bluuebunny/crossref_metadata_2025 huggingface dataset:
The rpoS gene from Yersinia enterocolitica and its influence on expression of virulence factors
Authors: Iriarte M , Stainier I , Cornelis G R
Published: January 2020

The chromosome of Yersinia enterocolitica encodes a heat-stable enterotoxin called Yst and a surface antigen called Myf, which closely resembles enterotoxin-associated fimbriae. Both factors could act in conjunction to produce diarrhea. Production of the enterotoxin is regulated by temperature, osmolarity, and pH and occurs only when bacteria reach the stationary phase. Myf production is regulated by temperature and pH and, as we show in this work, also occurs after the exponential growth phase. In an attempt to understand the late-phase expression of yst and myf, we cloned, sequenced, and mutagenized the gene encoding RpoS, an alternative sigma factor of the RNA polymerase involved in expression of stationary-phase genes in other enterobacteria. An intact rpoS gene was necessary for full expression of yst in the stationary phase but not for the expression of myf and of pYV-encoded virulence determinants.
Evidence implicating phospholipase as a virulence factor of Candida albicans
Authors: Ibrahim A S , Mirbod F , Filler S G , Banno Y , Cole G T , Kitajima Y , Edwards J E , Nozawa Y , Ghannoum M A
Published: January 2020

Three different approaches were used to investigate the role of extracellular phospholipases in the pathogenicity of Candida albicans. First, we compared 11 blood isolates of this yeast with an equal number of commensal strains isolated from the oral cavities of healthy volunteers. Blood isolates produced significantly more extracellular phospholipase activity than the commensal strains did. Second, two clinical isolates of C. albicans that differed in their levels of virulence in a newborn mouse model were compared for their ability to secrete phospholipases. The invasive strain produced significantly more extracellular phospholipase activity than the noninvasive strain did. Third, nine blood isolates were characterized for their phospholipase and proteinase production, germ tube formation, growth, and adherence to and damage of endothelial cells in vitro. These factors were analyzed subsequently to determine whether they predicted mortality in a mouse model of hematogenously disseminated candidiasis. By proportional hazard analysis, the relative risk of death was 5.6-fold higher (95% confidence interval, 1.672 to 18.84 [P < 0.005]) in the mice infected with the higher-phospholipase-secreting strains than in the low-phospholipase secretors. None of the other putative virulence factors predicted mortality. Characterization of phospholipases secreted by three of the blood isolates showed that these strains secreted both phospholipase B and lysophospholipase-transacylase activities. These results implicate extracellular phospholipase as a virulence factor in the pathogenesis of hematogenous infections caused by C. albicans.
Local and systemic antibody responses to dextran-cholera toxin B subunit conjugates
Authors: Bergquist C , Lagergård T , Lindblad M , Holmgren J
Published: January 2020

This study was designed to test local and systemic immunity following mucosal immunization with a polysaccharide-protein conjugate. After preparing and characterizing dextran-cholera toxin B subunit (CTB) conjugates, we studied their immunogenicity in mice following systemic or mucosal immunizations. Dextran was chosen as a model polysaccharide antigen and conjugated via adipic acid dihydrazide and N-succinimidyl-3-(2-pyridyldithio)propionate to CTB. Mice were immunized either subcutaneously, intranasally, or perorally three times, and cholera toxin was used as an adjuvant for the mucosal immunizations. Three conjugates with different molecular weights for dextran (40,000 and 76,000) or varying dextran/CTB molar ratios were tested. Peroral immunizations with all conjugates evoked local immunoglobulin A (IgA) antibody responses against dextran in the small intestine, and intranasal immunizations did the same in the lung. Intranasal immunizations also elicited serum antibody titers that were significantly higher than or equal to those after subcutaneous immunizations. Intranasal immunizations evoked serum IgG antidextran titers which were dependent on the dextran/CTB molar ratio and inversely related to the local IgA response, which was not the case for subcutaneous immunizations. This is the first study of local and systemic immunity following mucosal immunization with a polysaccharide-protein conjugate. The results show that it is possible to evoke a local as well as a systemic antibody response against a polysaccharide by conjugating it to CTB and using an appropriate route of immunization.
Listeria monocytogenes p60 supports host cell invasion by and in vivo survival of attenuated Salmonella typhimurium
Authors: Hess J , Gentschev I , Szalay G , Ladel C , Bubert A , Goebel W , Kaufmann S H
Published: January 2020

The extracellular protein p60 is a major virulence factor of the intracellular bacterium Listeria monocytogenes. Its roles in pathogen survival in vivo and host cell invasion in vitro were studied. To this end, Salmonella typhimurium SL7207 was used as carrier for secreted p60-HlyA fusion protein by Escherichia coli HlyB and HlyD transport proteins. C57BL/6 mice infected intravenously with this strain suffered from increased bacterial numbers in livers and spleens compared with the p60-nonexpressing control strain, but only transiently. In vitro experiments showed that p60 promotes invasion of recombinant S. typhimurium SL7207 p60 into hepatocytes and resting macrophages independent from complement. Moreover, the uptake of wild-type L. monocytogenes EGD and L. monocytogenes BUG 8, an internalin-deficient strain, into hepatocytes was partially blocked by anti-p60 antibodies. The impaired invasion of dissociated bacterial chains of L. monocytogenes RIII, a p60 expression mutant, into hepatocytes and macrophages was partially restored by addition of p60- or p60-HlyA-enriched bacterial supernatants. These data suggest that the L. monocytogenes surface-associated proteins, p60 and internalin, act in concert to achieve optimal uptake into nonprofessional phagocytes and macrophages. Together, these experiments reveal a substantial impact of p60 on cell invasion and virulence and thus emphasize the importance of the intracellular habitat for survival of L. monocytogenes in the host.
Immunoglobulin subclass distribution and dynamics of Shigella-specific antibody responses in serum and stool samples in shigellosis
Authors: Islam D , Wretlind B , Ryd M , Lindberg A A , Christensson B
Published: January 2020

