bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of extracellular phospholipase B, lysophospholipase, and acyltransferase produced by Cryptococcus neoformans
Authors: Chen S C , Wright L C , Santangelo R T , Muller M , Moran V R , Kuchel P W , Sorrell T C
Published: January 2020

We recently identified phospholipase activity as a potential virulence factor of Cryptococcus neoformans. We have now defined the nature of the phospholipase activity produced by a clinical isolate of C. neoformans var. neoformans, under native conditions, by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy and thin-layer chromatography (TLC) of radiolabelled substrates. Glycerophosphocholine was identified by NMR spectroscopy as the sole phospholipid degradation product of the reaction between substrate phosphatidylcholine (PC) and cryptococcal culture supernatants indicating the presence of phospholipase B (PLB). No lysophosphatidylcholine (lyso-PC) or products indicative of phospholipase C, phospholipase D, or other lipase activity were identified. Use of PC and lyso-PC containing radiolabelled acyl chains and separation of products by TLC confirmed the PLB and lysophospholipase (LPL) activities. Lysophospholipase transacylase (LPTA) activity was identified by the formation of radioactive PC from lyso-PC. Extracellular enzyme production was maximal after 6 to 10 h in fresh medium. Assay conditions were optimized for pH, linearity with time, enzyme concentration, and saturation by substrates to allow comparison with phospholipases from other organisms. LPL activity was 10- to 20-fold greater than PLB activity, with mean (+/- standard deviation) specific activities of 34.9 +/- 7.9 and 3.18 +/- 0.2 micromol of substrate hydrolyzed per min per mg of protein, respectively. The response of PLB to increasing substrate concentrations was bimodal, whereas inhibition of LPL and LPTA activities occurred at concentrations of substrate lyso-PC greater than 200 microM. Enzyme activities were stable at acid pH (3.8), with pH optima of 3.5 to 4.5. Activities were unchanged in the presence of exogenous serine protease inhibitors, divalent cations, and EDTA. We conclude that C. neoformans produces highly active extracellular PLB, LPL, and LPTA under native conditions.
Pathogenesis of Acanthamoeba keratitis: carbohydrate-mediated host-parasite interactions
Authors: Yang Z , Cao Z , Panjwani N
Published: January 2020

Acanthamoeba keratitis is a sight-threatening corneal infection. In a recent study, the saccharide mannose has been shown to inhibit the binding of Acanthamoeba organisms to the epithelium of the cornea (L. D. Morton, G. L. McLaughlin, and H. E. Whiteley, Infect. Immun. 59:3819-3822, 1991). In an attempt to determine the molecular mechanism by which acanthamoebae adhere to the surface of the cornea, the present study was designed to determine whether Acanthamoeba castellanii derived from an infected human cornea (i) binds to mannose-containing glycoproteins (mannose-GPs) of corneal epithelium and (ii) expresses one or more mannose-binding proteins. Mannose-GPs of primary cell cultures of rabbit corneal epithelium were isolated by using three different agarose-conjugated, mannose-specific lectins. By electrophoresis blot-overlay assays, 35S-labeled acanthamoebae were shown to bind to mannose-GPs of corneal epithelium and to a neoglycoprotein, mannose-bovine serum albumin (mannose-BSA). 35S-labeled acanthamoebae also bound to microtiter wells coated with mannose-BSA in a concentration-dependent manner. The binding of amoebae to mannose-GPs was blocked by free methyl-alpha-D-mannopyranoside. The parasites did not bind to galactose-BSA or to many other proteins lacking mannose residues. A membrane-associated mannose-binding protein (136 kDa) of A. castellanii was isolated by affinity chromatography of detergent extracts of unlabeled parasites and of cell surface biotin-labeled parasites on a p-aminophenyl alpha-D-mannopyranoside-agarose column. The affinity-purified protein of the amoeba was shown to bind specifically to mannose-BSA. In summary, a mannose-binding protein is present on the surface membranes of Acanthamoeba, and corneal epithelial cells express Acanthamoeba-reactive GPs. One of the mechanisms of Acanthamoeba adhesion to the corneal surface may involve interactions between the mannose-binding protein of Acanthamoeba and mannose-GPs on the surface of corneal epithelium.
Helicobacter pylori lipopolysaccharide can activate 70Z/3 cells via CD14
Authors: Kirkland T , Viriyakosol S , Perez-Perez G I , Blaser M J
Published: January 2020

Helicobacter pylori persistently colonizes the human gastrointestinal tract and is associated with chronic gastritis and, in some cases, peptic ulcer disease or gastric neoplasms. One factor in the persistence of this organism may be its inability to elicit a strong inflammatory response. Lipopolysaccharide (LPS) is a proinflammatory substance found in the cell walls of all gram-negative bacteria. H. pylori LPS has been found by several different measures to be less active than LPS from Enterobacteriaceae. This study addresses the role of CD14 and LPS-binding protein in the cellular response to H. pylori LPS. We report that H. pylori LPS activates mammalian cells expressing CD14 at much lower LPS concentrations than those for control cells not expressing CD14. The maximal activation of CD14-70Z/3 cells by H. pylori LPS also requires LPS-binding protein. H. pylori LPS at concentrations as high as 30 microg/ml does not elicit an interleukin-8 (IL-8) response from the epithelial cell line SW620 in the presence of CD14; 10 ng of Escherichia coli LPS per ml elicits a maximal IL-8 response. Furthermore, in contrast to some other types of LPS with little activity, H. pylori LPS does not inhibit the CD14-70Z/3 cell response to E. coli LPS. From these studies, we conclude that H. pylori LPS, though much less active than E. coli LPS, stimulates cells via CD14.
The central variable V2 region of the CS31A major subunit is involved in the receptor-binding domain
Authors: Di Martino P , Girardeau J P , Der Vartanian M , Joly B , Darfeuille-Michaud A
Published: January 2020

