bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fibrinogen Cleavage by the
Streptococcus pyogenes
Extracellular Cysteine Protease and Generation of Antibodies That Inhibit Enzyme Proteolytic Activity
Authors: Matsuka Yury V. , Pillai Subramonia , Gubba Siddeswar , Musser James M. , Olmsted Stephen B. 
Published: December 2019

ABSTRACT

The extracellular cysteine protease from
Streptococcus pyogenes
is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen α chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH
2
-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein.
Host Responses to Recombinant Hemagglutinin B ofPorphyromonas gingivalisin an Experimental Rat Model
Authors: Katz Jannet , Black Kelley P. , Michalek Suzanne M. 
Published: December 2019

ABSTRACTPorphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Several hemagglutinin genes have been identified, cloned, and expressed inEscherichia coli. The purpose of this study was to characterize host responses to purified recombinant hemagglutinin B (rHag B), using the conventional Fischer rat as the experimental animal model. The effectiveness of immunization with rHag B on protection against experimental periodontal bone loss following infection withP. gingivaliswas also evaluated. Groups of rats were immunized by the subcutaneous route with rHag B in complete Freund’s adjuvant, immunized with rHag B and orally infected withP. gingivalis, nonimmunized and noninfected, or orally infected withP. gingivalisonly. Serum and saliva samples were collected throughout the experiment and evaluated for serum immunoglobulin G (IgG) and IgM and salivary IgA antibody activity by enzyme-linked immunosorbent assay. No salivary IgA anti-Hag B activity was detected in the various groups of rats. A slight serum IgM response similar to that seen in preimmune samples was observed. Serum IgG antibody activity to Hag B was detected only in samples from rats immunized with rHag B. This response was primarily of the IgG1 and IgG2a subclasses, followed by IgG2b and low levels of IgG2c. Supernatants from rHag B-stimulated splenic lymphoid cell cultures from immunized rats contained high levels of gamma interferon, followed by interleukin-2 (IL-2), IL-10, and then IL-4. These results are consistent with the induction of T helper type 1 (Th1)- and Th2-like responses. Western blot analysis of sera derived from rHag B-immunized rats reacted with trichloroacetic acid (TCA) precipitates ofP. gingivalis33277, 381, A7A1-28, and W50, revealing a 50-kDa band reflective of Hag B. However, sera derived from rats immunized withP. gingivaliswhole cells or from rats infected withP. gingivalisonly did not react with rHag B but did react with TCA precipitates ofP. gingivalisstrains. Finally, radiographic measurements of periodontal bone loss indicated that rats immunized with rHag B had less bone loss than those infected withP. gingivalisonly. These results demonstrate the effectiveness of purified rHag B in inducing a protective immune response and support the potential usefulness of this component ofP. gingivalisin the development of a vaccine against adult periodontitis.
Characterization of the Interaction between
Yersinia enterocolitica
Biotype 1A and Phagocytes and Epithelial Cells In Vitro
Authors: Grant Travis , Bennett-Wood Vicki , Robins-Browne Roy M. 
Published: December 2019

ABSTRACT

Yersinia enterocolitica
strains of biotype 1A are increasingly being recognized as etiological agents of gastroenteritis. However, the mechanisms by which these bacteria cause disease differ from those of highly invasive, virulence plasmid-bearing
Y. enterocolitica
strains and are poorly understood. We have investigated several biotype 1A strains of diverse origin for their ability to resist killing by professional phagocytes. All strains were rapidly killed by polymorphonuclear leukocytes but persisted within macrophages (activated with gamma interferon) to a significantly greater extent (survival = 40.5% ± 17.4%) than did
Escherichia coli
HB101 (9.3% ± 0.7%;
P
= 0.0001). Strains isolated from symptomatic patients were significantly more resistant to killing by macrophages (survival = 48.9% ± 19.5%) than were strains obtained from food or the environment (survival = 32.1% ± 10.3%;
P
= 0.04). Some strains which had been ingested by macrophages or HEp-2 epithelial cells showed a tendency to reemerge into the tissue culture medium over a period lasting several hours. This phenomenon, which we termed “escape,” was observed in 14 of 15 strains of clinical origin but in only 3 of 12 nonclinical isolates (
P
= 0.001). The capacity of bacteria to escape from cells was not directly related to their invasive ability. To determine if escape was due to host cell lysis, we used a variety of techniques, including lactate dehydrogenase release, trypan blue exclusion, and examination of infected cells by light and electron microscopy, to measure cell viability and lysis. These studies established that biotype 1A
Y. enterocolitica
strains were able to escape from macrophages or epithelial cells without causing detectable cytolysis, suggesting that escape was achieved by a process resembling exocytosis. The observations that biotype 1A
Y. enterocolitica
strains of clinical origin are significantly more resistant to killing by macrophages and significantly more likely to escape from host cells than are strains of nonclinical origin suggest that these properties may account for the virulence of these bacteria.
Legionella pneumophilaInvasion of MRC-5 Cells Induces Tyrosine Protein Phosphorylation
Authors: Susa Milorad , Marre Reinhard 
Published: December 2019

