bluuebunny/crossref_metadata_2025 huggingface dataset:
Probing the Function ofBordetella bronchisepticaAdenylate Cyclase Toxin by Manipulating Host Immunity
Authors: Harvill Eric T. , Cotter Peggy A. , Yuk Ming Huam , Miller Jeff F.
Published: December 2019

We have examined the role of adenylate cyclase-hemolysin (CyaA) by constructing an in-frame deletion in theBordetella bronchiseptica cyaAstructural gene and comparing wild-type andcyaAdeletion strains in natural host infection models. Both the wild-type strain RB50 and its adenylate cyclase toxin deletion (ΔcyaA) derivative efficiently establish persistent infections in rabbits, rats, and mice following low-dose inoculation. In contrast, an inoculation protocol that seeds the lower respiratory tract revealed significant differences in bacterial numbers and in polymorphonuclear neutrophil recruitment in the lungs from days 5 to 12 postinoculation. We next explored the effects of disarming specific aspects of the immune system on the relative phenotypes of wild-type and ΔcyaAbacteria. SCID, SCID-beige, or RAG-1−/−mice succumbed to lethal systemic infection following high- or low-dose intranasal inoculation with the wild-type strain but not the ΔcyaAmutant. Mice rendered neutropenic by treatment with cyclophosphamide or by knockout mutation in the granulocyte colony-stimulating factor locus were highly susceptible to lethal infection by either wild-type or ΔcyaAstrains. These results reveal the significant role played by neutrophils early inB. bronchisepticainfection and by acquired immunity at later time points and suggest that phagocytic cells are a primary in vivo target of theBordetellaadenylate cyclase toxin.
Pseudomonas aeruginosa
Exoenzyme S Is a Biglutamic Acid ADP-Ribosyltransferase
Authors: Radke Jay , Pederson Kristin J. , Barbieri Joseph T.
Published: December 2019

Kinetic analysis of two mutations within
Pseudomonas aeruginosa
exoenzyme S (ExoS) showed that a E379D mutation inhibited expression of ADP-ribosyltransferase activity but had little effect on the expression of NAD glycohydrolase activity while a E381D mutation inhibited expression of both activities. These data identify ExoS as a biglutamic acid ADP-ribosyltransferase, where E381 is the catalytic residue and E379 contributes to the transfer of ADP-ribose to the target protein.
Strains of
Actinomyces naeslundii
and
Actinomyces viscosus
Exhibit Structurally Variant Fimbrial Subunit Proteins and Bind to Different Peptide Motifs in Salivary Proteins
Authors: Li Tong , Johansson Ingegerd , Hay Donald I. , Strömberg Nicklas
Published: December 2019

Oral strains of
Actinomyces
spp. express type 1 fimbriae, which are composed of major FimP subunits, and bind preferentially to salivary acidic proline-rich proteins (APRPs) or to statherin. We have mapped genetic differences in the
fimP
subunit genes and the peptide recognition motifs within the host proteins associated with these differential binding specificities. The
fimP
genes were amplified by PCR from
Actinomyces viscosus
ATCC 19246, with preferential binding to statherin, and from
Actinomyces naeslundii
LY7, P-1-K, and B-1-K, with preferential binding to APRPs. The
fimP
gene from the statherin-binding strain 19246 is novel and has about 80% nucleotide and amino acid sequence identity to the highly conserved
fimP
genes of the APRP-binding strains (about 98 to 99% sequence identity). The novel FimP protein contains an amino-terminal signal peptide, randomly distributed single-amino-acid substitutions, and structurally different segments and ends with a cell wall-anchoring and a membrane-spanning region. When agarose beads with CNBr-linked host determinant-specific decapeptides were used,
A. viscosus
19246 bound to the Thr
42
Phe
43
terminus of statherin and
A. naeslundii
LY7 bound to the Pro
149
Gln
150
termini of APRPs. Furthermore, while the APRP-binding
A. naeslundii
strains originate from the human mouth,
A. viscosus
strains isolated from the oral cavity of rat and hamster hosts showed preferential binding to statherin and contained the novel
fimP
gene. Thus,
A. viscosus
and
A. naeslundii
display structurally variant
fimP
genes whose protein products are likely to interact with different peptide motifs and to determine animal host tropism.
Role of Polymorphonuclear Neutrophils in a Murine Model ofChlamydia psittaci-Induced Abortion
Authors: Buendía Antonio J. , Montes de Oca Roberto , Navarro Jose A. , Sánchez Joaquín , Cuello Francisco , Salinas Jesús
Published: December 2019

To assess the role of polymorphonuclear neutrophils (PMNs) inChlamydia psittaciinfection in a pregnant mouse model, pregnant and nonpregnant Swiss OF1 mice were depleted of PMNs by treatment with the RB6-8C5 monoclonal antibody before intraperitoneal infection withC. psittaciserotype 1. Nondepleted mice served as infection controls. Depleted mice aborted earlier and had a much higher mortality rate than nondepleted mice. Bacteriological analysis showed that the number of chlamydiae isolated from the spleens of depleted mice at 5 and 7 days postinfection was 100 times greater than that isolated from nondepleted mice. Histopathological analysis of the placentas of depleted mice showed widespread necrosis of the uteroplacental units, with weak immunoreaction to chlamydial antigen, while the placentas of nondepleted mice showed substantial neutrophil infiltration but no large areas of necrosis, with moderate to strong immunoreaction to chlamydial antigen. The livers of depleted mice showed numerous chlamydial inclusions in the hepatocytes, delayed microgranuloma formation, and in the pregnant animals extensive coagulative periportal necrosis. The livers of nondepleted mice displayed multiple small foci of PMNs and mononuclear cells with microgranuloma formation. Among this group of mice, the pregnant animals always had more hepatic damage than nonpregnant animals. Our results suggest that PMNs play an essential role in the response toC. psittaciprimary infection, preventing the uncontrolled multiplication of chlamydiae in the liver and spleen.
Bacterium-Dependent Induction of Cytokines in Mononuclear Cells and Their Pathologic Consequences In Vivo
Authors: Jiang Yanling , Magli Luciano , Russo Michael
Published: December 2019

