bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet receptors for the Streptococcus sanguis adhesin and aggregation-associated antigens are distinguished by anti-idiotypical monoclonal antibodies
Authors: Gong K , Wen D Y , Ouyang T , Rao A T , Herzberg M C
Published: January 2020

Platelets aggregate in response to an adhesin and the platelet aggregation-associated protein (PAAP) expressed on the cell surfaces of certain strains of Streptococcus sanguis. We sought to identify the corresponding PAAP receptor and accessory adhesin binding sites on platelets. Since the adhesion(s) of S. sanguis for platelets has not been characterized, an anti-idiotype (anti-id) murine monoclonal antibody (MAb2) strategy was developed. First, MAb1s that distinguished the adhesin and PAAP antigens on the surface of S. sanguis I 133-79 were selected. Fab fragments of MAb1.2 (immunoglobulin G2b [IgG2b]; 70 pmol) reacted with 5 x 10(7) cells of S. sanguis to completely inhibit the aggregation of human platelets in plasma. Under similar conditions, MAb1.1 (IgG1) inhibited the adhesion of S. sanguis cells to platelets by a maximum of 34%, with a comparatively small effect on platelet aggregation. Together, these two MAb1s inhibited S. sanguis-platelet adhesion by 63%. In Western immunoblots, both MAb1s reacted with S. sanguis 133-79 87- and 150-kDa surface proteins and MAb1.2 also reacted with purified type I collagen. The hybridomas producing MAb1.1 and MAb1.2 were then injected into BALB/c mice. Enlarged spleens were harvested, and a panel of MAb2 hybridomas was prepared. To identify anti-ids against the specific MAb1s, the MAb2 panel was screened by enzyme-linked immunosorbent assay for reaction with rabbit polyclonal IgG antibodies against the 87- and 150-kDa antigens. The reactions between the specific rabbit antibodies and anti-ids were inhibited by the 87- and 150-kDa antigens. When preincubated with platelets, MAb2.1 (counterpart of MAb1.1) inhibited adhesion to platelets maximally by 46% and MAb2.2 (anti-MAb1.2) inhibited adhesion to platelets maximally by 35%. Together, both MAb2s inhibited the adhesion of S. sanguis to platelets by 81%. MAb2.2 also inhibited induction of platelet aggregation. MAb2.2 immunoprecipitated a biotinylated platelet membrane antigen of 170 kDa (unreduced); MAb2.1 precipitated membrane antigens of 175- and 230-kDa (unreduced). Therefore, platelet binding sites and the receptor for the S. sanguis adhesin and PAAP, respectively, are distinguished by the anti-id MAb2s.
Immunogenicity in Peruvian volunteers of a booster dose of oral cholera vaccine consisting of whole cells plus recombinant B subunit
Authors: Begue R E , Castellares G , Cabezas C , Sanchez J L , Meza R , Watts D M , Taylor D N
Published: January 2020

Forty-nine subjects received two doses of oral cholera vaccine consisting of whole cells plus recombinant B subunit; this was followed by a booster dose one year later. After the primary series, a significant (greater than twofold) increase in the levels of vibriocidal, anti-cholera toxin immunoglobulin G and anti-cholera toxin immunoglobulin A antibodies occurred in 54, 88, and 81% of the subjects, respectively. Within one year, titers decreased to levels close to baseline. A booster dose then induced rises to those which occurred after the initial vaccination. The results suggest that 1-year booster doses may be necessary to maintain immunity against cholera in Latin America.
Intracellular growth and cytotoxicity of Mycobacterium haemophilum in a human epithelial cell line (Hec-1-B)
Authors: Fischer L J , Quinn F D , White E H , King C H
Published: January 2020

We developed an in vitro model to study the temperature-regulated cytotoxicity and intracellular growth of Mycobacterium haemophilum in cultured human epithelial and endothelial cells. M. haemophilum associated with human epithelial and endothelial cells at similar rates when incubated at 33 and 37 degrees C, but only the epithelial cell line supported the multiplication of this organism. M. haemophilum grew equally well with epithelial cells at both temperatures. The aminoglycoside antibiotic amikacin was used to study the intracellular growth of M. haemophilum in the epithelial cells at 33 and 37 degrees C. Although an approximately equal number of bacteria were found within cells after 2 days of incubation at both temperatures, intracellular replication of M. haemophilum was 1,000-fold greater at 33 than at 37 degrees C. This intracellular multiplication was associated with destruction of the monolayers at 33 but not at 37 degrees C, and only culture filtrates from infected monolayers incubated at 33 degrees C were cytotoxic to fresh epithelial cell monolayers. This strain of M. haemophilum also produced contact-dependent hemolysis of sheep erythrocytes, demonstrating the possible presence of a cytolysin. These studies suggest that M. haemophilum has a preference for growth with cultured human epithelial cells. In addition, intracellular growth is best at 33 degrees C in epithelial cells, and this correlated with cytotoxicity at this temperature. This phenotype may be caused by induction of a soluble cytotoxic component, possibly a hemolytic cytolysin.
Reconstitution of B-cell-depleted mice with B cells restores Th2-type immune responses during Plasmodium chabaudi chabaudi infection
Authors: Taylor-Robinson A W , Phillips R S
Published: January 2020