CS31A is a K88-related capsule-like surface protein that mediates Escherichia coli and Klebsiella pneumoniae adhesion to the human Caco-2 and Intestine-407 cell lines. In this study, we demonstrate that ClpG, the major subunit of CS31A, contains the adhesive domain of the polymerized structure. We mapped this domain within the ClpG protein by performing adhesion inhibition experiments with Intestine-407 cells with nine synthetic peptides (CLP1 to CLP9) covering the dominant antigenic regions of ClpG and with the corresponding rabbit antipeptide antibodies. The peptides CLP1 (amino acid positions in parentheses) (5-18), CLP2 (44-56), CLP3 (82-96), CLP7 (174-190), CLP8 (185-199), and CLP9 (235-249) and corresponding antipeptide antibodies targeting parts of the N- and C-terminal regions of ClpG had no effect on the adhesion of the TCFF15 recombinant strain expressing CS31A. Only the CLP5 (132-146) peptide, corresponding to the central part of the protein, and relevant antibodies inhibited bacterial adhesion to intestinal epithelial cells. Anti-CLP4 (97-109) and anti-CLP6 (148-162) antibodies targeting regions surrounding the CLP5 sequence also inhibited bacterial adhesion. Site-directed mutagenesis experiments inducing changes in the amino acid sequence of the ClpG protein corresponding to the CLP5 peptide resulted in the expression of nonadhesive CS31A antigen. These findings indicate that the ClpG receptor-binding domain is located in the central variable V2 region.
Induction of biologically active interleukin-8 from lung epithelial cells by Burkholderia (Pseudomonas) cepacia products
Authors: Palfreyman R W , Watson M L , Eden C , Smith A W
Published: January 2020

The frequency of isolation of Burkholderia cepacia from the sputum of cystic fibrosis (CF) patients is increasing. Using the human A549 lung epithelial cell line, we have investigated the ability of B. cepacia exoproducts to stimulate interleukin-8 (IL-8) release. Cell-free supernatants from a panel of CF clinical, non-CF clinical, and nonclinical B. cepacia isolates were found to stimulate IL-8 release, with levels ranging from 11.8 +/- 2.8 to 80.0 +/- 3.5 ng/ml. A similar pattern was seen at the level of the IL-8 mRNA. The bioactivity of the IL-8 was confirmed by examining its effect on the intracellular free calcium in neutrophils and inhibition by a neutralizing anti-IL-8 antibody. B. cepacia lipopolysaccharide, which was able to stimulate IL-8 release from monocytes, did not release IL-8 from the A549 cells. Furthermore, the stimulating ability of the bacterial cell-free supernatant was not diminished by polymyxin B, was markedly reduced by boiling, and appeared unrelated to N-acylhomoserine lactones. The ability of B. cepacia to elicit IL-8 release from epithelial cells may be important in the pathology of CF.
Protegrin structure and activity against Neisseria gonorrhoeae
Authors: Qu X D , Harwig S S , Shafer W M , Lehrer R I
Published: January 2020

Protegrin 1 (PG-1) is a broad-spectrum antimicrobial peptide that contains 18 amino acid residues (RG GRLCYCRRRFCVCVGR) and has two intramolecular cystine disulfide bonds. To determine the minimal structure responsible for protegrin-mediated activity against Neisseria gonorrhoeae, we synthesized 15 protegrin variants and tested them against two well-characterized gonococcal strains. The MICs of PG-1 were 0.61 microM (1.31 microg/ml) for the serum-sensitive strain F 62 and 0.98 microM (2.11 microg/ml) for the serum-resistant strain FA 19. Six amino acid residues (Arg1, Gly2, Gly3, Arg4, Gly17, and Arg18) and either disulfide bond could be deleted from PG-1 without impairing its potency against strain F 62. In contrast, only Gly17 and Arg18 could be removed without decreasing its activity against FA 19. Protegrin congener 64a (PC-64a; LTYCRRRFCVTV), a variant of PG-1 with 12 amino acid residues and one disulfide bond, displayed MICs of 0.45 microM (0.68 microg/ml) for strain F 62 and 1.37 microM (2.07 microg/ml) for strain FA 19, which approximated those of intact PG-1. Serum-sensitive sac-1+ and sac-3+ transformants of N. gonorrhoeae FA 19 and two FA 19 derivatives with truncated lipooligosaccharide structures were more susceptible to PG-1 and variants with altered disulfide structures. These data suggest that structurally simpler protegrin variants, such as PC-64a, could be used as topical microbicides for N. gonorrhoeae. They also suggest that the cystine-stabilized antiparallel beta-sheet formed by PG-1 residues 5 to 16 is principally responsible for its activity against gonococci.
Selection of Opa+ Neisseria gonorrhoeae by limited availability of normal human serum
Authors: Bos M P , Hogan D , Belland R J
Published: January 2020