Viridans streptococci are a heterogeneous group of gram-positive bacteria that are normal inhabitants of the mouth. These organisms are thought to contribute significantly to the etiology of infective endocarditis, although recently they have been implicated in serious infections in other settings. Another group of oral bacteria, gram-negative anaerobes, is associated with chronic dental infections, such as periodontal diseases or endodontic lesion formation. We evaluated the ability of the oral pathogensStreptococcus mutansandPorphyromonas endodontalisto induce a pathogenic response in vivo, with the goal of quantifying the inflammatory response in soft tissue by measuring leukocyte recruitment and hard tissues by measuring osteoclastogenesis.S. mutansinduced a strong inflammatory response and was a potent inducer of osteoclast formation, whileP. endodontaliswas not. To further study the mechanisms by whichP. endodontalisandS. mutanselicit significantly different levels of inflammatory responses in vivo, we tested the capacity of each to induce production of cytokines by mononuclear cells in vitro.S. mutansstimulated high levels of interleukin-12 (IL-12), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α), all of which are associated with inflammation, enhanced monocyte function, and generation of a Th1 response. In contrast,P. endodontalisstimulated production of IL-10 but not of TNF-α, IL-12, or IFN-γ. These results demonstrate that oral pathogens differ dramatically in their abilities to induce inflammatory and immunoregulatory cytokines. Moreover, there is a high degree of correlation between the cytokine profile induced by these bacteria in vitro and their pathogenic capacity in vivo.
Levels of Antibody to Conserved Parts ofPlasmodium falciparumMerozoite Surface Protein 1 in Ghanaian Children Are Not Associated with Protection from Clinical Malaria
Authors: Dodoo Daniel , Theander Thor G. , Kurtzhals Jorgen A. L. , Koram Kojo , Riley Eleanor , Akanmori Bartholomew D. , Nkrumah Francis K. , Hviid Lars
Published: December 2019

The 19-kDa conserved C-terminal part of thePlasmodium falciparummerozoite surface protein 1 (PfMSP119) is a malaria vaccine candidate antigen, and human antibody responses to PfMSP119have been associated with protection against clinical malaria. In this longitudinal study carried out in an area of stable but seasonal malaria transmission with an estimated parasite inoculation of about 20 infective bites/year, we monitored 266 3- to 15-year-old Ghanaian children clinically and parasitologically over a period of 18 months. Blood samples were collected at the beginning of the study before the major malaria season in April and after the season in November. Using enzyme-linked immunosorbent assay, we measured antibody responses to recombinant gluthathioneS-transferase–PfMSP119fusion proteins corresponding to the Wellcome and MAD20 allelic variants in these samples. Prevalence of antibodies recognizing the Wellcome 19 construct containing both epidermal growth factor (EGF)-like motifs in Wellcome type PfMSP119was about 30%. Prevalence of antibodies to constructs containing only the first EGF domain from either Wellcome or MAD20 type PfMSP119was about 15%, whereas antibodies recognizing a construct containing only the second EGF domain of MAD20 type PfMSP119was found in only about 4% of the donors. Neither the prevalence nor the levels of any of the antibody specificities varied significantly with season, age, or sex. Significantly, and in contrast to previous reports from other parts of West Africa, we found no evidence of an association between antibody responses to PfMSP119and clinical protection against malaria.
Immunization with Recombinant Transferrin Binding Protein B Enhances Clearance of Nontypeable
Haemophilus influenzae
from the Rat Lung
Authors: Webb Dianne C. , Cripps Allan W.
Published: December 2019

Nontypeable
Haemophilus influenzae
(NTHI) is an opportunistic pathogen, and heterogeneity in the surface-exposed immunodominant domains of NTHI proteins is thought to be associated with the failure of an infection to stimulate an immune response that is cross-protective against heterologous NTHI strains. The aim of this study was to assess the vaccine potential of a surface-exposed component of the NTHI human transferrin receptor, TbpB, and to determine if the antibody response elicited was cross-reactive with heterologous strains of NTHI. The efficacy of immunization with a recombinant form of TbpB (rTbpB) was determined by assessing the pulmonary clearance of viable bacteria 4 h after a live challenge with NTHI. There was a significant reduction in the number of viable bacteria in both the bronchoalveolar lavage fluid (34% for the 20-μg dose and 58% for the 40-μg dose) and lung homogenates (26% for the 20-μg dose and 60% for the 40-μg dose) of rats immunized with rTbpB compared to the control animals. While rTbpB-specific antibodies from immunized rats were nonspecific in the recognition of TbpB from six heterologous NTHI strains on Western blots, these antibodies differed in their ability to block transferrin binding to heterologous strains and to cross-react in bactericidal assays. If bactericidal antibodies are key indicators of the efficacy of the immune response in eliminating NTHI, this data suggests that while immunization with rTbpB stimulates protective responses against the homologous isolate, variability in the recognition of TbpB from heterologous isolates may limit the potential of rTbpB as an NTHI vaccine component.
Shiga Toxin 1 fromEscherichia coliBlocks Activation and Proliferation of Bovine Lymphocyte Subpopulations In Vitro
Authors: Menge C. , Wieler L. H. , Schlapp T. , Baljer G.
Published: December 2019

