bluuebunny/crossref_metadata_2025 huggingface dataset:
A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins
Authors: Sirard J C , Weber M , Duflot E , Popoff M R , Mock M
Published: January 2020

The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines.
Construction and characterization of a live attenuated vaccine candidate against Shigella dysenteriae type 1
Authors: Klee S R , Tzschaschel B D , Fält I , Kärnell A , Lindberg A A , Timmis K N , Guzmán C A
Published: January 2020

Vaccine candidates against Shigella dysenteriae type 1, which is associated with the most severe cases of bacillary dysentery, were constructed. The rfp and rfb gene clusters, which code for S. dysenteriae 1 O antigen biosynthesis, were randomly integrated into either the chromosome or the virulence plasmid of the rough attenuated Shigella flexneri aroD strain SFL124-27 with a minitransposon carrying an arsenite resistance selection marker. The recombinant clones efficiently expressed the recombinant O antigen, exhibited a normal growth pattern, were able to invade and survive within eukaryotic cells to the same extent as the parental strain, and expressed the recombinant antigen within invaded cells. A clone was selected as the vaccine candidate, which was demonstrated to be immunogenic and safe in animal models, leading to 47% full protection and 53% partial protection against challenge with the wild-type strain.
Synergistic effect of mutations in invA and lpfC on the ability of Salmonella typhimurium to cause murine typhoid
Authors: Bäumler A J , Tsolis R M , Valentine P J , Ficht T A , Heffron F
Published: January 2020

Penetration of the intestinal mucosa at areas of Peyer's patches is an important first step for Salmonella typhimurium to produce lethal systemic disease in mice. However, mutations in genes that are important for intestinal invasion result in only moderately decreased virulence of S. typhimurium for mice. Here we report that combining mutations in invA and lpfC, two genes necessary for entry into Peyer's patches, results in a much stronger attenuation of S. typhimurium than inactivation of either of these genes alone. An S. typhimurium invA lpfC mutant was 150-fold attenuated by the oral route of infection but was fully virulent when the intestine was bypassed by intraperitoneal challenge of mice. During mixed-infection experiments, the S. typhimurium invA lpfC mutant showed a strong defect in colonizing Peyer's patches and mesenteric lymph nodes. These data suggest that mutations in invA and lpfC deactivate distinct pathways for intestinal penetration and colonization of Peyer's patches. While the inv-mediated pathway is widely distributed, the lpf operon is absent from many phylogenetic groups within the genus Salmonella. To investigate how acquisition of the lpf-mediated pathway for mucosal penetration contributed to evolution of virulence, we studied the relationship between the presence of the lpf operon and the pathogenicity for mice of 18 isolates representing 14 Salmonella serotypes. Only strains possessing the lpf operon were able to cause lethal infection in mice. These data show that both the invA- and lpfC-mediated pathways of intestinal perforation are conserved in mouse virulent Salmonella serotypes.
Distinct mechanisms of immunosuppression as a consequence of major surgery
Authors: Hensler T , Hecker H , Heeg K , Heidecke C D , Bartels H , Barthlen W , Wagner H , Siewert J R , Holzmann B
Published: January 2020

Altered host defense mechanisms after major surgery or trauma are considered important for the development of infectious complications and sepsis. In the present study, we demonstrate that major surgery results in a severe defect of T-lymphocyte proliferation and cytokine secretion in response to coligation of the antigen receptor complex and CD28. During the early postoperative course, reduced cytokine secretion was observed for interleukin-2 (IL-2), gamma interferon, and tumor necrosis factor alpha, which are associated with the Th1 phenotype of helper T lymphocytes, and for IL-4, the index cytokine of Th2 cells. During the late postoperative course, T-cell cytokine secretion increased to normal levels. Production of the anti-inflammatory cytokine IL-10 was altered, with different kinetics being selectively elevated during the late postoperative course. In contrast, the capacity of peripheral blood monocytes to present bacterial superantigens and to stimulate T-cell proliferation was normal or enhanced after surgery despite a significant loss of cell surface HLA-DR molecules. Thus, the level of major histocompatibility complex class II protein expression does not appear to predict the antigen-presenting capacity of monocytes obtained from surgical patients with uneventful postoperative recovery. Secretion of IL-1beta and IL-10 by endotoxin-stimulated peripheral blood monocytes was increased at different time points after surgery. Major surgery therefore results in a distinct pattern of immune defects with a predominant defect in the T-cell response to T-cell receptor- and CD28 coreceptor-mediated signals rather than an impaired monocyte antigen-presenting capacity. Suppression of T-cell effector functions during the early phase of the postoperative course may define a state of impaired defense against pathogens and increased susceptibility to infection and septic complications.
The role of tyrosine phosphorylation in lipopolysaccharide- and zymosan-induced procoagulant activity and tissue factor expression in macrophages
Authors: Dackiw A P , Grinstein S , Brisseau G F , McGilvray I D , Nathens A B , McGuire J A , Romanek R , Cheung P Y , Rotstein O D
Published: January 2020

