bluuebunny/crossref_metadata_2025 huggingface dataset:
Isotype Switching Increases Efficacy of Antibody Protection againstCryptococcus neoformansInfection in Mice
Authors: Yuan Rui Rong , Spira Gadi , Oh Jin , Paizi Melia , Casadevall Arturo , Scharff Matthew D.
Published: December 2019

The isotype and epitope specificities of antibodies both contribute to the efficacy of antibodies that mediate immunity toCryptococcus neoformans, but the relationship between these properties is only partially understood. In this study, we analyzed the efficacy of protection of two sets of immunoglobulin G (IgG) isotype switch variants from two IgG3 monoclonal antibodies (MAbs) which are either not protective or disease enhancing, depending on the mouse model used. The two IgG3 MAbs 3E5 and 4H3 have different epitope specificities. Protection experiments were done with A/JCr mice infected intravenously withC. neoformansand administered with 3E5 IgG3 and its IgG1, IgG2a, and IgG2b switch variants. These experiments revealed that IgG1, IgG2b, and IgG2a were each more effective than IgG3. For 4H3 IgG3 and its IgG1 and IgG2b switch variants, the relative efficacy was IgG2b > IgG1 >> IgG3. The combination of 3E5 IgG3 and 4H3 IgG3 was more deleterious than either IgG3 alone. All IgG isotypes were opsonic for mouse bronchoalveolar cells, with the relative efficacy being IgG2b > IgG2a > IgG1 > IgG3. These results (i) confirm that a nonprotective IgG3 MAb can be converted to a protective MAb by isotype switching, (ii) indicate that the efficacy of protection of an IgG1 MAb can be increased by isotype switching to another subclass, (iii) show that protective and nonprotective IgG MAbs are opsonic, and (iv) provide additional evidence for the concept that the efficacy of the antibody response toC. neoformansis dependent on the type of MAb elicited.
Chimeric Clostridial Cytotoxins: Identification of the N-Terminal Region Involved in Protein Substrate Recognition
Authors: Hofmann Fred , Busch Christian , Aktories Klaus
Published: December 2019

Clostridium sordellii
lethal toxin is a member of the family of large clostridial cytotoxins that glucosylate small GTPases. In contrast to
Clostridium difficile
toxins A and B, which exclusively modify Rho subfamily proteins,
C. sordellii
lethal toxin also glucosylates Ras subfamily proteins. By deletion analysis and construction of chimeric fusion proteins of
C. sordellii
lethal toxin and
C. difficile
toxin B, we localized the enzyme activity of the lethal toxin to the N terminus of the holotoxin and identified the region involved in protein substrate specificity. The toxin fragment of the N-terminal 546 amino acid residues of
C. sordellii
lethal toxin glucosylated Rho and Ras subfamily proteins, as the holotoxin did. Deletion of a further 30 amino acid residues from the C terminus of this active fragment drastically reduced glucotransferase activity and blocked glucohydrolase activity. Exchange of amino acid residues 364 through 516 of lethal toxin for those in the active toxin B fragment (1 to 546) allowed glucosylation of Ras subfamily proteins. In contrast, the chimera with amino acids 1 to 364 from toxin B, 365 to 468 from lethal toxin, and 469 to 546 from toxin B exhibited markedly reduced modification of Ras subfamily proteins, whereas modification of Rac and Cdc42 was hardly changed. The data indicate that the region of amino acid residues 364 through 516 primarily defines the substrate specificity of
C. sordellii
lethal toxin.
Biased T-Cell Antigen Receptor Repertoire in Lyme Arthritis
Authors: Roessner Karen , Trivedi Harsh , Gaur Lakshmi , Howard Diantha , Aversa John , Cooper Sheldon M. , Sigal Leonard H. , Budd Ralph C.
Published: December 2019

A common concern with many autoimmune diseases of unknown etiology is the extent to which tissue T-lymphocyte infiltrates, versus a nonspecific infiltrate, reflect a response to the causative agent. Lyme arthritis can histologically resemble rheumatoid synovitis, particularly the prominent infiltration by T lymphocytes. This has raised speculation about whether Lyme synovitis represents an ongoing response to the causative spirochete,Borrelia burgdorferi, or rather a self-perpetuating autoimmune reaction. In an effort to answer this question, the present study examined the repertoire of infiltrating T cells in synovial fluid from nine Lyme arthritis patients, before and after stimulation withB. burgdorferi. Using a highly sensitive and consistent quantitative PCR technique, a comparison of the T-cell antigen receptor (TCR) β-chain variable (Vβ) repertoires of the peripheral blood and synovial fluid showed a statistically significant increase in expression of Vβ2 and Vβ6 in the latter. This is remarkably similar to our previous findings in studies of rheumatoid arthritis and to other reports on psoriatic skin lesions. However, stimulation of synovial fluid T cells withB. burgdorferiprovoked active proliferation but not a statistically significant increase in expression of any TCR Vβ, including Vβ2 and Vβ6. Collectively, the findings suggest that the skewing of the TCR repertoire of fresh synovial fluid in Lyme arthritis may represent more a synovium-tropic or nonspecific inflammatory response, similar to that occurring in rheumatoid arthritis or psoriasis, rather than a specificBorreliareaction.
Chemokine Production by a Human Alveolar Epithelial Cell Line in Response toMycobacterium tuberculosis
Authors: Lin Yuanguang , Zhang Ming , Barnes Peter F.
Published: December 2019