Experimental infections of human male volunteers with Neisseria gonorrhoeae have provided valuable insights into the early stages of gonorrheal disease. Bacterial variants expressing outer membrane opacity (Opa) proteins appear to be selected from the inoculum during a period in which total recoverable numbers of bacteria decrease rapidly. This apparent survival advantage occurs simultaneously with the onset of an inflammatory response, characterized by local production of interleukin 6 (IL-6) and IL-8 and the appearance of leukocytes in urine. Since the inflammatory response may also result in the presence of serum factors on the mucosal surface, we investigated the possibility that killing in normal human serum (NHS) leads to the selection of Opa+ variants. We therefore studied killing of separate populations and mixtures of Opa- and Opa+ N. gonorrhoeae MS11mk in NHS. Expression of an Opa protein conferred a survival advantage upon the organism; i.e., the Opa+ variants were more serum resistant than their isogenic Opa- counterparts, resulting in a selection for Opa+ phenotypes when a mixture of Opa+ and Opa- gonococci (GC) was exposed to submaximal doses of NHS. This selection was observed in three different lipooligosaccharide (LOS) backgrounds, indicating that it was not due to a difference in LOS expression between Opa- and Opa+ phenotypes. Incubation in NHS of sialylated GC resulted in a similar selection for Opa+ variants. The presence of normal human urine during the serum killing assay had no effect on the selection phenomenon but drastically depleted NHS of bactericidal activity, which was found to be at least partly due to complement inhibition. The results suggest that serum killing may contribute to the transition from Opa- to Opa+ phenotypes during the early stages of infection of the male urethra.
Role of SefA subunit protein of SEF14 fimbriae in the pathogenesis of Salmonella enterica serovar Enteritidis
Authors: Ogunniyi A D , Kotlarski I , Morona R , Manning P A
Published: January 2020

In this study, the role of the SefA subunit protein of SEF14 fimbriae in the pathogenesis of Salmonella enterica serovar Enteritidis was investigated. This was accomplished by mutating the sefA gene in the chromosome of two strains of S. enterica serovar Enteritidis by allelic exchange with a copy that has been inactivated by interruption with a nonpolar kanamycin resistance (aphA-3) cassette. The effect of this mutation on the ability of the S. enterica serovar Enteritidis strains to colonize the intestinal epithelium and to invade other tissues was assessed in BALB/c mice and in vitro by adherence and invasion of HeLa cells. Our results show that an avirulent S. enterica serovar Enteritidis vaccine strain, 11RX (no somatic antigen; flagellum antigen phase 1, g,m; flagellum antigen phase 2, -), colonized better and persisted longer in the Peyer's patches of these mice than did its SefA-deficient counterpart. However, no such difference was observed between a highly virulent S. enterica serovar Enteritidis strain, 7314 (somatic antigen, O1, O9, O12; flagellum antigen phase 1, g,m; flagellum antigen phase 2 [1,7]), and its SefA-deficient isogenic mutant. These findings were correlated with in vitro adherence and invasion of HeLa cells. Furthermore, we could not demonstrate a role for SefA in the virulence of S. enterica serovar Enteritidis as assessed by 50% lethal dose determinations. The implications of these findings are discussed.
Mycobacterium marinum causes both long-term subclinical infection and acute disease in the leopard frog (Rana pipiens)
Authors: Ramakrishnan L , Valdivia R H , McKerrow J H , Falkow S
Published: January 2020

Mycobacterium marinum grows at an optimal temperature of 33 degrees C, far lower than that for M. tuberculosis. Consequently, M. marinum infection of mammals is restricted largely to the cooler surfaces of the body, such as the extremities, but it causes a systemic infection in a large number of poikilothermic animals. Here, we describe a laboratory animal model for M. marinum disease in the leopard frog (Rana pipiens), a natural host species. M. marinum causes a chronic granulomatous, nonlethal disease in immunocompetent frogs. Immunosuppression of the frogs with hydrocortisone results in an acute, fulminant, lethal disease. This animal model, in which a spectrum of tuberculosis-like disease can be produced, will be useful for the dissection of the genetic basis of mycobacterial pathogenesis.
Avirulence of Candida albicans FAS2 mutants in a mouse model of systemic candidiasis
Authors: Zhao X J , McElhaney-Feser G E , Sheridan M J , Broedel S E , Cihlar R L
Published: January 2020

Disruption of both alleles of the Candida albicans FAS2 gene abolishes the ability of the organism to establish infection in a murine model of systemic candidiasis. Within 72 h all mice inoculated with 10(6) CFU of the parental C. albicans strain had died. In contrast, all animals inoculated with the mutant strain CFD2 survived for the course of the experiment (21 days). Animals infected with either mutant strain CFD1 or CFD3, in which only one FAS2 allele was disrupted, also succumbed to infection, but mortality was not observed until 4 days postinfection and survivors remained for up to 20 days postinfection. The results demonstrate that FAS2 is required for successful C. albicans infection.
Variables affecting production of monocyte chemotactic factor 1 from human leukocytes stimulated with Cryptococcus neoformans
Authors: Levitz S M , North E A , Jiang Y , Nong S H , Kornfeld H , Harrison T S
Published: January 2020