To assess the humoral immunological responses at the subclass level in shigellosis, specific antibody responses against Shigella dysenteriae 1 lipopolysaccharide (LPS), S. flexneri Y LPS, invasion plasmid-coded protein antigens (Ipa), and Shiga toxin were analyzed. Antibody responses of 41 patients with S. dysenteriae 1 infection (SDIP) and 15 patients with S. flexneri infection (SFIP) were compared with those of controls (n = 40). The levels of total immunoglobulin G (IgG), IgA, IgM, and albumin in serum and stool samples were analyzed. In addition, total IgA (t-IgA), secretory IgA (s-IgA), and antigen-specific s-IgA in fecal samples were analyzed to evaluate the specificities and magnitudes of the mucosal immune responses. By comparing the relative increases in optical density for each IgG subclass separately, it was determined that the anti-LPS (homologous) response initially increased in the order IgG2 > IgG1 > IgG3 > IgG4 and that this order changed to IgG2 > IgG3 > IgG1 > IgG4 later in the disease. The IgG subclass response against protein antigens initially showed the order IgG1 > IgG3 > IgG2 > IgG4, which changed to IgG3 > IgG1 > IgG2 > IgG4 later in the disease. A significant increase in the proportion of IgA2 among t-IgA compared with that in controls was seen in both SDIP and SFIP, while significant changes in the proportions of IgG1 and IgG2 among t-IgG compared with controls was seen only in SDIP. The anti-LPS IgA2 response was more prominent in SDIP than in SFIP. We found an early peak of antigen-specific s-IgA in fecal samples, with a shorter duration than the corresponding response in serum samples. The simultaneous increase of serum IgA, fecal t-IgA, and s-IgA in SDIP compared with those in SFIP suggests that there is a massive increase in the local IgA production, giving an increase in systemic IgA concomitant with an extensive gut mucosal inflammation leading to an increased loss of albumin, IgG, and IgA with a high ratio of t-IgA to s-IgA.
Impact of antigen-presenting cells on cytokine profiles of human Th clones established after stimulation with Mycobacterium tuberculosis antigens
Authors: Conradt P , Kaufmann S H
Published: January 2020

Human T cells reactive with mycobacterial antigens are generally considered to correlate with a Th1 cytokine profile. Our data show that, in addition, Th0 and Th2 clones develop in bulk culture with appropriate antigen-presenting cells before cloning. CD4+ blasts activated by mycobacterial antigens were cloned, and their mRNA patterns for the interleukins (IL) IL-2, IL-4, IL-5, IL-6, and IL-10 and gamma interferon were characterized by reverse-transcribed PCR. Nonadherent, nonrosetting, enriched peripheral blood mononuclear cells promoted development of Th0; after further depletion of monocytes and natural killer cells, Th2 clones were also found. Epstein-Barr virus-transformed B cells, with specificity for the stimulating antigen, increased the proportion of Th2 clones.
High levels of interleukin 10 in serum are associated with fatality in meningococcal disease
Authors: Lehmann A K , Halstensen A , Sørnes S , Røkke O , Waage A
Published: January 2020

Interleukin 10 (IL-10) suppresses the production of proinflammatory cytokines in vitro and in murine models of endotoxemia and has been suggested as a candidate for treatment of bacterial septicemia. To investigate the role of IL-10 in meningococcal disease, a sandwich IL-10 enzyme-amplified sensitivity immunoassay was used to quantitate IL-10 in serum and cerebrospinal fluid samples from 41 patients with meningococcal bacteremia or meningitis with or without septic shock. High levels of IL-10 were demonstrated in sera from patients with meningococcal septic shock (mean, 21,221 pg/ml; range, 25 to 64,500 pg/ml). All cases involving fatalities had IL-10 levels in serum of > or = 1,000 pg/ml (mean, 23,058 pg/ml; range, 1,000 to 64,500 pg/ml). Patients with meningococcal meningitis without septic shock had comparably low concentrations of IL-10 in serum (mean, 119 pg/ml; range, 0 to 1,050 pg/ml) but exhibited compartmentalized release of IL-10 in cerebrospinal fluid. Concentrations of IL-10 in serum were positively correlated with the previously reported concentrations of tumor necrosis factor alpha, IL-6, and IL-8 in serum in the same patients. We conclude that IL-10 is extensively activated along with the proinflammatory cytokines during the initial phase of meningococcal septic shock and that IL-10 is associated with fatality in meningococcal disease.
Affinity, conservation, and surface exposure of hemopexin-binding proteins in Haemophilus influenzae
Authors: Wong J C , Patel R , Kendall D , Whitby P W , Smith A , Holland J , Williams P
Published: January 2020