To investigate the role of chemokines during the initial local response toMycobacterium tuberculosisin the human lung, we studied chemokine production by the human alveolar epithelial cell line A549 after infection withM. tuberculosis. M. tuberculosis-infected A549 cells produced mRNAs and protein for monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) but not mRNAs for macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and RANTES. Chemokine production in response toM. tuberculosiswas not dependent on production of tumor necrosis factor alpha, IL-1β, or IL-6. Two virulent clinicalM. tuberculosisisolates, the virulent laboratory strain H37Rv, and the avirulent strain H37Ra elicited production of comparable concentrations of MCP-1 and IL-8, whereas killedM. tuberculosisand threeMycobacterium aviumstrains did not. The three virulentM. tuberculosisstrains grew more rapidly than the avirulentM. tuberculosisstrain in the alveolar epithelial cell line, and the threeM. aviumstrains did not grow intracellularly. These findings suggest that intracellular growth is necessary for mycobacteria to elicit production of MCP-1 and IL-8 by alveolar epithelial cells but that virulence and the rate of intracellular growth do not correlate with chemokine production. Alveolar epithelial cells may contribute to the local inflammatory response in human tuberculosis by producing chemokines which attract monocytes, lymphocytes, and polymorphonuclear cells.
SalmonellaFlagellin Induces Tumor Necrosis Factor Alpha in a Human Promonocytic Cell Line
Authors: Ciacci-Woolwine Federica , Blomfield Ian C. , Richardson Stephen H. , Mizel Steven B.
Published: December 2019

During infection of the gastrointestinal tract, salmonellae induce cytokine production and inflammatory responses which are believed to mediate tissue damage in the host. In a previous study, we reported that salmonellae possess the ability to stimulate tumor necrosis factor alpha (TNF-α) accumulation in primary human monocytes, as well as in the human promonocytic cell line U38. In this model system, cytokine upregulation is not due to lipopolysaccharide but is mediated by a released protein. In the present study, TnphoAtransposon mutagenesis was used to identify the TNF-α-inducing factor. A mutantSalmonellastrain which lacks the ability to induce TNF-α was isolated from a TnphoAlibrary. Genetic analysis of this mutant demonstrated that thehnsgene has been interrupted by transposon insertion. Thehnsgene product is a DNA-binding protein that regulates the expression of a variety of unrelated genes in salmonellae. One of the known targets of histone-like protein H1 isflhDC, the master operon which is absolutely required for flagellar expression. Analysis of other nonflagellated mutantSalmonellastrains revealed a correlation between the ability to induce TNF-α and the expression of the phase 1 filament subunit protein FliC. Complementation experiments demonstrated that FliC is sufficient to restore the ability of nonflagellated mutantSalmonellastrains to upregulate TNF-α, whereas the phase 2 protein FljB appears to complement to a lesser extent. In addition,SalmonellaFliC can confer the TNF-α-inducing phenotype onEscherichia coli, which otherwise lacks the activity. Furthermore, assembly of FliC into complete flagellar structures may not be required for induction of TNF-α.
Human Platelets DamageAspergillus fumigatusHyphae and May Supplement Killing by Neutrophils
Authors: Christin Laurent , Wysong Deborah R. , Meshulam Tova , Hastey Ryan , Simons Elizabeth R. , Diamond Richard D.
Published: December 2019

Neutropenia is considered a significant risk factor for invasive aspergillosis but is almost always associated with concurrent thrombocytopenia. Studies determined that platelets, like neutrophils, attached to cell walls of the invasive hyphal form ofAspergillus fumigatus. Organisms were damaged as shown by loss of cell wall integrity in scanning laser confocal microscopy and release of defined hyphal surface glycoproteins. Rapid expression appearance of surface antigen CD63 and release of markers of platelet degranulation confirmed activation during attachment to hyphae. Optimal platelet activation required opsonization of hyphae with fresh or heat-inactivated whole plasma. These effects of opsonization with whole plasma could not be duplicated by pooled human serum, immunoglobulin G, or fibrinogen, whether used separately or combined. Thus, platelets in the presence of whole plasma have the potential to play an important role in normal host defenses against invasive aspergillosis.
Identification of Macrophage-Specific Infectivity Loci (
mil
) of
Legionella pneumophila
That Are Not Required for Infectivity of Protozoa
Authors: Gao Lian-Yong , Harb Omar S. , Kwaik Yousef Abu
Published: December 2019