Shiga toxin-producingEscherichia coli(STEC) is widespread in the cattle population, but the clinical significance of Shiga toxins (Stx’s) for the bovine species remains obscure. Since Stx’s exert immunomodulating effects in other species, we examined the effect of purified Stx1 on a bovine B lymphoma cell line (BL-3) and peripheral blood mononuclear cells (PBMC) isolated from adult bovine blood by viability assays and flow cytometry analysis. Stx1 markedly induced apoptosis in stimulated BL-3 cells. The susceptibility of this B-cell-derived cell line was induced only by either lipopolysaccharide (LPS) or pokeweed mitogen, while cultures stimulated with T-cell mitogens were unaffected by the toxin. In contrast, Stx1 did not induce cellular death—neither apoptosis nor necrosis—in primary cultures of PBMC but hindered the mitogen-induced increase in metabolic activity. The influence of Stx1 on single PBMC subpopulations varied with the type of mitogenic stimulus applied. Stimulation with phytohemagglutinin P particularly induced the proliferation of bovine CD8-expressing (BoCD8+) cells, and this proliferative response was blocked by Stx1. On the other hand, Stx1 reduced the portion of viable B cells in the presence of LPS. Modulation of activation marker expression (BoCD25 and BoCD71) by Stx1 indicated that the toxin hindered the proliferation of cells by blocking their activation. In conclusion, we assume that Stx1 contributes to the pathogenesis of STEC-associated diarrhea in calves by suppressing the mucosa-associated immune response. The usefulness of cattle as a model in which to study Stx-induced immunomodulation is discussed.
Functional Expression of Nramp1 In Vitro in the Murine Macrophage Line RAW264.7
Authors: Govoni Gregory , Canonne-Hergaux François , Pfeifer Cheryl G. , Marcus Sandra L. , Mills Scott D. , Hackam David J. , Grinstein Sergio , Malo Danielle , Finlay B. Brett , Gros Philippe
Published: December 2019

Mutations at theNramp1locus in vivo cause susceptibility to infection by unrelated intracellular microbes.Nramp1encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-typeNramp1G169cDNA into RAW 264.7 macrophages (which bear a homozygous mutantNramp1D169allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane ofSalmonella typhimuriumandYersinia enterocoliticacontaining phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication ofS. typhimurium. Studies with a replication-defectiveS. typhimuriummutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant ofS. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection ofNramp1cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.
Replication ofToxoplasma gondii, but NotTrypanosoma cruzi, Is Regulated in Human Fibroblasts Activated with Gamma Interferon: Requirement of a Functional JAK/STAT Pathway
Authors: Cerávolo Isabela Penna , Chaves Andréa C. L. , Bonjardim Cláudio A. , Sibley David , Romanha Alvaro J. , Gazzinelli Ricardo T.
Published: December 2019

To study the role of tryptophan degradation by indoleamine 2,3-dioxygenase (INDO) in the control ofTrypanosoma cruziorToxoplasma gondiireplication, we used human fibroblasts and a fibrosarcoma cell line (2C4). The cells were cultured in the presence or absence of recombinant gamma interferon (rIFN-γ) and/or recombinant tumor necrosis factor alpha (rTNF-α) for 24 h and were then infected with eitherT. cruziorT. gondii. Intracellular parasite replication was evaluated 24 or 48 h after infection. Treatment with rIFN-γ and/or rTNF-α had no inhibitory effect onT. cruzireplication. In contrast, 54, 73, or 30% inhibition ofT. gondiireplication was observed in the cells treated with rIFN-γ alone, rIFN-γ plus rTNF-α, or TNF-α alone, respectively. The replication ofT. gondiitachyzoites in cytokine-activated cells was restored by the addition of extra tryptophan to the culture medium. Similarly,T. gondiitachyzoites transfected with bacterial tryptophan synthase were not sensitive to the microbiostatic effect of rIFN-γ. We also investigated the basis of the cytokine effect on parasite replication by using the three mutant cell lines B3, B9, and B10 derived from 2C4 and expressing defective STAT1α (signal transducer and activator of transcription), JAK2 (Janus family of cytoplasmic tyrosine kinases), or JAK1, respectively, three important elements of a signaling pathway triggered by rIFN-γ. We found that rTNF-α was able to induce low levels expression of INDO mRNA in the parental cell line, as well as the cell line lacking functional JAK2. In contrast to the parental cell line (2C4), rIFN-γ was not able to induce the expression of INDO mRNA or microbiostatic activity in any of the mutant cell lines. These findings indicate the essential requirement of the JAK/STAT pathway for the induction of high levels of INDO mRNA, tryptophan degradation, and the anti-Toxoplasmaactivity inside human nonprofessional phagocytic cells.
Altered Immune Response of Interferon Regulatory Factor 1-Deficient Mice againstPlasmodium bergheiBlood-Stage Malaria Infection
Authors: Tan Rosemary Sok-Pin , Feng Chiguang , Asano Yoshihiro , Kara Anna Ursula
Published: December 2019