Haemophilus influenzae can acquire heme from hemopexin for use as a source of both essential porphyrin and iron. In classical ligand-binding studies, we observed time-dependent, saturable, and displaceable binding of human 125I-labelled hemopexin to intact cells of H. influenzae type b (Hib) strain 760705 grown in an iron-restricted medium. From these experiments, which demonstrate that hemopexin associates with a single class of binding site, the affinities (Kds) and receptor numbers were calculated for heme-hemopexin (Kd, 205 nM; 3,200 receptors per cell) and apohemopexin (Kd, 392 nM; 4,400 receptors per cell). Thus, Hib expresses a specific hemopexin receptor which shows some preference for the heme-protein complex. Affinity chromatography on hemopexin-Sepharose 4B of detergent-solubilized membranes from Hib strain 760705 results in the copurification of three proteins with molecular masses of 57, 38, and 29 kDa. Trypsinization of whole cells of Hib 760705 abolishes hemopexin binding and correlates with the disappearance of the 57-kDa hemopexin-binding protein and appearance of a 52-kDa species which does not bind either hemopexin in ligand blot assays or a monoclonal antibody (MAbT11-30) raised against the 57-kDa protein. From immunoblotting assays and NH2-terminal amino acid sequence analysis, the 38-kDa protein isolated following hemopexin affinity chromatography was identified as the porin protein P2. These data, taken together with the receptor-binding studies which support a single class of hemopexin-binding site, suggest that P2 and the 29-kDa protein function as accessory proteins to the 57-kDa hemopexin-binding protein to facilitate the uptake of heme from receptor-bound hemopexin. To determine whether hemopexin binding and the 57-kDa protein are conserved in Haemophilus strains, whole-cell dot blots and immunoblots of the outer membrane proteins prepared from strains belonging to each of 21 different Hib outer membrane protein subtypes, six nontypeable strains, and five Haemophilus parainfluenzae strains were probed with either hemopexin or MAbT11-30. Only the H. parainfluenzae strains which lack the 57-kDa protein do not bind hemopexin. Since H. influenzae has also been shown to produce a soluble 100-kDa hemopexin-binding protein, cell-free culture supernatants were also examined for the presence of this protein. Apart from Hib 760705 and H. parainfluenzae, the 100-kDa hemopexin-binding protein was detected in all the other Haemophilus strains. The abilities of Hib 760705 to both bind and acquire heme from hemopexin without expressing a 100-kDa soluble hemopexin-binding protein show that in strain 760705, this 100-kDa protein is not essential for the utilization of heme from hemopexin.
Involvement of cysteine residues in the biological activity of the active fragments of guinea pig neutrophil cationic peptides
Authors: Yomogida S , Nagaoka I , Yamashita T
Published: January 2020

Guinea pig neutrophil cationic peptides (GNCPs) are single-chain polypeptides with 31 amino acid residues containing six cysteine residues, which exhibit both antibacterial and histamine-releasing activities in vitro. In this study, the role of the sulfhydryl groups in defining the antibacterial and histamine-releasing activities of the active fragments of GNCP-1 (Arg-1 to Tyr-14 [Arg-1-Tyr-14] and Arg-15-Tyr-27 peptides) was examined by using peptides containing alkylated or nonalkylated sulfhydryl groups. Alkylation slightly increased the histamine-releasing activity of the Arg-15-Tyr-27 (RRLGTCIFQNRVY) peptide but abrogated the antibacterial activity. Alkylation of the Arg-1-Tyr-14 (RRCICTTRTCRFPY) peptide similarly reduced the antibacterial activity of this fragment but had minimal effect on the histamine-releasing activity. These findings suggest that cysteine residues with free sulfhydryl groups play an important role in the expression of the antibacterial activity of the active fragments of GNCP-1.
Clonal diversity of the taxon Porphyromonas gingivalis assessed by random amplified polymorphic DNA fingerprinting
Authors: Ménard C , Mouton C
Published: January 2020

A total of 97 strains of the periopathogen Porphyromonas gingivalis were collected. This collection included laboratory strains and clinical isolates of human origin with diverse clinical and geographical origins. Biological diversity was further increased by including 32 strains isolated from the oral cavities of nine different animal species. Genomic fingerprints of the 129 strains were generated as random amplified polymorphic DNAs (RAPDs) by the technique of PCR amplification with a single primer of arbitrary sequence. Four nonameric oligonucleotides were used as single primers, and the banding patterns of the DNA products separated on agarose gels were compared after ethidium ethidium bromide staining. Distance coeffients based on the positions of the major DNA fragments were calculated, and dendrograms were generated. We identified 102 clonal types (CTs) that could be assembled into three main groups by cluster analysis by the unweighted pair group method with mathematic averages. Group I (n = 79 CTs) included all 97 human strains and 6 monkey isolates. The strains in group II (n = 22 CTs) and III (n = 1 CT) were strongly differentiated from those in group I and included only strains of animal origin; they likely represent two cryptic species within the present P. gingivalis taxon. We observed that strains from Old World monkeys clustered together with the human genotype, whereas strains from New World monkeys clustered with the animal genotype. Our results with human strains also indicated that (i) the population structure is basically clonal, (ii) no dominant or widespread CT could be observed, and (iii) no relationship could be established between specific clusters of CTs and the periodontal status of the host. Our results corroborate previous findings by B. G. Loos, D. W. Dyer, T. S. Whittam, and R. K. Selander (Infect. Immun. 61:204-212, 1993) and suggest that P. gingivalis should be considered a commensal of the oral cavity acting as an opportunistic pathogen. Our results are not consistent with the hypothesis that only a few virulent clones of P. gingivalis are associated with disease.
Binding of lipopolysaccharide (LPS) to an 80-kilodalton membrane protein of human cells is mediated by soluble CD14 and LPS-binding protein
Authors: Schletter J , Brade H , Brade L , Krüger C , Loppnow H , Kusumoto S , Rietschel E T , Flad H D , Ulmer A J
Published: January 2020

