bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effects of the streptococcal fibronectin-binding protein, FBP54, on adhesion of group A streptococci to human buccal cells and HEp-2 tissue culture cells
Authors: Courtney H S , Dale J B , Hasty D I
Published: January 2020

We have previously demonstrated that fibronectin mediates streptococcal adhesion to host cells and that streptococci interact primarily with the N-terminal domain of fibronectin. FBP54 is a 54-kDa protein from group A streptococci that binds fibronectin. In this report, we show that the N-terminal domain of fibronectin reacts with FBP54 and preferentially blocks streptococcal adhesion to buccal epithelial cells. FBP54 blocked adhesion to human buccal epithelial cells by 80% in a dose-related fashion. In contrast, FBP54 had little effect on adhesion of group A streptococci to HEp-2 tissue culture cells. The fibronectin-binding domain of FBP54 has been localized to the first 89 N-terminal residues of the protein. Experiments using affinity-purified antibodies to this region indicated that the N terminus of FBP54 is exposed on the surface of streptococci in a manner that can interact with immobilized receptors. Analysis of sera from patients with post-streptococcal glomerulonephritis and acute rheumatic fever indicated that FBP54 is expressed in vivo and is immunogenic in the human host. These data indicate that FBP54 is a streptococcal adhesin that is expressed in the human host and that preferentially mediates adhesion to certain types of human cells.
Coinoculation with Hartmannella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires' disease
Authors: Brieland J , McClain M , Heath L , Chrisp C , Huffnagle G , LeGendre M , Hurley M , Fantone J , Engleberg C
Published: January 2020

The effect of inhaled amoebae on the pathogenesis of Legionnaires' disease was investigated in vivo. A/J mice, which are susceptible to replicative Legionella pneumophila infections, were inoculated intratracheally with L. pneumophila (10(6) bacteria per mouse) or were coinoculated with L. pneumophila (10(6) bacteria per mouse) and Hartmannella vermiformis (10(6) amoebae per mouse). The effect of coinoculation with H. vermiformis on bacterial clearance, histopathology, cellular recruitment into the lung, and intrapulmonary levels of cytokines including gamma interferon and tumor necrosis factor alpha was subsequently assessed. Coinoculation with H. vermiformis significantly enhanced intrapulmonary growth of L. pneumophila in A/J mice. Histopathologic and flow cytometric analysis of lung tissue demonstrated that while A/J mice inoculated with L. pneumophila alone develop multifocal pneumonitis which resolves with minimal mortality, mice coinoculated with H. vermiformis develop diffuse pneumonitis which is associated with diminished intrapulmonary recruitment of lymphocytes and mononuclear phagocytic cells and significant mortality. Furthermore, coinoculation of mice with H. vermiformis resulted in a fourfold enhancement in intrapulmonary levels of gamma interferon and tumor necrosis factor alpha compared with mice infected with L. pneumophila alone. The effect of H. vermiformis on intrapulmonary growth of L. pneumophila in a resistant host (i.e., BALB/c mice) was subsequently evaluated. While BALB/c mice do not develop replicative L. pneumophila infections following inoculation with L. pneumophila alone, there was an eightfold increase in intrapulmonary L. pneumophila in BALB/c mice coinoculated with H. vermiformis. These studies, demonstrating that intrapulmonary amoebae potentiate replicative L. pneumophila lung infection in both a susceptible and a resistant host, have significant implications with regard to the potential role of protozoa in the pathogenesis of pulmonary diseases due to inhaled pathogens and in the design of strategies to prevent and/or control legionellosis.
Epitope mapping of B-cell determinants on the 15-kilodalton lipoprotein of Treponema pallidum (Tpp15) with synthetic peptides
Authors: Baughn R E , Demecs M , Taber L H , Musher D M
Published: January 2020

The antigenicity of the 15-kDa lipoprotein of Treponema pallidum (Tpp15 or TpN15) was comprehensively evaluated in epitope-scanning studies with overlapping deca- and octapeptides and polygonal rabbit and human infant immunoglobulins (Igs) and antisera. This approach enabled us to identify potentially important regions and to determine the optimal dilutions of Igs or antisera for use in further studies. IgM and IgG from both species were capable of recognizing multiple, continuous epitopes. A total of 13 peptides, principally clustered in the central regions of the protein, were recognized by all syphilitic sera and Ig fractions. On the basis of window analyses, frequency profiles, and alanine substitution studies, five heptapeptides were selected for mimetic studies. Two of these five immunodominant, continuous epitopes initially appeared to be species specific; however, antisera elicited against mimetics of all five epitopes were polyspecific, recognizing similar motifs on several other treponemal proteins, including those of avirulent organisms. The only mimetic which yielded positive reactions with infant IgM and syphilitic sera in the absence of cross-reactions with rabbit antisera to avirulent treponemes was the variant of the VMYASSG motif. These findings are relevant to the development of simple, inexpensive assays for the serodiagnosis of active syphilis.
Analysis of epithelial cell stress response during infection by Shigella flexneri
Authors: Mantis N , Prévost M C , Sansonetti P
Published: January 2020