The expression of surface procoagulants by exudative macrophages represents an important mechanism underlying local fibrin deposition at sites of extravascular inflammation. The present studies investigated the contribution of tyrosine phosphorylation to the generation of macrophage procoagulant activity (PCA) and tissue factor expression in response to proinflammatory stimuli. Both lipopolysaccharide (LPS) and zymosan rapidly stimulated tyrosine phosphorylation in elicited murine peritoneal macrophages. This effect was prevented by the tyrosine kinase inhibitors genistein and herbimycin and augmented by the addition of the phosphotyrosine phosphatase inhibitor vanadate. The vanadate-mediated rise in phosphotyrosine accumulation was abrogated by the use of diphenylene iodonium, an inhibitor of the respiratory burst oxidase, suggesting a role for peroxides of vanadate as contributors to the tyrosine phosphorylation. This notion was supported by the finding that vanadyl hydroperoxide markedly increased the accumulation of phosphotyrosine residues. To define the role of tyrosine phosphorylation in the induction of macrophage PCA by LPS, the effects of tyrosine kinase inhibition by genistein and herbimycin were investigated. Both agents inhibited the expression of macrophage PCA. Further, Northern blot analysis with the cDNA probe for murine tissue factor indicated that the inhibition occurred at the mRNA level or earlier. Since vanadate augmented phosphotyrosine accumulation, it was hypothesized that it might enhance generation of macrophage products. However, vanadate reduced induction of PCA in response to LPS. By contrast, vanadate augmented basal prostaglandin E2 (PGE2) release and stimulated PGE2 release by macrophages. Indomethacin prevented the increase in PGE2 but only partially restored normal levels of PCA. The effect of vanadate on tissue factor expression appeared to be posttranscriptional. These studies thus demonstrate, by functional Western blotting and Northern blotting techniques, that tyrosine phosphorylation plays a role in the regulation of macrophage PCA and tissue factor expression in response to proinflammatory stimuli.
Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae
Authors: Harvey H A , Ketterer M R , Preston A , Lubaroff D , Williams R , Apicella M A
Published: January 2020

In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected.
Characterization of Shigella type 1 fimbriae: expression, FimA sequence, and phase variation
Authors: Snellings N J , Tall B D , Venkatesan M M
Published: January 2020

This study documents the presence of type 1 fimbriae on Shigella and confirms these mannose-sensitive adherence structures to be bona fide components of the Shigella surface. While laboratory-passaged Shigella strains and lyophilized clinical isolates failed to express type 1 fimbriae, 6 of 20 recent clinical isolates, including 4 Shigella flexneri strains, 1 Shigella boydii strain, and 1 Shigella dysenteriae strain, produced type 1 fimbriae as detected by mannose-sensitive hemagglutination (MSHA) and electron microscopy. Optimal production of a predominantly Fim+ population required serial passage every 48 to 72 h in unshaken brain heart infusion broth at 37 degrees C. Fim+ Shigella cultures were capable of reversibly switching to a non-MSHA, afimbriated phase during serial aerobic cultivation on tryptic soy agar plates. The amino acid sequence of S. flexneri type 1 FimA contained 18 substitutions compared to that of Escherichia coli fimbrillin. Indirect immunoelectron microscopy suggested the presence of both shared and unique epitopes on E. coli and S. flexneri type 1 fimbriae. Random phase variation between fimbriated and afimbriated states in Shigella was accompanied by the genomic rearrangement associated with phase variation in E. coli.
Internalization of Escherichia coli by human renal epithelial cells is associated with tyrosine phosphorylation of specific host cell proteins
Authors: Palmer L M , Reilly T J , Utsalo S J , Donnenberg M S
Published: January 2020

