bluuebunny/crossref_metadata_2025 huggingface dataset:
Infection of the ferret stomach by isogenic flagellar mutant strains of Helicobacter mustelae
Authors: Andrutis K A , Fox J G , Schauer D B , Marini R P , Li X , Yan L , Josenhans C , Suerbaum S
Published: January 2020

Helicobacter mustelae, like Helicobacter pylori, possesses two flagellin proteins, FlaA and FlaB. Isogenic mutant strains of H. mustelae have been constructed by disruption of the flaA or flaB gene with a kanamycin resistance cassette or by introduction of both a kanamycin and a chloramphenicol resistance gene to produce a double mutant. To determine whether one or both flagellin proteins are necessary for colonization and persistence of infection with H. mustelae, 19 ferrets, specific pathogen free for H. mustelae, were given either the HMF1 flaA::km (weakly motile), ATCC 43772 flaB::km (moderately motile), or HMF1 flaA::cat flaB::km (non-motile) mutant strain, the wild-type parent strains, or sterile broth. Gastric tissue samples were obtained during sequential gastric biopsies beginning at 3 weeks postinoculation and ending at necropsy at 3 months postinoculation. H. mustelae infection status was determined by culture, histology, and serology. The wild-type parent strains of H. mustelae infected all ferrets at all time points. The double-mutant strain was unable to colonize; the flaA and flaB single-mutant strains were able to initially colonize at a low level and establish persistent infection with increasing numbers of organisms over time. The severity of gastritis produced by infection with these strains of H. mustelae correlated with the number of organisms present in the gastric mucosa. Flagellar motility is an important virulence factor for colonization and pathogenesis in the H. mustelae ferret model.
Virulence properties and clonal structures of strains of Escherichia coli O119 serotypes
Authors: Gonçalves A G , Campos L C , Gomes T A , Rodrigues J , Sperandio V , Whittam T S , Trabulsi L R
Published: January 2020

A total of 110 Escherichia coli strains of serogroup O119 were examined for the presence of virulence properties characteristic of enteropathogenic E. coli (EPEC). Three virulence patterns were distinguished based on the detection of a chromosomal gene mediating intimate attachment (eaeA) and plasmid DNA involved in localized adherence (EAF and bfpA). The first pattern, represented by strains which hybridized with three gene probes, was the most common (68%) and, with a single exception, included only O119:H6 strains. Of these strains, 90% showed a typical localized adherence (LA) pattern in HEp-2 cells and 96% were positive for intimate attachment in a fluorescent-actin staining test with a 3-h incubation period. The second pattern was represented by strains which hybridized with the eaeA gene only. Most (89.5%) of these strains showed the LA phenotype but only after 6 h of incubation (LA-like phenotype). The third pattern consisted of strains which were positive for eaeA and bfpA but did not hybridize with the EAF probe. Most (80%) of these strains exhibited the LA-like phenotype. Analysis of several eaeA+ bfpA+ strains for the expression of the pilin subunit (BfpA) of the bundle-forming pili demonstrated that all LA strains expressed BfpA whereas the LA-like strains did not. The study of the clonal relationships, carried out by multilocus enzyme electrophoresis in 79 representative strains, defined 11 distinct electrophoretic types (ETs). ET1 included 66% of the strains, most of which displayed the eaeA+ bfpA+ EAF+ pattern and were serotyped as O119:H6 or O119:H-. The remaining 10 ETs were each represented by no more than five strains and, with the exception of ET8, included strains of a single serotype. The genetic relatedness of the ETs revealed two main clusters, with most strains in cluster A having the eaeA+ bfpA+ EAF+ combination and a O119:H6 serotype. Cluster B was represented by atypical EPEC strains with only the eaeA+ and the eaeA+ bfpA+ virulence pattern.
Intranasal immunization with C5a peptidase prevents nasopharyngeal colonization of mice by the group A Streptococcus
Authors: Ji Y , Carlson B , Kondagunta A , Cleary P P
Published: January 2020

Early inflammatory events are initiated by phased production of C5a and interleukin-8 in tissue. Most serotypes of group A streptococci express a surface-bound peptidase (SCPA) which specifically cleaves mouse and human C5a chemotaxins. This study investigates the impact of SCPA on colonization of the nasopharyngeal mucosa of mice and evaluates its potential to induce protective immunity. Two strains, serotypes M6 and M49, which contain insertion and deletion mutations in the SCPA gene (scpA) and represent the two major subdivisions of group A streptococci, were characterized and compared in a mouse intranasal infection model. In this model, SCPA mutants were more rapidly cleared from the nasopharynges of inoculated mice compared with wild-type strains. A 2,908-bp fragment of scpA49 gene, obtained by PCR, was ligated to the expression vector pGEX-4T-1 and expressed in Escherichia coli. The affinity-purified deltaSCPA49 protein proved to be highly immunogenic in mice and rabbits. Although the purified deltaSCPA49 immunogen lacked enzymatic activity, it induced high titers of rabbit antibodies which were able to neutralize peptidase activity associated with M1, M6, M12, and M49 streptococci in vitro. This result confirmed that antipeptidase antibodies lack serotype specificity. Intranasal immunization of mice with the deleted form of the SCPA49 protein stimulated significant levels of specific salivary secretory immunoglobulin A (IgA) and serum IgG antibodies and reduced the potential of wild-type M1, M2, M6, M11, and M49 streptococci to colonize. These experiments suggest a new approach to vaccine development for prevention of streptococcal pharyngitis.
Pleiotropic effects of a mutation in rfaC on Escherichia coli hemolysin
Authors: Bauer M E , Welch R A
Published: January 2020