Shigella flexneri-infected macrophage cells undergo an apoptotic-like death as early as one hour after infection (A. Zychlinsky, M. C. Prévost, and P. J. Sansonetti, Nature [London] 358:167-168, 1992). To determine the fate of infected epithelial cells, we characterized the viability, morphology, and several metabolic activities of HeLa cells after treatment with M90T, an invoffve isolate of S. flexneri serotype 5, or BS176, a noninvasive isolate cured of the 220-kb virulence plasmid. Using standard assays, we found that for at least 4 h after infection with M90T, HeLa cells remained viable and did not detach or lyse. The ultrastructural morphology of HeLa cells heavily infected with M90T was free of hallmarks associated with cells undergoing apoptosis. Consistent with the idea that intracellular bacterial growth is metabolically stressful to the host cell, we observed that, compared with BS176 treated-HeLa cells, M90T-treated HeLa cells showed (i) a significant decrease in the total pool size of nucleoside triphosphates, (ii) a reduced ability to incorporate extracellular radiolabeled methionine into the soluble and insoluble cell fractions, and (iii) a stimulation of glucose uptake. However, there was no detectable increase in expression of the stress-inducible hsp70 gene in M90T-infected HeLa cells or activation of the anaerobic metabolic pathway as determined by measuring total lactate levels. These results demonstrate clearly that the fate of S.flexneri-infected cells can vary dramatically between cell types and agree with the hypothesis that the destruction of epithelial cells observed in experimental models of shigellosis is due to the host inflammatory response and probably not bacterial intracellular multiplication per se.
Cytotoxic-T-lymphocyte responses to epitopes of listeriolysin O and p60 following infection with Listeria monocytogenes
Authors: Bouwer H G , Hinrichs D J
Published: January 2020

In order to test the influence of the cell surface density of a specific H2-Kd-presented epitope on the subsequent level of the cytotoxic-T-lymphocyte (CTL) response directed against the epitope, we investigated the CTL response to two secreted products of Listeria monocytogenes from mice immunized with viable L. monocytogenes. We determined the response to the H2-Kd-presented amino acid 91 to 99 (aa91-99) immunodominant peptide of listeriolysin O (LLO) and to the aa217-225 immunodominant peptide of p60. The p60-derived peptide appears at the cell surface as an H2-Kd-complexed peptide at a level sixfold higher than that of LLO aa91-99. CTL frequency analysis of anti-LLO- or anti-p60-specific CTLs from mice immunized with wild-type L. monocytogenes showed that the numbers of immune spleen cell-derived CTLs specific for the two peptides were essentially equivalent. We have also found that Listeria-specific CTL populations lyse target cells pulsed with the p60 aa217-225 peptide with a magnitude of the lytic response markedly less than that for targets pulsed with the LLO aa91-99 peptide. Additionally, immunization with mutants of L. monocytogenes which do not stimulate anti-LLO-specific CTLs does not alter the CTL frequency of anti-p60-specific effector cells, with levels of anti-p60-specific CTLs similar to those seen in mice immunized with wild-type L. monocytogenes. These results suggest that the relative cell surface density of major histocompatibility complex class I-presented L. monocytogenes-derived epitopes is but one of the criteria which determine the magnitude of the cytotoxic effector cell response that develops in antilisterial immunity.
Identification of Legionella pneumophila mutants that have aberrant intracellular fates
Authors: Swanson M S , Isberg R R
Published: January 2020

After uptake by macrophages, Legionella pneumiophila evades phagosome-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum. A collection of bacterial mutants defective for growth in macrophages were isolated, and the intracellular fate of each mutant strain was analyzed by fluorescence microscopy. To measure intracellular replication, bacteria inside macrophages were stained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI). Evasion of the endocytic pathway was quantified by immunofluorescence localization of lp120 [correction of IgpI20] (LAMP-1), a membrane protein of late endosomes and lysosomes, or by measuring colocalization of bacteria with a fluorescent tracer, Texas red-ovalbumin, preloaded into lysosomes. Replication vacuoles were quantified by immunofluorescence localization of BiP, an endoplasmic reticulum protein. By these approaches, four phenotypic groups of mutants were classified. One class formed replication vacuoles less efficiently than the wild type did; another formed replication vacuoles, but replication was abortive; in another class, most phagosomes containing bacteria acquired markers of the endocytic pathway but a minority formed replication vacuoles and the bacteria replicated; finally, a fourth class, the one most defective for intracellular growth, occupied vacuoles that acquired markers of the endocytic pathway.
Biofunctional domains of the Mycoplasma pneumoniae P30 adhesin
Authors: Dallo S F , Lazzell A L , Chavoya A , Reddy S P , Baseman J B
Published: January 2020

The P30 adhesin genes of spontaneous, hemadsorption-negative (HA-) class II Mycoplasma pneumoniae mutants that displayed P30 adhesin-deficient protein profiles were analyzed. One subclass of P30-deficient mutants possessed the entire p3O structural gene without alterations (825 nucleotides, encoding 275 amino acids with a predicted molecular mass of 29,743 Da [S. F. Dallo, A. Chavoya, and J. B. Baseman, Infect. Immun. 58:4163-4165, 1990]). However, the second mutant subclass contained a deletion in p3O resulting in the expression of a 25-kDa peptide (681 nucleotides, encoding 227 amino acids with a calculated molecular mass of 24,823 Da). This P25-truncated peptide lacked 8 of the 13 proline-rich amino acid repeat sequences at the carboxy terminus. Whole-cell radioimmunoprecipitation of M. pneumoniae with antibodies directed against the proline-rich repeat sequences located in the carboxy terminus demonstrated their surface accessibility. In contrast, antibodies generated against N-terminal amino acid sequences upstream of the repeats did not bind to intact mycoplasmas. The amino acid sequence homologies exhibited by the P30 adhesin and eucaryotic structural proteins were corroborated by cross-reactive epitopes shared between the P30 adhesin and fibrinogen, keratin, and myosin. These data reinforce the importance of the P30 protein in cytadherence and virulence and provide a molecular basis for postinfectious autoimmunity associated with M. pneumoniae-mediated pathologies.
The urease enzyme of Helicobacter pylori does not function as an adhesin
Authors: Clyne M , Drumm B
Published: January 2020