Human renal epithelial cells are capable of internalizing Escherichia coli regardless of whether the bacteria are isolated from individuals with pyelonephritis or from healthy volunteers. In this study, we investigated the role of host cell tyrosine kinase activity in internalization. We found that internalization of both fecal and pyelonephritis isolates is blocked by tyrosine kinase inhibitors. We found increased intensity of two tyrosine-phosphorylated proteins, with relative mobilities of approximately 123,000 and 110,000, in Western blots of extracts from human renal epithelial cells infected with E. coli. The increased intensity of these tyrosine-phosphorylated proteins was observed only in the Triton X-100-insoluble fraction, suggesting that these proteins could be associated with the cytoskeleton. Increased tyrosine phosphorylation of these proteins upon E. coli infection was observed in both transformed and primary human renal epithelial cells and in cells infected with several different strains of E. coli isolated from the feces of healthy individuals or from the blood or urine of patients with pyelonephritis. The increased tyrosine phosphorylation of these proteins required live bacteria and was blocked by tyrosine kinase inhibition but not by protein synthesis inhibitors or cytochalasin D. These experiments establish a strong link between E. coli internalization and host cell signaling through tyrosine kinases in human kidney cells and provide evidence that specific proteins are involved in these processes.
Modification of the Staphylococcus aureus fibronectin binding phenotype by V8 protease
Authors: McGavin M J , Zahradka C , Rice K , Scott J E
Published: January 2020

The amount of cell surface fibronectin (Fn)-binding protein (FnBP) adhesin expressed by Staphylococcus aureus is maximal during exponential growth but disappears rapidly as the culture progresses into stationary phase. To identify factors responsible for the loss of cell surface FnBP, a culture of S. aureus L170, which shows high levels of Fn binding, was supplemented at the time of inoculation with concentrated stationary-phase supernatant from S. aureus L530, a strain which binds Fn poorly. The resulting exponential-phase cells were devoid of FnBP. The factor responsible for this activity was purified from the culture supernatant and identified as V8 protease. When cultured with 375 ng of exogenous V8 protease ml(-1), exponential-phase cells of S. aureus L170 were devoid of cell surface FnBP, and concentrations as low as 23 ng x ml(-1) resulted in reduced amounts of FnBP. Addition of the protease inhibitor alpha2-macroglobulin to the culture medium prevented the growth-phase-dependent loss of cell surface FnBP, whereas growth with exogenous V8 protease resulted in reduced adherence to the solid-phase N-terminal fragment of Fn and to the extracellular matrix synthesized by fetal rabbit lung fibroblasts. Although FnBP was extremely sensitive to V8 protease, exogenous protease did not exert a significant influence on the amount of cell surface protein A. However, a limited number of other high-molecular-weight cell surface proteins were also sensitive to V8 protease. Therefore, both the adhesive phenotype and cell surface protein profile of S. aureus can be modified by V8 protease activity.
Role of bacterial Mn-cofactored superoxide dismutase in oxidative stress responses, nasopharyngeal colonization, and sustained bacteremia caused by Haemophilus influenzae type b
Authors: D'Mello R A , Langford P R , Kroll J S
Published: January 2020

Haemophilus influenzae type b, a causative agent of bacterial sepsis and meningitis in young children, contains a single superoxide dismutase (SOD), a cytoplasmic MnSOD. To study the role of this enzyme, a chromosomal sodA::lacZ mutant (M-2) was constructed. M-2 had an increased sensitivity towards oxygen and the redox-active agent paraquat. A 3.4-fold increase in sodA-lacZ expression was found in M-2 grown with oxygen supply rates between 3 and 36 mmol of O2/liter/h. In similar experiments with the wild type, assaying SodA activity, a 3.1-fold increase was found. Both the wild type and M-2 grew best at the lowest oxygen supply rate tested, consistent with the notion that H. influenzae prefers a more anaerobic environment. In the infant rat model of infection, the ability of M-2 to colonize the nasopharynx was found to be impaired, but its ability to cause invasive disease was unaffected. This suggests that after invasion, the growth disadvantage imposed by a SodA- phenotype is not limiting.
Clostridium difficile toxin A induces the release of neutrophil chemotactic factors from rat peritoneal macrophages: role of interleukin-1beta, tumor necrosis factor alpha, and leukotrienes
Authors: Rocha M F , Maia M E , Bezerra L R , Lyerly D M , Guerrant R L , Ribeiro R A , Lima A A
Published: January 2020