ABSTRACTAfter uptake and intracellular multiplication ofLegionella pneumophilain MRC-5 lung fibroblasts, important cytoskeletal filament structures, like actin, tubulin, or vimentin, and a cell membrane-associated fibronectin were rearranged during early infection, resulting in a loss of cell adhesion and collapse of the cytoskeleton. Dysregulation of the cellular phosphorylation and dephosphorylation cascade may contribute to the observed changes and may support intracellular survival and multiplication ofL. pneumophila. We therefore studied expression of phosphoproteins during intracellular growth ofL. pneumophila. By using an anti-tyrosine phosphoprotein antibody we showed that proteins phosphorylated on tyrosine residues accumulated progressively during late infection exclusively around or in phagosomes filled with bacteria. In contrast, expression of serine/threonine phosphoproteins did not change. To discern the origin of phosphorylated proteins, the host cells were treated with cycloheximide, an inhibitor of eukaryotic protein synthesis. The newly synthesized proteins were labeled metabolically with [35S]methionine-cysteine and immunoprecipitated with a phosphotyrosine-specific antibody. Sodium dodecyl sulfate gel electrophoresis gave evidence for synthesis of at least three protein clusters (160 to 200, 35 to 60, and 19 to 28 kDa) ofLegionellaorigin that were phosphorylated on tyrosine residues 24 h after infection. Treatment of infected host cells with genistein, a tyrosine kinase inhibitor, revealed that tyrosine protein phosphorylation was not important for bacterial uptake but contributed to intracellular growth ofL. pneumophila. Bacterial tyrosine phosphoproteins and the observed intracellular structural changes may be important to understanding the process involved in intracellular growth ofL. pneumophila.
Invasive Ability of an
Escherichia coli
Strain Isolated from the Ileal Mucosa of a Patient with Crohn’s Disease
Authors: Boudeau Jerome , Glasser Anne-Lise , Masseret Estelle , Joly Bernard , Darfeuille-Michaud Arlette 
Published: December 2019