In mice depleted of B cells from birth by treatment with anti-immunoglobulin M(mu) antibodies, progression from a Th1- to a Th2-regulated immune response during primary infection with Plasmodium chabaudi chabaudi fails to occur. While Th1-type immunity limits parasitemia, in the absence of B cells, chronic low-grade infections persist. Here, we show that reconstituting immune, and to a lesser extent naive, B cells to mice rendered deficient in B-cell function through anti-immunoglobulin M(mu) pretreatment restores the CD4+ T-cell response to the Th2 type later in P. c. chabaudi infection and with it the capacity to eliminate infection. This finding provides clear evidence that B cells are required for switching the balance of immune regulation between CD4+ T cells from Th1 to Th2 during P.c. chabaudi infection and supports the concept that B cells, through antibody production, are needed for effective antimalarial immunity.
Localization of biologically important regions on toxic shock syndrome toxin 1
Authors: Murray D L , Earhart C A , Mitchell D T , Ohlendorf D H , Novick R P , Schlievert P M
Published: January 2020

Toxic shock syndrome toxin 1 (TSST-1) contains a long central alpha helix that forms the base of two grooves on opposite sides of the molecule. Previous studies indicated that residues 132, 135, and 140 along the back of the central alpha helix are important in the biological activities. We made mutations of additional central alpha-helix residues exposed along this groove on the back of TSST-1. The proteins were purified, shown not to have gross alteration in structure, and tested for both superantigenicity and ability to elicit lethal TSS, using the superantigenicity, likely to because of alteration in T-cell receptor binding. Mutants H135A, Q136A, and E132K/ Q136K lost the ability to induce lethal TSS. The mutant Q136A was most increasing because it was superantigenic, yet nonlethal.
Expression of the inlAB operon by Listeria monocytogenes is not required for entry into hepatic cells in vivo
Authors: Gregory S H , Sagnimeni A J , Wing E J
Published: January 2020

Listeria monocytogenes injected intravenously into mice is taken up in the liver, where hepatocytes serve as the principal site of intracellular replication. The factors effecting entry of L. monocytogenes into hepatic cells remain to be determined. Others have shown that the protein products of the inlAB (internalin) operon are required for maximum entry of L. monocytogenes into a number of cell lines in vitro. Likewise, we report here that expression of the inlAB operon was required for maximum uptake of L. monocytogenes by primary cultures of mouse hepatocytes. Uptake of an inlAB mutant strain of L. monocytogenes was approximately 10-fold less than that of the isogenic wild-type control. In contrast, inlAB expression was not a factor in (i) clearance of L. monocytogenes injected intravenously into mice and taken up in the liver, (ii) the distribution of L. monocytogenes among hepatocytes and nonparenchymal cells in the liver, or (iii) internalization of L. monocytogenes by hepatic cells in vivo. These latter findings suggest that infection of hepatic cells by L. monocytogenes in vivo does not require the protein products of the inlAB operon.
The hemagglutinin gene A (hagA) of Porphyromonas gingivalis 381 contains four large, contiguous, direct repeats
Authors: Han N , Whitlock J , Progulske-Fox A
Published: January 2020

Porphyromonas gingivalis is a gram-negative anaerobic bacterial species strongly associated with adult periodontitis. One of its distinguishing characteristics and putative virulence properties is the ability to agglutinate erythrocytes. We have previously reported the cloning of multiple hemagglutinin genes from P. gingivalis 381. Subsequent sequencing of clone ST 2 revealed that the cloned fragment contained only an internal portion of the gene which lacked both start and stop codons. We here report the cloning and sequencing of the entire gene, designated hagA, as well as its relationship to other genes of this species. By use of inverse PCR technology and the construction of several additional genomic libraries, the complete open reading frame of hagA was found to be 7,887 bp in length, encoding a protein of 2,628 amino acids with a molecular mass of 283.3 kDa, which is among the largest genes ever cloned from a prokaryote to date. Within its open reading frame, four large, contiguous, direct repeats (varying from 1,318 to 1,368 bp) were identified. The repeat unit (HArep), which is assumed to contain the hemagglutinin domain, is also present in other recently reported protease and hemagglutinin genes in P. gingivalis. Thus, we propose that hagA and the other genes which share the HArep sequence form a multigene family with hagA as a central member.
Role of Porphyromonas gingivalis protease activity in colonization of oral surfaces
Authors: Tokuda M , Duncan M , Cho M I , Kuramitsu H K
Published: January 2020

Cysteine proteases, including Arg-gingipain of Porphyromonas gingivalis, have been implicated as important virulence factors in periodontal diseases. These enzymes are also involved in the hemagglutinating activity of the organisms. In order to determine the role of proteases in the colonization of the gingival margin, we have compared the attachment properties of P. gingivalis 381 with those of its Arg-gingipain-defective mutant, G-102. Interactions with gram-positive bacteria, human oral epithelial cells, extracellular matrix proteins, and type I collagen were evaluated. In all cases, mutant G-102 was deficient in attachment relative to the parental strain. The mutant's defects could be explained, in part, by the weak autoaggregation displayed by the mutant, which appeared to result from altered fimbrial expression. Both Western blot (immunoblot) and Northern (RNA) blot analyses indicated reduced expression of the major 43-kDa fimbrillin subunit in the mutant. These results suggest that Arg-gingipain may play both direct and indirect roles in the colonization of the gingival margin. In addition, fimbriae may play a direct role in interacting with some host surfaces.
Soluble invasion plasmid antigen C (IpaC) from Shigella flexneri elicits epithelial cell responses related to pathogen invasion
Authors: Marquart M E , Picking W L , Picking W D
Published: January 2020