Nitric oxide (NO) is a short-lived biological mediator which can be induced in various cell types and is able to cause many metabolic changes in target cells. Inhibition of tumor cell growth and antimicrobial activity has been attributed to the stimulation of NO production by transcriptional upregulation of inducible nitric oxide synthase. In the present study, we used mice devoid of functional interferon regulatory factor 1 by targeted gene disruption (IRF-1−/−) to investigate the role of NO in the host immune response against blood-stagePlasmodium bergheiANKA infection. IRF-1−/−mice survived longer with a later onset of and a lower peak parasitemia despite the inability to produce appreciable levels of NO. The administration of exogenous interleukin-12 (IL-12) was able to prolong survival in the wild-type mice with an upregulation in the expression of both gamma interferon (IFN-γ) and NO. However, the administration of IL-12 did not improve the survival of IRF-1−/−mice. These studies indicate that while IL-12 is able to mediate protection via an IFN-γ- and NO-dependent pathway in the wild-type mice, such a protective mechanism may not be functional in the IRF-1−/−mice. Our results suggest that NO may not be essential for host immunity to the parasite and that IRF-1−/−mice are able to induce an IFN-γ- and NO-independent mechanism againstP. bergheiinfection.
Relationship between Cell Surface Carbohydrates and Intrastrain Variation on Opsonophagocytosis ofStreptococcus pneumoniae
Authors: Kim Jean O. , Romero-Steiner Sandra , Sørensen Uffe B. Skov , Blom Jens , Carvalho M. , Barnard S. , Carlone George , Weiser Jeffrey N.
Published: December 2019

Streptococcus pneumoniaeundergoes spontaneous phase variation between a transparent and an opaque colony phenotype, the latter being more virulent in a murine model of sepsis. Opaque pneumococci have previously been shown to express lower amounts of C polysaccharide (cell wall teichoic acid) and in this study were shown to have a higher content of capsular polysaccharide by immunoelectron microscopy. This report then examined the relationship between expression of these two cell surface carbohydrate structures and their relative contribution to the increased virulence of opaque variants. Comparison of genetically related strains showed that the differential content of capsular polysaccharide did not affect the amount of teichoic acid as measured by a capture enzyme-linked immunosorbent assay (ELISA). In contrast, when the teichoic acid structure was altered by replacing choline in the growth medium with structural analogs, the quantity of capsular polysaccharide as measured by a capture ELISA was decreased, demonstrating a linkage in the expression of the two surface carbohydrate structures. A standardized assay was used to assess the relative contribution of cell surface carbohydrates to opsonophagocytosis. The opaque variants required 1.2- to 30-fold more immune human serum to achieve 50% opsonophagocytic killing than did related transparent variants (types 6B and 9V). The opsonophagocytic titer was proportional to the quantity of capsular polysaccharide rather than teichoic acid. The major factor in binding of the opsonin, C-reactive protein (CRP), was also the amount of capsular polysaccharide rather than the teichoic acid ligand. Only for the transparent variant (type 6B), which bound more CRP, was there enhanced opsonophagocytic killing in the presence of this serum protein. Increased expression of capsular polysaccharide, therefore, appeared to be the major factor in the decreased opsonophagocytic killing of opaque pneumococci.
Zinc-Regulated Biosynthesis of Immunodominant Antigens from
Aspergillus
spp
Authors: Segurado Mónica , López-Aragón Raquel , Calera José Antonio , Fernández-Abalos José Manuel , Leal Fernando
Published: December 2019

ASPND1 and ASPF2 are immunodominant antigens from
Aspergillus nidulans
and
A. fumigatus
, respectively, that are readily synthesized in infections in the human host, as demonstrated by their reactivity with more than 80% of sera from patients with aspergilloma or allergic bronchopulmonary aspergillosis. We demonstrate here that both antigens are exclusively produced under situations of low bioavailability of free Zn
2+
. Addition of micromolar concentrations of Zn
2+
to the culture medium strongly stimulated
Aspergillus
growth but totally inhibited ASPND1 or ASPF2 production. This effect was specific, since other divalent metals had no effect. Removal of endogenous Zn
2+
by a chelator also stimulated ASPND1 production, and the effect was specifically reversed by Zn
2+
. These results suggest a possible role of these antigens in the survival of the fungus in the lungs.
Enterohemorrhagic
Escherichia coli
O157:H7 Produces Tir, Which Is Translocated to the Host Cell Membrane but Is Not Tyrosine Phosphorylated
Authors: DeVinney Rebekah , Stein Markus , Reinscheid Dieter , Abe Akio , Ruschkowski Sharon , Finlay B. Brett
Published: December 2019

Intimate attachment to the host cell leading to the formation of attaching and effacing (A/E) lesions is an essential feature of enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 pathogenesis. In a related pathogen, enteropathogenic
E. coli
(EPEC), this activity is dependent upon translocation of the intimin receptor, Tir, which becomes tyrosine phosphorylated within the host cell membrane. In contrast, the accumulation of tyrosine-phosphorylated proteins beneath adherent EHEC bacteria does not occur, leading to questions about whether EHEC uses a Tir-based mechanism for adherence and A/E lesion formation. In this report, we demonstrate that EHEC produces a functional Tir that is inserted into host cell membranes, where it serves as an intimin receptor. However, unlike in EPEC, in EHEC Tir is not tyrosine phosphorylated yet plays a key role in both bacterial adherence to epithelial cells and pedestal formation. EHEC, but not EPEC, was unable to synthesize Tir in Luria-Bertani medium but was able to secrete Tir into M9 medium, suggesting that Tir synthesis and secretion may be regulated differently in these two pathogens. EHEC Tir and EPEC Tir both bind intimin and focus cytoskeletal rearrangements, indicating that tyrosine phosphorylation is not needed for pedestal formation. EHEC and EPEC intimins are functionally interchangeable, but EHEC Tir shows a much greater affinity for EHEC intimin than for EPEC intimin. These findings highlight some of the differences and similarities between EHEC and EPEC virulence mechanisms, which can be exploited to further define the molecular basis of pedestal formation.
Immune Response toNocardia brasiliensisAntigens in an Experimental Model of Actinomycetoma in BALB/c Mice
Authors: Salinas-Carmona Mario C. , Torres-Lopez Ernesto , Ramos Alma I. , Licon-Trillo Angel , Gonzalez-Spencer Daniel
Published: December 2019