Helicobacter pylori urease is essential for colonization of the gastric mucosa irrespective of whether the stomach is acidic or hypochlorhydric. It has therefore been speculated that the enzyme functions as an adhesin. The aim of this study was to compare the adherence of H. pylori N6 with the adherence of an isogenic urease-negative mutant, strain N6(ureB::TnKm), to gastric cells. Strain N6 originated from a patient with gastritis. Strain N6(ureB::TnKm) is specifically modified in the gene which encodes the large subunit of urease, UreB, and hence does not form a UreA-UreB enzyme complex. We have used flow cytometry to assess the adherence of H. pylori to the cells. We have also used phase-contrast microscopy to assess the adherence of the organism to Kato III cells. In the absence of urea both strains bound to Kato III cells and to primary gastric cells. Binding of both strains to the cells occurred rapidly. The presence of urea in the incubation medium decreased the binding of strain N6 to the cells. This was due to a rise in the pH of the incubation medium, which caused loss of viability of the organism. Urea had no effect on the adherence of strain N6(ureB::TnKm). We conclude that the urease of H. pylori does not play a role in the adherence of the organism to gastric cells.
The GafD protein of the G (F17) fimbrial complex confers adhesiveness of Escherichia coli to laminin
Authors: Saarela S , Westerlund-Wikström B , Rhen M , Korhonen T K
Published: January 2020

Escherichia coli IHE11088(pRR-5) expressing the G (F17) fimbria adhered to immobilized laminin as well as to reconstituted basement membranes. No adhesion was seen with the plasmidless strain IHE11088 or with the deletion derivative IHE11088(pHUB110), which expresses the G-fimbrial filament with a defective GafD lectin and lacks N-acetyl-D-glucosamine-specific binding. Adhesion of IHE11088(pRR-5) to laminin and to reconstituted basement membranes was specifically inhibited by N-acetyl-D-glucosamine, and adhesion was abolished after N-glycosidase F treatment of laminin. The results show that the GafD lectin binds to laminin carbohydrate and suggest a novel function for the F17 fimbria in binding to mammalian basement membranes.
Antibody-independent effector mechanisms in resistance to the intestinal nematode parasite Trichuris muris
Authors: Else K J , Grencis R K
Published: January 2020

The development of a strong Th2-type immune response is essential in resistance to the intestinal nematode parasite Trichuris muris. Although the underlying cell-regulatory mechanisms important in protective immunity are well defined, the actual Th2-controlled effector mechanisms culminating in worm expulsion are uncharacterized. Using methodology involving the selective reconstitution of severe combined immunodeficiency mice with highly pure populations of CD4+ T cells from immune BALB/c donors, we show here that antibody is not an essential component in resistance to T. muris. Thus, CD4+ T cells purified from BALB/c donor mice at a time point when Th2 cells are in dominance (days 19 to 21 postinfection) confer resistance to infection on recipient severe combined immunodeficiency mice in the complete absence of an antibody response.
Counterprotective effect of erythrocytes in experimental bacterial peritonitis is due to scavenging of nitric oxide and reactive oxygen intermediates
Authors: Kim Y M , Hong S J , Billiar T R , Simmons R L
Published: January 2020

Erythrocytes (RBC) in the peritoneal cavity significantly increase the lethality of bacterial peritonitis. The lethality is known to be associated with, and perhaps due to, increased bacterial counts in the peritoneal cavity. The mechanism is unknown. In this study, we investigated the hypothesis that RBC scavenge reactive oxygen intermediates (ROI) and nitric oxide (NO), so that the counterprotective effect is due to a loss of the microbiostatic activity of both ROI and NO. To study this effect, rats were subjected to a peritoneal inoculation of live Escherichia coli without RBC (nonlethal dose) or with RBC (lethal dose). The adjuvant effect of RBC was not modified by NG-monomethyl-L-arginine (NMA, an NO synthase inhibitor), superoxide dismutase, catalase, mannitol, or a combination of these agents. Furthermore, the increased number of bacteria in the peritoneal cavity in the presence of RBC was unaffected by these treatments. The administration of NMA with bacteria alone (no RBC) converted a nonlethal model into a lethal one associated with higher intraperitoneal bacterial counts. A similar effect was seen with superoxide dismutase and catalase but not with mannitol. During bacterial peritonitis in the absence of RBC, superoxide and NO formation (determined by the total nitrite plus nitrate formed) was detected in the ascites and inducible NO synthase mRNA expression was present in the peritoneal cells. In the absence of RBC, superoxide was detected and oxidation of dihydrorhodamine to rhodamine was observed, indicating that peroxynitrite was produced. Both were blocked by the inclusion of RBC. Preinjection with a low inoculum of killed bacteria protected the rats from a subsequent lethal peritoneal bacterial challenge; this effect was reversed by scavenging ROI and NO. The protective effect of killed bacterial pretreatment was lost when RBC were placed in the peritoneal cavity. In vitro bactericidal activity of NO- and ROI-generating macrophages was also inhibited by RBC or by inhibiting ROI and NO formation. Taken together, these data are consistent with the hypothesis that RBC can impair bacterial clearance by removing both NO and ROI, suggesting that NO in combination with superoxide may be important to the antimicrobial defenses of the peritoneal cavity.
Influence of agr on fibrinogen binding in Staphylococcus aureus Newman
Authors: Wolz C , McDevitt D , Foster T J , Cheung A L
Published: January 2020

