bluuebunny/crossref_metadata_2025 huggingface dataset:
Borrelia burgdorferi
Erp Proteins Are Immunogenic in Mammals Infected by Tick Bite, and Their Synthesis Is Inducible in Cultured Bacteria
Authors: Stevenson Brian , Bono James L. , Schwan Tom G. , Rosa Patricia
Published: December 2019

Borrelia burgdorferi
, the causative agent of Lyme disease, can contain multiple genes encoding different members of the Erp lipoprotein family. Some arthropod-borne bacteria increase the synthesis of proteins required for transmission or mammalian infection when cultures are shifted from cool, ambient air temperature to a warmer, blood temperature. We found that all of the
erp
genes known to be encoded by infectious isolate B31 were differentially expressed in culture after a change in temperature, with greater amounts of message being produced by bacteria shifted from 23 to 35°C than in those maintained at 23°C. Mice infected with B31 by tick bite produced antibodies that recognized each of the Erp proteins within 4 weeks of infection, suggesting that the Erp proteins are produced by the bacteria during the early stages of mammalian infection and may play roles in transmission from ticks to mammals. Several of the B31 Erp proteins were also recognized by antibodies from patients with Lyme disease and may prove to be useful antigens for diagnostic testing or as components of a protective vaccine.
Effects of Granulocyte-Macrophage Colony-Stimulating Factor and Tumor Necrosis Factor Alpha onTrypanosoma cruziTrypomastigotes
Authors: Fontt Elizabeth Olivares , De Baetselier Patrick , Heirman Carlo , Thielemans Kris , Lucas Ralph , Vray Bernard
Published: December 2019

We have previously shown that the addition of exogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) to nonactivated mouse peritoneal macrophages (MPM) limitsTrypanosoma cruziinfections in vitro (E. Olivares Fontt and B. Vray, Parasite Immunol. 17:135–141, 1995). Lower levels of infection were correlated with a higher level of production of tumor necrosis factor alpha (TNF-α) in the absence of nitric oxide (NO) release. These data suggested that GM-CSF and/or TNF-α might have a direct parasitocidal effect onT. cruzitrypomastigotes, independently of NO release. To address this question,T. cruzitrypomastigotes were treated with recombinant murine GM-CSF (rmGM-CSF), recombinant murine TNF-α (rmTNF-α), or both cytokines in a cell-free system. Treatment with rmGM-CSF but not rmTNF-α caused morphological changes in the parasites, and most became spherical after 7 h of incubation. Both cytokines exerted a cytolytic activity on the trypomastigotes, yet the trypanolytic activity of rmTNF-α was more effective than that of rmGM-CSF. Viable rmGM-CSF- and rmTNF-α-treated parasites were less able to infect MPM than untreated parasites, and this reduction in infectivity was greatest for rmGM-CSF. Treatments with both cytokines resulted in more lysis and almost complete inhibition of infection. The direct parasitocidal activity of rmTNF-α was inhibited by carbohydrates and monoclonal antibodies specific for the lectin-like domain of TNF-α. Collectively, these results suggest that cytokines such as GM-CSF and TNF-α may directly control the level ofT. cruzitrypomastigotes at least in vitro and so could determine the outcome of infection in vivo.
Effects of Toxin A fromClostridium difficileon Mast Cell Activation and Survival
Authors: Calderón Gloria M. , Torres-López Javier , Lin Tong-Jun , Chavez Bibiana , Hernández Manuel , Muñoz Onofre , Befus A. Dean , Enciso J. Antonio
Published: December 2019

Toxins A and B fromClostridium difficileare the main cause of antibiotic-associated diarrhea and pseudomembranous colitis. They cause fluid accumulation, necrosis, and a strong inflammatory response when inoculated in intestinal loops. Since mast cells are a rich source of inflammatory mediators, abundant in the gut, and known to be involved inC. difficile-induced enteritis, we studied the in vitro effect of toxin A on isolated mast cells. Normal rats sensitized by infection withNippostrongilus brasiliensiswere used to isolate peritoneal mast cells (PMC). PMC from naive rats were stimulated with calcium ionophore A23187 as a model of antigen-independent activation, and PMC from sensitized rats were stimulated withN. brasiliensisantigens to study immunoglobulin E-dependent mast cell activation. After 4 h, toxin A did not induce release of nitric oxide or histamine in naive PMC. However, 10 ng of toxin per ml caused a significant release of tumor necrosis factor alpha (TNF-α). In contrast, 1 μg of toxin per ml inhibited antigen or A23187-induced histamine release by PMC. Toxin A at 1 μg/ml for 4 h caused disruption of actin which aggregated in the cytoplasm and around the nucleus. After 24 h, chromatin condensation, cytoplasmic blebbing, and apoptotic-like vesicles were observed; DNA fragmentation was documented also. These results suggest that mast cells may participate in the initial inflammatory response toC. difficileinfection by releasing TNF-α upon interaction with toxin A. However, longer exposure to toxin A affects the release of inflammatory mediators, perhaps because of the alteration of the cytoskeleton and induction of apoptosis. The impaired functions and survival of mast cells byC. difficiletoxin A could hamper the capacity of these cells to counteract the infection, thus prolonging the pathogenic effects ofC. difficiletoxins.
Experimental Infection of Pregnant Ewes with
Chlamydia pecorum
Authors: Philips Helen L. , Clarkson Michael J.
Published: December 2019