Activation of cells by bacterial lipopolysaccharide (LPS) plays a key role in the pathogenesis of gram-negative septic shock. The 55-kDa glycoprotein CD14 is known to bind LPS and initiate cell activation. However, there must be additional LPS receptors because CD14 is linked by a glycosylphosphatidyl inositol anchor to the cell membrane and therefore unable to perform transmembrane signalling. Searching for potential LPS receptors, we investigated the binding of LPS to membrane proteins of the human monocytic cell line Mono-Mac-6. Membrane proteins were electrophoretically separated under reducing conditions, transferred to nitrocellulose, and exposed to LPS, which was visualized with anti-LPS antibody. Smooth- and rough-type LPS, as well as free lipid A, bound to a variety of proteins in the absence of serum. However, in the presence of serum, additional or preferential binding to a protein of approximately 80-kDa was observed. Experiments with differently acylated lipid A structures showed that the synthetic tetraacyl compound 406 was still able to bind, whereas no binding was detected with the bisacyl compound 606. The 80-kDa membrane protein was also detected on human peripheral blood monocytes and endothelial cells. The serum factors mediating the binding of lipid A to the 80-kDa membrane protein were identified as soluble CD14 and LPS-binding protein. From these results, we conclude that this 80-kDa protein is a candidate for the hypothetical molecule for LPS and/or LPS-CD14 recognition and signal transduction.
Human peripheral blood CD4+ and CD8+ T cells express Th1-like cytokine mRNA and proteins following in vitro stimulation with heat-inactivated Brucella abortus
Authors: Zaitseva M B , Golding H , Betts M , Yamauchi A , Bloom E T , Butler L E , Stevan L , Golding B
Published: January 2020

Defining the pattern of lymphokine production associated with Brucella abortus is critical for advancing the development of B. abortus as a vaccine carrier. In the present study we investigated the ability of heat-inactivated B. abortus or lipopolysaccharide from B. abortus to induce lymphokine production from purified human T cells in vitro. Gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 induction was assayed by mRNA-specific PCR and by enzyme-linked immunosorbent assay and bioassay for protein production. Following depletion of monocytes and B cells, B. abortus increased IFN-gamma and IL-2 mRNA expression in purified T cells compared with expression in unstimulated cells. In contrast, no IL-5 mRNA expression and only transient low-level IL-4 mRNA expression and no IL-4 protein secretion were detected. Phytohemagglutinin or phorbol myristate acetate plus ionomycin induced mRNA and protein for all these cytokines. Similar results were obtained with LPS purified from B. abortus. Removal of NK cells did not reduce lymphokine production, and enriched NK cells did not express IFN-gamma mRNA or secrete IFN-gamma protein in response to B. abortus, indicating that NK cells were not the responding population. Both CD4+ and CD8+ populations produced IFN-gamma and IL-2 in response to B. abortus. Preincubation of resting T cells with B. abortus or LPS from B. abortus for 7 days induced their differentiation into Th1-like cells as judged by their subsequent lymphokine response to phorbol myristate acetate plus ionomycin. These results suggest that B. abortus can induce differentiation of Th0 into Th1-type cells.
Characterization of intestinal invasion by Salmonella typhimurium and Salmonella dublin and effect of a mutation in the invH gene
Authors: Watson P R , Paulin S M , Bland A P , Jones P W , Wallis T S
Published: January 2020

The relative levels of invasiveness of two bovine isolates each of Salmonella typhimurium and Salmonella dublin and of invH mutants of S. typhimurium were determined in MDCK and Int 407 cultured-cell assays and in bovine ileal loops. S. dublin was found to be significantly less invasive in cultured cells than S. typhimurium, but this difference was not observed in bovine intestines. The invH mutants exhibited a significant reduction in invasion in both cultured cells and bovine intestines. The invasive phenotypes of the strains were confirmed by fluorescent microscopy and scanning and transmission electron microscopy. The wild-type strains were observed in the laminae propriae of the intestinal villi, while in contrast the invH mutants were generally associated with the enterocyte layer. The degree of damage in the bovine ileum was related to the magnitude of the invasion. There was no difference in the amount of S. typhimurium or S. dublin recovered from the bovine ileum either with or without Peyer's patches 3 h after inoculation of the loop.
Salmonella typhimurium displays normal invasion of mice with defective epidermal growth factor receptors
Authors: McNeil A , Dunstan S J , Clark S , Strugnell R A
Published: January 2020

The role of the epidermal growth factor (EGF) receptor in cell invasion by Salmonella typhimurium was examined in vitro and in vivo by using waved-2 mice which express an EGF receptor with reduced kinase activity. S. typhimurium invaded fibroblasts from waved-2 mice as efficiently as fibroblasts from wild-type control animals. In vivo, S. typhimurium both invaded the gastrointestinal tract and penetrated through to the spleen of waved-2 mice. Our studies suggest that the EGF receptor has only a limited role, if any, in cell invasion by S. typhimurium.
Different forms of streptolysin O produced by Streptococcus pyogenes and by Escherichia coli expressing recombinant toxin: cleavage by streptococcal cysteine protease
Authors: Pinkney M , Kapur V , Smith J , Weller U , Palmer M , Glanville M , Messner M , Musser J M , Bhakdi S , Kehoe M A
Published: January 2020