The ability of Staphylococcus aureus to bind fibrinogen is believed to be important in promoting bacterial adherence to both intravascular catheters and host tissues during infection. We investigated the influence of the global regulator agr on the fibrinogen binding capacity and its relationship to the expression of coagulase (encoded by coa) and clumping factor (encoded by clfA) in strain Newman. Strains were obtained by transducing site-specific mutations of clfA, coa, and agr into strain Newman to obtain single, double, and triple mutants of the respective genes. As expected, the clfA mutant bound less soluble 125I-labeled fibrinogen than the corresponding coa mutant in agr+ strains; however, with agr mutant strains, the upregulation in fibrinogen binding capacity correlated mostly with the increased expression and transcription of coagulase as shown by Western (immunoblot) and Northern (RNA) blot analysis. In particular, the coa agr double mutant resulted in a significant reduction in fibrinogen binding compared with that of the agr mutant. The contribution of clfA to fibrinogen binding in agr-negative strains was less than that of coa (32,740 +/- 1,189 versus 18,141 +/- 334 and 38,919 +/- 1,021 cpm for clfA agr, coa agr, and the single agr mutant, respectively). Thus, coagulase is a major binding protein for soluble fibrinogen in the agr-negative background. In in vitro microtiter and catheter adherence assays with solid-phase fibrinogen, clumping factor, but not coagulase, plays a major role in binding to immobilized fibrinogen. coa transcription was negatively modulated by agr and occurred mainly during the exponential growth phase. In contrast, clfA transcription was agr independent and was strongest during the postexponential phase. Although an agr coa clfA triple mutant bound less soluble fibrinogen than the agr coa double mutant (8,504 +/- 831 versus 18,141 +/- 334 cpm), significant residual fibrinogen binding capacity remained in the triple mutant, thus suggesting an additional fibrinogen binding component. By using direct ligand affinity blotting with 125I-fibrinogen, we could identify coagulase and an additional unidentified 52-kDa protein as a fibrinogen binding component in cell extracts. This band was absent in the extract of the coa clfA double mutant.
Pathogenicity island evaluation in Escherichia coli K1 by crossing with laboratory strain K-12
Authors: Bloch C A , Rode C K
Published: January 2020

In bacterial pathogens, strain-specific chromosomal segments often contain genes encoding strain-specific traits, and because these genes often appear to be dedicated to pathogenic interactions with eucaryotic hosts, the segments containing them may be considered so-called pathogenicity islands (G. Blum, M. Ott, A. Lischewski, A. Ritter, H. Imrich, H. Tschape, and J. Hacker, Infect. Immun. 62:606-614, 1994). We evaluated the contribution to pathogenesis of a recently identified strain-specific chromosomal segment from an Escherichia coli K1 mammalian-newborn sepsis strain: transfer of E. coli K-12 DNA sequences near 64 min, by P1 transduction, into K1 strain RS218 resulted in an RS218-K-12 chimera that (i) contained a shortened NotIotl restriction fragment (relative to wild-type RS218) encompassing the 64-min region; (ii) lacked invasiveness in newborn rats; and (iii) grew in vitro, in both rich and minimal laboratory media, indistinguishably from strain RS218. In addition, genomic DNA from the chimera failed to hybridize with sequences of the K1 capsule genes from strain RS218, suggesting that the chromosomal segment near 64 min which was lost contained these sequences and indeed contained K1-specific virulence genes. Transfer of K-12 sequences resulting in deletion of E. coli pathogen-specific chromosomal segments may afford a general method of detecting genes encoding virulence and/or other distinguishing traits.
Biochemical and molecular analysis of phospholipase C and phospholipase D activity in mycobacteria
Authors: Johansen K A , Gill R E , Vasil M L
Published: January 2020

Resurgence of mycobacterial infections in the United States has led to an intense effort to identify potential virulence determinants in the genus Mycobacterium, particularly ones that would be associated with the more virulent species (e.g., Mycobacterium tuberculosis). Thin-layer chromatography (TLC) using radiolabeled phosphatidylcholine and sphingomyelin as substrates indicated that cell extracts of M. tuberculosis contain both phospholipase C (PLC) and phospholipase D (PLD) activities. In contrast, only PLD activity was detected in cell extracts of M. smegmatis. Neither activity was detected in cell-free culture supernatants from these organisms. We and others recently identified two open reading frames in M. tuberculosis with the potential to encode proteins which are highly homologous to the nonhemolytic (PlcN) and hemolytic (PlcH) phospholipase C enzymes of Pseudomonas aeruginosa. In contrast to the plc genes in P. aeruginosa, which are considerably distal to each other (min 34 and 64 on the chromosome), the mycobacterial genes, designated mpcA and mpcB, are tandemly arranged in the same relative orientation and separated by only 191 bp. Both the mpcA and the mpcB genes were individually cloned in M. smegmatis, and PLC activity was expressed from each gene in this organism. Hybridization experiments using the mpcA and the mpcB genes as probes under conditions of moderate stringency identified sequences homologous to these genes in M. bovis, M. bovis BCG, and M. marinum but not in several other Mycobacterium species, including M. smegmatis, M. avium, and M. intracellulare. TLC analysis using radiolabeled substrates indicated that M. bovis and M. marinum cell extracts contain PLC and PLD activities, but only PLD activity was detected in M. bovis BCG cell extracts. Sphingomyelinase activity was also detected in whole-cell extracts of M. tuberculosis, M. marinum, M. bovis, and M. bovis BCG, but this activity was not detected in extracts of M. smegmatis. Sphingomyelinase activity was detected in cell extracts from M. smegmatis harboring either recombinant mpcA or mpcB. These data indicate that PLC and sphingomyelinase activities are associated with the most virulent mycobacterial species, while PLD activity was detected in both virulent and saprophytic strains.
Role of nitric oxide in host defense against an extracellular, metazoan parasite, Brugia malayi
Authors: Rajan T V , Porte P , Yates J A , Keefer L , Shultz L D
Published: January 2020