The chemokine monocyte chemoattractant protein 1 (MCP-1) is produced predominantly by mononuclear phagocytes and stimulates recruitment into infected tissues of blood monocytes and T cells. These cell types are thought to be critical to host defenses against infections due to Cryptococcus neoformans, a major cause of disease in persons with AIDS and other disorders of cell-mediated immunity. Accordingly, in the present study, we examined the conditions under which human monocytes and bronchoalveolar macrophages (BAM) are stimulated by C. neoformans to produce MCP-1. C. neoformans was a potent inducer of MCP-1 release from monocytes, with levels of chemokine secreted similar to that seen following stimulation with lipopolysaccharide (LPS). BAM, in contrast, were stimulated by LPS, but not by C. neoformans, to secrete MCP-1. A peak in MCP-1 mRNA was seen 8 h following cryptococcal stimulation of monocytes. Nine strains of C. neoformans stimulated monocytes to release MCP-1, and there was only modest variation between strains. However, when an individual strain was used, the capacity of C. neoformans to stimulate monocyte MCP-1 release did vary, depending upon the conditions used to grow the fungal stimuli. Finally, C. neoformans stimulated comparable quantities of MCP-1 release in monocytes from donors with and without human immunodeficiency virus infection. These data establish C. neoformans as a potent stimulator of MCP-1 in monocytes, but not in BAM. The failure of C. neoformans to stimulate MCP-1 in BAM, if occurring in vivo, could result in a diminished cell-mediated inflammatory response following inhalation of airborne fungi.
Increase of glycocalyx and altered lectin agglutination profiles of Pasteurella haemolytica A1 after incubation in bovine subcutaneous tissue chambers in vivo or in ruminant serum in vitro
Authors: Brogden K , Clarke C
Published: January 2020

Pasteurella haemolytica serotype A1 (bovine strain OK) was incubated for 2 and 6 h in bovine subcutaneous tissue chambers in vivo, and ovine strain 82-25 and bovine strain L011 were incubated in vitro for 2 h in heat-inactivated ovine or bovine serum from which gamma globulin had been depleted by protein G affinity chromatography to assess changes in morphology and lectin agglutination profiles (strains 82-25 and L101 only). Cells, removed from chambers after 2 h, were covered with an extensive, dense glycocalyx extending approximately 0.5 microm from the cell surface. In many cells, the glycocalyx was separated from the cell surface by a clear, electron-transparent area. Cells, removed at 6 h, were covered with a sparse glycocalyx of fine fibers 0.2 to 0.3 microm from the cell surface. Strains 82-25 and L101, incubated for 2 h in heat-inactivated ovine or bovine serum or in heat-inactivated ovine or bovine serum depleted of gamma globulin by protein G affinity chromatography, were also covered with a glycocalyx. The glycocalyx did not bind protein A-colloidal gold and therefore did not contain aggregates of accumulated antibody. Strains 82-25 and L101 were incubated individually for 2 h in 10 mM sodium phosphate buffer (pH 7.2) containing 0.14 M NaCl, 0.5 mM CaCl2, and 0.15 mM MgCl2 or with this buffer and either 25% heat-inactivated, gamma globulin-depleted ovine serum or 25% heat-inactivated, gamma globulin-depleted bovine serum. Agglutination profiles were then determined with 17 lectins in 10 mM HEPES-buffered saline (pH 8.4) with 0.1 mM CaCl2 and 0.08% sodium azide. Profiles did not vary with 10 of 17 lectins. However, profiles did vary with peanut agglutinin, Phaseolus vulgaris leucoagglutinin, Sophora japonica agglutinin, Maackia amurensis lectin II, Narcissus pseudonarcissus (daffodil) lectin, Griffonia simplicifolia lectin I, and Pisum sativum agglutinin. Altered profiles indicate a change in the bacterial cell surface, possibly by adsorption or alteration of surface carbohydrate moieties by serum constituents.
Porcine polymorphonuclear leukocytes generate extracellular microbicidal activity by elastase-mediated activation of secreted proprotegrins
Authors: Panyutich A , Shi J , Boutz P L , Zhao C , Ganz T
Published: January 2020

Antimicrobial peptides of several structural classes have been found in phagocytes and epithelial cells of many animals. The broadly microbicidal protegrins (PG1, -2, and -3) were originally isolated as 16 to 18-amino-acid peptides from pig neutrophil lysates, but the corresponding cDNA sequences encoded much larger precursors that belonged to the cathelicidin family of antimicrobial peptides. We explored the storage, secretion, and microbicidal activation of protegrins in porcine neutrophils and in a model system consisting of recombinant proprotegrin 3 (pPG3) and various serine proteases and their inhibitors. Protegrins were stored in neutrophils as inactive proforms that were cleaved by neutrophil elastase to mature protegrins during the preparation of granule lysate and during phorbol myristate acetate-stimulated granule secretion from intact neutrophils. Recombinant pPG3 was efficiently cleaved by trace amounts of human neutrophil elastase or equivalent amounts of elastase activity from porcine neutrophils, but pPG3 was relatively resistant to porcine pancreatic elastase or human neutrophil cathepsin G. The recombinant pPG3 and neutrophil proprotegrins lacked microbicidal activity, but the mature protegrins generated in the elastase-mediated cleavage reaction were as active against Listeria monocytogenes as the chemically synthesized protegrin. The secretion and elastase-mediated activation of proprotegrins accounted for much of the stable microbicidal activity of porcine neutrophil secretions against L. monocytogenes. Secreted proprotegrins and trace amounts of elastase constitute a binary microbicidal system that is likely to contribute to the antimicrobial activity of porcine inflammatory fluids.
Intervention in endotoxin shock by sulfatide (I3SO3-GalCer) with a concomitant reduction in tumor necrosis factor alpha production
Authors: Higashi H , Suzuki Y , Mukaida N , Takahashi N , Miyamoto D , Matsushima K
Published: January 2020