To resolve apparent discrepancies in the literature, N-terminal sequences of the active high- and low-molecular-weight (high- and low-M(r)) forms of native streptolysin O (nSLO) purified from Streptococcus pyogenes culture supernatants and of the similar-size high- and low-M(r) forms of recombinant SLO (rSLO) found in the periplasm of Escherichia coli expressing a cloned slo gene were determined. The high-M(r) forms of nSLO and rSLO are identical, reflecting removal of a 31-residue signal peptide, but the similar-size low-M(r) forms are very different. Removal of C-terminal sequences by proteases in the E. coli periplasm produces an inactive low-M(r) form of rSLO. In contrast, an active low-M(r) form of nSLO is produced by proteolytic cleavage between the N-terminal residues Lys-77 and Leu-78, which was shown to correspond to an extremely sensitive cleavage site for the pyrogenic exotoxin B-derived streptococcal cysteine protease.
Cloning and characterization of a protective outer membrane lipoprotein of Actinobacillus pleuropneumoniae serotype 5
Authors: Bunka S , Christensen C , Potter A A , Willson P J , Gerlach G F
Published: January 2020

The gene encoding an outer membrane lipoprotein (omlA) of Actinobacillus pleuropneumoniae serotype 5 was cloned, and the protein was expressed in Escherichia coli. One open reading frame of 1,104 bp was detected that encoded a protein (OmlA) with a predicted molecular mass of 40 kDa. A comparison with the omlA gene and the corresponding protein of A. pleuropneumoniae serotype 1 (G.-F. Gerlach, C. Anderson, S. Klashinsky, A. Rossi-Kampos, A.A. Potter, and P.J. Wilson, Infect. Immun. 61:565-572, 1993) revealed that the nucleic acid sequences had an overall sequence identity of 62.9% and the deduced amino acid sequences showed a sequence agreement of 57.3%. Both proteins were antigenically distinct. In a Western blot (immunoblot) analysis using a specific antiserum against A. pleuropneumoniae serotype 5 OmlA, a homologous protein was detected in the reference strains of A. pleuropneumoniae serotypes 5A, 5B, and 10. Pigs immunized with this recombinant protein were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 5 isolate.
Comparative immunogenicity of conjugates composed of Escherichia coli O111 O-specific polysaccharide, prepared by treatment with acetic acid or hydrazine, bound to tetanus toxoid by two synthetic schemes
Authors: Gupta R K , Egan W , Bryla D A , Robbins J B , Szu S C
Published: January 2020

Escherichia coli O111, of various H types and virulence factors, causes enteritis throughout the world, especially in young children. This O type is found rarely in healthy individuals. Serum antibodies to the O-specific polysaccharide of O111 lipopolysaccharide (LPS) protect mice and dogs against infection with this E. coli serotype. The O111 O-specific polysaccharide is composed of a pentasaccharide repeat unit with two colitoses bound to the C-3 and C-6 of glucose in a trisaccharide backbone; this structure is identical to that of Salmonella adelaide (O35), another enteric pathogen. Nonpyrogenic O111 O-specific polysaccharide was prepared by treatment of its LPS with acetic acid (O-SP) or the organic base hydrazine (DeA-LPS). The O-SP had a reduced concentration of colitose. These products were derivatized with adipic acid dihydrazide (ADH) or thiolated with N-succinimidyl-3(2-pyridyldithio) propionate (SPDP). The four derivatives were covalently bound to tetanus toxoid (TT) by carbodiimide-mediated condensation or with SPDP to form conjugates. Immunization of BALB/c and general-purpose mice by a clinically acceptable route showed that DeA-LPS-TTADH, of the four conjugates, elicited the highest level of LPS antibodies. Possible reasons to explain this differential immunogenicity between the four conjugates are discussed.
A novel synthetic lipid A analog with low endotoxicity, DT-5461, prevents lethal endotoxemia
Authors: Sato K , Yoo Y C , Fukushima A , Saiki I , Takahashi T A , Fujihara M , Tono-Oka S , Azuma I
Published: January 2020

Bacterial endotoxin (lipopolysaccharide [LPS]) causes severe damage to the host organism as a result of excessive release of inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), from mononuclear phagocytes during gram-negative bacterial infection. We evaluated the ability of a novel synthetic lipid A analog with low endotoxicity, DT-5461, to antagonize LPS-induced IL-1 and TNF-alpha production in cells of monocyte/macrophage lineage and examined the protective effect of DT-5461 against lethal endotoxic shock in mice. The IL-1- or TNF-alpha-inducing activity of DT-5461 is 100,000 to 10,000 times less active than that of Escherichia coli LPS (EcLPS) or synthetic lipid A. DT-5461 significantly inhibited EcLPS-induced IL-1 and TNF-alpha release when murine peritoneal macrophages were incubated with DT-5461 2 h prior to EcLPS stimulation at the same concentration (1 microgram/ml). The antagonistic effect of DT-5461 on the production of IL-1 and TNF-alpha induced by EcLPS occurred in a concentration-dependent manner. DT-5461 also inhibited IL-1 and TNF-alpha induction when murine peritoneal macrophages were stimulated by LPS from Salmonella typhimurium or synthetic lipid A, as well as by EcLPS, but not by muramyl dipeptides. This indicated that DT-5461 specifically antagonized the action of LPS. DT-5461 also antagonized EcLPS-mediated activation of human peripheral blood monocytes. DT-5461 blocked the binding of fluorescein isothiocyanate-labelled LPS to murine peritoneal macrophages as well as it did the binding of EcLPS and synthetic lipid A, i.e., in a concentration-dependent fashion. Injection of DT-5461 2 h before EcLPS challenge prevented the production of serum IL-1 and TNF-alpha in D-galactosamine-treated mice. Furthermore, this treatment modality protected mice against LPS-induced lethal toxicity. This study suggests that DT-5461 possesses a potent LPS antagonistic effect and may be useful in a protective strategy against lethal endotoxemia caused by gram-negative bacterial infection.
Humoral immunity to aerosolized staphylococcal enterotoxin B (SEB), a superantigen, in monkeys vaccinated with SEB toxoid-containing microspheres
Authors: Tseng J , Komisar J L , Trout R N , Hunt R E , Chen J Y , Johnson A J , Pitt L , Ruble D L
Published: January 2020