The mechanisms by which mammalian hosts eliminate microparasites such as bacteria and viruses are well established. In viral infections, these mechanisms include the interferons, neutralizing and opsonizing antibodies, and cytotoxic T lymphocytes. In bacterial infections, polymorphonuclear leukocytes and macrophages, often facilitated by opsonizing antibodies, ingest the infectious agent and mediate host defense. In addition, complement, in the presence of specific antibodies directed against surface antigens, can lyse certain bacterial pathogens. In contrast, our understanding of the host defenses against metazoan, extracellular parasites is less well grounded. We obtained data by two different approaches to document the role of nitric oxide (NO) as a mediator of host defense against a human nematode parasite. First, treatment of immunocompetent, nonpermissive mice with an inhibitor of NO synthase abrogated resistance to Brugia malayi, one of the causative agents of human lymphatic filariasis. Second, treatment of permissive, immunodeficient mice with a compound that releases NO conferred resistance to infection. These data reinforce studies by James and her coworkers (I. P. Oswald, T. A. Wynn, A. Sher, and S. L. James, Comp. Biochem. Physiol. Pharmacol. Toxicol. Endocrinol. 108:11-18, 1994) on the role of NO in defense against trematode parasites and of Kanazawa et al. (T. Kanazawa, H. Asahi, H. Hata; K. Machida, N. Kagei, and M. J. Stadecker, Parasite Immunol. 15: 619-623, 1993) on cestode parasites.
Distinct characteristics of initiation of the classical and alternative complement pathways by Candida albicans
Authors: Kozel T R , Weinhold L C , Lupan D M
Published: January 2020

Candida albicans is a potent activator of the complement system. The objective of this study was to characterize factors that influence the kinetics for activation of C3 and binding of C3 fragments to C. albicans. Factors that were examined included the surface properties of the yeast and contributions of the classical and alternative complement pathways. The results showed that incubation of hydrophobic, hydrophilic, or germinating yeast cells in normal human serum (NHS) containing radiolabeled C3 led to immediate accumulation of C3 on all three cell types, although the rate of accumulation of C3 on germinating cells was lower. An examination of the sites for early C3 binding showed that classical pathway initiation led to immediate, synchronous binding over the entire cell surface. A blockade of the classical pathway by absorption of putative classical pathway initiators or by chelation of calcium limited activation to the alternative pathway. Binding of C3 solely via the alternative pathway was characterized by a significant lag in the initial binding kinetics. In the absence of classical pathway initiation, the early cellular sites for C3 binding appeared as random, asynchronous foci of C3 that appeared to expand with time. The factor(s) mediating rapid deposition of C3 that was characteristic of the classical pathway initiation was reciprocally cross-absorbed by hydrophilic and hydrophobic C. albicans but was not removed by absorption of NHS with Saccharomyces cerevisiae, encapsulated Cryptococcus neoformans, or nonencapsulated C. neoformans. Delayed binding of C3 produced by absorption of serum was largely reversed by addition to the absorbed serum of immunoglobulin G isolated from NHS, indicating a significant role for a naturally occurring anti-C. albicans immunoglobulin C. in classical pathway initiation.
Interactions between Campylobacter jejuni and lipids
Authors: Szymanski C M , Armstrong G D
Published: January 2020

We previously showed that motility plays several key roles in Campylobacter jejuni pathogenesis, including increasing the efficiency of C. jejuni attachment to host epithelial cells. To further characterize C. jejuni attachment, we first examined the role of carbohydrates. Experiments with Chinese hamster ovary (CHO) cell mutants with defined defects in complex carbohydrate biosynthesis revealed that oligosaccharide sequences probably play a subordinate role in C. jejuni attachment to eukaryotic cells. Simple sugars such as mannose, fucose, glucose, N-acetylglucosamine, maltose, and galactose also did not significantly alter the binding of C. jejuni to CHO cells. Thin-layer chromatography overlay analysis with lipids extracted from CHO cells suggested that C. jejuni binds to lipids. Lipid binding was further investigated using a receptor-based enzyme-linked immunosorbent assay. Hydrophobic interactions were determined to play a minor role in binding, since tetramethylurea, a strong inhibitor of hydrophobic interactions, did not significantly decrease binding between C. jejuni and lipids. The interaction was dissected further by comparing the binding of C. jejuni to lipids and their derivatives. The results showed that binding was greatest to the entire lipid structure and decreased in affinity when portions of the lipid were removed. Thin-layer chromatography overlay analysis showed that lipids with unsaturated fatty acids were bound with the highest affinity. Our results suggest that C. jejuni may interact with lipids in host cell membranes. However, lipids only partially inhibited C. jejuni binding to CHO cells, suggesting that multiple interactions occur between the bacteria and host cells.
The Escherichia coli K-12 gntP gene allows E. coli F-18 to occupy a distinct nutritional niche in the streptomycin-treated mouse large intestine
Authors: Sweeney N J , Klemm P , McCormick B A , Moller-Nielsen E , Utley M , Schembri M A , Laux D C , Cohen P S
Published: January 2020