Accumulating evidence indicates that tumor necrosis factor alpha (TNF-alpha) is a principal mediator of endotoxin shock. We previously reported that the action as well as the production of TNF requires the adhesion of leukocytes to the endothelium through integrin beta2 and intercellular adhesion molecule 1. In order to elucidate the roles of the initial interaction of the leukocytes with the endothelium through the selectins, we have examined the effects of a ligand for L- and P-selectins, sulfatide, on endotoxin shock in mice. Consistent with previous reports, a single injection of a high dose of endotoxin caused acute lethality, marked hypotension, leukopenia, and elevation in serum TNF-alpha levels. Pretreatment with sulfatide prevented acute lethality and hypotension, but not leukopenia, with a concomitant reduction in the increase in serum TNF-alpha levels. Moreover, pretreatment with sulfatide inhibited lipopolysaccharide (LPS)-induced TNF-alpha production by a human monocytic cell line, THP-1, in a dose-dependent manner. These results suggest either that selectin is critically involved in conferring the responsiveness of leukocytes to LPS or that sulfatide interferes with the intracellular signaling pathway which leads to TNF-alpha gene activation.
Naturally occurring deletions in the centisome 63 pathogenicity island of environmental isolates of Salmonella spp
Authors: Ginocchio C C , Rahn K , Clarke R C , Galán J E
Published: January 2020

We have identified several environmental isolates of Salmonella senftenberg and S. litchfield which carry a deletion encompassing a vast segment of the centisome 63 region of the Salmonella chromosome. The deletion includes the entire inv, spa, and hil loci, which are required for entry of Salmonella spp. into mammalian cells. Consequently, these isolates were found to be markedly deficient in the ability to enter cultured epithelial cells. In contrast, no deletions were found in the corresponding regions of the chromosomes of clinical isolates of these serovars; consequently, these isolates were found to be highly invasive for cultured epithelial cells. These data confirm the importance of the centisome 63 region of the Salmonella chromosome in mediating the entry of these organisms into cultured mammalian cells and indicate that additional entry pathways are presumably not utilized by these environmental isolates. These results are also consistent with the notion that this region constitutes a pathogenicity island which remains unstable in certain Salmonella serotypes.
Borrelia burgdorferi migrates into joint capsules and causes an up-regulation of interleukin-8 in synovial membranes of dogs experimentally infected with ticks
Authors: Straubinger R K , Straubinger A F , Härter L , Jacobson R H , Chang Y F , Summers B A , Erb H N , Appel M J
Published: January 2020

Twenty 6-week-old specific-pathogen-free beagles were infected with Borrelia burgdorferi by tick challenge, and five uninfected dogs served as controls. During the study, all dogs were monitored for infection, clinical signs, and antibody response against B. burgdorferi. During episodes of lameness or postmortem, synovial fluids from each dog were examined for volume, cell number, polymorphonuclear leukocyte (PMN) content, cell viability, and chemotactic activity. Twenty-five tissues collected postmortem from each dog were tested for interleukin-8 (IL-8) mRNA, tumor necrosis factor alpha (TNF-alpha) mRNA, presence of live spirochetes, and histopathological changes. Thirteen infected dogs (group A), which seroconverted rapidly (maximum titers within 50 to 90 days), developed acute and severe mono- or oligoarthritis almost exclusively in the limb closest to the tick bite (median incubation period, 66 days). Synovial fluids of the arthritic joints collected during episodes of lameness had significantly elevated volume, cell count, PMN proportion, cell viability, and chemotactic activity for PMNs. The remaining joints of the same animals contained synovial fluids with elevated chemotactic activity and cell viability. Twelve dogs tested positive for IL-8 mRNA in multiple tissues (synovia, pericardium, and peritoneum), and 10 dogs expressed TNF-alpha mRNA, but only in the tributary lymph nodes of the inflamed joints. Histological examinations revealed severe poly- or oligoarthritis and moderate to severe cortical hyperplasia in draining lymph nodes of the inflamed joints in all 13 dogs. Seven infected dogs with mild or no clinical signs (group B) seroconverted slowly (peak titers after 90 days), and only some joint fluids showed chemotactic activity, which on average was lower than that in inflamed and noninflamed joints from dogs in group A. Four dogs expressed IL-8 mRNA (in the synovia and pericardium), and three dogs had TNF-alpha mRNA in tributary lymph nodes. Histologically, nonsuppurative arthritis was found in multiple joints, and mild to moderate cortical hyperplasia was found in draining lymph nodes. Five uninfected dogs without lameness (group C) had normal synovial fluids and tissues. In all infected dogs, live spirochetes were demonstrated more frequently in tissues of the somatic quadrant closest to the tick bite than in tissues further from the site of infection, suggesting that dissemination of B. burgdorferi occurs more by migration than by blood-borne spread. From these studies employing a canine model of B. burgdorferi infection, we conclude that IL-8 is involved in the pathogenesis of acute Lyme arthritis.
Glucose up-regulates expression of the differentiation-associated brush border binding site for enterotoxigenic Escherichia coli colonization factor antigen I in cultured human enterocyte-like cells
Authors: Bernet-Camard M F , Duigou F , Kernéis S , Coconnier M H , Servin A L
Published: January 2020