Nine- to twelve-week-old BALB/c mice were injected in footpads with 107CFU of aNocardia brasiliensiscell suspension. Typical actinomycetoma lesions, characterized by severe local inflammation with abscess and fistula formation, were fully established by day 28 after infection. These changes presented for 90 days, and then tissue repair with scar formation slowly appeared, with complete healing after 150 days of infection. Some animals developed bone destruction in the affected area. Histopathology showed an intense inflammatory response, with polymorphonuclear cells and hyaloid material around the colonies of the bacteria, some of which were discharged from draining abscesses. Sera from experimental animals were analyzed by Western blotting, and immunodominant antigens P61 and P24 were found as major targets for antibody response. Anti-P24 immunoglobulin M (IgM) isotype antibodies were present as early as 7 days, IgG peaking 45 days after infection. Lymphocyte proliferation with spleen and popliteal lymph node cells demonstrated thymidine incorporation at 7 days after infection, the stimulation index decreasing by day 60. Levels of interleukin-1 (IL-1), IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon (IFN-γ) were determined by enzyme-linked immunosorbent assay in the sera of infected animals. The circulating levels of IFN-γ increased more than 10 times the basal levels; levels of IL-4, IL-6 and IL-10 also increased during the first 4 days of infection.
Insertional Inactivation of Genes Responsible for the
d
-Alanylation of Lipoteichoic Acid in
Streptococcus gordonii
DL1 (Challis) Affects Intrageneric Coaggregations
Authors: Clemans Daniel L. , Kolenbrander Paul E. , Debabov Dmitri V. , Zhang Qunying , Lunsford R. Dwayne , Sakone Holly , Whittaker Catherine J. , Heaton Michael P. , Neuhaus Francis C.
Published: December 2019

Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci. Two genes relevant to these intrageneric coaggregations were identified by transposon Tn
916
mutagenesis of
Streptococcus gordonii
DL1 (Challis). A 626-bp sequence flanking the left end of the transposon was homologous to
dltA
and
dltB
of
Lactobacillus rhamnosus
ATCC 7469 (formerly called
Lactobacillus casei
). A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of
S. gordonii
DL1. This DNA encoded
d
-alanine-
d
-alanyl carrier protein ligase that was expressed in
Escherichia coli
from the fosmid clone. The cloned streptococcal
dltA
was disrupted by inserting an
ermAM
cassette, and then it was linearized and transformed into
S. gordonii
DL1 for allelic replacement. Erythromycin-resistant transformants containing a single insertion in
dltA
exhibited a loss of
d
-alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation. This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C. J. Whittaker, D. L. Clemans, and P. E. Kolenbrander, Infect. Immun. 64:4137–4142, 1996). The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations. The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs. The natural DNA transformation frequency was reduced 10-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the
dltA
probe revealed the presence of this gene in most viridans streptococci. Thus, it is hypothesized that
d
-alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin to mediate intrageneric coaggregation.
Expression of Interleukin-6 in the Cornea in Response to Infection with Different Strains ofPseudomonas aeruginosa
Authors: Cole Nerida , Bao Shisan , Willcox Mark , Husband Alan J.
Published: December 2019

Strains ofPseudomonas aeruginosacausing keratitis can be either cytotoxic (6206) or invasive (6294), while a strain (Paer1) causing contact lens-induced acute red eye has been shown to be neither. In situ hybridization was used to examine the location and identity of cells expressing interleukin-6 (IL-6) mRNA in the murine cornea and changes in expression in response to infection with different strains ofP. aeruginosa. The number of IL-6-positive cells was determined by image analysis. IL-6 protein levels were measured by an enzyme-linked immunosorbent assay. BALB/c mice were challenged by use of the wounded-cornea model withP. aeruginosa6294, 6206, or Paer1 (2 × 106CFU). At time intervals up to 24 h, postchallenge corneal tissue was probed for IL-6 mRNA. IL-6 mRNA expression was rapidly elevated in the epithelium in response to strains 6294 and 6206. At the conclusion of the experiments, infiltrating inflammatory cells also stained positively for IL-6 mRNA. In contrast, corneas challenged with strain Paer1 showed significant upregulation of IL-6 mRNA only at 4 h postchallenge. Three distinct patterns of IL-6 mRNA expression in the mouse cornea occur in response to these three ocular isolates ofP. aeruginosa. The data obtained for mRNA expression in the cornea for all three strains ofP. aeruginosacorrelated well with IL-6 protein analysis of whole-eye homogenates. Differences in the cytokine responses to these strains correlate with differences in the pathology associated with each strain and may offer an opportunity to develop strategies for the improved management of ocular inflammation.
Local Secretory Immunoglobulin A and Postimmunization Gastritis Correlate with Protection againstHelicobacter pyloriInfection after Oral Vaccination of Mice
Authors: Goto Takayuki , Nishizono Akira , Fujioka Toshio , Ikewaki Junko , Mifune Kumato , Nasu Masaru
Published: December 2019