ABSTRACT

Crohn’s disease (CD) is an inflammatory bowel disease in which
Escherichia coli
strains have been suspected of being involved. We demonstrated previously that ileal lesions of CD are colonized by
E. coli
strains able to adhere to intestinal Caco-2 cells but devoid of the virulence genes so far described in the pathogenic
E. coli
strains involved in gastrointestinal infections. In the present study we compared the invasive ability of one of these strains isolated from an ileal biopsy of a patient with CD, strain LF82, with that of reference enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteraggregative (EAggEC), enterohemorrhagic (EHEC), and diffusely adhering (DAEC)
E. coli
strains. Gentamicin protection assays showed that
E. coli
LF82 was able to efficiently invade HEp-2 cells. Its invasive level was not significantly different from that of EIEC and EPEC strains (
P
> 0.5) but significantly higher than that of ETEC (
P
< 0.03), EHEC (
P
< 0.005), EAggEC (
P
< 0.004) and DAEC (
P
< 0.02) strains. Strain LF82 also demonstrated efficient ability to invade intestinal epithelial cultured Caco-2, Intestine-407, and HCT-8 cells. Electron microscopy examination of infected HEp-2 cells revealed the presence of numerous intracellular bacteria located in vacuoles or free in the host cell cytoplasm. In addition, the interaction of strain LF82 with epithelial cells was associated with the elongation of microvillar extensions that extruded from the host cell membranes and engulfed the bacteria. This internalization mechanism strongly resembles
Salmonella
- or
Shigella
-induced macropinocytosis. The use of cytochalasin D and colchicine showed that the uptake of strain LF82 by HEp-2 cells was mediated by both an actin microfilament-dependent mechanism and microtubule involvement. In addition, strain LF82 survived for at least 24 h in HEp-2 and Intestine-407 cells and efficiently replicated intracellularly in HEp-2 cells. PCR and hybridization experiments did not reveal the presence of any of the genetic determinants encoding EIEC, EPEC, or ETEC proteins involved in bacterial invasion. Thus, these findings show that LF82, which colonized the ileal mucosa of a patient with CD, is a true invasive
E. coli
strain and suggest the existence of a new potentially pathogenic group of
E. coli
, which we propose be designated adherent-invasive
E. coli
.
Resistance to Both Complement Activation and Phagocytosis in Type 3 Pneumococci Is Mediated by the Binding of Complement Regulatory Protein Factor H
Authors: Neeleman Chris , Geelen Sibyl P. M. , Aerts Piet C. , Daha Mohammed R. , Mollnes Tom E. , Roord John J. , Posthuma George , van Dijk Hans , Fleer André 
Published: December 2019

ABSTRACTTo study the role of surface-associated proteins in the virulence ofStreptococcus pneumoniae, we used two serotype 3 strains, ATCC 6303 and WU2, and two PspA-negative mutants of WU2, an encapsulated one, JY1123 (Caps+/PspA−), and an unencapsulated one, DW3.8 (Caps−/PspA−). ATCC 6303 and WU2 were highly virulent in mice, while the virulence of JY1123 was slightly decreased (50% lethal doses [LD50s], 24, 6, and 147 CFU/mouse, respectively); DW3.8 was avirulent (LD50, 2 × 108CFU). In vitro, ATCC 6303, WU2, and JY1123 (Caps+/PspA−) strongly resisted complement activation and complement-dependent opsonophagocytosis, whereas DW3.8 (Caps−/PspA−) was easily phagocytized in fresh serum. Trypsin treatment of ATCC 6303, WU2, and JY1123 (Caps+/PspA−) resulted in enhanced complement activation and complement-dependent opsonophagocytosis. Trypsin had no deleterious effect on the polysaccharide capsule. In addition, trypsin pretreatment of ATCC 6303 strongly reduced virulence upon intraperitoneal challenge in mice. This indicated that surface proteins play a role in the resistance to complement activation and opsonophagocytosis and contribute to the virulence of type 3 pneumococci. In subsequent experiments, we could show that the modulation of complement activation was associated with surface components that bind complement regulator factor H; binding is trypsin sensitive and independent of prior complement activation. Immunoblotting of cell wall proteins of the virulent strain ATCC 6303 with anti-human factor H antibody revealed three factor H-binding proteins of 88, 150, and 196 kDa. Immunogold electron microscopy showed a close association of factor H-binding components with the outer surface of the cell wall. The role of these factor H-binding surface proteins in the virulence of pneumococci is interesting and warrants further investigation.
Replication-Defective Adenovirus Infection ReducesHelicobacter felisColonization in the Mouse in a Gamma Interferon- and Interleukin-12-Dependent Manner
Authors: Jiang Bo , Jordana Manel , Xing Zhou , Smaill Fionna , Snider Denis P. , Borojevic Rajka , Steele-Norwood Darlene , Hunt Richard H. , Croitoru Kenneth 
Published: December 2019