Several genes involved in the lipopolysaccharide (LPS) biosynthetic pathway have been shown to affect the expression or activity of Escherichia coli hemolysin (Hly), a secreted cytotoxin that is the prototype of the RTX family of toxins. To further study this relationship, E. coli K-12 strains harboring mutations in the LPS biosynthetic genes rfaS, rfaQ, rfaJ, rfaP, and rfaC were transformed with a recombinant plasmid harboring the hlyCABD operon and examined for their effects on extracellular expression and hemolytic activity. A mutation in rfaC that affected both extracellular expression and activity of Hly was studied in greater detail. This mutation led to a growth-phase-dependent decrease up to 16-fold in the steady-state level of extracellular HlyA, although transcription and secretion of HlyA were decreased no more than 2-fold. Specific hemolytic activity in toxin produced from the rfaC mutant strain was significantly reduced, in a growth-phase-dependent manner. With the rfaC gene supplied in trans, both the decreased expression and activity of Hly were restored to wild-type levels. Hly from the rfaC mutant strain exhibited much slower kinetics of hemolysis, a more rapid rate of decay of activity, and greater formation of apparently inactive HlyA-containing aggregates in culture supernatants than was exhibited in the wild-type strain. A model is proposed for a physical interaction between LPS and Hly in which LPS with intact inner core participates in forming or maintaining an active conformation of Hly and helps to protect it from aggregation or degradation.
Expression of botulinum toxin binding sites in Xenopus oocytes
Authors: Bakry N M , Kamata Y , Simpson L L
Published: January 2020

The binding of iodinated botulinum toxin type B to nerve membranes was studied by using rat and mouse preparations. The toxin was examined both in the single-chain and in the proteolytically processed dichain form, and binding sites both in the spinal cord and in various brain regions were assayed. Rat and mouse brains possessed specific binding sites for botulinum toxin type B. The average Kd values for the various rat and mouse membrane preparations examined were 4.2 +/- 0.7 nM and 3.7 +/- 0.9 nM, respectively. The average Bmax values for the same tissue preparations were 7.3 +/- 0.7 pmol/mg of protein and 7.5 +/- 1.9 pmol/mg protein, respectively. The binding of botulinum toxin type B to rat brain membranes was not antagonized by a polyclonal antibody against the cytosolic domain of synaptotagmin 1 or by a monoclonal antibody directed against the luminal domain of synaptotagmin 1. In addition, these antibodies did not protect the mouse phrenic nerve-hemidiaphragm from toxin-induced neuromuscular blockade. Extraction of whole-brain mRNA and injection into Xenopus oocytes led to expression of binding sites for botulinum toxin. Extraction and injection of cerebellar mRNA led to expression of a higher density of binding sites. The number of binding sites was not diminished when oocytes were pretreated with antibodies against the cytosolic and luminal domains of synaptotagmin 1. These findings are likely to aid in the isolation, characterization, and reconstitution of toxin binding sites.
Prelytic and lytic conformations of erythrocyte-associated Escherichia coli hemolysin
Authors: Moayeri M , Welch R A
Published: January 2020

Flow cytometry was developed as a method to assess the conformation of erythrocyte-bound Escherichia coli hemolysin polypeptide (HlyA). Topology of membrane-associated hemolysin (HlyA(E)) was investigated by testing surface accessibility of HlyA regions in lytic and nonlytic bound states, using a panel of 12 anti-HlyA monoclonal antibodies (MAbs). Hemolysin associates nonlytically with erythrocytes at 0 to 2 degrees C. To test the hypothesis that the nonlytic HlyA(E) conformation at 0 to 2 degrees C differs from the lytic conformation at 23 degrees C, MAb epitope reactivity profiles at the two temperatures were compared by flow cytometry. Four MAbs have distinctly increased reactivity at 0 to 2 degrees C compared to 23 degrees C. HlyA requires HlyC-dependent acylation at lysine residues 563 and 689 for lytic function. Toxin with cysteine substitution mutations at each lysine (HlyA(K563C) and HlyA(K689C)) as well as the nonacylated form of hemolysin made in a HlyC-deficient strain were examined by flow cytometry at 0 to 2 and 23 degrees C. The three mutants bind erythrocytes at wild-type toxin levels, but there are conformational changes reflected by altered MAb epitope accessibility for six of the MAbs. To test further the surface accessibility of regions in the vicinity of MAb-reactive epitopes, HlyA(E) was proteolytically treated prior to testing for MAb reactivity. Differences in protease susceptibility at 0 to 2 degrees and 23 degrees C for the reactivities of three of the MAbs further support the model of two distinct conformations of cell-associated toxin.
Depletion of alveolar macrophages exacerbates respiratory mycoplasmosis in mycoplasma-resistant C57BL mice but not mycoplasma-susceptible C3H mice
Authors: Hickman-Davis J M , Michalek S M , Gibbs-Erwin J , Lindsey J R
Published: January 2020