We have recently shown that many mutants of
Legionella pneumophila
exhibit similar defective phenotypes within both U937 human-derived macrophages and the protozoan host
Acanthamoeba
(L.-Y. Gao, O. S. Harb, and Y. Abu Kwaik, Infect. Immun. 65:4738–4746, 1997). These observations have suggested that many of the mechanisms utilized by
L. pneumophila
to parasitize mammalian and protozoan cells are similar, but our data have not excluded the possibility that there are unique mechanisms utilized by
L. pneumophila
to survive and replicate within macrophages but not protozoa. To examine this possibility, we screened a bank of 5,280 miniTn
10
::kan transposon insertion mutants of
L. pneumophila
for potential mutants that exhibited defective phenotypes of cytopathogenicity and intracellular replication within macrophage-like U937 cells but not within
Acanthamoeba polyphaga
. We identified 32 mutants with various degrees of defects in cytopathogenicity, intracellular survival, and replication within human macrophages, and most of the mutants exhibited wild-type phenotypes within protozoa. Six of the mutants exhibited mild defects in protozoa. The defective loci were designated
mil
(for macrophage-specific infectivity loci). Based on their intracellular growth defects within macrophages, the
mil
mutants were grouped into five phenotypic groups. Groups I to III included the mutants that were severely defective in macrophages, while members of the other two groups exhibited a modestly defective phenotype within macrophages. The growth kinetics of many mutants belonging to groups I to III were also examined, and these were shown to have a similar defective phenotype in peripheral blood monocytes and a wild-type phenotype within another protozoan host,
Hartmannella vermiformis
. Transmission electron microscopy of
A. polyphaga
infected by three of the
mil
mutants belonging to groups I and II showed that they were similar to the parent strain in their capacity to recruit the rough endoplasmic reticulum (RER) around the phagosome. In contrast, infection of macrophages showed that the three mutants failed to recruit the RER around the phagosome during early stages of the infection. None of the
mil
mutants was resistant to NaCl, and the
dot
or
icm
NaCl
r
mutants are severely defective within mammalian and protozoan cells. Our data indicated that in addition to differences in mechanisms of uptake of
L. pneumophila
by macrophages and protozoa, there were also genetic loci required for
L. pneumophila
to parasitize mammalian but not protozoan cells. We hypothesize that
L. pneumophila
has evolved as a protozoan parasite in the environment but has acquired loci specific for intracellular replication within macrophages. Alternatively, ecological coevolution with protozoa has allowed
L. pneumophila
to possess multiple redundant mechanisms to parasitize protozoa and that some of these mechanisms do not function within macrophages.
Regulation of Periodontal Ligament Cell Functions by Interleukin-1β
Authors: Agarwal Sudha , Chandra Charu S. , Piesco Nicholas P. , Langkamp Herman H. , Bowen Lathe , Baran Coskun
Published: December 2019

Periodontal ligament (PDL) cells maintain the attachment of the tooth to alveolar bone. These cells reside at a site in which they are challenged frequently by bacterial products and proinflammatory cytokines, such as interleukin-1β (IL-1β), during infections. In our initial studies we observed that IL-1β down-regulates the osteoblast-like characteristics of PDL cells in vitro. Therefore, we examined the functional significance of the loss of the PDL cell’s osteoblast-like characteristics during inflammation. In this report we show that, during inflammation, IL-1β can modulate the phenotypic characteristics of PDL cells to a more functionally significant lipopolysaccharide (LPS)-responsive phenotype. In a healthy periodontium PDL cells exhibit an osteoblast-like phenotype and are unresponsive to gram-negative bacterial LPS. Treatment of PDL cells with IL-1β inhibits the expression of their osteoblast-like characteristics, as assessed by the failure to express transforming growth factor β1 (TGF-β1) and proteins associated with mineralization, such as alkaline phosphatase and osteocalcin. As a consequence of this IL-1β-induced phenotypic change, PDL cells become responsive to LPS and synthesize proinflammatory cytokines. The IL-1β-induced phenotypic changes in PDL cells were transient, as removal of IL-1β from PDL cell cultures resulted in reacquisition of their osteoblast-like characteristics and lack of LPS responsiveness. The IL-1β-induced phenotypic changes occurred at concentrations that are frequently observed in tissue exudates during periodontal inflammation (0.05 to 5 ng/ml). The results suggest that, during inflammation in vivo, IL-1β may modulate PDL cell functions, allowing PDL cells to participate directly in the disease process by assuming LPS responsiveness at the expense of their normal structural properties and functions.
MP1
Encodes an Abundant and Highly Antigenic Cell Wall Mannoprotein in the Pathogenic Fungus
Penicillium marneffei
Authors: Cao Liang , Chan Che-man , Lee Cindy , Sai-yin Wong Samson , Yuen Kwok-yung
Published: December 2019

We cloned the
MP1
gene, which encodes an abundant antigenic cell wall mannoprotein from the dimorphic pathogenic fungus
Penicillium marneffei. MP1
is a unique gene without homologs in sequence databases. It codes for a protein, Mp1p, of 462 amino acid residues, with a few sequence features that are present in several cell wall proteins of
Saccharomyces cerevisiae
and
Candida albicans
. It contains two putative N glycosylation sites, a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. Specific anti-Mp1p antibody was generated with recombinant Mp1p protein purified from
Escherichia coli
to allow further characterization of Mp1p. Western blot analysis with anti-Mp1p antibody revealed that Mp1p has predominant bands with molecular masses of 58 and 90 kDa and that it belongs to a group of cell wall proteins that can be readily removed from yeast cell surfaces by glucanase digestion. In addition, Mp1p is an abundant yeast glycoprotein and has high affinity for concanavalin A, a characteristic indicative of a mannoprotein. Furthermore, ultrastructural analysis with immunogold staining indicated that Mp1p is present in the cell walls of the yeast, hyphae, and conidia of
P. marneffei
. Finally, it was observed that infected patients develop a specific antibody response against Mp1p, suggesting that this protein represents a good cell surface target for host humoral immunity.
Glycosphingolipids as Novel Targets for T-Cell Suppression by the B Subunit of Recombinant Heat-Labile Enterotoxin
Authors: Truitt Robert L. , Hanke Carrie , Radke Jay , Mueller Reinhold , Barbieri Joseph T.
Published: December 2019