Staphylococcal enterotoxin B (SEB) toxoid-containing microspheres were tested for efficacy in rhesus monkeys as a vaccine candidate for respiratory SEB toxicosis and toxic shock. Forty monkeys were randomly separated into 10 groups of four monkeys each: 9 groups were vaccinated with the microspheres via combinations of mucosal and nonmucosal routes, and 1 group served as nonvaccinated controls. Both vaccinated and nonvaccinated monkeys were then challenged with a high lethal dose of SEB aerosol. Monkeys primed with an intramuscular dose of the microspheres followed by an intratracheal booster all survived the SEB challenge. Overall, monkeys with an intratracheal booster generally had the highest antibody levels, which is consistent with their high survival rate and lower rate of illness. Protective immunity was correlated with antibody levels in both the circulation and the respiratory tract. The protection was not due to the depletion or anergy of SEB-reactive T cells, since SEB-induced proliferation in cultures of circulating lymphocytes was not significantly reduced after the microsphere vaccination. It is evident that the nonsurvivors did not die of systemic anaphylaxis or hypersensitivity because the monkeys did not die immediately after SEB challenge and there were no significant differences in histamine levels between the vaccinated and control monkeys before and after SEB challenge. The antibodies seemed to neutralize the SEB that got into the airway and the circulation.
Shigella infection induces cellular activation of T and B cells and distinct species-related changes in peripheral blood lymphocyte subsets during the course of the disease
Authors: Islam D , Bardhan P K , Lindberg A A , Christensson B
Published: January 2020

Immunophenotypic changes in peripheral blood lymphocytes (T, B, and NK cells) in patients during shigellosis was characterized by using triple-color flow cytometry. Eleven Shigella dysenteriae 1-infected adult patients (SDIP), 11 Shigella flexneri-infected adult patients (SFIP), 15 age- and sex-matched healthy controls from Bangladesh (C-B), and 15 healthy volunteers from Sweden (V-S) were studied. In SDIP and SFIP, a significant increase in the CD45RO+ cells in both CD4+ and CD8+ T cells were seen. We found evidence for sequential T-cell activation, as shown by increased proportions of CD25 and CD4+ cells; HLA-DR and CD38 on CD8+ cells, and CD54 on CD4+ and CD8+ cells. We found differences in the lymphocyte activation and subset patterns related to the infecting Shigella species. Thus, a decrease in CD45 expression was seen in SFIP; this decrease progressed further during the disease. The proportions of NK cells (CD56+ cells) and CD3- CD8+ cells out of the total CD8+ cells were increased in SFIP but not in SDIP. The CD3+ CD8+ CD57+ T-cell subset was significantly lower in SDIP than in C-B. The proportion of B-lymphocyte-expressing activation markers CD80 and CD23 was higher in patients than in C-B. There was a significant increase in the proportion of CD4+ T cells and a significant decrease in the percentages of total B cells, the CD3+ CD8+ CD57+ T-cell subset, and the CD56+ CD16+ NK-cell subset for V-S compared with C-B. Our results indicate that distinct subset changes and activation patterns are elicited in SDIP compared with SFIP and also that the degree of activation is related to disease severity. In addition, a common sequence of cell activation was seen during the disease course. The difference in the subset patterns seen in C-B and V-S may be related to differences in the levels or spectra of infectious agents in the environment.
Characterization of lbpA, the structural gene for a lactoferrin receptor in Neisseria gonorrhoeae
Authors: Biswas G D , Sparling P F
Published: January 2020

Neisseria gonorrhoeae acquires iron (Fe) efficiently from lactoferrin (LF). A 103-kDa gonococcal outer membrane LF-binding protein (Lbp) was identified previously. We isolated the structural gene lbpA for Lbp1 by screening a gonococcal library for a clone that could repair an LF- receptor mutant. An mTnCm3 transposon insertion mutant of lbpA was unable to use LF-bound Fe for growth, unable to bind LF to whole cells, and unable to express Lbp1. The DNA sequence of lbpA predicted a protein that shared 94% identity with the meningococcal LF receptor protein, Lbp, and was closely related to Tbp1, one of the transferrin receptor proteins. Clinical isolates of gonococci are frequently unable to acquire Fe from LF, and LF- isolates do not have a functional LF receptor. The wild-type lbpA gene transformed most tested LF- clinical isolates to LF+, indicating that lbpA is defective in many clinical isolates.
A system for generalized mutagenesis of Haemophilus ducreyi
Authors: Stevens M K , Cope L D , Radolf J D , Hansen E J
Published: January 2020