Indirect evidence suggests that innate immune mechanisms involving alveolar macrophages (AMs) are of major importance in antimycoplasmal defense. We compared the effects of AM depletion on intrapulmonary killing of Mycoplasma pulmonis during the early phase of infection in mycoplasma-resistant C57BL/6NCr (C57BL) and mycoplasma-susceptible C3H/HeNCr (C3H) mice. More than 80% of AMs were depleted in both strains of mice by intratracheal insufflation of liposome-encapsulated dichloromethylene bisphosphonate (L-Cl2MBP), compared to no significant AM depletion in either strain following insufflation of liposome-encapsulated phosphate-buffered saline (L-PBS), PBS alone, or no treatment. AM-depleted (L-Cl2MBP) and control (L-PBS) mice were infected intranasally with 10(5) CFU of M. pulmonis UAB CT, and their lungs were quantitatively cultured to assess intrapulmonary killing at 0, 8, 12, and 48 h postinfection. AM depletion exacerbated the infection in C57BL mice by reducing killing of the organism to a level comparable to that in C3H mice without AM depletion. In contrast, AM depletion did not alter killing in C3H mice. These results directly identify the AM as the main effector cell in early pulmonary antimycoplasmal defense and suggest that differences in mycoplasmal killing by AMs may explain the resistance of C57BL mice and the susceptibility of C3H mice to mycoplasmal infection.
Localization of alpha/beta and gamma/delta T lymphocytes in Cryptosporidium parvum-infected tissues in naive and immune calves
Authors: Abrahamsen M S , Lancto C A , Walcheck B , Layton W , Jutila M A
Published: January 2020

The nature of the host's T-lymphocyte population within the intestinal villi following Cryptosporidium parvum infection was characterized with a bovine model of cryptosporidiosis. In naive animals, infection with C. parvum resulted in substantial increases in the numbers of alpha/beta T cells, both CD4+ (150%) and CD8+ (60%), and of gamma/delta T cells (70%) present within the intestinal villi of the infected ileum. In immune animals, the host T-lymphocyte response to a challenge infection with C. parvum was restricted to alpha/beta T cells. The number of CD4+ T cells within the Peyer's patch of the ileum increased dramatically; however, there was little change in the number or localization of CD4+ T cells within the intestinal villi. In contrast, the number of CD8+ T cells within the intestinal villi increased following a challenge infection. In addition, the CD8+ T cells were found to be intimately associated with the epithelial cells of the intestinal villi. The precise correlation between the accumulation of CD8+ T cells and the normal site of parasite development suggests an important role for CD8+ T cells in the immune animal.
Distribution of endosomal, lysosomal, and major histocompatability complex markers in a monocytic cell line infected with Chlamydia psittaci
Authors: Ojcius D M , Hellio R , Dautry-Varsat A
Published: January 2020

The intracellular fate of Chlamydia psittaci during infection of a monocytic cell line, THP1, was characterized. Cytochalasin D inhibited phagocytosis of latex beads but had no effect on infection by C. psittaci, and vacuoles expressed the transferrin receptor, suggesting accessibility to the endocytic pathway. Early Chlamydia-containing vacuoles expressed major histocompatibility complex (MHC) class I molecules, and most vacuoles fused with host cell lysosomes, since they expressed LAMP-1 and had acidic pHs. In cells prestimulated with gamma interferon, vacuoles also expressed MHC class II molecules, suggesting that the monocytes might effectively process Chlamydia-derived antigens for presentation by MHC class I and class II molecules.
Avirulence of LT2 strains of Salmonella typhimurium results from a defective rpoS gene
Authors: Swords W E , Cannon B M , Benjamin W H
Published: January 2020

In order to identify the genetic basis for the attenuation of Salmonella typhimurium LT2 strains, experiments were performed to identify a gene(s) which restores virulence to an avirulent LT2 strain. These and further experiments confirmed that an rpoS mutation is the sole determinant of the attenuation of S. typhimurium LT2.
Conversion of M serotype 24 of Streptococcus pyogenes to M serotypes 5 and 18: effect on resistance to phagocytosis and adhesion to host cells
Authors: Courtney H S , Liu S , Dale J B , Hasty D L
Published: January 2020

In this study, we utilized recombinant strains expressing the M5 and M18 proteins and M- mutants of group A streptococci to compare the abilities of these M proteins to confer resistance to phagocytosis and to mediate adhesion to host cells. The data indicate that the M5 and M18 proteins can confer resistance to phagocytosis, that fibrinogen is required for this resistance, and that these M proteins can mediate adhesion to HEp-2 cells.
Role of neutrophils in experimental septicemia and septic arthritis induced by Staphylococcus aureus
Authors: Verdrengh M , Tarkowski A
Published: January 2020

We have previously described a murine model of hematogenously induced Staphylococcus aureus sepsis and arthritis. In this model, large numbers of granulocytes can be observed both in the circulation and locally in the inflamed synovium within 24 h after bacterial inoculation. To assess the role of neutrophils in this severe infection, mice were given granulocyte-depleting monoclonal antibody RB6-8C5 before being inoculated with S. aureus. All the control mice survived their intravenous injection with 3 x 10(7) CFU of S. aureus, whereas all the mice given RB6-8C5 antibody died of sepsis within 2 to 3 days. Even when the inoculum size was reduced sixfold (i.e., 6 x 10(6) CFU/mouse), 50% of the RB6-8C5-treated animals died within 6 days. The RB6-8C5-treated mice had a significantly higher burden of bacteria in their blood and kidneys 24 and 48 h after bacterial inoculation. In addition, when a suboptimal dose of bacteria was administered, the neutrophil-depleted animals displayed a higher frequency of arthritis than did the controls. The granulocyte-depleted animals exhibited increased levels of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-6, and gamma interferon, reflecting the severity of their disease. This is the first direct demonstration of neutrophils playing a crucial protective role in the early phase of S. aureus infection.
Synthesis and function of Actinomyces naeslundii T14V type 1 fimbriae require the expression of additional fimbria-associated genes
Authors: Yeung M K , Ragsdale P A
Published: January 2020