Pregnant ewes were infected in midpregnancy with three isolates of
Chlamydia pecorum
derived from the feces of healthy lambs from three different farms. Oral infection, alone or together with
Fasciola hepatica
, did not result in tissue invasion, since all placental and fecal samples were negative for chlamydiae. Intravenous infection resulted in placental infection in 16 of 18 ewes in that chlamydiae were cultured from placentas or vaginal swabs. Two ewes bore dead lambs after a shortened gestation time. The chlamydiae isolated were all
C. pecorum
. There were no significant differences between the weights of the lambs from the infected groups and those from uninfected control ewes. Most ewes showed no serological evidence of infection by the complement fixation test; therefore, it is unlikely that the enteric subtype of
C. pecorum
is responsible for the cross-reactions sometimes seen in flocks being tested for
C. psittaci
infection.
Helicobacter pylori
Disrupts Epithelial Barrier Function in a Process Inhibited by Protein Kinase C Activators
Authors: Terrés A. M. , Pajares J. M. , Hopkins A. M. , Murphy A. , Moran A. , Baird A. W. , Kelleher D.
Published: December 2019

Helicobacter pylori
colonizes the gastric mucosa, and the infection is related to the development of diverse gastric pathologies, possibly by directly or indirectly affecting epithelial-cell function. We analyzed the influence of the bacteria on transepithelial electrical resistance (TER) on a model tight epithelium, T84, grown to confluence in permeable filters.
H. pylori
sonicates produced a dramatic decrease in TER after 1 to 2 h of exposure, while sonicates from other bacteria did not induce a significant reduction of TER. The effect induced by sonicates was mimicked by a water-soluble fraction from the bacterial surface, was not reproducible with isolated lipopolysaccharide, and was concomitant with a significant increase in the paracellular permeability of the marker molecule [
14
C]mannitol. Furthermore,
H. pylori
sonicates also provoked a significant increase in permeability to [
14
C]mannitol across rat gastric mucosa in vitro. The sonicate-induced decrease in TER in T84 monolayers was inhibited by the protein kinase C (PKC) activator phorbol myristate acetate. As PKC is directly involved in tight junction regulation, we suggest that
H. pylori
may induce intracellular signalling events counteracting PKC effects. Following long-term
H. pylori
stimulation, epithelial monolayers regained baseline resistance values slowly after 24 h. The resistance recovery process was inhibited by cycloheximide, indicating its dependency upon protein synthesis. No association between resistance variation and E-cadherin protein levels was observed. These results indicate that
H. pylori
alters in vitro the barrier properties of the epithelium, probably by generating cell signalling events counteracting the normal function of PKC. This increased permeability may provide a potential mechanism by which
H. pylori
antigens can reach the gastric lamina propria, thereby activating the mucosal immune system.
Trypanosoma cruziInfection in Tumor Necrosis Factor Receptor p55-Deficient Mice
Authors: Castaños-Velez Esmeralda , Maerlan Stephanie , Osorio Lyda M. , Åberg Frederik , Biberfeld Peter , Örn Anders , Rottenberg Martín E.
Published: December 2019

Tumor necrosis factor receptor p55 (TNFRp55) mediates host resistance to several pathogens by allowing microbicidal activities of phagocytes. In the studies reported here, TNFRp55−/−mice infected with the intracellular parasiteTrypanosoma cruzishowed clearly higher parasitemia and cumulative mortality than wild-type (WT) controls did. However, gamma interferon (IFN-γ)-activated macrophages from TNFRp55−/−mice produced control levels of nitric oxide and killed the parasite efficiently in vitro. Trypanocidal mechanisms of nonphagocytic cells (myocardial fibroblasts) from both TNFRp55−/−and WT mice were also activated by IFN-γ in a dose-dependent way. However, IFN-γ-activated TNFRp55−/−nonphagocytes showed less effective killing ofT. cruzithan WT control nonphagocytes, even when interleukin 1β (IL-1β) was added as a costimulator. In vivo,T. cruzi-infected TNFRp55−/−mice and WT mice released similar levels of NO and showed similar levels of IFN-γ mRNA and inducible nitric oxide synthase mRNA in their tissues. Instead, increased susceptibility toT. cruziof TNFRp55−/−mice was associated with reduced levels of parasite-specific immunoglobulin G (IgG) (but not IgM) antibodies during infection, which is probably linked to abnormal B-cell differentiation in secondary lymphoid tissues of the mutant mice. Surprisingly,T. cruzi-infected TNFRp55−/−mice showed increased inflammatory and necrotic lesions in several tissues, especially in skeletal muscles, indicating that TNFRp55 plays an important role in controlling the inflammatory process. Accordingly, levels of Mn2+superoxide dismutase mRNA, a TNF-induced enzyme which protects the cell from the toxic effects of superoxide, were lower in mutant than in WT infected mice.
Formation of Ring-Shaped Structures on Erythrocyte Membranes after Treatment with Botulinolysin, a Thiol-Activated Hemolysin from
Clostridium botulinum
Authors: Sekiya Kachiko , Danbara Hirofumi , Futaesaku Yutaka , Haque Abdul , Sugimoto Nakaba , Matsuda Morihiro
Published: December 2019

Damage to erythrocyte membranes by botulinolysin (BLY) was studied by electron microscopy, which revealed ring-shaped structures with inner diameters and widths of approximately 32 and 6.7 nm, respectively. BLY bound to membranes at 0°C, but subsequent treatment with glutaraldehyde prevented ring formation during further incubation at 37°C. Zn
2+
ions inhibited ring formation but not binding of BLY to membranes.
Molecular Characterization of
Treponema pallidum mcp2
, a Putative Chemotaxis Protein Gene
Authors: Greene Shermalyn R. , Stamm Lola V.
Published: December 2019