Heat-labile enterotoxin subunit B (LTB) is a noncatalytic protein derived fromEscherichia colithat binds to ganglioside GM1, a glycosphingolipid on the surface of mammalian cells. In this study, the effects of recombinant LTB (rLTB) on murine lymphocytes were examined in vitro. T and B cells readily bound fluorescein isothiocyanate-labeled rLTB. CD8+T cells bound twice as much as CD4+T cells and B cells. Exposure of T-cell subsets and B cells to rLTB abrogated mitogen-driven proliferation. CD8+T cells were more susceptible to rLTB than either CD4+T cells or B cells. There were differences in the sensitivity of lymphocytes from various strains of mice to rLTB. This was attributed to qualitative and quantitative differences in the CD4+T cells. rLTB induced apoptosis in both T-cell subsets, but the level was significantly higher in CD8+T cells. Apoptosis peaked at around 8 h after exposure to rLTB and incubation at 37°C. Binding to ganglioside GM1was essential for suppression, since rLTB/G33D, a mutant which does not bind GM1, failed to inhibit proliferation or induce apoptosis. Naive T cells, which were acutely sensitive to rLTB, became more resistant after activation. Conversely, activated T cells regained their sensitivity to rLTB when they reverted back to a resting state. A 1-h pulse with rLTB was sufficient to inhibit T-cell proliferation and cytotoxic-T-lymphocyte generation in primary mixed lymphocyte reaction cultures. CD8+T cells were preferentially depleted in these cultures. rLTB also induced functional modifications in T cells as indicated by inhibition of gamma interferon secretion after polyclonal activation. Thus, rLTB may have immunomodulatory properties independent of its ability to induce apoptosis.
Molecular Cloning and Characterization of the Genes Coding for the Highly Immunogenic Cluster of 90-Kilodalton Envelope Proteins from theChlamydia psittaciSubtype That Causes Abortion in Sheep
Authors: Longbottom David , Russell Mary , Dunbar Susanna M. , Jones Gareth E. , Herring Alan J.
Published: December 2019

Proteins present in the outer membrane of chlamydiae that are involved in mucosal epithelial cell infection must clearly be identified and characterized if we are to understand and modify the pathogenic mechanisms utilized by these organisms. We have identified and isolated a family of four genes encoding putative outer membrane proteins (POMPs), a group of proteins of approximately 90 kDa present in the outer membrane of the subtype ofChlamydia psittacithat causes ovine enzootic abortion (strain S26/3). These proteins, although minor components, are major immunogens, as shown by the immunoblotting of chlamydial outer membrane complexes with postabortion sheep sera, and are therefore potential diagnostic and/or protective antigen candidates. Immunoblotting of the expressed amino- and carboxy-terminal halves of one of the POMPs with postabortion sheep sera showed that the major humoral immune response appeared to be directed solely against the amino-terminal half. This result, in combination with the positive immunofluorescence staining of S26/3-infected cells using POMP-specific (specific to the amino-terminal half of the proteins) monoclonal antibodies, suggests the probable surface localization of the POMPs and, more specifically, the surface exposure of the amino-terminal half of these proteins. The fourpompgenes are highly homologous, sharing 82 to 100% similarity with each other (two of the genes are identical). Genes with strong and weak homologies were also detected inC. psittaciavian and feline pneumonitis strains, respectively. Nopomphomologs were found in strains ofC. trachomatisandC. pneumoniae, but this does not preclude their existence. The absence of homology with various subtypes ofC. pecorum, which complicate the diagnosis of the ovine abortion subtype, indicates the possible suitability of the these 90-kDa proteins as serodiagnostic antigens.
Elastase Is the Only Human Neutrophil Granule Protein That Alone Is Responsible for In Vitro Killing of
Borrelia burgdorferi
Authors: Garcia Rodolfo , Gusmani Laura , Murgia Rossella , Guarnaccia Corrado , Cinco Marina , Rottini Giandomenico
Published: December 2019

Phagocytosis of
Borrelia burgdorferi
by human polymorphonuclear leukocytes triggers oxygen-dependent and -independent mechanisms of potentially cidal outcome. Nevertheless, no factor or process has yet been singled out as being borreliacidal. We have studied the
B. burgdorferi
-killing ability of the myeloperoxidase-H
2
O
2
-chloride system and that of primary and secondary granule components in an in vitro assay. We found that neither secondary granule acid extracts nor the chlorinating system could kill these microorganisms, while primary granule extracts were effective. The
Borrelia
-killing factor was purified to homogeneity and demonstrated to be elastase. Its cidal activity was found to be independent of its proteolytic activity.
Enhanced Protective Antibody Responses to PspA after Intranasal or Subcutaneous Injections of PspA Genetically Fused to Granulocyte-Macrophage Colony-Stimulating Factor or Interleukin-2
Authors: Wortham Charles , Grinberg Luba , Kaslow David C. , Briles David E. , McDaniel Larry S. , Lees Andrew , Flora Michael , Snapper Clifford M. , Mond James J.
Published: December 2019