The nucleotide sequence of the chromosomal DNA flanking the Actinomyces naeslundii (formerly A. viscosus) T14V type 1 fimbrial structural subunit gene (fimP) was determined. Six open reading frames (ORFs), in the order 5' ORF3, ORF2, ORF1,fimP, ORF4, ORF5, ORF6 3', were identified. ORF1 encoded a protein of 408 amino acid residues (Mr = 39,270) and had significant sequence homology with the A. naeslundii T14V type 1 and A. naeslundii WVU45 type 2 fimbrial structural subunits. An in-frame fusion of ORF1 to the malE gene of the expression vector, pMAL-c2, yielded a protein that was immunostained with antibodies raised against the maltose binding protein and A. naeslundii T14V whole bacteria. Digestion of the fusion protein with factor Xa released a protein (apparent molecular mass of 34 kDa) that was immunostained only with the antibody directed against A. naeslundii T14V whole bacterial cells. Integration plasmids carrying a kanamycin resistance gene (kan) that was used to substitute for ORF1 or for DNA fragments internal to the coding region of the other five ORFs were used to transform A. naeslundii T14V. Neither type 1 fimbriae nor the 65-kDa fimbrial structural subunit was detected in mutants obtained by allelic replacement of ORF1 or ORF2. Mutants obtained by allelic replacement of ORF3 or ORF4 expressed only the 65-kDa fimbrial structural subunit. These mutants did not bind, in vitro, to proline-rich proteins that serve as the receptors for Actinomyces type 1 fimbriae. In contrast, a mutant in which the integration plasmid DNA had been inserted at a site close to the carboxyl terminus of ORF6 expressed type 1 fimbriae and had adherence properties similar to those observed in the wild-type strain. These results demonstrate the existence of additional genes near fimP that are likely to be involved in the synthesis and function of cell surface fimbriae of A. naeslundii T14V.
Hyperlipoproteinemia enhances susceptibility to acute disseminated Candida albicans infection in low-density-lipoprotein-receptor-deficient mice
Authors: Netea M G , Demacker P N , de Bont N , Boerman O C , Stalenhoef A F , van der Meer J W , Kullberg B J
Published: January 2020

Recent studies have suggested the use of lipoproteins as an adjuvant treatment of lethal gram-negative infections. However, other important microorganisms for the etiology of sepsis, such as Candida species, grow better in lipid-rich environments. We investigated the effect of hyperlipoproteinemia on systemic candidiasis in low-density-lipoprotein-receptor-deficient (LDLR-/-) mice, in which the loss of the receptor results in a seven- to ninefold-higher plasma LDL level than that in their wild-type littermates (C57BL/6J). LDLR-/- mice died earlier, and the outgrowth of Candida albicans in the kidneys and livers of LDLR-/- mice was significantly higher compared with that of controls. After infection, circulating cytokine concentrations were significantly higher in LDLR-/- mice. In vitro, C. albicans grew better in plasma samples of LDLR-/- mice than in control plasma samples and peritoneal macrophages of LDLR-/- mice challenged with heat-killed C. albicans produced more cytokines than did those of controls. This latter phenomenon was probably due to increased binding of yeast cells to macrophages of LDLR-/- mice. These data suggest that hyperlipoproteinemia is deleterious in systemic candidiasis.
Sialic acid-dependent recognition of laminin and fibrinogen by Aspergillus fumigatus conidia
Authors: Bouchara J P , Sanchez M , Chevailler A , Marot-Leblond A , Lissitzky J C , Tronchin G , Chabasse D
Published: January 2020

In an attempt to define the molecular basis of the adherence of Aspergillus fumigatus conidia to the host tissues, a step which might be mediated by the recognition of basement membrane laminin or fibrinogen, we analyzed the binding of these glycoproteins by flow cytometry and a microtiter plate adherence assay. Flow cytometry revealed that the binding of fluorescein isothiocyanate-labeled laminin to conidia was saturable and specific. Moreover, the ability of conidia to bind laminin increased with their maturation. Competition experiments showed a cross-reactivity between laminin and fibrinogen binding and a lack of interactions with glycosaminoglycans. In addition, the binding of laminin was not inhibited by the different adhesive synthetic peptides tested. Furthermore, the microtiter plate assay of adherence to chymotrypsin degradation products of laminin or fibrinogen purified by gel filtration suggested a unique binding site common to sequential degradation fragments or the presence of multiple binding sites on the two ligands. Therefore, the role of carbohydrates in the recognition process was investigated. Among the carbohydrates tested, constitutive of the conidial wall or of the oligosaccharide side chains of laminin and fibrinogen, only N-acetylneuraminic acid and sialyllactose inhibited the binding of these glycoproteins to conidia. In conclusion, these results strengthen the idea that the laminin and fibrinogen receptors in A. fumigatus are identical and suggest an interaction mediated by a sialic acid-specific lectin of the conidial wall.
Utilization of iron-catecholamine complexes involving ferric reductase activity in Listeria monocytogenes
Authors: Coulanges V , Andre P , Ziegler O , Buchheit L , Vidon D J
Published: January 2020