Escherichia coli F-18 is a human fecal isolate that makes type 1 fimbriae, encoded by the fim gene cluster, and is an excellent colonizer of the streptomycin-treated mouse intestine. E. coli F-18 fimA::tet, lacking type 1 fimbriae, was constructed by bacteriophage P1 transduction of the fim region of the E. coli K-12 strain ORN151, containing the tetracycline resistance gene from Tn10 inserted in the fimA gene, into E. coli F-18. E. coli F-18 fimA::tet was found to occupy a distinct niche in the streptomycin-treated mouse intestine when fed in small numbers (10(4) CFU) to mice, along with large numbers (10(10) CFU) of E. coli F-18, as defined by the ability of the E. coli F-18 fimA::tet strain to grow and colonize only 1 order of magnitude below E. coli F-18. The same effect was observed when mice already colonized with E. coli F-18 were fed small numbers of E. coli F-18 fimA::tet. Experiments which show that the E. coli K-12 gene responsible for this effect is not fim::tet but gntP, which maps immediately downstream of the fim gene cluster, are presented. gntP encodes a high-affinity gluconate permease, suggesting that the distinct niche in the mouse large intestine is defined by the presence of gluconate. The data presented here support the idea that small numbers of an ingested microorganism can colonize the intestine as long as it can utilize an available nutrient better than any of the other resident species can.
Decreased interleukin-4 and increased gamma interferon production by peripheral blood mononuclear cells of patients with Lyme borreliosis
Authors: Oksi J , Savolainen J , Pène J , Bòusquet J , Laippala P , Viljanen M K
Published: January 2020

Spontaneous and Borrelia burgdorferi-stimulated proliferation of peripheral blood mononuclear cells (PBMCs) and their interleukin-4 (IL-4), gamma interferon (IFN-gamma), and NO production were measured in 36 patients with second- or third-stage Lyme borreliosis (LB) and 11 control subjects. Spontaneous proliferation of PBMCs was significantly higher (P = 0.0003) in the LB patients than in the control subjects. Spontaneous production of IL-4 was significantly lower in patients than in control subjects (P = 0.0007), but spontaneous production of IFN-gamma was slightly higher in patients. The proliferative response of PBMCs to stimulation with B. burgdorferi was significantly higher (P = 0.01) in patients. The B. burgdorferi-induced production of IFN-gamma (P = 0.002) was also significantly higher in patients. The spontaneous and B. burgdorferi-induced production of NO showed no significant difference between patients and control subjects. These findings indicate that the activation of PBMCs in patients with late LB is enhanced in vivo. Furthermore, the production of IL-4 is effectively suppressed spontaneously, whereas the production of IFN-gamma by PBMCs is slightly increased spontaneously and significantly enhanced during stimulation with B. burgdorferi in vitro. The "spontaneous" or disease-induced alterations in cytokine levels of patients, in this case suppression of a Th2-type cytokine production and activation of a Th1-type cytokine production, may contribute to the pathogenesis of LB.
Characterization of a P1-deficient strain of Streptococcus mutans that expresses the SpaA protein of Streptococcus sobrinus
Authors: Kuykindoll R J , Holt R G
Published: January 2020

The Streptococcus sobrinus SpaA protein and the Streptococcus mutans P1 protein share 66% sequence homology at the amino acid level. To determine if the SpaA protein can be expressed in S. mutans and functionally replace the P1 protein, the spaA gene of S. sobrinus 6715 was isolated from plasmid pX1303 and inserted into the Escherichia coli-Streptococcus shuttle vector pVA838. The resulting plasmid pX1600 was transformed into the P1-deficient strain S. mutans 834 that has defects in saliva-mediated aggregation and in the ability to adhere to saliva-coated hydroxyapatite surfaces. Western blot (immunoblot) analysis of cellular protein fractions of S. mutans 834 (pX1600) detected in mutanolysin-solubilized cell walls a major protein of 210 kDa with an electrophoretic mobility similar to that of S. sobrinus SpaA protein and a minor 210-kDa protein and a major 64-kDa protein in the extracellular protein fraction. Analysis of virulence traits showed that expression of SpaA protein by S. mutans 834(pX1600) cells had restored the ability of the S. mutans 834 cells to aggregate in the presence of saliva or salivary agglutinin but not to adhere to saliva-coated hydroxyapatite. This cell aggregation was inhibited specifically by antisera to S. sobrinus SpaA protein. These results indicate that SpaA plays a role in the virulence of S. sobrinus by specifically interacting with fluid-phase salivary agglutinin to mediate cell aggregation.
Identification of an outer membrane protein involved in utilization of hemoglobin-haptoglobin complexes by nontypeable Haemophilus influenzae
Authors: Maciver I , Latimer J L , Liem H H , Muller-Eberhard U , Hrkal Z , Hansen E J
Published: January 2020