Shigella flexneri invades colonic epithelial cells by pathogen-induced phagocytosis. The three proposed effectors of S. flexneri internalization are invasion plasmid antigens B (IpaB), IpaC, and IpaD, which are encoded on the pathogen's 230-kb virulence plasmid and translocated to the extracellular milieu via the Mxi-Spa translocon. To date, there are no definitive functional data for any purified Ipa protein. Here, we describe the first characterization of highly purified recombinant IpaC, which elicits numerous epithelial cell responses related to events that take place during pathogen invasion. 125I-labeled IpaC binds cultured Henle 407 intestinal cells with an apparent dissociation constant in the low micromolar range. Moreover, incubation of epithelial cells with IpaC results in general changes in cellular phosphoprotein content, demonstrating this protein's ability to influence cellular protein kinase activities. These results contrast dramatically with those seen for recombinant IpaD, which does not bind to or induce detectable changes in the normal activities of cultured epithelial cells. In addition to influencing host cell activities, preincubation of epithelial cells with purified IpaC enhances uptake of S. flexneri by host cells. A similar result is seen when the cells are preincubated with a highly concentrated water extract of virulent S. flexneri 2a (strain 2457T). Interestingly, soluble IpaC also appears to promote uptake of the noninvasive S. flexneri 2a strain BS103. Purified IpaD failed to enhance the uptake of virulent S. flexneri and did not facilitate uptake of BS103. Taken together, the data suggest that IpaC is a potential effector of the host cell biological activities and may be responsible for entry of S. flexneri into target cells.
Genetic and physiological analysis of the lethal effect of L-(+)-lactate dehydrogenase deficiency in Streptococcus mutans: complementation by alcohol dehydrogenase from Zymomonas mobilis
Authors: Hillman J D , Chen A , Snoep J L
Published: January 2020

CH4ts is a previously isolated recombinant mutant of Streptococcus mutans NG8 which produces a thermolabile L-(+)-lactate dehydrogenase (LDH) activity. It does not grow at 42 degrees C under a variety of cultivation conditions. In this study, we show that a batch culture of CH4ts shifted from 30 to 42 degrees C underwent rapid cessation of growth and accelerated cell death. The mutant grew at 42 degrees C in continuous culture under glucose-limiting conditions. Under these conditions, lactate production was replaced by production of ethanol and, to a smaller extent, acetoin. The cloned Zymomonas mobilis gene for alcohol dehydrogenase II, placed under the control of the S. mutans spaP regulatory signals, complemented LDH deficiency. The alcohol dehydrogenase-complemented mutant grew as well or better than NG8 on a variety of carbon sources at 42 degrees C and produced significant amounts of ethanol in place of lactic acid. These results are in accord with other approaches indicating that S. mutans has other enzymatic activities, including pyruvate formate-lyase and pyruvate dehydrogenase, for pyruvate metabolism. However, at high glucose concentrations, the levels of activity of these enzymes are apparently insufficient to compensate for the absence of LDH.
Ubiquitin-like epitopes associated with Candida albicans cell surface receptors
Authors: Sepulveda P , Lopez-Ribot J L , Gozalbo D , Cervera A , Martinez J P , Chaffin W L
Published: January 2020

We have recently reported the cloning of a Candida albicans polyubiquitin gene and the presence of ubiquitin in the cell wall of this fungus. The polyubiquitin cDNA clone was isolated because of its reactivity with antibodies generated against the candidal 37-kDa laminin-binding protein. In the present study, we have further investigated the relationship between ubiquitin and cell wall components displaying receptor-like activities, including the 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. Two-dimensional electrophoretic analysis and immunoblot experiments with antibodies against ubiquitin and the individually purified receptor-like molecules confirmed that these cell surface components are ubiquitinated. In an enzyme-linked immunosorbent assay, polyclonal antisera to each receptor reacted with ubiquitin, thus demonstrating that the purified receptor preparations used as immunogens contained ubiquitin-like epitopes. It is proposed that ubiquitin may play a role in modulating the activity of these receptors and in the interaction of C. albicans cells with host structures.
Expression of the human cell surface glycoconjugates Lewis x and Lewis y by Helicobacter pylori isolates is related to cagA status
Authors: Wirth H P , Yang M , Karita M , Blaser M J
Published: January 2020

Monoclonal antibodies were used in an enzyme-linked immunosorbent assay for the detection of human Lewis immunodeterminants in the lipopolysaccharide of Helicobacter pylori. In 94 H. pylori isolates, expression of Lewis(x) (Le(x)) and Le(y) was a stable phenotypic marker independent of the growth medium and cell age; 46 (49%) of the isolates expressed both and 34 (36%) of the isolates expressed either Le(x) or Le(y); 14 (15%) were negative for both determinants. Twelve (13%) isolates expressed Le(b), 3 (3%) expressed Le(a), and 2 (2%) expressed sialyl-Le(x). H. pylori isolates positive for both Le(x) and Le(y) were predominantly cagA+ (P < 0.001) and possessed the s1 signal sequence (P < 0.05) and the m1 midregion type (P = 0.033) of vacA. Isogenic mutants of H. pylori CPY3401 were created by interruption of the cagA, picB, or ureA gene. The cagA-ablated strain (but not the picB- and ureA-ablated mutant strains) had significantly (P < 0.01) diminished expression of Le(y) compared with that of the wild-type strain; for all four strains, expression of Le(x) was similar. In conclusion, 89% of H. pylori isolates express Le determinants in their lipopolysaccharide, mimicking human cell surface glycoconjugates. Strong expression of Le(x) and Le(y) by cagA+ isolates could counterbalance their enhanced proinflammatory activities and thereby facilitate persistence.
Restoration of lipopolysaccharide-mediated B-cell response after expression of a cDNA encoding a GTP-binding protein
Authors: Kang A D , Wong P M , Chen H , Castagna R , Chung S W , Sultzer B M
Published: January 2020