Antibody to pneumococcal surface protein A (PspA) has been shown to be protective forStreptococcus pneumoniaeinfections in mice. In an attempt to define a model for inducing protective antibody to PspA in the absence of adjuvant, we designed two genetic fusions, PspA–interleukin-2 [IL-2]) and PspA–granulocyte-macrophage colony-stimulating factor (GM-CSF). These constructs maintained high cytokine function in vitro, as tested by their activity on IL-2 or GM-CSF-dependent cell lines. While intranasal immunization with PspA induced no detectable anti-PspA response, both PspA–IL-2 and PspA–GM-CSF stimulated high immunoglobulin G1 (IgG1) antibody responses. Interestingly, only the PspA–IL-2, not the PspA–GM-CSF, construct stimulated IgG2a antibody responses, suggesting that this construct directed the response along a TH1-dependent pathway. Comparable enhancement of the anti-PspA response with similar isotype profiles was observed after subcutaneous immunization as well. The enhancement observed with PspA–IL-2 was dependent on IL-2 activity in that it was not seen in IL-2 receptor knockout mice, while PspA in alum induced high-titer antibody in these mice. The antibody was tested for its protective activity in a mouse lethality model usingS. pneumoniaeWU-R2. Passive transfer of 1:90 dilutions of sera from mice immunized with PspA–IL-2 and PspA–GM-CSF elicited protection of CBA/N mice against intravenous challenge with over 170 50% lethal doses of capsular type 3 strain WU2. Only 0.17 μg or less of IgG antibody to PspA was able to provide passive protection against otherwise fatal challenge withS. pneumoniae. The data demonstrate that designing protein-cytokine fusions may be a useful approach for mucosal immunization and can induce high-titer systemic protective antibody responses.
Vaccination with Plasmid DNA Encoding Mycobacterial Antigen 85A Stimulates a CD4+and CD8+T-Cell Epitopic Repertoire Broader than That Stimulated byMycobacterium tuberculosisH37Rv Infection
Authors: Denis Olivier , Tanghe Audrey , Palfliet Kamiel , Jurion Fabienne , van den Berg Thierry-P. , Vanonckelen Albert , Ooms Josette , Saman Eric , Ulmer Jeffrey B. , Content Jean , Huygen Kris
Published: December 2019

Vaccination of mice with plasmid DNA carrying the gene for the major secreted mycobacterial antigen 85A (Ag85A) fromMycobacterium tuberculosisis a powerful technique for generating robust specific Th1 helper T-cell responses, CD8+-mediated cytotoxicity, and protection againstM. tuberculosischallenge (K. Huygen et al., Nat. Med. 2:893–898, 1996). We have now analyzed in more detail the antigen-specific immune CD4+- and CD8+-T-cell responses induced in BALB/c mice vaccinated with Ag85A DNA and have compared these responses to those generated by intravenous infection withM. tuberculosis. T-cell-epitope mapping, as measured by interleukin-2 and gamma interferon secretion from splenic T cells restimulated in vitro with synthetic 20-mer peptides spanning the complete mature sequence of Ag85A, demonstrated that DNA vaccination stimulated a stronger and broader T-cell response than didM. tuberculosisinfection. Moreover, elevated cytotoxic T lymphocyte (CTL) activity against Ag85A-transfected and peptide-pulsed P815 target cells could be generated exclusively by vaccination with plasmid DNA, not followingM. tuberculosisinfection. By using DNA vaccination, three Ag85A CTL epitopes with predicted major histocompatibility complex class I binding motifs were defined. One of them was previously reported as a dominant, promiscuously recognized T-cell epitope in healthy humans with primary infections. These data strengthen the potential of DNA vaccination with respect to inducing antituberculous immunity in humans.
Helper T-Cell Epitopes Encoded by theBabesia bigemina rap-1Gene Family in the Constant and Variant Domains Are Conserved among Parasite Strains
Authors: Brown Wendy C. , McElwain Terry F. , Hötzel Isidro , Suarez Carlos E. , Palmer Guy H.
Published: December 2019

Among important candidates for babesial vaccines are apical complex proteins, including rhoptry-associated protein 1 (RAP-1) fromBabesia bovisandB. bigemina, which have been shown to induce partial immunity. Four variantB. bigemina rap-1transcripts identified in a clone of the Mexico strain have highly conserved sequence in the central region but vary in sequence at the amino and carboxy termini (NT and CT) of the predicted proteins, resulting in different combinations of NT and CT domains in the individual gene products. Cattle were immunized with native protein consisting of the RAP-α1 variant, which contains NT-1 and CT-1 domains, and T-cell responses were characterized. We previously reported the identification of two T helper (Th) cell epitopes inB. bigeminaRAP-1α1 protein (I. Hötzel, W. C. Brown, T. F. McElwain, S. D. Rodriguez, and G. H. Palmer, Mol. Biochem. Parasitol. 81:89–99, 1996). One epitope mapped to the constant domain of RAP-1 (amino acids [aa] 144 to 187), and one mapped to the CT-1 variable domain (aa 386 to 480). Th1-like clones responding to these epitopes proliferated differentially to different strains ofB. bigemina, raising the possibilities that the T-cell epitopes may vary antigenically and that CT-1 may be differentially expressed with respect to the other RAP-1 CT domains in the different strains. In this report, we definitively map the T-cell epitope identified in the constant domain of RAP-1 to aa 159 to 187 (FVVSLLKKNVVRDPESNDVENFASQYFYM) and show that the predicted amino acid sequence is completely conserved among seven strains. The T-cell epitope in the CT-1 domain was mapped to aa 436 to 465 (VNSEKVDADDAGNAETQQLPDAENEVRADD), which is also completely conserved among eight strains ofB. bigemina. We further show that the RAP-1α1-immunized cattle were protected against homologousB. bigeminachallenge, thus suggesting an association between protective immunity and the helper T-cell response against the two epitopes. The immunogenic and highly conserved nature of these T-cell epitopes and their ability to stimulate functionally relevant Th cells that express gamma interferon support their inclusion in a vaccine.
Copy Number of Pilus Gene Clusters in
Haemophilus influenzae
and Variation in the
hifE
Pilin Gene
Authors: Read Timothy D. , Satola Sarah W. , Opdyke Jason A. , Farley Monica M.
Published: December 2019