Listeria monocytogenes is a ubiquitous potentially pathogenic organism requiring iron for growth and virulence. Although it does not produce siderophores, L. monocytogenes is able to obtain iron by using either exogenous siderophores produced by various microorganisms or natural catechol compounds widespread in the environment. In the presence of tropolone, an iron-chelating agent, growth of L. monocytogenes is completely inhibited. However, the growth inhibition can be relieved by the addition of dopamine or norepinephrine under their different isomeric forms, while the catecholamine derivatives 4-hydroxy-3-methoxyphenylglycol and normetanephrine did not relieve the inhibitory effect of tropolone. Preincubation of L. monocytogenes with chlorpromazine and yohimbine did not antagonize the growth-promoting effect of catecholamines in iron-complexed medium. In addition, norepinephrine stimulated the growth-promoting effect induced by human transferrin in iron-limited medium. Furthermore, dopamine and norepinephrine allowed 55Fe uptake by iron-deprived bacterial cells. The uptake of iron was energy dependent, as indicated by inhibition of 55Fe uptake at 0 degrees C as well as by preincubating the bacteria with KCN. Inhibition of 55Fe uptake by L. monocytogenes was also observed in the presence of Pt(II). Moreover, when assessed by a whole-cell ferric reductase assay, reductase activity of L. monocytogenes was inhibited by Pt(II). These data demonstrate that dopamine and norepinephrine can function as siderophore-like compounds in L. monocytogenes owing to their ortho-diphenol function and that catecholamine-mediated iron acquisition does not involve specific catecholamine receptors but acts through a cell-bound ferrireductase activity.
Differences in the association of Chlamydia trachomatis serovar E and serovar L2 with epithelial cells in vitro may reflect biological differences in vivo
Authors: Davis C H , Wyrick P B
Published: January 2020

Chlamydia trachomatis serovar E is one of the most common bacterial sexually transmitted pathogens. Since it is an obligate intracellular bacterium, efficient colonization of genital mucosal epithelial cells is crucial to the infectious process. Serovar E elementary bodies (EB) metabolically radiolabeled with 35S-Cys-Met and harvested from microcarrier bead cultures, which significantly improves the infectious EB-to-particle ratio, provided a more accurate picture of the parameters of attachment of EB to human endometrial epithelial cells (HEC-1B) than did less infectious 14C-EB harvested from flask cultures. Binding of serovar E EB was (i) equivalent at 35 and 4 degrees C, (ii) decreased by preexposure of EB to heat or the topical microbicide C31G, (iii) comparable among common eukaryotic cell lines (HeLa, McCoy), and (iv) significantly increased to the apical surfaces of polarized cells versus nonpolarized cells. In parallel experiments with C. trachomatis serovar L2, serovar E attachment was not affected by heparin or heparan sulfate whereas these glucosaminoglycans dramatically reduced serovar L2 attachment. These data were confirmed by competitive inhibition of serovar E binding and infectivity by excess unlabeled live and UV-inactivated serovar E EB but not by excess serovar L2 EB. The noninvasive serovar E strains in the lumen of the genital tract enter and exit the apical domains of target columnar epithelial cells to spread canalicularly in an ascending fashion from the lower to the upper genital tract. In contrast, the invasive serovar L2 strains are primarily submucosal pathogens and likely use the glucosaminoglycans concentrated in the extracellular matrix to colonize the basolateral domains of mucosal epithelia to perpetuate the infectious process.
Lymphocyte proliferative responses of goats vaccinated with Brucella melitensis 16M or a delta purE201 strain
Authors: Olsen S C , Cheville N F , Stevens M G , Houng H H , Drazek E S , Hadfield T L , Warren R L , Hoover D L
Published: January 2020

The response to a Brucella melitensis purEK deletion mutant, delta purE201 (referred to as strain 201), was compared with the response to its parental strain, 16M, in juvenile goats. Proliferative responses to gamma-irradiated bacteria were detected earlier in strain 201-infected goats. Lymphocytes from strain 16M- or 201-infected goats proliferated in response to one-dimensional polyacrylamide gel electrophoresis-separated proteins of similar mass isolated from strain 16M or Brucella abortus RB51. Data from this study suggest that some antigens stimulating cell-mediated responses are conserved among Brucella species, as 201- and 16M-infected goats recognized similar proteins expressed by RB51 and 16M.
Genetic diversity among clinical and environmental isolates of Aspergillus fumigatus
Authors: Debeaupuis J P , Sarfati J , Chazalet V , Latgé J P
Published: January 2020

To determine if cases of invasive aspergillosis (IA) were caused by strains of Aspergillus fumigatus with unique characteristics, strains from immunosuppressed patients with IA were compared to strains obtained from sputa of patients with cystic fibrosis and to strains from the environment. An extremely high genomic diversity was observed among the 879 strains typed by Southern blotting with a retrotransposon-like element from A. fumigatus (C. Neuvéglise, J. Sarfati, J. P. Latgé, and S. Paris, Nucleic Acids Res. 24:1428-1434, 1996). Analysis of Southern blot hybridization patterns showed the absence of clustering between environmental isolates and clinical isolates from patients with IA or cystic fibrosis. In addition, strains could not be clustered depending on their geographical location. This study implies that practically any strain of A. fumigatus is potentially pathogenic and can provoke a case of IA when it encounters a favorable environment in an immunosuppressed host.
Active sites of salivary proline-rich protein for binding to Porphyromonas gingivalis fimbriae
Authors: Kataoka K , Amano A , Kuboniwa M , Horie H , Nagata H , Shizukuishi S
Published: January 2020