Previous analysis of hybrid progeny derived from lipopolysaccharide (LPS) responder and nonresponder inbred mouse strains demonstrated that a single genetic locus controlled responsiveness to LPS. Using a differential functional screening approach, we report the isolation of a cDNA that has sequence homology to a GTP-binding protein. Expression of the cDNA in splenic B cells of C3H/HeJ nonresponder, endotoxin-resistant mice resulted in polyclonal B-cell activation in response to LPS stimulation. Thus a GTP-binding protein may be involved in LPS stimulation in B cells and perhaps other cell types.
A riboflavin auxotroph of Actinobacillus pleuropneumoniae is attenuated in swine
Authors: Fuller T E , Thacker B J , Mulks M H
Published: January 2020

Actinobacillus pleuropneumoniae is the etiological agent of a highly contagious and often fatal pleuropneumonia in swine. A riboflavin-requiring mutant of A. pleuropneumoniae serotype 1, designated AP233, was constructed by deleting a portion of the riboflavin biosynthetic operon (ribGBAH) and replacing it with a gene cassette encoding kanamycin resistance. The genes affected included both the alpha- and beta-subunits of riboflavin synthase as well as a bifunctional enzyme containing GTP cyclohydrase and 3,4-dihydroxy-2-butanone-4-phosphate synthase activities. AP233 was unable to grow in the absence of exogenous riboflavin but otherwise was phenotypically identical to the parent wild-type strain. Experimental infection studies with pigs demonstrated that the riboflavin-requiring mutant was unable to cause disease, on the basis of mortality, lung pathology, and clinical signs, at dosages as high as 500 times the normal 50% lethal dose for the wild-type parent. This is the first demonstration of the attenuation of A. pleuropneumoniae by introduction of a defined mutation in a metabolic gene and the first demonstration that mutations in the genes required for riboflavin biosynthesis can lead to attenuation in a bacterial pathogen.
Cavitary tuberculosis produced in rabbits by aerosolized virulent tubercle bacilli
Authors: Converse P J , Dannenberg A M , Estep J E , Sugisaki K , Abe Y , Schofield B H , Pitt M L
Published: January 2020

Liquefaction of solid caseous tuberculous lesions and the subsequent cavity formation are probably the most dangerous processes in the pathogenesis of human pulmonary tuberculosis. In liquefied caseum, the tubercle bacilli grow extracellularly for the first time since the onset of the disease and can reach such large numbers that mutants with antimicrobial resistance may develop. From a cavity, the bacilli enter the bronchial tree and spread to other parts of the lung and also to other people. Of the commonly used laboratory animals, the rabbit is the only one in which cavitary tuberculosis can be readily produced. This report is the first to describe and analyze the complete course of cavitary tuberculosis, produced by aerosolized virulent bovine-type tubercle bacilli in commercially available New Zealand white rabbits. After the inhalation of 220 to 880 bacillary units, all of the rabbits were overtly well until they were sacrificed at 33 weeks. After the inhalation of 3,900 to 5,800 bacillary units, half of the rabbits died of progressive tuberculosis between 5 and 9 weeks and the other half lived until they were sacrificed at 18 weeks. Pulmonary cavities developed in both low- and high-dose groups, some beginning as early as 6 weeks. Bacilli from primary cavities sometimes caused nearby secondary cavities, but more frequently, they ascended the bronchial escalator, were swallowed, and caused secondary tubercles in the lymphoid tissue of the appendix and ileocecal junction. Histologically, and by culture, the number of bacilli found in the liquefied caseum varied from many to comparatively few. Strong tuberculin reactions at 4 weeks after infection were associated with fewer primary lesions, while strong tuberculin reactions at 33 weeks were associated with more cavitary lesions. In the tuberculous granulation tissue surrounding caseous and liquefied pulmonary foci and cavities, we found many mature epithelioid macrophages that contained high levels of the proteinase cathepsin D. Therefore, cathepsin D probably plays a major role in the liquefaction of solid caseous material and in the subsequent cavity formation.
Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus
Authors: Bilge S S , Vary J C , Dowell S F , Tarr P I
Published: January 2020