A recombinant plasmid containing a 6.5-kb fragment of nontypeable Haemophilus influenzae (NTHI) chromosomal DNA was shown to confer a hemoglobin-haptoglobin-binding phenotype on Escherichia coli. Use of a mini-Tn10kan transposon for random insertion mutagenesis of this recombinant plasmid allowed localization of the NTHI DNA responsible for this hemoglobin-haptoglobin-binding phenotype to a 3.5-kb PstI-XhoI fragment within the 6.5-kb NTHI DNA insert. When this mutagenized NTHI DNA fragment was used to transform the wild-type NTHI strain, the resultant kanamycin-resistant mutant exhibited significantly decreased abilities to bind hemoglobin-haptoglobin and utilize it as a source of heme for aerobic growth in vitro. This mutant also lacked expression of a 115-kDa outer membrane protein that was present in the wild-type parent strain. Transformation of this mutant with wild-type NTHI chromosomal DNA restored the abilities to bind and utilize hemoglobin-haptoglobin and to express the 115-kDa outer membrane protein. Nucleotide sequence analysis of the relevant NTHI DNA revealed the presence of a gene, designated hhuA, that encoded a predicted 117,145-Da protein. The HhuA protein exhibited features typical of a TonB-dependent outer membrane receptor and had significant identity with the hemoglobin receptors of both Haemophilus ducreyi and Neisseria meningitidis.
Gastric invasion by Trypanosoma cruzi and induction of protective mucosal immune responses
Authors: Hoft D F , Farrar P L , Kratz-Owens K , Shaffer D
Published: January 2020

Trypanosoma cruzi is an intracellular parasite transmitted from a reduviid insect vector to humans by exposure of mucosal surfaces to infected insect excreta. We have used an oral challenge murine model that mimics vector-borne transmission to study T. cruzi mucosal infection. Although gastric secretions have microbicidal activity against most infectious pathogens, we demonstrate that T. cruzi can invade and replicate in the gastric mucosal epithelium. In addition, gastric mucosal invasion appears to be the unique portal of entry for systemic T. cruzi infection after oral challenge. The mucosal immune responses stimulated by T. cruzi gastric infection are protective against a secondary mucosal parasite challenge. This protective mucosal immunity is associated with increased numbers of lymphocytes that secrete parasite-specific immunoglobulin A. Our results document the first example of systemic microbial invasion through gastric mucosa and suggest the feasibility of a mucosal vaccine designed to prevent infection with this important human pathogen.
Activation of human monocytic cells by Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides proceeds via a pathway distinct from that of lipopolysaccharide but involves the transcriptional activator NF-kappa B
Authors: Norgard M V , Arndt L L , Akins D R , Curetty L L , Harrich D A , Radolf J D
Published: January 2020

There is increasing evidence that lipoproteins of Treponema pallidum and Borrelia burgdorferi are key inflammatory mediators during syphilis and Lyme disease. A principal objective of the present study was to identify more precisely similarities and divergences among lipopolysaccharide (LPS)- and lipoprotein-lipopeptide-induced immune cell signaling events. Like LPS, purified native B. burgdorferi OspA and synthetic analogs of OspA, OspB, and two T. pallidum lipoproteins (Tpp47 and Tpp17) all induced NF-kappa B translocation in THP-1 human monocytoid cells. Acylation of OspA and the synthetic peptides was requisite for cell activation. Polymyxin B abrogated only the response to LPS. By using 70Z/3-derived pre-B-cell lines either lacking or expressing human CD14 (the LPS receptor), it was observed that expression of human CD14 imparted responsiveness to LPS but not to OspA or spirochetal lipopeptides (assessed by induction of NF-kappa B and expression of surface immunoglobulin M). Finally, the biological relevance of the observation that T. pallidum lipoproteins-lipopeptides induce both NF-kappa B and cytokine production in monocytes was supported by the ability of the synthetic analogs to promote human immunodeficiency virus replication in chronically infected U1 monocytoid cells; these observations also suggest a potential mechanism whereby a syphilitic chancre can serve as a cofactor for human immunodeficiency virus transmission. The combined data lend additional support to the proposal that spirochetal lipoproteins and LPS initiate monocyte activation via different cell surface events but that the signaling pathways ultimately converge to produce qualitatively similar cellular responses.
Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo
Authors: Sánchez J , Buendía A J , Salinas J , Bernabé A , Rodolakis A , Stewart I J
Published: January 2020

Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells.
Analysis of T-cell responses in cattle immunized against heartwater by vaccination with killed elementary bodies of Cowdria ruminantium
Authors: Totté P , McKeever D , Martinez D , Bensaid A
Published: January 2020

Cattle were successfully immunized against heartwater with a lysate of Cowdria ruminantium formulated in Freund's adjuvant. Vaccinated animals proved fully resistant to virulent challenge 3 and 10 months after vaccination. For the first time a helper T lymphocyte response to Cowdria antigens was observed and characterized. Cowdria-specific T-cell lines generated from vaccinated animals by in vitro restimulation with Cowdria lysates are 95 to 100% CD4+, are MHC class II restricted, and produce gamma interferon. They proliferate in response to autologous monocytes infected with live Cowdria but not in response to uninfected monocytes. These T-cell lines will facilitate the search for Cowdria antigens that are immunogenic for T cells and will therefore be of relevance in the development of a subunit vaccine against the disease.
Role of tumor necrosis factor alpha in pathogenesis of pneumococcal pneumonia in mice
Authors: Takashima K , Tateda K , Matsumoto T , Iizawa Y , Nakao M , Yamaguchi K
Published: January 2020