C57BL/6 mice were orally immunized with five weekly doses of 2 mg, 200 μg, or 2 μg ofHelicobacter pylori(Sydney strain) whole-cell sonicate combined with cholera toxin. One week after the last vaccination, mice were challenged with 5 × 107CFU of liveH. pylorithree times at 2-day intervals. At 6 or 18 weeks after the challenge, mice were sacrificed and bacterial cultures and histological studies of the stomach were performed. Vaccination with 2 mg/session or 200 μg/session inhibitedH. pyloricolonization by 90 and 100%, respectively. These mice were considered protected. Lower levels ofH. pylori-specific immunoglobulin A (IgA) were detected in fecal and saliva samples before challenge. However, a significant increase in IgA secretion in mucosal tissue and a higher labeling index for IgA-positive lumina of pyloric glands were noted in these mice in response to challenge and in a vaccine dose-dependent manner. In protected mice, however, severe gastritis characterized by marked infiltration of inflammation mononuclear cells was noted at 6 weeks after challenge, compared with the gastritis seen in unprotected mice or nonvaccinated, ordinarily infected mice. Marked expression of gamma interferon mRNA was detected in the stomach of all protected mice, and 50% of these mice expressed interleukin 4 (IL-4) or IL-5 mRNA. Our findings suggest that local secretory IgA antibody and severe postimmunization gastritis correlate well with protection of mice againstH. pyloriinfection.
Lipopolysaccharide Induction of Tissue Factor Expression in Rabbits
Authors: Erlich Jonathan , Fearns Colleen , Mathison John , Ulevitch Richard J. , Mackman Nigel
Published: December 2019

Tissue factor (TF) is the major activator of the coagulation protease cascade and contributes to lethality in sepsis. Despite several studies analyzing TF expression in animal models of endotoxemia, there remains debate about the cell types that are induced to express TF in different tissues. In this study, we performed a detailed analysis of the induction of TF mRNA and protein expression in two rabbit models of endotoxemia to better understand the cell types that may contribute to local fibrin deposition and disseminated intravascular coagulation. Northern blot analysis demonstrated that lipopolysaccharide (LPS) increased TF expression in the brain, lung, and kidney. In situ hybridization showed that TF mRNA expression was increased in cells identified morphologically as epithelial cells in the lung and as astrocytes in the brain. In the kidney, in situ hybridization experiments and immunohistochemical analysis showed that TF mRNA and protein expression was increased in renal glomeruli and induced in tubular epithelium. Dual staining for TF and vWF failed to demonstrate TF expression in endothelial cells in LPS-treated animals. These results demonstrate that TF expression is induced in many different cell types in LPS-treated rabbits, which may contribute to local fibrin deposition and tissue injury during endotoxemia.
Role of Interleukin-18 (IL-18) in Mycobacterial Infection in IL-18-Gene-Disrupted Mice
Authors: Sugawara Isamu , Yamada Hiroyuki , Kaneko Hirotaka , Mizuno Satoru , Takeda Kiyoshi , Akira Shizuo
Published: December 2019

Immunity to mycobacterial infection is closely linked to the emergence of T cells that secrete cytokines, gamma interferon (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-α), resulting in macrophage activation and recruitment of circulating monocytes to initiate chronic granuloma formation. The cytokine that mediates macrophage activation is IFN-γ, and, like IL-12, IL-18 was shown to activate Th1 cells and induce IFN-γ production by these cells. In order to investigate the role of IL-18 in mycobacterial infection, IL-18-deficient mice were infected withMycobacterium tuberculosisandMycobacterium bovisBCG Pasteur, and their capacities to control bacterial growth, granuloma formation, cytokine secretion, and NO production were examined. These mice developed marked granulomatous, but not necrotic, lesions in their lungs and spleens. Compared with the levels in wild-type mice, the splenic IFN-γ levels were low but the IL-12 levels were normal in IL-18-deficient mice. The reduced IFN-γ production was not secondary to reduced induction of IL-12 production. The levels of NO production by peritoneal macrophages of IL-18-deficient and wild-type mice did not differ significantly. Granulomatous lesion development by IL-18-deficient mice was inhibited significantly by treatment with exogenous recombinant IL-18. Therefore, IL-18 is important for the generation of protective immunity to mycobacteria, and its main function is the induction of IFN-γ expression.
Neutralization of Macrophage Inflammatory Protein 2 (MIP-2) and MIP-1α Attenuates Neutrophil Recruitment in the Central Nervous System during Experimental Bacterial Meningitis
Authors: Diab Asim , Abdalla Hana , Li Hu Lun , Shi Fu Dong , Zhu Jie , Höjberg Bo , Lindquist Lars , Wretlind Bengt , Bakhiet Moiz , Link Hans
Published: December 2019