Brazilian purpuric fever (BPF)-associated
Haemophilus influenzae
biogroup aegyptius strain F3031 contains two identical copies of a five gene cluster (
hifA
to
hifE
) encoding pili similar to well-characterized Hif fimbriae of
H. influenzae
type b. HifE, the putative pilus tip adhesin of F3031, shares only 40% amino acid sequence similarity with the same molecule from type b strains, whereas the other four proteins have 75 to 95% identity. To determine whether pilus cluster duplication and the
hifE
F3031
allele were special features of BPF-associated bacteria, we analyzed a collection of
H. influenzae
strains by PCR with
hifA
- and
hifE
-specific oligonucleotides, by Southern hybridization with a
hifC
gene probe, and by nucleotide sequencing. The presence of two pilus clusters was limited to some
H. influenzae
biogroup aegyptius strains. The
hifE
F3031
allele was limited to
H. influenzae
biogroup aegyptius. Two strains contained one copy of
hifE
F3031
and one copy of a variant
hifE
allele. We determined the nucleotide sequences of four
hifE
genes from
H. influenzae
biogroup aegyptius and
H. influenzae
capsule serotypes a and c. The predicted proteins produced by these genes demonstrated only 35 to 70% identity to the three published HifE proteins from nontypeable
H. influenzae
, serotype b, and BPF strains. The C-terminal third of the molecules implicated in chaperone binding was the most highly conserved region. Three conserved domains in the otherwise highly variable N-terminal putative receptor-binding region of HifE were similar to conserved portions in the N terminus of
Neisseria
pilus adhesin PilC. We concluded that two pilus clusters and
hifE
F3031
were not specific for BPF-causing
H. influenzae
, and we also identified portions of HifE possibly involved in binding mammalian cell receptors.
Decreased Antitoxic Activities among Children with Clinical Episodes of Malaria
Authors: Jakobsen Palle Høy , McKay Veronica , N’Jie Ramou , Olaleye Ben O. , D’Alessandro Umberto , Zhang Gui-Hang , Eggelte Teunis A. , Koch Claus , Greenwood Brian M.
Published: December 2019

Healthy Gambian children, children with clinical
Plasmodium falciparum
malaria, and children with asymptomatic
P. falciparum
infections were studied to investigate whether antitoxic activities may contribute to protection against malarial symptoms. Markers of inflammatory reactions, soluble tumor necrosis factor receptor I, and C-reactive protein were found in high concentrations in children with symptomatic
P. falciparum
malaria compared with levels in children with asymptomatic
P. falciparum
infections or in healthy children, indicating that inflammatory reactions are induced only in children with clinical symptoms. Concentrations of soluble tumor necrosis factor receptor I and C-reactive protein were associated with levels of parasitemia. We detected antitoxic activities in sera as measured by their capacity to block toxin-induced
Limulus
amoebocyte lysate (LAL) activation. Symptomatic children had decreased capacity to block induction of LAL activation by
P. falciparum
exoantigen. The decreased blocking activity was restored in the following dry season, when the children had no clinical malaria. Symptomatic children also had the highest immunoglobulin G (IgG) reactivities to conserved
P. falciparum
erythrocyte membrane protein 1 and “Pfalhesin” (band #3) peptides, indicating that such IgG antibodies are stimulated by acute disease but are lost rapidly after the disease episode. Half of the children with symptomatic infections had low levels of haptoglobin, suggesting that these children had chronic
P. falciparum
infections which may have caused symptoms previously. Only a few of the children with asymptomatic
P. falciparum
infections had high parasite counts, and antitoxic immunity in the absence of antiparasite immunity appears to be rare among children in this community.
Role of Adenylate Cyclase-Hemolysin in Alveolar Macrophage Apoptosis duringBordetella pertussisInfection In Vivo
Authors: Gueirard Pascale , Druilhe Anne , Pretolani Marina , Guiso Nicole
Published: December 2019

Bordetella pertussisinduces in vitro apoptosis of murine alveolar macrophages by a mechanism that is dependent on expression of bacterial adenylate cyclase-hemolysin. Using a murine respiratory model, we found in this study that intranasal infection with a parentalB. pertussisstrain, but not with an isogenic variant deficient in the expression of all toxins and adhesins, induced a marked neutrophil accumulation in the bronchoalveolar lavage fluid and an early decrease in macrophage numbers. These phenomena paralleled a time-dependent rise in the proportion of apoptotic nuclei, as detected by flow cytometry, and of macrophages which had engulfed apoptotic bodies. Apoptotic death of bronchopulmonary cells was observed exclusively following intranasal infection with bacteria reisolated from lungs of infected animals and not withB. pertussiscollected after in vitro subculture. Using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling technique coupled to fluorescence microscopy and morphological analysis, we established that the apoptotic cells in bronchoalveolar lavage fluids were neutrophils and macrophages. Histological analysis of the lung tissues fromB. pertussis-infected mice showed increased numbers of apoptotic cells in the alveolar compartments. Cellular accumulation in bronchoalveolar lavage fluids and apoptosis of alveolar macrophages were significantly attenuated in mice infected with a mutant deficient in the expression of adenylate cyclase-hemolysin, indicating a role of this enzyme in these processes.
Synthesis and Characterization of Lipooligosaccharide-Based Conjugates as Vaccine Candidates for
Moraxella
(
Branhamella
)
catarrhalis
Authors: Gu Xin-Xing , Chen Jing , Barenkamp Stephen J. , Robbins John B. , Tsai Chao-Ming , Lim David J. , Battey James
Published: December 2019