Clostridium difficile produces a potent enterotoxin and cytotoxin, toxins A and B, respectively, which appear to be responsible for pseudomenbranous colitis and antibiotic-associated diarrhea. In the present study we explored the neutrophil migration evoked by toxin A in the peritoneal cavities and subcutaneous air pouches of rats and examined the role of macrophages and their inflammatory mediators in this process. Toxin A causes a significant dose-dependent neutrophil influx into the peritoneal cavity, with a maximal response at 0.1 microg/ml and at 4 h. The depletion of macrophages by peritoneal washing prevents the toxin A-induced neutrophil migration into the peritoneal cavity. In contrast, an increase in macrophages induced by peritoneal injection of thioglycolate amplifies this toxin effect on neutrophil migration. Furthermore, the injection of supernatants from toxin A-stimulated macrophages into the rat peritoneal cavity causes significant neutrophil migration. Pretreatment of rats with BWA4C, nordihydroguaiaretic acid, mepacrine, or dexamethasone inhibits the neutrophil migration evoked by toxin A in the peritoneal cavities. However, pretreatment with the cyclooxygenase inhibitor indomethacin or the platelet-activating factor antagonist BN52021 fails to alter toxin A-induced neutrophil migration. Toxin A was also injected into air pouches of normal rats or rats pretreated with anti-interleukin-1beta (anti-IL-1beta) or anti-tumor necrosis factor alpha (anti-TNF-alpha) antibodies. Anti-TNF-alpha or anti-IL-1beta antibodies significantly reduce the neutrophil migration induced by toxin A. These data suggest that neutrophil migration evoked by toxin A is in part dependent on macrophage-derived cytokines, such as TNF-alpha and IL-1beta, and leukotrienes. These mediators may help to explain the intense inflammatory colitis caused by C. dificile toxin A in an experimental animal model of this disease.
Proinflammatory cytokine deficiency and pathogenesis of Pseudomonas aeruginosa keratitis in aged mice
Authors: Hobden J A , Masinick S A , Barrett R P , Hazlett L D
Published: January 2020

Corneal clarity in young adult Swiss (HSD:ICR) mice is restored after Pseudomonas aeruginosa infection. Previous data showed that this response involves a rapid up-regulation of constitutive intercellular cell adhesion molecule-1 (ICAM-1) and migration of inflammatory cells into the cornea. In contrast, in aged mice, there is no up-regulation of corneal ICAM-1, inflammatory cell infiltration into the cornea is delayed, and the cornea perforates. Therefore, the aim of this study was to test whether specific cytokines which up-regulate ICAM-1 expression differ in young and aged mice. Corneas of young (6- to 8-week-old) and aged (1- to 2-year-old) mice were scarified and inoculated with P. aeruginosa. The eyes were graded for pathologic changes (score 0 to +4); at 6, 12, 24, and 48 h postinfection (p.i.), six mice from each age group were sacrificed. Three corneas from each respective group were excised for quantitation of interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and gamma interferon (IFN-gamma) by enzyme-linked immunosorbent assay. The remaining three corneas from each age group were harvested for quantitation of viable bacteria by direct plate count determination and for infiltrating polymorphonuclear leukocytes (PMNs) by a myeloperoxidase (MPO) assay. Compared to those of young mice, the corneas of infected aged mice had less IL-1beta at 6 h p.i. (P < or = 0.04) and less IFN-gamma at 12 to 48 h p.i. (P < or = 0.05). Also, compared to those of young mice, corneas of aged mice had fewer PMNs (P < or = 0.008) by the MPO assay at 6 h p.i. and more viable bacteria (P < or = 0.01) per cornea by plate count determination at 24 h p.i. These data suggest that the lack of up-regulation of ocular ICAM-1 in aged mice may reflect a reduction in both IL-1beta and IFN-gamma levels in the infected cornea. Consequently, a sufficient number of PMNs and other inflammatory cells fail to rapidly migrate into the infected corneas of aged mice, the bacterial load is initially greater than that in young mice, and the cornea perforates.
The phagosomal environment protects virulent Mycobacterium avium from killing and destruction by clarithromycin
Authors: Fréhel C , Offredo C , de Chastellier C
Published: January 2020