The association of enterotoxigenic Escherichia coli expressing colonization factor antigen I (CFA/I) with the cultured human colon adenocarcinoma cell, a model of the mature enterocyte of the small intestine, is dependent on the binding of CFA/I to a brush border-associated component. Binding of the purified radiolabeled [125I]CFA/I- and 14C-labeled CFA/I-positive bacteria could be displaced by an increasing concentration of unlabeled CFA/I. Moreover, we showed that expression of the specific CFA/I binding developed as a function of cell differentiation in Caco-2 cells, whereas expression of the nonspecific binding did not. Expression of the brush border differentiation-associated component acting as a binding site for CFA/I was up-regulated by glucose. Indeed, the enterocyte-like HT-29 glc- cell subpopulation not expressing the CFA/I binding site when cultured in dialyzed serum and hexose-free medium regained the ability to bind CFA/I when the cells were returned to culture medium containing glucose. Furthermore, expression of the brush border-associated CFA/I binding site in the enterocyte-like Caco-2 cells was repressed when the cells were cultured in hexose-free conditions.
Bacterial porins stimulate bone resorption
Authors: Meghji S , Henderson B , Nair S P , Tufano M A
Published: January 2020

Porins are abundant outer membrane proteins of gram-negative bacteria involved in transport of low-molecular-mass molecules. During the past decade, porins from a number of bacteria have also been shown to have proinflammatory activities including inducing the synthesis of proinflammatory mediators (cytokines, platelet-activating factor, and nitric oxide) in cultured cells and inducing inflammation in vivo. With this range of actions, it was possible that porins could also interact with bone cells to cause aberrant bone remodeling and that this could contribute to the bone destruction seen in gram-negative bone infections. By using purified preparations of Salmonella typhimurium and Pseudomonas aeruginosa porins, in the presence of polymyxin B, it was possible to induce concentration-dependent loss of calcium from cultured murine calvaria at porin concentrations in the range of 1 to 10 nM. The mechanism of action of the porins was determined by the inclusion of inhibitors of cyclooxygenase or inflammatory cytokines in the culture media. The bone-resorbing activity of both porins was not inhibited by the cyclooxygenase inhibitor indomethacin or by neutralizing the activity of tumor necrosis factor. Indeed, relatively high concentrations of these agents produced an unexpected increase in the bone resorption induced by the porins. In contrast, porin-induced bone resorption could be inhibited by relatively high concentrations of the natural inhibitor of interleukin-1 (IL-1 receptor antagonist). It appears that these porins stimulate bone resorption by a mechanism distinct from that of lipopolysaccharide, and the possibility therefore exists that porins play a role in bone destruction in gram-negative bacterial infections of bone.
Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women
Authors: Kozlowski P A , Cu-Uvin S , Neutra M R , Flanigan T P
Published: January 2020

To determine which mucosal immunization routes may be optimal for induction of antibodies in the rectum and female genital tract, groups of women were immunized a total of three times either orally, rectally, or vaginally with a cholera vaccine containing killed Vibrio cholerae cells and the recombinant cholera toxin B (CTB) subunit. Systemic and mucosal antibody responses were assessed at 2-week intervals by quantitation of CTB-specific antibodies in serum and in secretions collected directly from mucosal surfaces of the oral cavity, rectum, cervix, and vagina with absorbent wicks. The three immunization routes increased levels of specific immunoglobulin G (IgG) in serum and specific IgA in saliva to similar extents. Rectal immunization was superior to other routes for inducing high levels of specific IgA and IgG in rectal secretions but was least effective for generating antibodies in female genital tract secretions. Only vaginal immunization significantly increased both specific IgA and specific IgG in both the cervix and the vagina. In addition, local production of CTB-specific IgG in the genital tract could be demonstrated only in vaginally immunized women. Vaginal immunization did not generate antibodies in the rectum, however. Thus, generation of optimal immune responses to sexually transmitted organisms in both the rectal and the genital mucosae of women may require local immunization at both of these sites.
Unmasking of intestinal epithelial lateral membrane beta1 integrin consequent to transepithelial neutrophil migration in vitro facilitates inv-mediated invasion by Yersinia pseudotuberculosis
Authors: McCormick B A , Nusrat A , Parkos C A , D'Andrea L , Hofman P M , Carnes D , Liang T W , Madara J L
Published: January 2020

Idiopathic intestinal disease states characterized by active inflammation associated with transepithelial migration of neutrophils may, paradoxically, be associated with an increased risk of infection by enteric pathogens. Although the specific ligands with which various intestinal pathogens associate remain largely unknown, it is thought that many reside on the basolateral membrane. For example, beta1 integrin, a basolateral membrane protein, mediates the specific interaction between epithelial cells and the inv gene product (invasin) on the surface of Yersinia pseudotuberculosis. Our observations indicate that neutrophil migration across model T84 cell intestinal epithelia produced transient separation of epithelial cells at sites of neutrophil migration, resulting in microdiscontinuities that remained unsealed for several hours. We hypothesized that such sites of microdiscontinuities would yield a potential route for luminal pathogens to gain access to basolateral ligands and, thus, provide a window of risk for enteric infection. The surface biotinylation and fluorescence localization studies reported here revealed that, as in natural intestinal epithelia, beta1 integrin was strictly polarized to the basolateral membrane in confluent T84 monolayers. However, the transient microdiscontinuities resulting from neutrophil migration permitted access to beta1 integrin from the apical reservoir. Coincident with such basolateral exposure of beta1 integrin, monolayers became susceptible to invasion by Y. pseudotuberculosis. Fluorescence localization indicated that Y. pseudotuberculosis selectively associated with monolayers at sites where small discontinuities resulting from neutrophil transmigration were found. An increased risk for Y. pseudotuberculosis infection was specifically related to exposure of beta1 integrin (normally concealed by tight junctions) to the apical compartment, as Y. pseudotuberculosis cells lacking the inv gene were unable to invade following neutrophil transepithelial migration. Following closure of the microdiscontinuities associated with neutrophil migration, a small pool of beta1 integrin remained apically localized, presumably due to incomplete repolarization. However, this small apical pool of beta1 integrin was insufficient to support a detectable increased risk of Yersinia infection. Together, these observations indicate that by transiently perturbing monolayer continuity, neutrophil transepithelial migration is associated with a window of risk in which luminal pathogens can access basolateral ligands such as beta1 integrin.
Ehrlichia chaffeensis inclusions are early endosomes which selectively accumulate transferrin receptor
Authors: Barnewall R E , Rikihisa Y , Lee E H
Published: January 2020