The nucleotide sequence of the
Treponema pallidum mcp2
gene was determined.
mcp2
encodes a 45.8-kDa protein whose deduced amino acid sequence has significant homology with the C-terminal region of bacterial methyl-accepting chemotaxis proteins (MCPs). The Mcp2 N terminus lacks the hydrophobic transmembrane regions present in most MCPs. An Mcp2 fusion protein was strongly reactive with antibody (HC23) to the highly conserved domain of MCPs and with rabbit syphilitic serum. Antibody HC23 reacted with six
T. pallidum
proteins, including a 45-kDa protein that may correspond to Mcp2. This protein was present in the aqueous phase from
T. pallidum
cells that were solubilized with Triton X-114 and phase partitioned.
Human Antibody Response toHelicobacter pyloriLipopolysaccharide: Presence of an Immunodominant Epitope in the Polysaccharide Chain of Lipopolysaccharide
Authors: Yokota Shin-ichi , Amano Ken-ichi , Hayashi Shunji , Kubota Toru , Fujii Nobuhiro , Yokochi Takashi
Published: December 2019

We have examined the antibody response toHelicobacter pylorilipopolysaccharides (LPS) in humans. We used sera from patients with gastroduodenal diseases and healthy adults infected or not infected withH. pylori. Data from the experiments for antibody binding to LPS suggested that the polysaccharide chains from manyH. pyloristrains showed high immunogenicity in humans. Sera from most (above 70%)H. pylori-infected individuals contained immunoglobulin G (IgG) antibodies against the polysaccharide region highly immunogenicH. pyloriLPS. The IgG titers of individual serum samples that reacted strongly with highly immunogenic LPS were quite similar (r2= 0.84 to 0.98). The results suggest wide distribution amongH. pyloristrains of a highly antigenic epitope in the polysaccharide moieties of their LPS. Also, the similarity in the titers of individual serum samples against highly immunogenic LPS points to the existence of epitopes sharing a common structural motif. However, some strains showed low antigenicity, even those with polysaccharide-carrying LPS. The dominant subclass of IgG that reacted with the highly immunogenic LPS was IgG2, which was preferentially raised against polysaccharide antigens. Recently, a structure that mimics that of the Lewis antigens was identified in the O-polysaccharide fraction ofH. pyloriLPS; however, no correlation between antigenicity of the polysaccharide chain in humans and the presence of Lewis antigens was found. The IgA and IgM titers againstH. pyloriLPS seemed to be mostly nonspecific and directed against lipid A. In a few cases, however, sera from individuals infected withH. pylorigave strong IgA and IgM titers against the highly immunogenic polysaccharide. In conclusion, the LPS of manyH. pyloristrains possess an antigenic epitope in their polysaccharide regions that is immunogenic in humans. However, our results show that the antigenic epitope is unlikely to be immunologically related to structures mimicking Lewis antigens.
Inhibition of Human Peripheral Blood Mononuclear Cell Proliferation byStreptococcus pyogenesCell Extract Is Associated with Arginine Deiminase Activity
Authors: Degnan B. A. , Palmer J. M. , Robson T. , Jones C. E. D. , Fischer M. , Glanville M. , Mellor G. D. , Diamond A. G. , Kehoe M. A. , Goodacre J. A.
Published: December 2019

Streptococcus pyogenes(group AStreptococcus) cell extracts (CE) have a remarkably powerful and dose-dependent inhibitory effect on antigen, superantigen, or mitogen-stimulated human peripheral blood mononuclear cell (PBMC) proliferation in vitro. Purification of the inhibitory component present inS. pyogenestype M5 (Manfredo strain) CE by anion-exchange chromatography followed by gel filtration chromatography showed that the inhibitor had an approximate native molecular mass of 100 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inhibitory fractions followed by silver staining gave a single band with an approximate molecular mass of 47 kDa, indicating that the inhibitor is composed of two identical subunits. NH2-terminal sequencing of the protein revealed that it was identical to the previously characterized streptococcal acid glycoprotein (SAGP); this protein possesses between 31.5 and 39.0% amino acid identity with arginine deiminase (AD) fromMycoplasma hominis,Mycoplasma arginini,Pseudomonas putida, andPseudomonas aeruginosa. AD enzyme activity was present in unfractionated CE prepared from a range of streptococcal strains, and partially purified inhibitory fractions of Manfredo CE also had high levels of activity. The inhibitory effect of Manfredo CE was overcome by the addition ofl-arginine to proliferation assays in which human PBMC were stimulated with phytohemagglutinin. We conclude that SAGP, or its homolog, possesses AD activity and that the potent inhibition of proliferation of human T cells by streptococcal CE is due to activity of this enzyme.
Effect of Mild Acid Treatment on the Survival, Enteropathogenicity, and Protein Production in
Vibrio parahaemolyticus
Authors: Wong Hin-chung , Peng Po-Yen , Han Jun-Ming , Chang Chia-Yu , Lan Shang-Lun
Published: December 2019