Shiga-toxigenic Escherichia coli strains belonging to serotype O157 are important human pathogens, but the genetic basis of expression of the O157 antigen and the role played by the lipopolysaccharide O side chain in the adherence of this organism to epithelial cells are not understood. We performed TnphoA mutagenesis on E. coli O157:H7 strain 86-24 to identify a mutant (strain F12) deficient in O-antigen expression. Nucleotide sequence analysis demonstrated that the transposon inserted within an open reading frame with significant homology to rfbE of Vibrio cholerae O1 (U. H. Stroeher, L. E. Karageorgos, R. Morona, and P. A. Manning, Proc. Natl. Acad. Sci. USA 89:2566-2570, 1992), which is postulated to encode perosamine synthetase. This open reading frame was designated rfbE(EcO157:H7). The guanine-plus-cytosine fraction (0.35) suggests that rfbE(EcO157:H7) may have originated in a species other than E. coli. rfbE(EcO157:H7) is conserved in nontoxigenic E. coli O157 strains expressing a variety of other flagellar antigens but is not found in E. coli O55:H7 strains, which are more closely related to E. coli O157:H7. Strain F12 was significantly more adherent to HeLa cells in a quantitative adherence assay than was its E. coli O157:H7 parent, but they did not differ in other phenotypes. Restoration of the expression of the O side chain by complementation of the TnphoA mutation in strain F12 by a plasmid expressing intact rfbE(EcO157:H7) reduced the adherence of the hyperadherent strain F12. We conclude that rfbE(EcO157:H7) is necessary for the expression of the O157 antigen, that acquisition of E. coli rfb genes occurred independently in E. coli O157:H7 and unrelated O157 strains, and that the O side chain of E. coli O157:H7 lipopolysaccharide interferes with the adherence of E. coli O157:H7 to epithelial cells.
CD4+ T-helper-cell responses in mice with low-level Candida albicans infection
Authors: Mencacci A , Spaccapelo R , Del Sero G , Enssle K H , Cassone A , Bistoni F , Romani L
Published: January 2020

Resistance and susceptibility to Candida albicans infection have been shown to be dependent upon the activation of CD4+ T helper (Th) type 1 or Th2 cells, respectively. To study the type, kinetics, and cytokine dependency of CD4+ Th-cell responses in low-level C. albicans infection, susceptible mice were infected with sublethal doses of C. albicans and assessed for parameters of CD4+ Th-dependent immunity. Interleukin (IL)-12 and gamma interferon were always produced early in infection regardless of the pathogen load. In contrast, production of IL-4, and hence Th2-cell reactivity, was strictly dose dependent, being induced at the higher dose of the fungus. Production of IL-12 correlated with a successful control of infection in mice exposed to the lower doses of C. albicans but not with the development of acquired immunity. An antigenic stimulus appeared to be required for IL-12 to induce a protective anticandidal response. Cytokine depletion in vivo revealed that neutralization of IL-4 was protective early but not late in infection, suggesting a different role for IL-4 in the induction versus maintenance of an ongoing anticandidal Th response. Late in infection, an exacerbative effect was also observed upon IL-12 neutralization. These results indicate that the fungal burden and timing of cytokine appearance greatly influence CD4+ Th induction and effector functions in mice with candidiasis.
Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinases and with hemoglobin
Authors: Dalton J P , McGonigle S , Rolph T P , Andrews S J
Published: January 2020

Two cathepsin L proteinases, cathepsin L1 and cathepsin L2, secreted by liver flukes may be involved in tissue penetration, nutrition, and protection from immune attack. To ascertain the immunoprophylactic potential of these proteinases, and of another molecule, liver fluke hemoglobin (Hb), we performed vaccine trials in cattle. In the first vaccine trial various doses of cathepsin L1 were tested. The mean protection level obtained was 53.7%. In a second vaccine trial cathepsin L1 and Hb elicited 42.5 and 43.8% protection levels, respectively, while a combination of the two molecules induced a significantly higher level of protection (51.9%). Cathepsin L2 was not examined alone; however, vaccination of cattle with a combination of cathepsin L2 and Hb elicited the highest level of protection (72.4%). The animals that received cathepsin L1-Hb or cathepsin L2-Hb showed reduced liver damage as assessed by serum glutamic dehydrogenase and gamma-glutamyl transferase levels. Furthermore, a reduced viability was observed for fluke eggs recovered from all vaccine groups. This anti-embryonation effect of vaccination was particularly evident in the group that received cathepsin L2-Hb where >98% of the eggs recovered did not embryonate to miracidia. Although all vaccine preparations induced high antibody titers which were boosted following the challenge infection, there was no correlation between antibody titers and protection. The results of these trials demonstrate that cathepsin Ls and Hb could form the basis of a molecular vaccine that would not only reduce parasite burden but would also prevent transmission of liver fluke disease.
Effector mechanisms responsible for gamma interferon-mediated host resistance to Legionella pneumophila lung infection: the role of endogenous nitric oxide differs in susceptible and resistant murine hosts
Authors: Heath L , Chrisp C , Huffnagle G , LeGendre M , Osawa Y , Hurley M , Engleberg C , Fantone J , Brieland J
Published: January 2020