Ehrlichia chaffeensis is an obligatory intracellular bacterium which infects macrophages and monocytes. Double immunofluorescence labeling was used to characterize the nature of E. chaffeensis inclusion in the human promyelocytic leukemia cell line THP-1. E. chaffeensis was labeled with dog anti-E. chaffeensis serum and fluorescein isothiocyanate-conjugated anti-dog immunoglobulin G (IgG). Lissamine rhodamine-conjugated anti-mouse IgG was used to label various mouse monoclonal antibodies. Ehrlichial inclusions did not fuse with lysosomes, since they were not labeled with anti-CD63 or anti-LAMP-1. The ehrlichial inclusions were slightly acidic, since they weakly accumulated 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and stained weakly positive for vacuolar type H+ ATPase. Some ehrlichial inclusions were labeled positive with antibodies against HLA-DR, HLA-ABC, and beta2 microglobulin, while other inclusions in the same cell were labeled negative. The inclusions were labeled strongly positive for transferrin receptors (TfRs) and negative for the clathrin heavy chain. Time course labeling for TfRs showed that up to 3 h postinfection, most of the ehrlichial inclusions were negative for TfRs. After 6 h postinfection, 100% of the ehrlichial inclusions became TfR positive and the intensity of labeling was increased during the subsequent 3 days. Reverse transcription-PCR showed a gradual increase in the level of TfR mRNA postinfection, which reached a peak at 24 h postinfection. These results suggest that ehrlichial inclusions are early endosomes which selectively accumulate TfRs and that the ehrlichiae up-regulate TfR mRNA expression.
Early gamma interferon responses in lethal and nonlethal murine blood-stage malaria
Authors: De Souza J B , Williamson K H , Otani T , Playfair J H
Published: January 2020

This study was undertaken to explore early differences in cytokine production during nonlethal and lethal blood-stage murine malaria infections. Cytokine analysis of spleens during these infections showed that the principal difference between two nonlethal and two lethal Plasmodium species was the production of gamma interferon 24 h after infection with nonlethal parasites. In contrast, no increases in interleukin-4 production were observed in the first 24 h and tumor necrosis factor alpha levels increased equally in both nonlethal and lethal infections. During the later phase of infection with nonlethal parasites, both gamma interferon and interleukin-4 levels increased markedly a few days before parasite clearance. Early increases in gamma interferon production in nonlethal infections of Plasmodium yoelii and Plasmodium chabaudi were dose related and increased significantly with the size of the inoculum. Studies with the nonlethal P. yoelii suggest that the early gamma interferon response is mediated by T cells and natural killer cells, as it was reduced in athymic mice and in mice depleted of their natural killer cells by treatment with specific antiserum. Infecting mice with increasing numbers of lethal P. yoelii and Plasmodium berghei parasites did not increase the amount of gamma interferon, interleukin-4, and tumor necrosis factor alpha produced in a dose-dependent fashion. We conclude that one consequence of the early production of gamma interferon and tumor necrosis factor-alpha, particularly after nonlethal P. yoelii infection, may be to adjust the balance of T-helper cell subset activation, and probably that of other immune responses, so as to enhance the mechanisms that are essential for elimination of the parasites. This suggests that a successful vaccine should contain antigens capable of inducing such responses.
Protective effect of antilipopolysaccharide monoclonal antibody in experimental Klebsiella infection
Authors: Rukavina T , Tícac B , Susa M , Jendrike N , Jonjíc S , Lucin P , Marre R , Doríc M , Trautmann M
Published: January 2020

An O-antigen-specific murine monoclonal antibody (MAb) directed against an immunodominant epitope expressed on Klebsiella O1, O6, and O8 lipopolysaccharides (LPS) was examined with respect to its binding to nonencapsulated and encapsulated bacterial cells and its ability to protect against lethal murine Klebsiella sepsis. While the MAb (clone Ru-O1, mouse immunoglobulin G2b) bound well to nonencapsulated organisms of the O1 serogroup, binding was significantly, but not completely, abolished by the presence of the K2 capsule. In a model of experimental Klebsiella peritonitis and sepsis induced by a virulent O1:K2 serogroup strain, higher doses of anti-LPS MAb Ru-O1 than of a previously described anticapsular MAb specific for the K2 capsular polysaccharide were needed to provide protection. However, high-dose (40 microg/g of body weight) pretreatment with anti-LPS MAb Ru-O1 significantly reduced bacterial dissemination to various organs as well as macroscopic and histologic pulmonary alterations. Thus, since the number of Klebsiella capsular antigens occurring in clinical material is too large to be completely "covered" by a K-antigen-specific hyperimmunoglobulin preparation, O-antigen-specific antibodies may supplement K-antigen-specific immunoprophylaxis and -therapy of clinical Klebsiella infection.
Elongated versions of Vlp surface lipoproteins protect Mycoplasma hyorhinis escape variants from growth-inhibiting host antibodies
Authors: Citti C , Kim M F , Wise K S
Published: January 2020