Vibrio parahaemolyticus
is an important food-borne enteropathogen that encounters various adverse conditions in its native environment or during infection. Effects of mild acid treatment on survival under stress conditions, enteropathogenicity, and protein production in this pathogen were investigated. Logarithmically grown cells, at pH 7.5 shifted to pH 5.0 for 30 min, were more resistant to subsequent acid challenge at pH 4.4. A two-phase adaptive procedure (pH 5.8 for 30 min; pH 5.0 for 30 min) was better than a single-phase procedure for enhancing the acid tolerance of this pathogen. The acid-adapted cells were cross-protected against the challenges of low salinity and thermal inactivation. One-dimensional polyacrylamide gel electrophoresis revealed that proteins with molecular masses of 6.4, 9.0, 13.6, 16.3, 18.9, 22.9, 24.4, 28.3, 33.9, 36.9, 41.2, 47.6, 58.1, 65.6, 80.5, 88.2, and 96.9 kDa were induced or significantly enhanced, while proteins of 25.3, 30.1, 30.7, and 91.7 kDa were significantly inhibited. Two-dimensional polyacrylamide gel electrophoresis revealed that 20 species of proteins were induced or significantly enhanced, while 26 species were inhibited. In assays conducted using the suckling mouse model, enteropathogenicity of the acid-adapted cells was significantly enhanced in terms of intestine/body weight ratio and in vivo recovery of infected cells.
Immunological and Molecular Characterization of Three Variant Subtype P1.14 Strains of
Neisseria meningitidis
Authors: Saunders Nancy B. , Brandt Brenda L. , Warren Richard L. , Hansen Brian D. , Zollinger Wendell D.
Published: December 2019

Epidemic outbreaks of group B meningococcal disease exhibit a clonal nature consisting of a common serotype-subtype. Subtype-specific monoclonal antibodies (MAbs) directed toward two variable regions (VR1 and VR2) of the class 1 protein of
Neisseria meningitidis
are used in this classification scheme. A new MAb was developed to classify a nonsubtypeable (NST) strain of
N. meningitidis
, 7967. This MAb bound to both the NST strain and the prototype subtype P1.14 strain, S3446, by dot blot analysis. However, a MAb produced to the prototype P1.14 strain did not bind to strain 7967. Sixteen additional strains were further identified as P1.14 with the prototype MAb; of these, 15 strains bound both MAbs. Differences in the characteristics of binding of both antibodies to the three apparently diverse P1.14 strains were studied further by using outer membrane complex proteins, immobilized peptides, and soluble peptides. Deduced amino acid analysis suggested that both MAbs bind to VR2 and that single amino acid changes within VR2 (KM, NM, or KK) might explain the differences in binding characteristics. These results demonstrated that minor variations which exist within subtype variable regions may be clearly identified only by a combination of molecular and immunologic testing. The impact of subtype variation will become more evident as subtype-specific vaccines are developed and tested for efficacy.
Cloning of the Gene Encoding the 44-Kilodalton Antigen of the Agent of Human Granulocytic Ehrlichiosis and Characterization of the Humoral Response
Authors: Ijdo Jacob W. , Sun Wei , Zhang Yan , Magnarelli Louis A. , Fikrig Erol
Published: December 2019

Antibodies in the sera of patients with human granulocytic ehrlichiosis (HGE) commonly recognize a 44-kDa antigen. We cloned the gene encoding the 44-kDa protein of the agent of HGE (aoHGE) by probing an aoHGE lambda ZAP II genomic DNA expression library with sera from aoHGE-infected mice. The gene,hge-44, is part of a multigene family, with sequence similarity to theAnaplasma marginale msp-2genes. RNA-PCR studies confirmed thathge-44is expressed by aoHGE cultured in HL-60 cells and by aoHGE during murine infection. Recombinant HGE-44, expressed and purified as a glutathione transferase fusion protein, was used as the substrate in immunoblots to help diagnose HGE. Antibodies in eight sera from eight patients with HGE and in two sera from two aoHGE-infected mice bound recombinant HGE-44. Antibodies in the sera of healthy individuals or patients withEhrlichia chaffeensisorBorrelia burgdorferiinfection did not recognize HGE-44. We conclude thathge-44is a member of a multigene family and thathge-44is expressed and elicits specific antibodies during infection.
Human and Murine Immune Responses to a NovelLeishmania majorRecombinant Protein Encoded by Members of a Multicopy Gene Family
Authors: Webb John R. , Campos-Neto Antonio , Ovendale Pamela J. , Martin Tricia I. , Stromberg Erika J. , Badaro Roberto , Reed Steven G.
Published: December 2019

Vaccination of BALB/c mice withLeishmania majorpromastigote culture filtrate proteins plusCorynebacterium parvumconfers resistance to infection withL. major. To define immunogenic components of this protein mixture, we used sera from vaccinated mice to screen anL. majoramastigote cDNA expression library. One of the immunoreactive clones thus obtained encoded a novel protein ofL. majorwith a molecular mass of 22.1 kDa. The predicted amino acid sequence of this clone exhibited significant homology to eukaryotic thiol-specific-antioxidant (TSA) proteins. Therefore, we have designated this proteinL. majorTSA protein. Southern blot hybridization analyses indicate that there are multiple copies of the TSA gene in all species ofLeishmaniaanalyzed. Northern blot analyses demonstrated that the TSA gene is constitutively expressed inL. majorpromastigotes and amastigotes. Recombinant TSA protein containing an amino-terminal six-histidine tag was expressed inEscherichia coliwith the pET17b system and was purified to homogeneity by affinity chromatography. Immunization of BALB/c mice with recombinant TSA protein resulted in the development of strong cellular immune responses and conferred protective immune responses against infection withL. majorwhen the protein was combined with interleukin 12. In addition, recombinant TSA protein elicited in vitro proliferative responses from peripheral blood mononuclear cells of human leishmaniasis patients and significant TSA protein-specific antibody titers were detected in sera of both cutaneous-leishmaniasis and visceral-leishmaniasis patients. Together, these data suggest that the TSA protein may be useful as a component of a subunit vaccine against leishmaniasis.
In Vivo Phase Variation of
Escherichia coli
Type 1 Fimbrial Genes in Women with Urinary Tract Infection
Authors: Lim Jean K. , Gunther Nereus W. , Zhao Hui , Johnson David E. , Keay Susan K. , Mobley Harry L. T.
Published: December 2019