The lack of a generalized mutagenesis system for Haemophilus ducreyi has hampered efforts to identify virulence factors expressed by this sexually transmitted pathogen. To address this issue, the transposable element Tn1545-delta 3, which encodes resistance to kanamycin, was evaluated for its ability to insert randomly into the H. ducreyi chromosome and produce stable, isogenic mutants. Electroporation of H. ducreyi with 1 microgram of plasmid pMS1 carrying Tn1545-delta 3 resulted in the production of 10(4) kanamycin-resistant transformants; Southern blot analysis of a number of these transformants indicated that insertion of the transposon into the chromosome occurred at a number of different sites. This pMS1-based transposon delivery system was used to produce an H. ducreyi mutant that expressed an altered lipooligosaccharide (LOS). Passage of this mutant in vitro in the presence or absence of kanamycin did not affect the stability of the transposon insertion. To confirm that the observed mutant phenotype was the result of the transposon insertion, a chromosomal fragment containing Tn1545-delta 3 was cloned from this H. ducreyi LOS mutant. Electroporation of the wild-type H. ducreyi strain with this DNA fragment yielded numerous kanamycin-resistant transformants, the majority of which had the same altered LOS phenotype as the original mutant. Southern blot analysis confirmed the occurrence of proper allelic exchange in the LOS-deficient transformants obtained in this backcross experiment. The ability of Tn1545-delta 3 to produce insertion mutations in H. ducreyi should facilitate genetic analysis of this pathogen.
Functional capacities of clonal antibodies to Haemophilus influenzae type b polysaccharide
Authors: Nahm M H , Kim K H , Anderson P , Hetherington S V , Park M K
Published: January 2020

Haemophilus influenzae type b (Hib) is an important pathogen for young children, and children can be protected with antibodies (Abs) to Hib polysaccharide (PS) capsule, a linear polymer of ribosyl ribitol phosphate. The structure of anti-Hib-PS Abs has been well characterized at the molecular level; about two-thirds of anti-Hib-PS Abs use a V kappa gene named A2, and the remaining anti-Hib-PS Abs use one of many other VL genes. In order to understand the structural basis for the variability in the function of these Abs, we prepared 18 clonally pure Abs from adults and studied their affinity, avidity, bactericidal potency in vitro, and ability to reduce bacteremia in newborn rats. Affinities and avidities were determined as the inverse of the concentrations of short (3 repeating units) and long (20 repeating units) ligands which could bind 50% of anti-Hib-PS Ab in solution, respectively. No significant correlations between the protection of newborn rats and affinity (r = 0.02) or avidity (r = 0.16) were observed. The amount of Ab required to kill 50% of bacteria in vitro decreased with avidity (r = -0.32), as expected. However, Abs with high affinity were unexpectedly found to have less bactericidal activity (r = 0.38). This suggests that avidity may be a better predictor of Ab function than affinity. Affinity and avidity results were negatively correlated (r = 0.76, P = 0.0022), and Abs that had A2 V kappa gene products had higher avidity (P < 0.05) and lower affinity (P = 0.06) than Abs that had other VL genes. A possible explanation of these observations is that the epitope for Abs with the A2 gene is within the Hib-PS chain itself, whereas the epitope for Abs with a non-A2 gene is the terminus of Hib-PS.
Expansion of gamma delta+ T cells in BALB/c mice infected with Leishmania major is dependent upon Th2-type CD4+ T cells
Authors: Rosat J P , Conceiçao-Silva F , Waanders G A , Beermann F , Wilson A , Owen M J , Hayday A C , Huang S , Aguet M , MacDonald H R
Published: January 2020

T cells belong to either the alpha beta+ or gamma delta+ lineage as defined by their antigen receptor. Although both T-cell subsets have been shown to be involved in the immune response to the parasite Leishmania major, very little is known about possible interactions between these two populations. In this study, using a mouse model of infection with L. major, we showed that expansion of a subset of gamma delta+ T cells in vivo is dependent upon the presence of alpha beta+ CD4+ T cells. Moreover, this effect appears to be mediated via the secretion of lymphokines by CD4+ cells with a T-helper 2 (Th2) functional phenotype. Results showing that activation of Th2-type cells in mice treated with anti-immunoglobulin D antibodies or infected with Nippostrongylus brasiliensis also results in gamma delta+ T-cell expansion suggest that this effect of the Th2-type CD4+ cells is a general phenomenon not restricted to infection with L. major.
Antibody response of the mouse reservoir of Borrelia burgdorferi in nature
Authors: Brunet L R , Sellitto C , Spielman A , Telford S R
Published: January 2020

To determine whether the white-footed mouse reservoir host (Peromyscus leucopus) of the agent of Lyme disease (Borrelia burgdorferi) naturally mounts an immune response against the full range of antigens expressed by this zoonotic pathogen, we analyzed the pattern of immunoreactivity of these rodents at sites in which the intensity of transmission differs. Although the incidence of seroconversion within the reservoir population relates proportionally to the density of subadult deer ticks (Ixodes dammini), seroprevalence appears constant. About a fifth as many juvenile mice recognize spirachete antigens as do adult mice. Virtually all reservoir mice in nature recognize the p20, p35.5, p39, and p58 antigens, regardless of the intensity of transmission. Seropositive mice retain reactivity to a wide range of spirochetal antigens. Few mice recognize flagellin, OspB, and OspC. Although a third of serum samples include reactivity to a 31-kDa band, this reaction is irregular and may represent an uncharacterized antigen that comigrates with OspA. Mice captured where transmission is intense recognize the same spectrum of antigens as do mice captured where vector ticks are scarce.
Immunization of rats with synthetic peptide constructs from the glucan-binding or catalytic region of mutans streptococcal glucosyltransferase protects against dental caries
Authors: Taubman M A , Holmberg C J , Smith D J
Published: January 2020