Moraxella
(
Branhamella
)
catarrhalis
is an important cause of otitis media and sinusitis in children and of lower respiratory tract infections in adults. Lipooligosaccharide (LOS) is a major surface antigen of the bacterium and elicits bactericidal antibodies. Treatment of the LOS from strain ATCC 25238 with anhydrous hydrazine reduced its toxicity 20,000-fold, as assayed in the
Limulus
amebocyte lysate (LAL) test. The detoxified LOS (dLOS) was coupled to tetanus toxoid (TT) or high-molecular-weight proteins (HMP) from nontypeable
Haemophilus influenzae
through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratios of dLOS to TT and HMP conjugates were 19:1 and 31:1, respectively. The antigenicity of the two conjugates was similar to that of the LOS, as determined by double immunodiffusion. Subcutaneous or intramuscular injection of both conjugates elicited a 50- to 100-fold rise in the geometric mean of immunoglobulin G (IgG) to the homologous LOS in mice after three injections and a 350- to 700-fold rise of anti-LOS IgG in rabbits after two injections. The immunogenicity of the conjugate was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced antisera had complement-mediated bactericidal activity against the homologous strain and heterologous strains of
M. catarrhalis
. These results indicate that a detoxified LOS-protein conjugate is a candidate for immunization against
M. catarrhalis
diseases.
Genetic Control of Immune Response to Recombinant Antigens Carried by an AttenuatedSalmonella typhimuriumVaccine Strain:Nramp1Influences T-Helper Subset Responses and Protection against Leishmanial Challenge
Authors: Soo Shiu-Shing , Villarreal-Ramos Bernardo , Khan C. M. Anjam , Hormaeche Carlos E. , Blackwell Jenefer M.
Published: December 2019

Attenuated strains ofSalmonella typhimuriumhave been widely used as vehicles for delivery and expression of vaccine antigens in murine models of infectious disease. In mice, early bacterial replication following infection withS. typhimuriumis controlled by the gene (Nramp1, formerlyIty/Lsh/Bcg) encoding the natural-resistance-associated macrophage protein (Nramp1). Nramp1 regulates macrophage activation and has multiple pleiotropic effects, including regulation of tumor necrosis factor alpha, interleukin 1β (IL-1β), and major histocompatibility complex class II molecules, all of which influence antigen processing and presentation. Nramp1 also has a direct effect on antigen processing, possibly by regulating the activity of proteases in the late endosomal compartment. Hence, there are multiple ways (regulation of bacterial load or recombinant antigen dose, class II molecule expression, costimulatory or adjuvant activity, and antigen processing) that Nramp1 might influence responses to recombinant salmonella vaccines. To test the hypothesis that Nramp1 influences responses to vaccination, congenic mouse strains have been used to analyze immune responses to recombinant antigens (tetanus toxoid antigen and leishmanial gp63) carried by live attenuatedS. typhimurium aroA aroDmutants. Results show that congenic mice carrying the wild-type (S. typhimuriumresistance)Nramp1allele mount a predominantly T-helper-1 (IL-2 and gamma interferon) response to vaccination and show enhanced resolution of lesions following challenge infection withLeishmania major. In contrast, mice carrying mutant (S. typhimuriumsusceptibility)Nramp1mount a T-helper-2 (immunoglobulin E and IL-4) response and show exacerbated lesion growth upon challenge.
Campylobacter jejuni
Cytolethal Distending Toxin Causes a G
2
-Phase Cell Cycle Block
Authors: Whitehouse Chris A. , Balbo Paul B. , Pesci Everett C. , Cottle Daniel L. , Mirabito Peter M. , Pickett Carol L.
Published: December 2019

Cytolethal distending toxin (CDT) from the diarrheagenic bacterium
Campylobacter jejuni
was shown to cause a rapid and specific cell cycle arrest in HeLa and Caco-2 cells. Within 24 h of treatment, CDT caused HeLa cells to arrest with a 4
N
DNA content, indicative of cells in G
2
or early M phase. Immunofluorescence studies indicated that the arrested cells had not entered M phase, since no evidence of tubulin reorganization or chromatin condensation was visible. CDT treatment was also shown to cause HeLa cells to accumulate the inactive, tyrosine-phosphorylated form of CDC2. These results indicated that CDT treatment results in a failure to activate CDC2, which leads to cell cycle arrest in G
2
. This mechanism of action is novel for a bacterial toxin and provides a model for the generation of diarrheal disease by
C. jejuni
and other diarrheagenic bacteria that produce CDT.
Identification of Epitope and Surface-Exposed Domains of
Shigella flexneri
Invasion Plasmid Antigen D (IpaD)
Authors: Turbyfill K. Ross , Mertz Jennifer A. , Mallett Corey P. , Oaks Edwin V.
Published: December 2019

Transport and surface expression of the invasion plasmid antigens (Ipa proteins) is an essential trait in the pathogenicity of
Shigella
spp. In addition to the type III protein secretion system encoded by the
mxi/spa
loci on the large virulence plasmid, transport of IpaB and IpaC into the surrounding medium is modulated by IpaD. To characterize the structural topography of IpaD, the Geysen epitope-mapping system was used to identify epitopes recognized by surface-reactive monoclonal and polyclonal antibodies produced against purified recombinant IpaD or synthetic IpaD peptides. Surface-exposed epitopes of IpaD were confined to the first 180 amino acid residues, whereas epitopes in the carboxyl-terminal half were not exposed on the
Shigella
surface. By using convalescent-phase sera from 10
Shigella flexneri
-infected monkeys, numerous epitopes were mapped within a surface-exposed region of IpaD between amino acid residues 14 and 77. Epitopes were also identified in the carboxyl-terminal half of IpaD with a few convalescent-phase sera. Comparison of IpaD epitope sequences with
Salmonella
SipD sequences indicated that very similar epitopes may exist in the carboxyl-terminal region of each protein whereas the IpaD epitopes in the surface-exposed amino-terminal region were unique for the
Shigella
protein. Although the IpaD and SipD homologs may play similar roles in transport, the dominant serum antibody response to IpaD is against the unique region of this protein exposed on the surface of the pathogen.
Identification of a Homolog of CcpA Catabolite Repressor Protein in
Streptococcus mutans
Authors: Simpson Christine L. , Russell Roy R. B.
Published: December 2019

