methionyl-leucyl-phenylalanine receptor was still functioning. Hence, the blocking effect was restricted to certain receptors and their signaling pathways. The use of different Yop mutant strains revealed that the protein tyrosine phosphatase YopH was responsible for the inhibition. This virulence determinant has previously been implicated in very rapidYersinia-mediated effects on target cells as the key effector in the blockage of phagocytic uptake. The present finding, thatY. pseudotuberculosis, via YopH, specifically inhibits a self-induced immediate-early Ca2+signal in neutrophils, offers more-detailed information concerning the effectiveness of this virulence effector and implies an effect on Ca2+-dependent, downstream signals.
Molecular Characterization of a
Brucella
Species Large DNA Fragment Deleted in
Brucella abortus
Strains: Evidence for a Locus Involved in the Synthesis of a Polysaccharide
Authors: Vizcaíno Nieves , Cloeckaert Axel , Zygmunt Michel S. , Fernández-Lago Luis 
Published: December 2019

ABSTRACT

A
Brucella melitensis
16M DNA fragment of 17,119 bp, which contains a large region deleted in
B. abortus
strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified
omp31
gene, 14 hypothetical genes have been identified in the
B. melitensis
fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in
B. abortus
and that all the polysaccharides described in the
Brucella
genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel
Brucella
polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably not synthesized in
B. abortus
might be interesting for a better understanding of the pathogenicity and host preference differences observed between the
Brucella
species. However, the possibility that the genes described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class
Proteobacteria
, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class
Proteobacteria
, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in
B. abortus
strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the sequenced
B. melitensis
DNA fragment.
Construction of a Single-Chain Interleukin-12-Expressing Retroviral Vector and Its Application in Cytokine Gene Therapy against Experimental Coccidioidomycosis
Authors: Jiang Chengyong , Magee D. Mitchell , Cox Rebecca A. 
Published: December 2019

ABSTRACTT-cell-mediated immunity is an important determinant in protection against primary infection withCoccidioides immitis, a dimorphic fungal pathogen that causes the disease coccidioidomycosis. To determine if interleukin-12 (IL-12) gene therapy could potentiate host response againstC. immitis, we constructed a single-chain cDNA encoding the p40 and p35 subunits linked by a polylinker and, using a retroviral vector, transfected J774 macrophages with the construct. The transduced J774 cells expressed IL-12 in vitro, with a mean concentration of 28,440 pg from 106cells in 48 h as measured by an IL-12 (p75)-specific enzyme-linked immunosorbent assay. The secreted IL-12 was biologically active, as judged by its ability to induce the production of gamma interferon (IFN-γ) by spleen cells from BALB/c mice. Treatment of the highly susceptible BALB/c mouse strain with the IL-12-transduced J774 cells inhibitedC. immitisgrowth in tissues from mice challenged by a pulmonary route, as evidenced by 1.37-, 2.59-, and 1.22-log reductions in the number of CFU in the lungs, spleens, and livers, respectively, compared to the fungal load in mice given vector-transduced J774 cells. The protective effect of IL-12 gene therapy was accompanied by increased levels of IFN-γ in the lungs and sera of mice treated with IL-12-transduced J774 cells and the constitutive production of IFN-γ by their spleen cells cultured in vitro. These results suggest that IL-12 gene therapy could be used as adjunct therapy for coccidioidomycosis.