Chemokines are low-molecular-weight chemotactic cytokines that have been shown to play a central role in the perivascular transmigration and accumulation of specific subsets of leukocytes at sites of tissue damage. Using in situ hybridization (ISH), we investigated the mRNA induction of macrophage inflammatory protein 2 (MIP-2), MIP-1α, monocyte chemoattractant protein 1 (MCP-1), and RANTES. Challenge of infant rats’ brains withHaemophilus influenzaetype b intraperitoneally resulted in the time-dependent expression of MIP-2, MIP-1α, MCP-1, and RANTES, which was maximal 24 to 48 h postinoculation. Immunohistochemistry showed significant increases in neutrophils and macrophages infiltrating the meninges, the ventricular system, and the periventricular area. The kinetics of MIP-2, MIP-1α, MCP-1, and RANTES mRNA expression paralleled those of the recruitment of inflammatory cells and disease severity. Administration of anti-MIP-2 or anti-MIP-1α antibodies (Abs) resulted in significant reduction of neutrophils. Administration of anti-MCP-1 Abs significantly decreased macrophage infiltration. Combined studies of ISH and immunohistochemistry showed that MIP-2- and MIP-1α-positive cells were neutrophils and macrophages. MCP-1-positive cells were neutrophils, macrophages, and astrocytes. Expression of RANTES was localized predominantly to resident astrocytes and microglia. The present study indicates that blocking of MIP-2 or MIP-1α bioactivity in vivo results in decreased neutrophil influx. These data are also the first demonstration that the C-C chemokine MIP-1α is involved in neutrophil recruitment in vivo.
Antibody to Glucosyltransferase Induced by Synthetic Peptides Associated with Catalytic Regions of α-Amylases
Authors: Smith Daniel J. , Heschel Rhonda L. , King William F. , Taubman Martin A.
Published: December 2019

We examined the immunogenicity and induction of inhibitory activity of 19-mer synthetic peptides which contained putative catalytic regions that were associated with the β
5
(EAW) and β
7
(HDS) strand elements of the suggested (β,α)
8
catalytic barrel domain of
Streptococcus mutans
glucosyltransferase (GTF). Both peptides readily induced serum immunoglobulin G (IgG) and salivary IgA antipeptide activity which was reactive both with the inciting peptide and with intact
S. mutans
GTF. Antisera to each peptide construct also inhibited the ability of
S. mutans
GTF to synthesize glucan. These observations support the existence of catalytic subdomains containing glutamate and tryptophan (EAW) or aspartate and histidine (HDS) residues, each of which have been suggested to be involved with the catalytic activity of GTF. Furthermore, the epitopes defined in these sequences have significant immunogenicity and can induce immune responses which interfere with GTF-mediated glucan synthesis.
Binding of
Haemophilus ducreyi
to Extracellular Matrix Proteins
Authors: Bauer Margaret E. , Spinola Stanley M.
Published: December 2019

We developed an enzyme-linked immunosorbent assay-based assay to assess
Haemophilus ducreyi
binding to extracellular matrix (ECM) proteins.
H. ducreyi
35000HP bound to fibronectin, laminin, and type I and III collagen but not to type IV, V, or VI collagen or elastin. Isogenic strains with mutations in
ftpA
or
losB
bound as well as the parent, suggesting that neither pili nor full-length lipooligosaccharide is required for
H. ducreyi
to bind to ECM proteins.
T-Cell Epitope Mapping of the Three Most Abundant Extracellular Proteins ofMycobacterium tuberculosisin Outbred Guinea Pigs
Authors: Lee Bai-Yu , Horwitz Marcus A.
Published: December 2019

The three most abundant extracellular proteins ofMycobacterium tuberculosis, the 30-, 32-, and 16-kDa major extracellular proteins, are particularly promising vaccine candidates. We have mapped T-cell epitopes of these three proteins in outbred guinea pigs by immunizing the animals with each protein and assaying splenic lymphocyte proliferation against a series of overlapping synthetic peptides covering the entire length of the mature proteins. The 30-kDa protein contained nine immunodominant epitopes, the 32-kDa protein contained two immunodominant epitopes, and the 16-kDa protein contained a highly immunodominant region at its N terminus. The immunodominant epitopes of the 30- and 32-kDa proteins in outbred guinea pigs were frequently identified in healthy purified-protein-derivative-positive or BCG-vaccinated individuals in previous studies. The immunodominant epitopes of these major extracellular proteins have potential utility in an epitope-based vaccine against tuberculosis.
Production of β-Defensin Antimicrobial Peptides by the Oral Mucosa and Salivary Glands
Authors: Mathews Michael , Jia Hong Peng , Guthmiller Janet M. , Losh Garrett , Graham Scott , Johnson Georgia K. , Tack Brian F. , McCray Paul B.
Published: December 2019

β-Defensins are cationic peptides with broad-spectrum antimicrobial activity that are produced by epithelia at mucosal surfaces. Two human β-defensins, HBD-1 and HBD-2, were discovered in 1995 and 1997, respectively. However, little is known about the expression of HBD-1 or HBD-2 in tissues of the oral cavity and whether these proteins are secreted. In this study, we characterized the expression of HBD-1 and HBD-2 mRNAs within the major salivary glands, tongue, gingiva, and buccal mucosa and detected β-defensin peptides in salivary secretions. Defensin mRNA expression was quantitated by RNase protection assays. HBD-1 mRNA expression was detected in the gingiva, parotid gland, buccal mucosa, and tongue. Expression of HBD-2 mRNA was detected only in the gingival mucosa and was most abundant in tissues with associated inflammation. To test whether β-defensin expression was inducible, gingival keratinocyte cell cultures were treated with interleukin-1β (IL-1β) or bacterial lipopolysaccharide (LPS) for 24 h. HBD-2 expression increased ∼16-fold with IL-1β treatment and ∼5-fold in the presence of LPS. Western immunoblotting, liquid chromatography, and mass spectrometry were used to identify the HBD-1 and HBD-2 peptides in human saliva. Human β-defensins are expressed in oral tissues, and the proteins are secreted in saliva; HBD-1 expression was constitutive, while HBD-2 expression was induced by IL-1β and LPS. Human β-defensins may play an important role in the innate defenses against oral microorganisms.
Protection against Development of Otitis Media Induced by NontypeableHaemophilus influenzaeby Both Active and Passive Immunization in a Chinchilla Model of Virus-Bacterium Superinfection
Authors: Bakaletz Lauren O. , Kennedy Bobbie-Jo , Novotny Laura A. , Duquesne Guy , Cohen Joe , Lobet Yves
Published: December 2019