Porphyromonas gingivalis fimbriae specifically bind salivary acidic proline-rich protein 1 (PRP1) through protein-protein interactions. The binding domains of fimbrillin (a subunit of fimbriae) for PRP1 were analyzed previously (A. Amano, A. Sharma, J.-Y. Lee, H. T. Sojar, P. A. Raj, and R. J. Genco, Infect. Immun. 64:1631-1637, 1996). In this study, we investigated the sites of binding of the PRP1 molecules to the fimbriae. PRP1 (amino acid residues 1 to 150) was proteolysed to three fragments (residues 1 to 74 [fragment 1-74], 75 to 129, and 130 to 150). 125I-labeled fimbriae clearly bound fragments 75-129 and 130-150, immobilized on a polyvinylidene difluoride membrane; both fragments also inhibited whole-cell binding to PRP1-coated hydroxyapatite (HAP) beads by 50 and 83%, respectively. However, the N-terminal fragment failed to show any effect. Analogous peptides corresponding to residues 75 to 89, 90 to 106, 107 to 120, 121 to 129, and 130 to 150 of PRP1 were synthesized. The fimbriae significantly bound peptide 130-150, immobilized on 96-well plates, and the peptide also inhibited binding of 125I-labeled fimbriae to PRP1-coated HAP beads by almost 100%. Peptides 75-89, 90-106, and 121-129, immobilized on plates, showed considerable ability to bind fimbriae. For further analysis of active sites in residues 130 to 150, synthetic peptides corresponding to residues 130 to 137, 138 to 145, and 146 to 150 were prepared. Peptide 138-145 (GRPQGPPQ) inhibited fimbrial binding to PRP1-coated HAP beads by 97%. This amino acid sequence was shared in the alignment of residues 75 to 89, 90 to 106, and 107 to 120. Six synthetic peptides were prepared by serial deletions of individual residues from the N and C termini of peptide GRPQGPPQ. Peptide PQGPPQ was as inhibitory as peptide GRPQGPPQ. Further deletions of the dipeptide Pro-Gln from the N and C termini of peptide PQGPPQ resulted in significant loss of the inhibitory effect. These results strongly suggest that PQGPPQ is the minimal active segment for binding to P. gingivalis fimbriae and that the moiety of the Pro-Gln dipeptide plays a critical role in expressing binding ability.
Mechanisms involved in Helicobacter pylori-induced interleukin-8 production by a gastric cancer cell line, MKN45
Authors: Aihara M , Tsuchimoto D , Takizawa H , Azuma A , Wakebe H , Ohmoto Y , Imagawa K , Kikuchi M , Mukaida N , Matsushima K
Published: January 2020

Accumulating evidence suggests an important role of interleukin-8 (IL-8) in Helicobacter pylori infection-associated chronic atrophic gastritis and peptic ulcer. We observed in this study that a gastric cancer-derived cell line, MKN45, produced a massive amount of IL-8 upon coculture with live H. pylori but not with killed H. pylori, H. pylori culture supernatants, or live H. pylori separated by a permeable membrane, indicating that IL-8 production requires a direct contact between the cells and live bacteria. Moreover, the tyrosine kinase inhibitor herbimycin but neither a protein kinase C inhibitor (staurosporine) nor a protein kinase A inhibitor (H89) inhibited IL-8 production by MKN45 cells cocultured with live bacteria, suggesting the involvement of a tyrosine kinase(s) in H. pylori-induced IL-8 production. In addition, coculture of H. pylori induced IL-8 mRNA expression in MKN45 cells and an increase in luciferase activity in cells which were transfected with a luciferase expression vector linked with a 5'-flanking region of the IL-8 gene (bp -133 to +44), indicating that the induction of IL-8 production occurred at the transcriptional level. This region contain three cis elements important for induction of IL-8 gene expression: AP-1 (-126 to -120 bp), NF-IL6 (-94 to -81 bp), and NF-kappaB (-80 to -70 bp) binding sites. Mutation of the NF-kappaB binding site abrogated completely the induction of luciferase activity, whereas that of the AP-1 site partially reduced the induction. However, mutation of the NF-IL6 binding site resulted in no decrease in the induction of luciferase activity. Moreover, specific NF-kappaB complexes were detected in the nuclear proteins extracted from MKN45 cells which were infected with H. pylori. Collectively, these results suggest that H. pylori induced the activation of NF-kappaB as well as AP-1, leading to IL-8 gene transcription.
Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities
Authors: Chu L , Ebersole J L , Kurzban G P , Holt S C
Published: January 2020

A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa proteins were purified to homogeneity. Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(cat) = 12 s(-1). Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by beta-mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme, with activity of an alphaC-N and betaC-S lyase (cystathionase) type. Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule.
Prognostic value of anti-Plasmodium falciparum-specific immunoglobulin G3, cytokines, and their soluble receptors in West African patients with severe malaria
Authors: Sarthou J L , Angel G , Aribot G , Rogier C , Dieye A , Toure Balde A , Diatta B , Seignot P , Roussilhon C
Published: January 2020