Type 1 fimbriae, expressed by most
Escherichia coli
strains, are thought to attach to human uroepithelium as an initial step in the pathogenesis of urinary tract infections (UTI). Numerous reports using both in vitro and murine models support this role for type 1 fimbriae in colonization. Unfortunately, only a limited number of studies have directly examined the expression of fimbriae in vivo. To determine whether type 1 fimbrial genes are transcribed during an acute UTI, we employed a modification of an established method. The orientation (ON or OFF) of the invertible promoter element, which drives transcription of type 1 fimbrial genes, was determined by PCR amplification using primers that flank the invertible element, followed by
Sna
BI digestion. The orientation of the type 1 fimbrial switch was determined under three experimental conditions. First,
E. coli
strains from different clinical sources (acute pyelonephritis patients, cystitis patients, and fecal controls) were tested under different in vitro culture conditions (agar versus broth; aerated versus static). The genes in the more-virulent strains (those causing acute pyelonephritis) demonstrated a resistance, in aerated broth, to switching from OFF to ON, while those in fecal strains readily switched from OFF to ON. Second, bladder and kidney tissue from CBA mice transurethrally inoculated with
E. coli
CFT073 (an established murine model of ascending UTI) was assayed. The switches directly amplified from infected bladder and kidney tissues were estimated to be 33 and 39% ON, respectively, by using a standard curve. Finally, bacteria present in urine samples collected from women with cystitis were tested for type 1 fimbria switch orientation. For all 11 cases, an average of only 4% of the switches in the bacteria in the urine were ON. In 7 of the 11 cases, we found that all of the visible type 1 fimbrial switches were in the OFF position (upper limit of detection of assay, 98% OFF). Strains recovered from these urine samples, however, were shown after culture in vitro to be capable of switching the fimbrial gene to the ON position and expressing mannose-sensitive hemagglutinin. The results from experimental infections and cases of cystitis in women suggest that type 1 fimbrial genes are transcribed both in the bladder and in the kidney. However, those bacteria found in the urine and not attached to the uroepithelium are not transcriptionally active for type 1 fimbrial genes.
At Least Four Percent of the
Salmonella typhimurium
Genome Is Required for Fatal Infection of Mice
Authors: Bowe Frances , Lipps Craig J. , Tsolis Renee M. , Groisman Eduardo , Heffron Fred , Kusters Johannes G.
Published: December 2019

Salmonella typhimurium
infection of mice is an established model system for studying typhoid fever in humans. Using this model, we identified
S. typhimurium
genes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo. Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1,000 times that of the parental strain were identified. These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions. In addition, the corresponding transposon insertions were mapped within the
S. typhimurium
genome and the nucleotide sequence of the transposon-flanking DNA was obtained.
Salmonella
spp. and related bacteria were probed with flanking DNA for the presence of these genes. All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of these were previously described. Furthermore, the proportion of attenuated mutants obtained in this study suggests that mutations in about 4% of the
Salmonella
genome lead to 1,000-fold or greater attenuation in the mouse typhoid model of infection. Most of these genes appear to be required during the early stages of a natural infection.
Inhibition of Binding of Malaria-Infected Erythrocytes by a Tetradecasaccharide Fraction from Chondroitin Sulfate A
Authors: Beeson James G. , Chai Wengang , Rogerson Stephen J. , Lawson Alexander M. , Brown Graham V.
Published: December 2019

Adherence of parasite-infected erythrocytes (IEs) to the microvascular endothelium of various organs, a process known as sequestration, is a feature ofPlasmodium falciparummalaria. This event is mediated by specific adhesive interactions between parasite proteins, expressed on the surface of IEs, and host molecules.P. falciparumIEs can bind to purified chondroitin sulfate A (CS-A), to the proteoglycan thrombomodulin through CS-A side chains, and to CS-A present on the surface of brain and lung endothelial cells and placental syncytiotrophoblasts. In order to identify structural characteristics of CS-A important for binding, oligosaccharide fragments ranging in size from 2 to 20 monosaccharide units were isolated from CS-A and CS-C, following controlled chondroitin lyase digestion, and used as competitive inhibitors of IE binding to immobilized ligands. Inhibition of binding to CS-A was highly dependent on molecular size: a CS-A tetradecasaccharide fraction was the minimum length able to almost completely inhibit binding. The effect was dose dependent and similar to that of the parent polysaccharide, and the same degree of inhibition was not found with the CS-C oligosaccharides. There was no effect on binding of IEs to other ligands, e.g., CD36 and intercellular adhesion molecule 1. Hexadeca- and octadecasaccharide fractions of CS-A were required for maximum inhibition of binding to thrombomodulin. Analyses of oligosaccharide fractions and polysaccharides by electrospray mass spectrometry and high-performance liquid chromatography suggest that the differences between the activities of CS-A and CS-C oligosaccharides can be attributed to differences in sulfate content and sulfation pattern and that iduronic acid is not involved in IE binding.
Protection of Mice againstPlasmodium yoeliiSporozoite Challenge withP. yoeliiMerozoite Surface Protein 1 DNA Vaccines
Authors: Becker Sylvia I. , Wang Ruobing , Hedstrom Richard C. , Aguiar Joao C. , Jones Trevor R. , Hoffman Stephen L. , Gardner Malcolm J.
Published: December 2019