To facilitate identification of the effector mechanism(s) responsible for gamma interferon (IFN-gamma)-mediated host resistance to Legionella pneumophila, a murine model of legionellosis in BALB/c mice with a targeted disruption in the IFN-gamma gene (gamma knockout [GKO] mice) was developed. Immunocompetent BALB/c mice and GKO mice were inoculated intratracheally with virulent L. pneumophila (10(6) bacteria per mouse), and bacterial clearance and the pulmonary inflammatory response were assessed. L. pneumophila did not replicate in, and was rapidly cleared from, the lungs of immunocompetent BALB/c mice, demonstrating that immunocompetent BALB/c mice are resistant to replicative L. pneumophila pulmonary infections. In contrast, similarly infected GKO mice developed persistent, replicative intrapulmonary L. pneumophila infections with extrapulmonary dissemination of the bacteria to the spleen. Histopathologic and flow cytometric analysis of L. pneumophila-infected lung tissue demonstrated that while immunocompetent BALB/c mice develop multifocal pneumonitis which resolves, similarly infected GKO mice develop diffuse pneumonitis with persistent neutrophil recruitment into the lung. Intratracheal administration of exogenous IFN-gamma to L. pneumophila-infected GKO mice facilitated intrapulmonary clearance of the bacteria, confirming the pivotal role of IFN-gamma in innate host defenses to L. pneumophila lung infection in this murine host. The potential role of endogenous reactive nitrogen intermediates, including nitric oxide (NO), in IFN-gamma-mediated resistance to L. pneumophila pulmonary infections in immunocompetent BALB/c mice was subsequently assessed. Macrophage inducible nitric oxide synthetase (an enzyme responsible for the production of NO) was induced in alveolar cells from L. pneumophila-infected immunocompetent BALB/c mice (with maximal expression at 48 h postinfection) but was not induced in similarly infected GKO mice. However, administration of the NO synthetase inhibitor N-monomethyl-L-arginine did not significantly inhibit clearance of L. pneumophila from the lung of immunocompetent BALB/c mice (compared with that in similarly infected mice not administered N-monomethyl-L-arginine). In contrast, we have previously demonstrated that IFN-gamma-induced host resistance to replicative L. pneumophila lung infections in a susceptible murine host (A/J mice) is mediated, in part, by endogenous NO. Taken together, these studies identify a differing role of endogenous NO in IFN-gamma-mediated resistance to L. pneumophila pulmonary infection in susceptible and resistant murine hosts.
Influence of cloned tRNA genes from a uropathogenic Escherichia coli strain on adherence to primary human renal tubular epithelial cells and nephropathogenicity in rats
Authors: Susa M , Kreft B , Wasenauer G , Ritter A , Hacker J , Marre R
Published: January 2020

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) possesses pathogenicity islands which are incorporated into two tRNA genes, the selC and the leuX gene. The leuX gene influences the expression of different putative virulence factors. We demonstrate an effect of the leuX-specific tRNA on adherence and uropathogenicity.
Cloning and characterization of the gene encoding the OmpU outer membrane protein of Vibrio cholerae
Authors: Sperandio V , Bailey C , Girón J A , DiRita V J , Silveira W D , Vettore A L , Kaper J B
Published: January 2020

The OmpU outer membrane protein is a member of the ToxR regulon of Vibrio cholerae and has recently been shown to be a potential adherence factor for this species. Using PCR and degenerate oligonucleotide primers based on internal peptide sequences of purified OmpU, we have cloned and sequenced the gene encoding OmpU. The ompU gene is predicted to encode a 36,646-molecular-weight protein which is present in both cholera toxin-positive and -negative V. cholerae O1 and O139 strains.
Passive immunization with monoclonal antibodies against Porphyromonas gingivalis in patients with periodontitis
Authors: Booth V , Ashley F P , Lehner T
Published: January 2020

Selective inhibition of recolonization of Porphyromonas gingivalis was investigated by topical application of monoclonal antibody (MAb). To select a MAb to P. gingivalis with the potential for recognizing most strains of P. gingivalis, we examined seven MAbs, one of which (MAb 61BG 1.3) recognized all 22 laboratory strains and serotypes of P. gingivalis tested as well as 105 human clinical isolates. A comparative study of the number of P. gingivalis bacteria identified by conventional culture and immunofluorescence with MAb 61BG 1.3 showed a very significant correlation between the two methods (Spearman r = 0.85, P < 0.001). Fourteen patients with periodontitis, who harbored P. gingivalis in their subgingival plaque, were treated by root planing and with metronidazole to suppress any detectable P. gingivalis. In this double-blind study, the patients were then divided randomly into two groups; one was treated with MAb to P. gingivalis, and the other was treated with saline. Each patient had four subgingival applications of 3 micrograms of MAb (or saline) per tooth at 1, 3, 7, and 10 days after P. gingivalis was suppressed. The number of P. gingivalis bacteria was then monitored, and significantly less recolonization of the sites with the most severe periodontitis was found in the MAb-treated patients than in the control patients (P < 0.01). This was evident at 6 and 9 months after the application of MAb, but by 12 months, P. gingivalis, was also found to recolonize these sites in two of the MAb-treated patients. The effect of MAb was specific to P. gingivalis, since the numbers of spirochetes were not significantly different between the two groups. However, no significant difference in any clinical periodontal indices between the immunized and control patients at 6 and 12 months was observed. This is the first demonstration that a putative periodontal pathogen can be selectively prevented from recolonization for up to 9 months in sites with the most severe periodontitis. This strategy could be used to establish directly in humans whether a microorganism is involved in the pathogenesis of periodontitis, by repeated application of the corresponding MAb at about 6-month intervals and by comparing the clinical indices between the MAb-treated and control patients.
Orally administered microencapsulated Bordetella pertussis fimbriae protect mice from B. pertussis respiratory infection
Authors: Jones D H , McBride B W , Thornton C , O'Hagan D T , Robinson A , Farrar G H
Published: January 2020

Fimbriae from Bordetella pertussis have been encapsulated in poly(lactide-co-glycolide) microparticles of a size appropriate for uptake by the immune inductive tissues of the gastrointestinal tract. Mice were immunized by oral gavage with a single dose of 10 micrograms of microencapsulated fimbriae. The resulting immune responses were compared with those resulting from intraperitoneal injection of mice with equivalent amounts of fimbriae absorbed onto alhydrogel. The examination of serum and mucosal secretions, collected over a 6-week period, for specific antifimbrial antibodies clearly demonstrated that only orally immunized animals mounted measurable immune responses in external secretions. Six weeks after immunization, all immunized animals were protected against intranasal challenge with live B. pertussis.
Lack of cleavage of IcsA in Shigella flexneri causes aberrant movement and allows demonstration of a cross-reactive eukaryotic protein
Authors: d'Hauteville H , Dufourcq Lagelouse R , Nato F , Sansonetti P J
Published: January 2020