Forty-one African patients suffering from clinically defined severe malaria were studied in the intensive medical care unit of the main hospital in Dakar, Senegal, West Africa. All of these individuals lived in Greater Dakar, an area of low and seasonal Plasmodium falciparum endemicity. Twenty-seven patients (mean age +/- 1 standard deviation, 19.2 +/- 12.7 years) survived this life-threatening episode, but 14 (30.8 +/- 16.2 years old) died despite initiation of adequate treatment. On the day of admission (day 0) and 3 days later, one to two blood samples (i.e., approximately 10 to 15 ml) were obtained from each subject, and different biological parameters were evaluated in the two groups. Plasma samples were tested for their content in tumor necrosis factor alpha (TNF-alpha), soluble receptors I and II for TNF-alpha (TNF-alpha sRI and TNF-alpha sRII), interleukin-6 (IL-6), IL-6 sR, IL-10, and IL-2 sR. The concentrations of all these cytokines and/or their receptors was significantly elevated in patient plasma samples on day 0, and it rapidly decreased in the group of individuals who survived. By comparison, the mean concentration of the same parameters decreased slowly in the group of patients who died (except for IL-10, which dramatically fell in all patient plasma samples soon after initiation of antimalarial treatment). The TNF-alpha sRI level remained significantly elevated among the patients who died, and the highest levels of soluble TNF-alpha sRI receptor were found among the older patients. Parasite-specific immunoglobulin M (IgM), total IgG, IgG1, IgG2, IgG3, and IgG4 were evaluated by enzyme-linked immunosorbent assay using a crude extract of a local P. falciparum isolate as antigen and human class- and subclass-specific monoclonal antibodies. Parasite-specific IgM, total IgG, and IgG1 were detectable in the plasma samples of most of these African patients, whereas IgG2 and IgG4 mean values were low. The mean level of parasite-specific IgG3 was different (P = 0.024) at day 0, i.e., before initiation of intensive medical care, between the group of the 27 surviving subjects and the group of 14 patients dying of severe malaria. As a consequence, most of the African patients who died had only trace amounts or almost no detectable level of parasite-specific IgG3 at the time of admission. In contrast, the presence of even limited IgG3 activity at day 0 was found to be associated with a significantly increased probability of recovering from severe malaria. Therefore, in our study, both an elevated level of TNF-alpha sRI and absence of IgG3 activity were of bleak prognostic significance, whereas a favorable outcome was usually observed when parasite-specific IgG3 activity was detectable. This finding was strongly suggestive of a prime role for these parasite-specific immunoglobulins in the capacity to help recovery from severe malaria.
The endotoxin of Helicobacter pylori is a modulator of host-dependent gastritis
Authors: Sakagami T , Vella J , Dixon M F , O'Rourke J , Radcliff F , Sutton P , Shimoyama T , Beagley K , Lee A
Published: January 2020

Atrophic gastritis caused by Helicobacter pylori is the precursor lesion in the development of intestinal-type gastric adenocarcinoma. In animal models, atrophic gastritis induced by Helicobacter felis has been shown to be host dependent, developing in some mouse strains and not in others. The lipopolysaccharide (LPS) of H. pylori has been suggested to play a role in the induction of gastritis. The goal of this study was to compare the inflammation induced by long-term infection of the C3H/He and the C3H/HeJ strains of mice with H. felis. C3H/HeJ mice are unresponsive to LPS. Six months after infection, severe atrophic gastritis had developed in the body mucosae of all infected C3H/He mice, with replacement of parietal and chief cells. Atrophy was associated with a loss of the H. felis from the antral mucosa. In contrast, no atrophy was seen in the infected C3H/HeJ non-LPS responder animals, and heavy colonization of the antrum remained. There were no significant differences between both the quantitative and qualitative serum immunoglobulin G (IgG) and salivary IgA levels in both strains of mice. The main difference between the two strains of long-term-infected mice was a lack of macrophage infiltration in the lamina propria. Immunization induced good protective immunity to challenge with viable H. felis. Helicobacter-induced, host-dependent gastritis is likely to be cell mediated. The C3H/He and C3H/HeJ mouse model provides an excellent opportunity to investigate the cellular basis of atrophic gastritis.
Sensitization of human umbilical vein endothelial cells to Shiga toxin: involvement of protein kinase C and NF-kappaB
Authors: Louise C B , Tran M C , Obrig T G
Published: January 2020

Infection of humans with Shiga toxin-producing Escherichia coli O157:H7 and Shigella dysenteriae 1 is strongly associated with vascular endothelial cell damage and the development of hemolytic-uremic syndrome. The cytotoxic effect of Shiga toxins on vascular endothelial cells in vitro is enhanced by prior exposure to bacterial lipopolysaccharide (LPS) or either of the host cytokines tumor necrosis factor alpha (TNF) and interleukin-1beta (IL-1). The purpose of this study was to examine individual signal transduction components involved in the sensitization of human umbilical vein endothelial cells (HUVEC) to Shiga toxin 1. The results demonstrate that class I and II protein kinase C (PKC) isozymes are required for sensitization of HUVEC to Shiga toxin by phorbol myristate acetate (PMA) or LPS but not by TNF or IL-1. Thus, the specific competitive inhibitor of class I/II PKC, 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG), prevented only the action of PMA and LPS on HUVEC. Additional data obtained with ATP binding site inhibitors which affect all PKCs (i.e., classes I, II, and III) suggest that TNF may utilize class III PKC isozymes in the Shiga toxin sensitization of HUVEC. Transcriptional activator NF-kappaB did not appear to be involved in the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA. Thus, the specific serine protease inhibitor L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) did not inhibit the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA despite its ability to inhibit NF-kappaB activation and the induction of the NF-kappaB-dependent tissue factor gene by these agents. Finally, all-trans retinoic acid partially inhibited the sensitization of HUVEC to Shiga toxin, by unknown mechanisms which also appeared to be independent of NF-kappaB activation. These results indicate that PKC plays a role in the sensitization of HUVEC to Shiga toxin in response to some, but not all, sensitizing agents. In contrast, NF-kappaB activation appears not to be involved in the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA.
Local expression of tumor necrosis factor alpha in an experimental model of acute osteomyelitis in rats
Authors: Littlewood-Evans A J , Hattenberger M R , Lüscher C , Pataki A , Zak O , O'Reilly T
Published: January 2020