ABSTRACTHelicobacter infection leads to chronic inflammation of the stomach. Although the infection persists in spite of an immune response, animal studies have shown that adjuvant-based oral vaccines can protect against infection and even eliminate established infection. These vaccines are thought to induce a Th2 immune response, counterbalancing the Th1 response seen with natural infections. As a prelude to using adenovirus vectors carrying cytokine genes to modulate the immune response to establishedHelicobacter felisinfection, we first examined the effect of the replication-defective adenovirus (RDA) vector itself. C57BL/6 mice chronically infected withH. felis(8 to 10 weeks) received intramuscular injections of RDA. The effect of RDA on the severity ofH. feliscolonization and the degree of gastric inflammation was assessed 2 weeks later. RDA caused a significant decrease inH. feliscolonization without significantly altering the associated inflammation. RDA did not alter theH. felis-specific immunoglobulin G1 (IgG1), IgG2a, and IgA responses in the serum but was associated with an increase in gamma interferon (IFN-γ)-producing CD8+spleen cells. To determine if IFN-γ or Th1 cytokines were involved in the response to RDA, we examined RDA treatment ofH. felisinfection in mice lacking either IFN-γ or interleukin-12 (IL-12). RDA failed to alterH. feliscolonization in either of these two mouse strains. Thus, viral infection of mice chronically infected withH. felisled to a significant decrease inH. feliscolonization in an IFN-γ- and IL-12-dependent manner. These results demonstrate that Th1 responses associated with systemic viral infection can influence an establishedH. felisinfection.
Isolation of Peptides That Mimic Epitopes on a Malarial Antigen from Random Peptide Libraries Displayed on Phage
Authors: Adda Christopher G. , Tilley Leann , Anders Robin F. , Foley Michael 
Published: December 2019

ABSTRACT

The ring-infected erythrocyte surface antigen (RESA) is a dense-granule protein of
Plasmodium falciparum
which binds to the cytoskeletal structure of the erythrocyte after parasite invasion. It is currently under trial as a vaccine candidate. In an effort to characterize further the antibody responses to this antigen, we have panned two independent libraries of random peptides expressed on the surface of filamentous phage with a monoclonal antibody (MAb 18/2) against RESA. One library consisted of a potentially constrained 17-mer peptide fused with the gpVIII phage coat protein, and the other displayed an unconstrained 15-mer as a fusion with the minor phage coat protein gpIII. Several rounds of biopanning resulted in enrichment from both libraries clones that interacted specifically with MAb 18/2 in protein-blotting and enzyme-linked immunosorbent assay experiments. Nucleotide sequencing of the random oligonucleotide insert revealed a common predominant motif: (S/T)AVDD. Several other clones had related but degenerate motifs. Thus, a monoclonal antibody against a malarial antigen can select common mimotopes from different random peptide libraries. We envisage many uses for this technology in malaria research.
Antiphosphorylcholine Antibody Levels Are Elevated in Humans with Periodontal Diseases
Authors: Schenkein Harvey A. , Gunsolley John C. , Best Al M. , Harrison M. Travis , Hahn Chin-Lo , Wu Jiuhua , Tew John G. 
Published: December 2019

ABSTRACTHuman immunoglobulin G2 (IgG2) serum concentrations and the IgG2 antibody response toActinobacillus actinomycetemcomitanscan be influenced by genes, by environmental factors such as smoking, and by periodontal disease status. Examination of the IgG2 response to phosphorylcholine (PC), a response thought to be mainly induced by the C polysaccharide ofStreptococcus pneumoniae, suggested that periodontal disease status was also associated with this response. This prompted the hypothesis that PC is an important oral antigen associated with organisms in the periodontal flora and that anti-PC antibody is elevated as a consequence of periodontal disease. Subjects in various periodontal disease diagnostic categories in which attachment loss is exhibited were tested for anti-PC in serum. Those with adult periodontitis, localized juvenile periodontitis, generalized early-onset periodontitis, and gingival recession all had similar levels of anti-PC IgG2 serum antibody which were significantly greater than in the group of subjects with no attachment loss. Analysis of plaque samples from subgingival and supragingival sites in all diseases categories for reactivity with the anti-PC specific monoclonal antibody TEPC-15 revealed that a substantial proportion of the bacteria in dental plaque (30 to 40%) bear PC antigen; this antigen was not restricted to morphotypes resembling only cocci but was also present on rods and branched filamentous organisms. We found thatS. mitis,S. oralis, andS. sanguis, as well as oral actinomycetes, includingA. viscosus,A. odontolyticus, andA. israelii, incorporated substantial amounts of [3H]choline from culture media. Further analysis of antigens derived from these organisms by Western blot indicated thatS. oralis,S. sanguis,A. viscosus,A. odontolyticus, andA. israeliicontained TEPC-15-reactive antigens. The data show that many commonly occurring bacterial species found in dental plaque contain PC antigen and that immunization with plaque-derived PC antigens as a consequence of inflammation and periodontal attachment loss may influence systemic anti-PC antibody concentrations.
Role of Complement Receptors in Uptake ofMycobacterium aviumby Macrophages In Vivo: Evidence from Studies Using CD18-Deficient Mice
Authors: Bermudez Luiz E. , Goodman Joseph , Petrofsky Mary 
Published: December 2019