Once in the cytoplasm of mammalian cells, Shigella flexneri expresses a motile phenotype caused by polar directional assembly of actin. This process depends on accumulation of IcsA (VirG), a 120-kDa protein with ATPase activity, at the pole of the bacterium opposite to that at which ongoing septation occurs. IcsA is also secreted into the bacterial supernatant as a 95-kDa species, after cleavage at an SSRRASS sequence which, when mutagenized, blocks processing. MAbF15, an anti-IcsA monoclonal antibody, recognizes an epitope located within repeated Gly-rich boxes in the N-terminal half of the protein. We used this monoclonal antibody to visualize the location of a noncleavable 120-kDa IcsA mutant protein expressed in S. flexneri. We found that this noncleavable IcsA protein no longer localized exclusively to the pole of the bacterium but also could be detected circumferentially. Whereas the monoclonal antibody detected the wild-type cleavable form of IcsA in only 40% of the cells expressing this protein, the noncleavable was easily detectable in all the cells carrying the icsA mutant allele. Similar aberrant localization of the IcsA mutant protein on bacteria growing within the cytoplasm of HeLa cells was observed. The strains expressing the noncleavable IcsA protein expressed abnormal intracellular movement and were often observed moving in a direction perpendicular to their longitudinal axis. The putative protease which processes IcsA may therefore play a role in achieving polar expression of this protein and providing maximum asymmetry essential to directional movement. In addition, MAbF15 allowed us to identify a 70-kDa eukaryotic protein cross-reacting with IcsA. This protein accumulated in the actin tails of motile bacteria and in membrane ruffles of the cells.
Transferrin associated with the porcine intestinal mucosa is a receptor specific for K88ab fimbriae of Escherichia coli
Authors: Grange P A , Mouricout M A
Published: January 2020

Putative receptors of Escherichia coli K88 fimbriae are either tightly membrane bound or an integral part of membranes. Thus, proteins associated with piglet small intestinal mucosae were solubilized by a detergent (deoxycholate). A 74-kDa glycoprotein (GP74) purified from enterocyte and brush border membrane preparations was specifically detected in vitro by K88ab fimbriae. GP74 was recognized only in the mucosae of phenotypically adhesive animals. Metaperiodate treatment abolished the recognition, indicating that K88ab fimbriae-GP74 binding required the carbohydrate moiety. This glycoprotein belongs to the transferrin family and differed from the serum transferrin of the same adhesive-phenotype piglets. Unlike intestinal transferrin, serum transferrin was recognized independently of the adhesion phenotype. The glycan moieties of intestinal and serum transferrins differed in their molar compositions. Transferrin GP74 contained one monosialylated and monofucosylated glycan chain of the N-acetyllactosamine type. Intestinal holotransferrin exhibited pI values of 5.2, 5.3, 5.5, and 5.6, whereas serum holotransferrin pI values ranged between 5.4 and 6.2. Since mucosal transferrin was found intimately entrapped on membranes, we hypothesize that a K88ab fimbriae-transferrin-cell transferrin receptor complex might allow the bacteria to adhere to specific sites of the mucosa.
Persistence of serum and salivary antibody responses after oral immunization with a bacterial protein antigen genetically linked to the A2/B subunits of cholera toxin
Authors: Hajishengallis G , Michalek S M , Russell M W
Published: January 2020

Primary oral immunization of mice with a bacterial protein antigen genetically coupled to the A2 and B subunits of cholera toxin induced specific secretory immunoglobulin A and serum immunoglobulin G antibodies that persisted at substantial levels for at least 11 months. A subsequent single booster immunization did not further enhance the antibody responses. Long-term antibody persistence may be especially important in infections caused by common pathogens for which continuous immunity would be advantageous.
Lymphocyte proliferation in response to Brucella abortus RB51 and 2308 proteins in RB51-vaccinated or 2308-infected cattle
Authors: Stevens M G , Olsen S C , Cheville N F
Published: January 2020

Cattle vaccinated with Brucella abortus strain RB51 (SRB51) or infected with strain 2308 (S2308) had lymph node lymphocytes which proliferated most when incubated with 32-, 27-, 18-, or <18-kDa proteins of either SRB51 or S2308. Some S2308-infected cattle but no SRB51-vaccinated cattle had lymphocytes which proliferated in response to 80- and 49-kDa proteins of SRB51 and S2308. These results suggest that cattle vaccinated with SRB51 or infected with S2308 have lymphocytes which proliferate in response to most of the same S2308 proteins and that the immunodominant protein antigens of SRB51 and S2308 have similar molecular masses of 32, 27, 18, and <18 kDa.
Evidence of multiple extracellular phospholipase activities of Aspergillus fumigatus
Authors: Birch M , Robson G , Law D , Denning D W
Published: January 2020