Variation in Vlp surface proteins of Mycoplasma hyorhinis was evaluated in terms of its role in determining susceptibility of organisms to growth inhibition by host antibodies (Abs). High-frequency switching of Vlp surface lipoproteins has been studied in isogenic lineages of M. hyorhinis SK76. In these lineages, the products of three genes, vlpA, vlpB, and vlpC, are subject to phase and size variation in vitro, which occur through distinct mutator elements that independently govern the expression of each vlp gene (promoter mutations) or the size of the vlp gene product (by intragenic expansion or contraction of a 3' region containing tandem repeats). Isogenic clonal variants of M. hyorhinis SK76 expressing distinct profiles of Vlp products were assessed for their susceptibility to complement-independent growth inhibition by serum Abs of swine experimentally infected with the arthritigenic SK76 strain. Invariably, variants expressing longer versions of VlpA, VlpB, or VlpC (each expressed individually) were completely resistant to host immune serum Abs, whereas variants expressing shorter allelic versions of each Vlp were susceptible. The target of growth-inhibiting Abs was not the Vlp products, since removal of anti-Vlp Abs had no effect on the inhibitory activity of the host immune serum on susceptible variants. Escape variant populations derived by propagating susceptible variants in an immune (versus control) host serum revealed a strong selection for the long-Vlp phenotype, irrespective of the identity of the Vlp expressed. Apparent mutational pathways of acquiring the protective phenotype included mutational switches to express long vlp genes that had been transcriptionally silent or switches to elongate expressed vlp genes. These results suggest that a major function of the Vlp system is to shield the wall-less mycoplasma surface from host Abs capable of binding vital (and as-yet-unidentified) surface antigens of this organism.
Characterization of the nucleotide sequence of the groE operon encoding heat shock proteins chaperone-60 and -10 of Francisella tularensis and determination of the T-cell response to the proteins in individuals vaccinated with F. tularensis
Authors: Ericsson M , Golovliov I , Sandström G , Tärnvik A , Sjöstedt A
Published: January 2020

The groE operon of Francisella tularensis LVS, encoding the heat shock proteins chaperone-10 (Cpn10) and Cpn60, was sequenced and characterized, and the T-cell response of LVS-vaccinated individuals to the two proteins and the third major chaperone, Ft-DnaK, was assayed. The cpn10 and cpn60 genes were amplified by PCR with degenerate oligonucleotides derived from the N-terminal sequence of the two proteins. The sequence analysis revealed the expected two open reading frames, encoding proteins with estimated Mrs of 10,300 and 57,400. The deduced amino acid sequences closely resembled Cpn10 and Cpn60 proteins of other prokaryotes. The genes constituted a bicistronic operon, the cpn10 gene preceding the cpn60 gene. Upstream of the cpn10 gene, an inverted repeat and motifs similar to -35 and -10 sequences of sigma70-dependent but not of sigma32-dependent promoters of Escherichia coli were found. The inverted repeat of the operon resembled so-called hairpin loops identified in other characterized prokaryotic groE operons lacking sigma32-dependent promoters. Primer extension analysis disclosed one and the same transcription start, irrespective of the presence or absence of heat or oxidative stress. After separation of lysates of the F. tularensis LVS organism by two-dimensional gel electrophoresis, DnaK, Cpn60, and Cpn10 were extracted and used as antigens in T-cell tests. When compared to those from nonvaccinated individuals, T cells from individuals previously vaccinated with live F. tularensis LVS showed an increased proliferative response to DnaK and Cpn60 but not to Cpn10. The present data will facilitate further studies of the involvement of the heat shock proteins in protective immunity to tularemia.
Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker
Authors: Copass M , Grandi G , Rappuoli R
Published: January 2020

Research on Helicobacter pylori has been hindered by the lack of useful genetic tools. Using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows introduction of unmarked mutations in H. pylori. A kan-sacB cassette, consisting of the sacB gene expressed from the H. pylori flagellin promoter and the kanamycin resistance module, was introduced by homologous recombination into a target H. pylori gene. The resultant strains were sucrose sensitive and kanamycin resistant. Following transformation with a mutated allele, growth in sucrose-containing medium allowed the selection of strains that had lost the kan-sacB module and had integrated the unmarked allele. We have used this cassette to perform a site-directed modification of two histidine residues encoded by the vacA gene in a two-step procedure. This system should prove useful in the site-directed mutagenesis of H. pylori genes.
Characterization of an antigen from Leishmania amazonensis amastigotes able to elicit protective responses in a murine model
Authors: Beyrodt C G , Pinto A R , Freymüller E , Barbiéri C L
Published: January 2020

Lymphoproliferative responses to an antigen from Leishmania amazonensis amastigotes with an apparent molecular mass of 30 kDa, termed p30, were evaluated with BALB/c mice. The p30 antigen was purified after separation of parasite extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution. Lymphoproliferative responses to p30 were obtained by subcutaneous immunization of animals with L. amazonensis amastigote extracts, and maximal stimulation indices were observed at an antigen concentration of 5 microg/ml. Induction of lymphoproliferation by p30 is stage specific, and no differences in the responses to this antigen between mice susceptible and resistant to L. amazonensis were detected. The predominant T cells characterized in the lymphocyte cultures were CD4+. Lymphokine analysis of the supernatants from these cultures indicated that Th1 is the subset involved in the lymphoproliferative responses to the antigen. BALB/c mice immunized with p30 and challenged with L. amazonensis amastigotes showed a very low level of infection, indicating a protective role for p30 and a correlation between Th1 and protection. Further biochemical characterization studies showed that this antigen presents cysteine proteinase activity.