Previously, we have described peptide constructs from two regions of glucosyltransferase (GTF) of mutans streptococci. A putative catalytic site in the amino-terminal half of the molecule and a repeated glucan-binding site in the carboxyl-terminal half of GTF were the regions upon which sequences were based. The present study explored the effects of immunization with these peptide constructs (called CAT or GLU) and with streptococcal GTFs from Streptococcus sobrinus and S. mutans on immunological, microbiological, and disease parameters. Groups of immunized Sprague-Dawley rats were infected with either 10(8) S. sobrinus 6715 or 10(8) S. mutans SJ32 organisms. Serum immunoglobulin G antibody levels, determined by enzyme-linked immunosorbent assay, to the respective peptide constructs and to the appropriate streptococcal GTF were significantly increased (after immunization) prior to infection and at the end of the experiment. Also, serum antibody from CAT-, GLU-, and S. sobrinus GTF-immunized rats inhibited S. sobrinus GTF-mediated insoluble glucan synthesis (all) and S. mutans GTF-mediated soluble glucan synthesis (all except anti-GLU) from sucrose. Immunization with the CAT or GLU peptide construct resulted in significantly reduced smooth surface and sulcal caries after infection with S. sobrinus. Sulcal dental caries after infection with S. mutans SJ32 were also significantly reduced in CAT- and GLU-immunized rats. Thus, immunization with peptides whose sequences are based on putative functional domains of mutans streptococcal GTF are protective toward a cariogenic S. sobrinus or S. mutans infection.
Swine model of Haemophilus ducreyi infection
Authors: Hobbs M M , San Mateo L R , Orndorff P E , Almond G , Kawula T H
Published: January 2020

Haemophilus ducreyi is a strict human pathogen that causes sexually transmitted genital ulcer disease. We infected domestic swine with H. ducreyi 35000, resulting in the development of cutaneous ulcers histologically resembling human chancroid lesions. Intraepidermal lesions progressed from pustules to ulcers containing polymorphonuclear leukocytes and were accompanied by a dermal inflammatory infiltrate containing T cells and macrophages. H. ducreyi was recovered from lesions up to 17 days after inoculation, and pigs did not develop immunity to reinfection with the challenge strain. Features of the model include inoculation through abrasions in the epidermis, ambient housing temperatures for infected pigs, the ability to deliver multiple different inocula to a single host, and the availability of monoclonal antibodies against porcine immune cells permitting immunohistochemical characterization of the host immune response to H. ducreyi infection.
Identification of Babesia bovis merozoite antigens separated by continuous-flow electrophoresis that stimulate proliferation of helper T-cell clones derived from B. bovis-immune cattle
Authors: Brown W C , Logan K S , Zhao S , Bergman D K , Rice-Ficht A C
Published: January 2020

To characterize Babesia bovis merozoite antigens that stimulate anamnestic T helper (Th)-cell responses from B. bovis-immune cattle, B. bovis-specific Th-cell lines and clones, previously assigned to different antigenic groups (W. C. Brown, S. Zhao, A. C. Rice-Ficht, K. S. Logan, and V. M. Woods, Infect. Immun. 60:4364-4372, 1992), were tested in proliferation assays against fractionated merozoite antigens. The antigenic groups were determined by the patterns of response of Th clones to different parasite isolates and soluble or membrane forms of merozoite antigen. Soluble antigen fractionated by anion-exchange chromatography or gel filtration by using fast-performance liquid chromatography resolved two or three antigenic peaks, respectively. To enable fractionation of membrane-associated proteins and to resolve more precisely the proteins present in homogenized merozoites, a novel technique of continuous-flow electrophoresis was employed. Merozoite membranes or whole merozoites were homogenized and solubilized in sodium dodecyl sulfate-sample buffer, electrophoresed under reducing conditions on 15% or 10% acrylamide gels, eluted, and collected as fractions. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested for the ability to stimulate Babesia-specific CD4+ T-cell lines and clones. CD4+ Th-cell lines from two cattle displayed differential patterns of reactivity and detected numerous peaks of antigenic activity, ranging from < 14 to 76 kDa. Th-cell clones previously categorized into different antigenic groups detected antigenic peaks unique for clones representative of a given group. Antigens of 29, 51 to 52, and 85 to 95 kDa (group I), 40 kDa (group III), 20 kDa (group IV), 58 to 60 kDa (group VI), and 38, 45, and 83 kDa (group VII) were identified in the stimulatory fractions. Immunization of rabbits with selected fractions produced a panel of antisera that reacted specifically on Western blots (immunoblots) with merozoite antigens of similar sizes, leading to the tentative identification of candidate antigens of B. bovis merozoites with molecular masses of 20, 40, 44, 51 to 52 or 95, and 58 to 60 kDa that stimulate proliferation of Th clones representative of five different antigenic groups. These antisera may be useful for isolating recombinant proteins that are immunogenic for Th cells of immune cattle and therefore potentially useful for vaccine development.