The production and role of tumor necrosis factor alpha (TNF-alpha) in pneumococcal pneumonia were investigated in a mouse pneumonia model. When approximately 10(6) CFU of Streptococcus pneumoniae TUM19 were used to inoculate CBA/J mice intranasally, TNF-alpha levels in the lungs and serum began to increase from 1 and 3 days after infection, respectively, concomitantly with the increase in bacterial counts in the lungs. Anti-TNF-alpha antibody accelerated bacterial proliferation in the blood and the death of the mice. Although serum levels of immunoglobulin G antibody against the infecting bacteria were not affected by the anti-TNF-alpha antibody treatment, neutrophil counts in the blood were decreased by the treatment. These results suggest that TNF-alpha produced in the course of pneumococcal pneumonia prevents bacteremia by increasing the number of neutrophils in the blood.
Successful cultivation of a potentially pathogenic coccoid organism with trophism for gastric mucin
Authors: Lee S G , Kim C , Ha Y C
Published: January 2020

We have devised a procedure that permits the cultivation of a gram-positive coccoid species from biopsy material obtained from the antrum of the stomachs of patients with gastric disorders. Antibodies directed against surface proteins obtained from the coccoid isolates were detected in all patients with gastric disorders examined in this study, including both Helicobacter pylori-infected and H. pylori-uninfected patients. Several of these isolates, including a prototype designated strain SL100, have been characterized in some detail. Strain SL100 exhibits urease and exceptionally high catalase activities and assumes a variety of spherical morphologies as detected by electron microscopy. This isolate expresses an adhesin that binds to gastric mucin. The adhesin activity was detected only after the isolate was exposed to an acidic pH, suggesting that in the natural process of infection, the low pH of the stomach unmasks a cell surface component with adhesin activity. Strain SL100 grows best under a microaerophilic conditions (10% CO2, 5% O2, 85% N2), but it also grows quite well under aerobic conditions. Thus, this organism would be expected to proliferate outside of the human host as well as in the gastric mucosa. Oral infection of newborn piglets resulted in colonization of the gastric antrum and growth retardation. Preliminary taxonomic classification indicates similarity to the Staphylococcus DNA homology groups containing S. cohnii and S. xylosus. One of us (C.K.) apparently became infected with this organism as indicated by gastric symptoms and the subsequent presence of strain-specific antisera not present in other workers in the laboratory.
Induction of tumor necrosis factor production from monocytes stimulated with mannuronic acid polymers and involvement of lipopolysaccharide-binding protein, CD14, and bactericidal/permeability-increasing factor
Authors: Jahr T G , Ryan L , Sundan A , Lichenstein H S , Skjåk-Braek G , Espevik T
Published: January 2020

Well-defined polysaccharides, such as beta1-4-linked D-mannuronic acid (poly[M]) derived from Pseudomonas aeruginosa, induce monocytes to produce tumor necrosis factor (TNF) through a pathway involving membrane CD14. In this study we have investigated the effects of soluble CD14 (sCD14), lipopolysaccharide-binding protein (LBP), and bactericidal/permeability-increasing factor (BPI) on poly(M) binding to monocytes and induction of TNF production. We show that LBP increased the TNF production from monocytes stimulated with poly(M). Addition of sCD14 alone had only minor effects, but when it was added together with LBP, a rise in TNF production was seen. BPI was found to inhibit TNF production from monocytes stimulated with poly(M) in the presence of LBP, LBP-sCD14, or 10% human serum. Binding studies showed that poly(M) bound to LBP- and BPI-coated immunowells, while no significant binding of poly(M) to sCD14-coated wells in the absence of serum was observed. Binding of poly(M) to monocytes was also examined by flow cytometry, and it was shown that the addition of LBP or 10% human serum clearly increased the binding of poly(M) to monocytes. BPI inhibited the binding of poly(M) to monocytes in the presence of LBP, LBP-sCD14, or 10% human serum. Our data demonstrate a role for LBP, LBP-sCD14, and BPI in modulating TNF responses of defined polysaccharides.
Role of reactive nitrogen and oxygen intermediates in gamma interferon-stimulated murine macrophage bactericidal activity against Burkholderia pseudomallei
Authors: Miyagi K , Kawakami K , Saito A
Published: January 2020

We examined the contributions of reactive nitrogen and oxygen intermediates (RNI and ROI, respectively) in macrophage bactericidal activity against Burkholderia pseudomallei, the causative agent of melioidosis, in order to understand host defense mechanisms against infection caused by this bacterium. The bacteria multiplied in unstimulated murine macrophage cell line J774.1. However, a strong dose-dependent inhibition of intracellular bacterial growth was observed when gamma interferon (IFN-gamma)-activated macrophages were used. The induction of bactericidal activity correlated well with the production of nitric oxide (NO) by IFN-gamma-activated macrophages and was markedly suppressed by N(G)-monomethyl L-arginine (L-NMMA), a competitive inhibitor of NO synthesis. Superoxide dismutase (SOD) and catalase significantly inhibited macrophage bactericidal activity, and the combined addition of L-NMMA, SOD, and catalase resulted in the complete inhibition of IFN-gamma-stimulated activity. The bacteria were susceptible to the killing effects of chemically generated NO and superoxide anion in a macrophage-free system. Our results indicate that IFN-gamma-induced macrophage bactericidal activity against B. pseudomallei is mediated to a large extent by RNI killing mechanisms and to a lesser extent by ROI-dependent mechanisms.