Immunization of mice with DNA vaccines encoding the full-length form and C and N termini ofPlasmodium yoeliimerozoite surface protein 1 provided partial protection against sporozoite challenge and resulted in boosting of antibody titers after challenge. In C57BL/6 mice, two DNA vaccines provided protection comparable to that of recombinant protein consisting of the C terminus in Freund’s adjuvant.
Bartonella henselaeInvasion of Feline Erythrocytes In Vitro
Authors: Mehock Jane R. , Greene Craig E. , Gherardini Frank C. , Hahn Tae-Wook , Krause Duncan C.
Published: December 2019

Bartonella henselae, the causative agent of cat scratch disease, establishes long-term bacteremia in cats, in which it attaches to and invades feline erythrocytes (RBC). Feline RBC invasion was assessed in vitro, based on gentamicin selection for intracellular bacteria or by laser confocal microscopy and digital sectioning. Invasion rates ranged from 2 to 20% of the inoculum, corresponding to infection of less than 1% of the RBC. Invasion was a slow process, requiring >8 h before significant numbers of intracellular bacteria were detected. Pretreatment of the bacteria with trypsin, or of the RBC with trypsin or neuraminidase, had no effect, but pronase pretreatment of RBC resulted in a slight increase in invasion frequency. The ability to modelB. henselaeinvasion of feline RBC in vitro should permit identification of bacterial surface components involved in this process and elucidate the significance of RBC invasion to transmission and infection in cats.
Inducible Nitric Oxide Synthase-Deficient Mice Develop Enhanced Type 1 Cytokine-Associated Cellular and Humoral Immune Responses after Vaccination with AttenuatedSchistosoma mansoniCercariae but Display Partially Reduced Resistance
Authors: James Stephanie L. , Cheever Allen W. , Caspar Patricia , Wynn Thomas A.
Published: December 2019

High levels of nitric oxide (NO) are produced by inducible nitric oxide synthase (iNOS) in response to activating signals from Th1-associated cytokines and play an important role in cytotoxicity and cytostasis against many pathogenic microorganisms. In addition to its direct effector function, NO serves as a potent immunoregulatory factor. NO produced by gamma interferon-activated macrophages immobilizes and killsSchistosoma mansonilarvae, and several studies have indicated a role for this pathway in protective immunity against this parasite. The potential regulatory influence of NO in immunity toS. mansoniis less well understood. In this study, we have used iNOS-deficient mice to determine the role of NO in mice vaccinated with irradiated cercariae ofS. mansoni. We show by enzyme-linked immunosorbent assay and reverse transcriptase PCR analysis that vaccinated iNOS-deficient mice develop exacerbated type 1 cytokine responses in the lungs, the site where resistance to infection is primarily manifested. In addition, parasite-specific immunoglobulin G2a (IgG2a) and IgG2b antibody responses were significantly increased in vaccinated iNOS-deficient animals and total IgE antibody levels in serum were decreased relative to those in wild-type controls. Surprisingly, since resistance in this vaccine model is largely Th1 dependent and since Th1-related cellular and humoral immune responses were found to be exacerbated in vaccinated iNOS-deficient mice, vaccine-elicited protective immunity against challenge infection was found to be reduced. These findings demonstrate that iNOS plays a paradoxical role in immunity toS. mansoni, both in the effector mechanism of resistance and in the down regulation of the type 1 cytokine response, which is ultimately required for NO production.
Cytokine mRNA Expression and Proliferative Responses Induced by Pertussis Toxin, Filamentous Hemagglutinin, and Pertactin ofBordetella pertussisin the Peripheral Blood Mononuclear Cells of Infected and Immunized Schoolchildren and Adults
Authors: He Qiushui , Minh Nhu Nguyen Tran , Edelman Kati , Viljanen Matti K. , Arvilommi Heikki , Mertsola Jussi
Published: December 2019

Pertussis infection is increasingly recognized in older children and adults, indicating the need of booster immunizations in these age groups. We investigated the induction of pertussis-specific immunity in schoolchildren and adults after booster immunization and natural infection. The expression of mRNA of gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-5 in the peripheral blood mononuclear cells (PBMCs) was assayed by reverse transcription-PCR. The PBMCs of 17 children immunized with one dose of an acellular vaccine containing pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (PRN) significantly proliferated in vitro after stimulation with the vaccine antigens. The PBMCs of seven infected individuals markedly proliferated in the presence of PT and FHA, but the cells of only two of these subjects responded to PRN. At least one of the antigens induced mRNA for IL-4 and/or IL-5 in the cells of 93% of tested vaccinees and patients, and FHA induced IFN-γ mRNA in the cells of two-thirds of them. Expression of mRNA for IFN-γ correlated with the production of the cytokine protein. Anti-FHA immunoglobulin G antibodies significantly correlated with FHA-induced proliferative responses both before and after immunization. These results show that booster immunization with acellular pertussis vaccine induces both antibody- and cell-mediated immune responses in schoolchildren. Further, booster immunization and natural infection seem to induce the expression of mRNA of T-helper 1 (Th1) and Th2 type cytokines in similar manners. This observation supports the use of acellular pertussis vaccines for booster immunizations of older children, adolescents, and adults.
Characterization of a Neutralizing Monoclonal Antibody Directed at the Lipopolysaccharide of
Chlamydia pneumoniae
Authors: Peterson Ellena M. , de la Maza Luis M. , Brade Lore , Brade Helmut
Published: December 2019