The inflammatory response associated with Staphylococcus aureus osteomyelitis results in extensive bone damage characterized by apparent increases in bone resorption and formation. These results suggest an increased local release of agents capable of modulating bone remodelling. Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine proposed to play an important role both in normal bone remodelling and in bone diseases; however, its potential role in osteomyelitis is unclear. This study evaluated changes in bone TNF levels during infection, using a rat model of acute osteomyelitis due to S. aureus. Following direct tibial infection, bacterial counts in bone were persistently high (approximately 6 log10 CFU/g of bone over 63 days) and bone weights increased. TNF activity was undetectable in uninfected bone (<0.01 ng/g of bone) but dramatically higher in infected bone (up to 5.2 +/- 3.5 ng/g of bone). Although TNF-alpha mRNA was weakly detected in uninfected bone, osteomyelitis was associated with up to 37-fold increases in expression of both the 1.6- and 2.4-kb transcripts. Both TNF activity and mRNA transcript levels remained elevated throughout the course of infection. TNF-alpha mRNA detected by in situ hybridization was present in osteoblasts as well as in populations of marrow cells and/or inflammatory infiltrate cells. Histopathology of infected bone indicated extensive bone resorption and adjacent areas of formation that were associated with cells expressing TNF-alpha mRNA. These data suggest that the elevated TNF levels induced by experimental infection may be directly related to changes in the histology of bone during osteomyelitis.
Role of vacuolating cytotoxin in gastritis due to Helicobacter pylori in gnotobiotic piglets
Authors: Eaton K A , Cover T L , Tummuru M K , Blaser M J , Krakowka S
Published: January 2020

To investigate the role of the Helicobacter pylori cytotoxin in the pathogenesis of gastritis, gnotobiotic piglets were colonized with either toxigenic H. pylori or a nontoxigenic isogenic mutant. Only piglets given the toxigenic strain developed toxin-neutralizing antibodies (indicating that toxin is expressed in vivo), but there was no difference in bacterial colonization, epithelial vacuolation, or gastritis between the two groups of piglets.
Porphyromonas gingivalis lipopolysaccharide-stimulated bone resorption via CD14 is inhibited by broad-spectrum antibiotics
Authors: Miyata Y , Takeda H , Kitano S , Hanazawa S
Published: January 2020

In the present study, we examined mechanisms of Porphyromonas gingivalis lipopolysaccharide (P-LPS)-stimulated bone resorption via CD14, one of the lipopolysaccharide (LPS) receptors, and also assessed the inhibitory action of several kinds of antibiotics on the LPS-induced stimulation. First, we observed by using mouse embryonic calvarial cells that P-LPS stimulated bone resorption through the action of endogenous interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) via CD14 because (i) P-LPS-stimulated expression of IL-1beta and IL-6 genes in calvarial cells was inhibited by an anti-mouse CD14 antibody, (ii) stimulated bone resorption was markedly inhibited by both IL-1beta and IL-6 antibodies, and (iii) P-LPS-stimulated bone resorption was clearly neutralized by an anti-mouse CD14 antibody. Next, we examined the effects of several kinds of antibiotics on P-LPS-stimulated bone resorption via CD14. Two of them, chloramphenicol and erythromycin, inhibited P-LPS-stimulated bone resorption in a dose-dependent manner. In an additional experiment, we observed that chloramphenicol clearly inhibited P-LPS-stimulated expression of the CD14, IL-1beta, and IL-6 genes in calvarial cells. These results suggest that chloramphenicol might be a useful antibiotic as an anti-inflammatory agent against P-LPS-stimulated periodontal destruction occurring via CD14 in periodontal disease.
Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death
Authors: Kusters J G , Gerrits M M , Van Strijp J A , Vandenbroucke-Grauls C M
Published: January 2020

Helicobacter pylori can transform from its normal helical bacillary morphology to a coccoid morphology. Since this coccoid form cannot be cultured in vitro, it has been speculated that it is a dormant form potentially involved in the transmission of H. pylori and in a patient's relapse after antibiotic therapy. In this study we determined the effects of aging, temperature, aerobiosis, starvation, and antibiotics on the morphologic conversion rate and culturability of H. pylori. Aerobiosis and the addition of a bactericidal antibiotic to the culture medium resulted in the highest conversion rate. During the conversion to coccoid forms, the cultures always lost culturability at the stage where 50% of the organisms were still in bacillary form; this result indicated that culturability and coccoid morphology are two separate but related entities. Independent of the conditions used to induce the conversion into coccoids, the morphological conversion was accompanied by several marked antigenic and ultrastructural changes. Also, both the total amounts and the integrity of RNA and DNA were significantly reduced in coccoid forms. With the potential-sensitive probe diOC(5)-3, a clear loss of membrane potential in coccoid forms was observed. Inhibition of protein or RNA synthesis by the addition of bacteriostatic antibiotics did not prevent the conversion to coccoid forms but resulted in an increased conversion rate. Hence, we conclude that conversion of H. pylori from the bacillary to the coccoid form is a passive process that does not require protein synthesis. Our data suggest that the coccoid form of H. pylori is the morphologic manifestation of bacterial cell death.