Murine bone marrow-derived macrophages (Mphis) infected with virulent strains of Mycobacterium avium (TMC 724 and a human clinical isolate) or with an avirulent opaque variant that spontaneously dissociates from the virulent human clinical isolate were subjected to a prolonged and continuous treatment with clarithromycin added at the MIC. The efficiency of this antibiotic in terms of inhibition of bacterial growth and bacterial degradation was evaluated during a 21-day treatment period. Growth was assessed by determination of CFU of intracellular bacteria and by a quantitative ultrastructural analysis which allowed us also to determine the extent of bacterial degradation. A similar treatment was applied to the same strains growing in liquid medium. Our data show that in liquid medium, clarithromycin caused a 90% decrease in CFU within 7 days of treatment. When applied to Mphis infected with virulent M. avium, clarithromycin immediately arrested bacterial growth but was unable to fully kill and degrade intracellularly growing virulent bacteria. After 21 days of treatment, 25% of intracellular bacteria were still morphologically intact. These bacteria resumed growth upon removal of the antibiotic, with a normal replication rate. These bacteria had not become more resistant to the drug, since the MIC was unchanged as compared to the one determined for the initial stock used to infect Mphis. Our data therefore suggest that the intraphagosomal environment protects bacteria from degradation. We propose that the inability of the drug to completely destroy bacteria is the result of a limited accessibility of the drug due to prevention of fusions between the immature phagosomes in which virulent bacteria reside and lysosomes in which clarithromycin accumulates. In accord with our proposal, we show that the avirulent opaque variant, which does not prevent phagosome-lysosome fusions (unpublished data), is finally destroyed by clarithromycin even within the phagosomal environment.
Pathogenicity island sequences of pyelonephritogenic Escherichia coli CFT073 are associated with virulent uropathogenic strains
Authors: Kao J S , Stucker D M , Warren J W , Mobley H L
Published: January 2020

Urinary tract infection is the most frequently diagnosed kidney and urologic disease, and Escherichia coli is by far the most common etiologic agent. Defined blocks of DNA termed pathogenicity islands have been found in uropathogenic strains to carry genes not generally found in fecal strains. We have identified one of these regions of DNA within the chromosome of the highly virulent E. coli CFT073, isolated from the blood and urine of a woman with acute pyelonephritis. This strain, which is cytotoxic for cultured renal cells and causes acute pyelonephritis in transurethrally infected CBA mice, contains two distinct copies of the pap operon and is hemolytic. One pap operon was localized on a cosmid clone which was used to identify three overlapping cosmid clones. By using restriction mapping, DNA hybridization, sequencing, and PCR amplification, a region of approximately 50 kb was found to be present in this uropathogenic strain and to have no corresponding sequences in E. coli K-12. This gene block also carries hemolysin genes hlyCABD. The pathogenicity island begins 7 bp downstream of dadX (catabolic alanine racemase; 26.55 min) and ends at a position in the K-12 genome 75 bp downstream of the metV tRNA gene (62.74 min); this suggests that a chromosomal rearrangement has occurred relative to the K-12 linkage map. The junctions of the pathogenicity island were verified by PCR amplification directly from the genomic DNA of strain CFT073. DNA sequencing within the boundaries of the junctions revealed genes not previously identified in E. coli or in some cases bearing no known homologs. When used as probes for DNA hybridization, these sequences were found significantly more often in strains associated with the clinical syndromes of cystitis (82%) and acute pyelonephritis (79%) than in fecal strains (19%; P < 0.001).
Macrophages and enriched populations of T lymphocytes interact synergistically for the induction of severe, destructive Lyme arthritis
Authors: DuChateau B K , Jensen J R , England D M , Callister S M , Lovrich S D , Schell R F
Published: January 2020

Hamsters receiving both macrophages exposed to Formalin-inactivated Borrelia burgdorferi (Mphi-FBb) and enriched populations of either immune or naive T lymphocytes developed severe swelling of the hind paws when infected with B. burgdorferi. Swelling was detected 6 days after infection, peaked on day 10, and gradually decreased. Swelling was also observed in the hind paws of hamsters infused with only Mphi-FBb or only enriched populations of either immune or naive T lymphocytes after infection with B. burgdorferi. However, the swelling detected in these hamsters was less severe and of shorter duration. In addition, hamsters receiving both macrophages not exposed to Formalin-inactivated B. burgdorferi (Mphi-NFBb) and enriched populations of either immune or naive T lymphocytes failed to develop severe swelling after infection with B. burgdorferi. No swelling was also observed in hamsters infused with both Mphi-FBb and enriched populations of immune T lymphocytes and then inoculated with spirochetal growth medium. We further showed that macrophages and enriched populations of T lymphocytes did not interact synergistically for controlling B. burgdorferi infection, as spirochetes were readily recovered from the tissues of all cell transfer recipients infected with B. burgdorferi. These findings demonstrate that hamsters infused with both Mphi-FBb and enriched populations of either immune or naive T lymphocytes develop a more fulminate arthritis after infection with B. burgdorferi than recipients infused with either cell type alone. These findings suggest that macrophages and T lymphocytes interact synergistically for the induction of severe, destructive Lyme arthritis.
Effects of pH and salinity on the antimicrobial properties of clavanins
Authors: Lee I H , Cho Y , Lehrer R I
Published: January 2020