ABSTRACTMycobacterium aviumis an intracellular pathogen that has been shown to invade macrophages by using complement receptors in vitro, but mycobacteria released from one cell can enter a second macrophage by using receptors different from complement receptors. Infection of CD18 (β2integrin) knockout mice and the C57 BL/6 control mice led to comparable levels of tissue infection at 1 day, 2 days, 1 week, and 3 weeks following administration of bacteria. A histopathological study revealed similar granulomatous lesions in the two mouse strains, with comparable numbers of organisms. In addition, transmission electron microscopy of spleen tissues from both strains of mice showed bacteria inside macrophages. Our in vivo findings support the hypothesis thatM. aviumin the host is likely to use receptors other than CR3 and CR4 receptors to enter macrophages with increased efficiency.
Molecular Mechanism for the Spontaneous Generation of Pigmentless
Porphyromonas gingivalis
Mutants
Authors: Chen Wen , Kuramitsu Howard K. 
Published: December 2019

ABSTRACT

Porphyromonas gingivalis
is one of the pathogens associated with periodontal diseases, and its protease activity has been implicated as an important virulence factor. Kgp is the major Lys-gingipain protease of
P. gingivalis
and appears to be involved not only in enzyme activity but also in hemagglutination and the pigmented phenotype due to heme accumulation and/or hemoglobin binding. However, little information concerning the molecular mechanism for the spontaneous generation of pigmentless
P. gingivalis
mutants is currently available. In this study, several spontaneous pigmentless mutants of
P. gingivalis
were isolated and characterized. The results revealed that a portion of the
kgp
gene had been deleted from the chromosomes of the pigmentless mutants. This deletion appears to result from recombination between the highly homologous DNA sequences encoding the adhesin domains of the tandemly arranged
hagA
and
kgp
genes on the chromosomes of
P. gingivalis
strains.
Virulence ofMycobacterium tuberculosisCDC1551 and H37Rv in Rabbits Evaluated by Lurie’s Pulmonary Tubercle Count Method
Authors: Bishai William R. , Dannenberg Arthur M. , Parrish Nikki , Ruiz Rafael , Chen Ping , Zook Bernard C. , Johnson Walter , Boles James W. , Pitt M. Louise M. 
Published: December 2019

ABSTRACTThe virulence of the CDC1551 strain ofMycobacterium tuberculosiswas compared to that of H37Rv in a rabbit inhalation model. While rabbits that inhaled the two strains produced equal numbers of grossly visible primary tubercles, CDC1551 tubercles were smaller and contained fewer bacilli than H37Rv tubercles. These findings suggest that a miniepidemic near the Kentucky-Tennessee border caused by CDC1551 was due not to increased virulence but to increased transmissibility.
Outer Membrane Proteins Omp10, Omp16, and Omp19 of
Brucella
spp. Are Lipoproteins
Authors: Tibor Anne , Decelle Béatrice , Letesson Jean-Jacques 
Published: December 2019

ABSTRACT

The deduced sequences of the Omp10, Omp16, and Omp19 outer membrane proteins of
Brucella
spp. contain a potential bacterial lipoprotein processing sequence. After extraction with Triton X-114, these three proteins partitioned into the detergent phase. Processing of the three proteins is inhibited by globomycin, a specific inhibitor of lipoprotein signal peptidase. The three proteins were radioimmunoprecipitated from [
3
H]palmitic acid-labeled
Brucella abortus
lysates with monoclonal antibodies. These results demonstrate that Omp10, Omp16, and Omp19 are lipoproteins.