Three separate studies, two involving active-immunization regimens and one involving a passive-transfer protocol, were conducted to initially screen and ultimately more fully assess several nontypeableHaemophilus influenzaeouter membrane proteins or their derivatives for their relative protective efficacy in chinchilla models of otitis media. Initial screening of these antigens (P5-fimbrin, lipoprotein D, and P6), delivered singly or in combination with either Freund’s adjuvant or alum, indicated that augmented bacterial clearance from the nasopharynx, the middle ears, or both anatomical sites could be induced by parenteral immunization with P5-fimbrin combined with lipoprotein D, lipoprotein D alone, or the synthetic chimeric peptide LB1 (derived from P5-fimbrin), respectively. Data from a second study, wherein chinchillas were immunized with LB1 or lipoprotein D, each delivered with alum, again indicated that clearance of nontypeableH. influenzaecould be augmented by immunization with either of these immunogens; however, when this adjuvant was used, both antibody titers in serum and efficacy were reduced. A third study was performed to investigate passive delivery of antisera directed against either LB1, lipoprotein D, nonacylated lipoprotein D, or a unique recombinant peptide designated LPD-LB1(f)2,1,3. The last three antiserum pools were generated by using the combined adjuvant of alum plus monophosphoryl lipid A. Passive transfer of sera specific for LB1 or LPD-LB1(f)2,1,3to adenovirus-compromised chinchillas, prior to intranasal challenge with nontypeableH. influenzae, significantly reduced the severity of signs and incidence of otitis media which developed (P≤ 0.001). Collectively, these data indicate the continued merit of further developing LB1 and LPD-LB1(f)2,1,3as components of vaccines for otitis media.
Cross-Reactivity between the Rheumatoid Arthritis-Associated Motif EQKRAA and Structurally Related Sequences Found in
Proteus mirabilis
Authors: Tiwana Harmale , Wilson Clyde , Alvarez Alison , Abuknesha Ramadan , Bansal Sukhvinder , Ebringer Alan
Published: December 2019

Cross-reactivity or molecular mimicry may be one of the underlying mechanisms involved in the etiopathogenesis of rheumatoid arthritis (RA). Antiserum against the RA susceptibility sequence EQKRAA was shown to bind to a similar peptide ESRRAL present in the hemolysin of the gram-negative bacterium
Proteus mirabilis
, and an anti-ESRRAL serum reacted with EQKRAA. There was no reactivity with either anti-EQKRAA or anti-ESRRAL to a peptide containing the EDERAA sequence which is present in HLA-DRB1∗0402, an allele not associated with RA. Furthermore, the EQKRAA and ESRRAL antisera bound to a mouse fibroblast transfectant cell line (Dap.3) expressing HLA-DRB1∗0401 but not to DRB1∗0402. However, peptide sequences structurally related to the RA susceptibility motif LEIEKDFTTYGEE (
P. mirabilis
urease), VEIRAEGNRFTY (collagen type II) and DELSPETSPYVKE (collagen type XI) did not bind significantly to cell lines expressing HLA-DRB1∗0401 or HLA-DRB1∗0402 compared to the control peptide YASGASGASGAS. It is suggested here that molecular mimicry between HLA alleles associated with RA and
P. mirabilis
may be relevant in the etiopathogenesis of the disease.
Access of Antibody or Trypsin to an Integral Outer Membrane Protein (P66) ofBorrelia burgdorferiIs Hindered by Osp Lipoproteins
Authors: Bunikis Jonas , Barbour Alan G.
Published: December 2019

The outer membrane ofBorrelia burgdorferi, the Lyme disease agent, contains lipoproteins anchored by their lipid moieties and integral proteins with membrane-spanning regions. We used the techniques of in situ proteolysis, immunofluorescence, in vitro growth inhibition, and cross-linking with formaldehyde to characterize topological relationships between P66, an integral membrane protein, and selected Osp lipoproteins ofB. burgdorferi. Protease treatment of intact spirochetes cleaved P66 and Osp proteins but not the periplasmic flagellin or the BmpA protein of the cytoplasmic membrane. P66 of cells lacking OspA, OspB, and OspC was more susceptible to trypsin cleavage than was P66 of cells with these Osp proteins. A monoclonal antibody against the surface loop of P66 bound, agglutinated, and inhibited the growth of viable spirochetes lacking OspA, OspB, OspC, and OspD but not of the cells that expressed OspA, OspC, and/or OspD. When cells were fixed, the antibody bound to cells that express OspD and OspC but still not to cells with OspA. The close association of OspA and P66 was confirmed by the crosslinking of the two proteins by formaldehyde. These results show that Osp proteins, particularly OspA, limit the access of antibody or trypsin to the surface loop region of P66. The proximity and possible contact between P66 and OspA (or other Osp proteins) may hinder the effectiveness of antibodies to what otherwise would be an appropriate vaccine target.