Identification of protective epitopes is one of the first steps in the development of a subunit vaccine. One approach to accomplishing this is to identify structures or epitopes by using monoclonal antibodies (MAb) that can attenuate infectivity in vitro and in vivo. To date attempts to use this approach with
Chlamydia pneumoniae
have failed. This report is the first description of a MAb directed to the lipopolysaccharide (LPS) of
Chlamydia
that neutralizes both in vitro and in vivo the infectivity of
C. pneumoniae
. MAb CP-33, an immunoglobulin G2b (IgG2b), was identified from a fusion using splenocytes from mice immunized with
C. pneumoniae
TW-183. By Western blot analysis, MAb CP-33 exhibited genus-specific reactivity in that it recognized the LPSs of
C. pneumoniae
,
Chlamydia trachomatis
, and
Chlamydia psittaci
. MAb CP-33 did not react with 15 genera of gram-negative and gram-positive bacteria and
Candida albicans
. By using isolated LPS of Re mutants of
Escherichia coli
,
Salmonella enterica
serovar Minnesota, and recombinants expressing the 3-deoxy-
d
-
manno
-oct-2-ulosonic acid (Kdo) transferase gene
kdtA
of
C. trachomatis
, MAb CP-33 was shown to require for binding the presence of the genus-specific trisaccharide epitope αKdo(2→8)αKdo(2→4)αKdo. By employing synthetic oligosaccharides and neoglycoconjugates in an enzyme immunoassay (EIA) and EIA inhibition, it was further shown that MAb CP-33 differed from the extensively investigated prototype chlamydial LPS MAb S25-23. Most likely, MAb CP-33 recognizes a conformational epitope in which the αKdo(2→8)αKdo(2→4)αKdo trisaccharide is an essential structural component. When tested in an in vitro neutralization assay, MAb CP-33 gave a 50% neutralization titer of 8 ng/ml against
C. pneumoniae
TW-183. However, this MAb did not neutralize other
C. pneumoniae
strains,
C. trachomatis
, or
C. psittaci. C. pneumoniae
TW-183 was treated with either MAb CP-33 or a control IgG and then used to inoculate mice by the respiratory route. Five days after inoculation, there was a difference between the mice inoculated with the control IgG-treated inoculum and those inoculated with the MAb CP-33-treated organisms as to the number of mice infected as well as the number of inclusion-forming units recovered from lung cultures (
P
< 0.05). In summary, a
Chlamydia
-specific LPS MAb was able to neutralize in vitro the infectivity of
C. pneumoniae
TW-183.
Transgenic Expression of a Mosquito-Stage Malarial Protein, Pbs21, in Blood Stages of Transformed
Plasmodium berghei
and Induction of an Immune Response upon Infection
Authors: Margos Gabriele , van Dijk Melissa R. , Ramesar Jai , Janse Chris J. , Waters Andrew P. , Sinden Robert E.
Published: December 2019

Pbs21 is a surface protein of the ookinete of
Plasmodium berghei
, which can induce a potent transmission-blocking immune response. Pbs21 is normally expressed only by parasite stages in the mosquito, i.e., female gametes/zygotes, ookinetes, and oocysts. However, the Pbs21 gene is transcribed in female gametocytes which circulate in the bloodstream of the host, where translation of the resulting mRNA is totally repressed. Episomal transfection has been used to investigate whether expression of Pbs21 protein could be achieved in blood stages of the parasite. By using plasmid pMD221, the complete mRNA-encoding region of Pbs21, flanked only by 218 nucleotides (nt) of its promoter region and 438 nt of its 3′ region downstream from the polyadenylation site, was introduced into the blood stages of gametocyte-producing and non-gametocyte-producing clones of
P. berghei
. In both of these transformed parasite lines, Pbs21 protein was expressed in asexual trophozoites, schizonts, and, when present, in both male and female gametocytes. Hence, the flanking regions present are sufficient to allow transcription but lack the elements that exert natural control of sex- and stage-specific transcription. The mRNA and the protein expressed by transformed blood stages were indistinguishable from the wild-type forms by the criteria tested, and the protein was recognized by both conformation-dependent and conformation-independent monoclonal antibodies raised against native Pbs21. In mice infected with transformed non-gametocyte-producing parasites, a Pbs21-specific immune response was induced and characterized with respect to isotype (IgG2a/IgG2b) and quantity (11.5 ± 10 μg/ml) of antibody produced. However, as found in previous studies, these antibody levels were insufficient to inhibit development of the parasites in the mosquito. The ability to express mosquito midgut-stage antigens in blood-stage parasites will facilitate further investigations of molecular and immunological properties of these proteins.
Expression ofToxoplasma gondii-Specific Heat Shock Protein 70 during In Vivo Conversion of Bradyzoites to Tachyzoites
Authors: Silva Neide M. , Gazzinelli Ricardo T. , Silva Deise A. O. , Ferro Eloisa A. V. , Kasper Lloyd H. , Mineo Jose R.
Published: December 2019

Stage conversion between bradyzoites and tachyzoites was investigated in C57BL/6 mice chronically infected with the ME-49 strain ofToxoplasma gondii. In order to promote bradyzoite-tachyzoite conversion, mice were treated in vivo with neutralizing doses of anti-gamma interferon (IFN-γ) or anti-tumor necrosis factor alpha (TNF-α) antibodies. Expression of parasite-specific antigens SAG-1, SAG-2, and heat shock protein 70 (Hsp-70) was visualized in the central nervous system by immunocytochemistry and measured by photometric assay. The immunosuppressive effect of anti-IFN-γ or anti-TNF-α treatment was immediate, leading to parasite stage conversion as indicated by the increased expression of tachyzoite-specific antigens (SAG-1 and SAG-2) and by rapid parasite replication. We also observed expression of high levels of Hsp-70 during a short period of conversion of bradyzoites to tachyzoites. Our data suggest that Hsp-70 may have an important role in the process of bradyzoite-tachyzoite conversion during the reactivation of chronic toxoplasmosis.
Streptococcal Pyrogenic Exotoxin B Induces Apoptosis and Reduces Phagocytic Activity in U937 Cells
Authors: Kuo Chih-Feng , Wu Jiunn-Jong , Tsai Pei-Jane , Kao Fu-Jen , Lei Huan-Yao , Lin Ming T. , Lin Yee-Shin
Published: December 2019