Extracellular phospholipase activity has been implicated in the pathogenesis of several bacterial infections. Recently, extracellular phospholipase activity has been proposed as a virulence factor in the opportunistic yeast Candida albicans. Aspergillus fumigatus is the most pathogenic member of its genus, responsible for > 90% of infections. Previously, no specific virulence factors have been determined. We investigated the ability of A. fumigatus to produce extracellular phospholipases at 37 degrees C. Fast atom bombardment was used to compare lipid-containing media before and at 5-h intervals during shaking culture of A. fumigatus. Lipids were extracted and analyzed. Many anions corresponding to phospholipid breakdown products were identified. Specific anion species identified indicated phospholipase A, B, C (PLC), and D activities. PLC activity was further investigated by using the synthetic substrate p-nitrophenylphosphorylcholine. PLC activity was initially observed after 30 h of growth and accumulated in broth cultures up to 50 h. At 55 h, there was a sharp increase in PLC activity which coincided with cultures reaching the stationary phase. Activity of the PLC was measured at different temperatures, with greater activity occurring at 37 degrees C than at lower temperatures. Phospholipases could represent a virulence determinant in A. fumigatus.
Enhanced protection by use of a combination of anticapsule and antilipopolysaccharide monoclonal antibodies against lethal Escherichia coli O18K5 infection of mice
Authors: Frasa H , Benaissa-Trouw B , Tavares L , van Kessel K , Poppelier M , Kraaijeveld K , Verhoef J
Published: January 2020

To study antibody-mediated protection against Escherichia coli peritonitis in BALB/c mice, monoclonal antibodies (MAbs) were generated against the capsule (K5) and the lipopolysaccharide (O18) of E. coli. Flow cytometric analysis with two selected immunoglobulin M MAbs revealed that bacteria were antigenically heterogeneous. Arbitrarily, three subpopulations in E. coli O18K5 cultures could be distinguished by double immunofluorescence. A subpopulation bound only the anti-K5 MAb, and another subpopulation bound only the anti-O18 MAb. An intermediate subpopulation, however, bound both MAbs. In agreement with this result, combinations of both MAbs enhanced phagocytosis of fluorescein isothiocyanate-labeled bacteria by human polymorphonuclear leukocytes and mouse macrophage J774 cells as well. In protection experiments, combinations of both MAbs, preincubated with 3 50% lethal doses of E. coli O18K5, protected all mice upon intraperitoneal challenge. Relatively high doses of either MAb alone proved to be not fully protective in this infection model. Protection of mice by the combination of MAbs was associated with significantly lower (P < 0.02) tumor necrosis factor levels in serum 90 min after challenge compared with any other treatment group. Similarly, prophylactic administration of MAbs yielded significantly lower (P < 0.01) tumor necrosis factor levels in mice that received the combination of MAbs than in any other treatment group.
Intracellular survival of Burkholderia pseudomallei
Authors: Jones A L , Beveridge T J , Woods D E
Published: January 2020

Burkholderia pseudomallei is the causative agent of melioidosis, a disease being increasingly recognized as an important cause of morbidity and mortality in many regions of the world. Several features of melioidosis suggest that B. pseudomallei is a facultative intracellular pathogen. This study was designed to assess the ability of B. pseudomallei to invade and survive in eukaryotic cells. We have shown that B. pseudomallei has the capacity to invade cultured cell lines, including HeLa, CHO, A549, and Vero cells. We have demonstrated intracellular survival of B. pseudomallei in professional phagocytic cells, including rat alveolar macrophages. B pseudomallei was localized inside vacuoles in human monocyte-like U937 cells, a histiocytic lymphoma cell line with phagocytic properties. Additionally, electron microscopic visualization of B. pseudomallei-infected HeLa cells and polymorphonuclear leukocytes confirmed the presence of intracellular bacteria within membrane-bound vacuoles. B. pseudomallei was found to be resistant to the cationic peptide protamine and to purified human defensin HNP-1.
Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis
Authors: Heinzen R A , Scidmore M A , Rockey D D , Hackstadt T
Published: January 2020

Coxiella burnetii and Chlamydia trachomatis are bacterial obligate intracellular parasites that occupy distinct vacuolar niches within eucaryotic host cells. We have employed immunofluorescence, cytochemistry, fluorescent vital stains, and fluid-phase markers in conjunction with electron, confocal, and conventional microscopy to characterize the vacuolar environments of these pathogens. The acidic nature of the C. burnetii-containing vacuole was confirmed by its acquisition of the acidotropic base acridine orange (AO). The presence of the vacuolar-type (H+) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demonstrated by indirect immunofluorescence, and growth of C. burnetii was inhibited by bafilomycin A1 (Baf A), a specific inhibitor of the V-ATPase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamydial growth was not inhibited by Baf A or the lysosomotropic amines methylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. burnetii incorporated the fluorescent fluid- phase markers, fluorescein isothiocyanate-dextran (FITC-dex) and Lucifer yellow (LY), indicating trafficking between that vacuole and the endocytic pathway. Neither FITC-dex nor LY was sequestered by chlamydial inclusions. The late endosomal-prelysosomal marker cation-independent mannose 6-phosphate receptor was not detectable in the vacuolar membranes encompassing either parasite. However, the lysosomal enzymes acid phosphatase and cathepsin D and the lysosomal glycoproteins LAMP-1 and LAMP-2 localized to the C. burnetii vacuole but not the chlamydial vacuole. Interaction of C. trachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C6-NBD-ceramide, to the chlamydial inclusion and incorporation into the bacterial cell wall. Similar trafficking of C-NBD-ceramide was not evident in C. burnetii-infected cells. Collectively, the data indicate that C. trachomatis replicates within a nonacidified vacuole that is disconnected from endosome-lysosome trafficking but may receive lipid from exocytic vesicles derived from the trans-Golgi network. These observations are in sharp contrast to those for C. burnetii, which by all criteria resides in a typical phagolysosome.