A locus containing a gene with homology to
ccpA
of other bacteria has been cloned from
Streptococcus mutans
LT11, sequenced, and named
regM
. Upstream of the
regM
gene, on the opposite strand, is a gene encoding an X-Pro dipeptidase,
pepQ
. A 14-bp palindromic sequence with homology to the consensus catabolite-responsive element sequence lay in the promoter region between the two genes. To study the function of
regM
, the gene was inactivated by insertion of an antibiotic resistance marker. Diauxic growth of
S. mutans
on a number of sugars in the presence of glucose was not affected by disruption of
regM
. The loss of RegM increased glucose repression of α-galactosidase, mannitol-1-P dehydrogenase, and P-β-galactosidase activities. These results suggest that while RegM can affect catabolite repression in
S. mutans
, it does not conform to the model proposed for CcpA in
Bacillus subtilis.
Cloning of Syrian Hamster (Mesocricetus auratus) Cytokine cDNAs and Analysis of Cytokine mRNA Expression in Experimental Visceral Leishmaniasis
Authors: Melby Peter C. , Tryon Victor V. , Chandrasekar Bysani , Freeman Gregory L.
Published: December 2019

The Syrian golden hamster (Mesocricetus auratus) is uniquely susceptible to a variety of intracellular pathogens and is an excellent model for a number of human infectious diseases. The molecular basis for this high level of susceptibility is unknown, and immunological studies related to this model have been limited by the lack of available reagents. In this report we describe the cloning and sequence analysis of portions of the Syrian hamster interleukin 2 (IL-2), IL-4, gamma interferon (IFN-γ), tumor necrosis factor alpha, IL-10, IL-12p40, and transforming growth factor β cDNAs. In addition, we examined the cytokine response to infection with the intracellular protozoanLeishmania donovaniin this animal model. Sequence analysis of the hamster cytokines revealed 69 to 93% homology with the corresponding mouse, rat, and human nucleotide sequences and 48 to 100% homology with the deduced amino acid sequences. The hamster IFN-γ, compared with the mouse and rat homologs, had an additional 17 amino acids at the C terminus that could decrease the biological activity of this molecule and thus contribute to the extreme susceptibility of this animal to intracellular pathogens. The splenic expression of these genes in response to infection withL. donovani, the cause of visceral leishmaniasis (VL), was determined by Northern blotting. VL in the hamster is a progressive, lethal disease which very closely mimics active human disease. In this model there was pronounced expression of the Th1 cytokine mRNAs, with transcripts being detected as early as 1 week postinfection. Basal expression of IL-4 in uninfected hamsters was prominent but did not increase in response to infection withL. donovani. IL-12 transcript expression was detected at low levels in infected animals and paralleled the expression of IFN-γ. Expression of IL-10, a potent macrophage deactivator, increased throughout the course of infection and could contribute to the progressive nature of this infection. These initial studies are the first to examine the molecular immunopathogenesis of a hamster model of VL infection and indicate that progressive disease in this model of VL is not associated with early polarization of the splenic cellular immune response toward a Th2 phenotype and away from a Th1 phenotype.
Integrated Physical and Genetic Mapping of
Bacillus cereus
and Other Gram-Positive Bacteria Based on IS
231
A Transposition Vectors
Authors: Léonard Catherine , Zekri Omar , Mahillon Jacques
Published: December 2019

The genome structure of
Bacillus cereus
is relatively complex, its DNA being modulated between a size-varying chromosome and large plasmids. To study the genetic organization of the
B. cereus
type strain ATCC 14579, thermosensitive transposition vectors were designed on the basis of IS
231
A-derived cassettes containing uncommon restriction sites. A highly preferred insertion site for IS
231
A was detected in the chromosome by Southern blotting and pulsed-field gel electrophoresis (PFGE) analyses of independent insertion mutants. However, once this insertional hot spot was occupied, secondary IS
231
A insertions occurred randomly, as demonstrated by isolation of independent
B. cereus
auxotrophs at a frequency of approximately 0.6%. The hot-spot site, as well as several auxotrophic mutations, were mapped by using
Not
I,
Sfi
I, and
Asc
I PFGE restriction profiles. It was confirmed by sequencing that one of the insertions, generating an Ade
−
phenotype, had disrupted a gene of the purine synthesis pathway. These results showed that combined PFGE and sequencing analyses of mini-IS
231
A insertions enable the construction of integrated physical and genetic maps of
B. cereus
type strain. Moreover, the presence of the ultrarare I-
Sce
I restriction site in the mini-IS
231
A allowed the isolation, in double-insertion mutants, of contiguous and nonoverlapping large chromosomal fragments, convenient for direct sequencing. The system detailed in this report is therefore a powerful tool for comparative genetic studies among members of the
B. cereus
group (i.e.,
B. cereus
,
B. thuringiensis
,
B. mycoides
, and
B. anthracis
) and could also be applied to more distantly related gram-positive bacteria.