Treatment of U937 human monocyte-like cells withStreptococcus pyogenesled to an induction of apoptosis in these cells. A comparison between the wild-type strain and its isogenic protease-negative mutant indicated that the production of streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, caused a greater extent of apoptosis in U937 cells. Further study using purified SPE B showed that this protease alone could induce U937 cells to undergo apoptosis, which was characterized by morphologic changes, DNA fragmentation laddering on the gel, and an increase in the percentages of hypodiploid cells. The protease activity of SPE B was required for apoptosis to proceed, since treatment with cysteine protease inhibitor E64 or heat inactivation abrogated this death-inducing effect. The SPE B-induced apoptosis pathway was interleukin-1β converting enzyme (ICE) family protease dependent. Further experiments showed that the phagocytic activity of U937 cells was reduced by SPE B. Treatment with E64 and heat inactivation both abrogated this phagocytosis-inhibitory effect. Taken together, the present data show that SPE B not only possesses the ability to induce apoptosis in monocytic cells but also helps bacteria to resist phagocytosis by host cells.
Expression of Listeriolysin O and ActA by Intracellular and ExtracellularListeria monocytogenes
Authors: Moors Marlena A. , Levitt Brian , Youngman Philip , Portnoy Daniel A.
Published: December 2019

Listeria monocytogenesrequires listeriolysin O (LLO) and ActA, the products ofhlyandactA, respectively, to establish a productive intracellular infection. LLO is essential for vacuolar lysis and entry into the cytosol, while ActA is required for bacterial spread to adjacent cells. We have used a transcriptional reporter gene system to compare the expression ofactAandhlyduring intracellular growth to that during growth in broth cultures. ThehlyandactAgenes were transcriptionally fused toEscherichia coli lacZandBacillus pumilus cat-86 (cat), and the fusions were integrated in single copies into theL. monocytogeneschromosome. A chloramphenicol resistance assay indicated that thehlyfusion but not theactAfusion was significantly activated in Luria-Bertani (LB) broth, and this finding correlated with LLO and ActA levels detectable in broth cultures. Quantitation of promoter activity on the basis of β-galactosidase activity revealed up to 10-fold-higher level of expression of thehlyfusion relative to theactAfusion in LB broth. In contrast, both fusions were active in the cytosol of J774 cells, and the activity of theactAfusion was approximately 3-fold higher than that of thehlyfusion under these conditions. However, quantitative immunoprecipitation of ActA and LLO from infected J774 cells demonstrated approximately 70-fold more cytosolic ActA than cytosolic LLO. Finally, in comparison to induction in broth cultures,actAwas highly induced (226-fold) andhlywas moderately induced (20-fold) in J774 cells. Collectively, these results indicate thatactAandhlyare differentially regulated in response to the growth environment and that both genes are preferentially expressed during intracellular growth. Further, while the lower level of production of ActA than of LLO in broth can be accounted for by transcriptional regulation, the relative abundance of intracellular ActA compared to that of intracellular LLO is a function of additional, possibly host-mediated, factors.
Gamma Interferon Augments Macrophage Activation by Lipopolysaccharide by Two Distinct Mechanisms, at the Signal Transduction Level and via an Autocrine Mechanism Involving Tumor Necrosis Factor Alpha and Interleukin-1
Authors: Held Thomas K. , Weihua Xiao , Yuan Liang , Kalvakolanu Dhananjaya V. , Cross Alan S.
Published: December 2019

When given in the presence of gamma interferon (IFN-γ), otherwise nontoxic doses of lipopolysaccharide (LPS or endotoxin) become highly lethal for mice. The mechanisms of this synergistic toxicity are not known. We considered the possibility that an interaction between the LPS-induced NF-κB and IFN-γ-induced JAK-STAT pathways at the pretranscriptional level may enhance the LPS-induced signals. To test this hypothesis, we incubated murine macrophage RAW 264.7 cells with IFN-γ for 2 h before addition of different doses of LPS. Consistent with the synergistic induction of inducible nitric oxide synthase mRNA and nitric oxide production by a combination of LPS and IFN-γ, IFN-γ strongly augmented LPS-induced NF-κB activation and accelerated the binding of NF-κB to DNA to as early as 5 min. In agreement with this, IFN-γ pretreatment promoted rapid degradation of IκB-α but not that of IκB-β. Inhibition of protein synthesis during IFN-γ treatment suppressed LPS-initiated NF-κB binding. A rapidly induced protein appeared to be involved in IFN-γ priming. Preincubation of cells with antibodies to tumor necrosis factor alpha or the interleukin-1 receptor partially reduced the priming effect of IFN-γ. In a complementary manner, LPS enhanced the activation of signal-transducing activator of transcription 1 by IFN-γ. These data suggest novel mechanisms for the synergy between IFN-γ and LPS by which they cross-regulate the signal-transducing molecules. Through this mechanism, IFN-γ may transform a given dose of LPS into a lethal stimulus capable of causing sepsis. It may also serve a beneficial purpose by enabling the host to respond quickly to relatively low doses of LPS and thereby activating antibacterial defenses.