Clavanins are histidine-rich, amidated alpha-helical antimicrobial peptides that were originally isolated from the leukocytes (hemocytes) of a tunicate, Styela clava. The activities of clavanin A amide and clavanin A acid against Escherichia coli, Listeria monocytogenes, and Candida albicans were substantially greater at pH 5.5 than at pH 7.4. In contrast, clavanin AK, a synthetic variant of clavanin A acid containing 4 histidine-->lysine substitutions exerted substantial activity at both pH 7.4 and pH 5.5. Each of these three clavanins permeabilized the outer and inner membranes of E. coli very effectively at pH 5.5, but only clavanin AK did so at pH 7.4. Unlike magainin 1 and cecropin P1, alpha-helical antimicrobial peptides from frog skin and porcine intestine, respectively, clavanins were broadly effective against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, as well as gram-negative organisms. Because clavanins exert substantial antimicrobial activity in 0.1 to 0.3 M NaCl, they provide templates for designing broad-spectrum peptide antibiotics intended to function in extracellular environments containing normal or elevated NaCl concentrations. The pH-dependent properties of histidine-rich antimicrobial peptides may allow the design of agents that would function selectively in acidic compartments, such as the gastric lumen, or within phagolysosomes.
PlcR1 and PlcR2 are putative calcium-binding proteins required for secretion of the hemolytic phospholipase C of Pseudomonas aeruginosa
Authors: Cota-Gomez A , Vasil A I , Kadurugamuwa J , Beveridge T J , Schweizer H P , Vasil M L
Published: January 2020

The plcHR operon of Pseudomonas aeruginosa includes the structural gene for the hemolytic phospholipase C,plcH (previously known as plcS), and two overlapping, in-phase, genes designated plcR1 and plcR2. Hemolytic and phospholipase C (PLC) activities produced by Escherichia coli and P. aeruginosa T7 expression systems were measured in strains carrying both plcH and plcR genes and in strains carrying each gene separately. When plcH was expressed by itself in the E. coli T7 system, the area of the hemolytic zone on blood agar was less than twice the area of growth. By contrast, when plcR was coexpressed with plcH in this system, the area of the hemolytic zone was approximately 10 times that of the area of the growth on blood agar. Native polyacrylamide gel electrophoretic analyses of PlcH activity expressed in either the E. coli or the P. aeruginosa T7 system carrying plcH alone, or along with the plcR genes, suggest that PlcR either posttranslationally alters the physical or biochemical nature of PlcH or releases PlcH from a complex in the cell so that it can be secreted. The hypothesis that PlcR is involved in the secretion of PlcH is supported by the observation that the ratio of extracellular to cell-associated PlcH activity produced by P. aeruginosa strains containing an in-frame deletion in the chromosomal plcR genes is significantly reduced in comparison with this ratio seen with the wild-type parental strain. This defect in the secretion of PlcH can be complemented by the plcR genes in trans. Additional data suggest that PlcR does not directly affect the secretion of the nonhemolytic phospholipase C (PlcN). PlcR is highly similar to a calcium-binding protein (CAB) from Streptomyces erythraeus. PlcR and CAB contain typical motifs (EF hands) characteristic of eucaryotic calcium-binding proteins, including calmodulin. P. aeruginosa naturally produces membrane vesicles (MVs) containing extracellular proteins including PLC. MVs from the PAO1WT strain contained at least 10-fold more PLC specific activity than those isolated from a strain carrying a deletion of plcR (PAO1 deltaR). Immunogold electron microscopy of PAO1WT and PAO1 deltaR whole cells revealed a distribution of PlcH in these strains consistent with the hypothesis that PlcR is required for the secretion of PlcH.
Granuloma cytokines in murine cysticercosis
Authors: Robinson P , Atmar R L , Lewis D E , White A C
Published: January 2020

Neurocysticercosis, caused by Taenia solium, is one of the most common causes of seizures worldwide. The symptoms result from granulomatous inflammation associated with dying cyst forms of the parasite. Although the invasive larvae can be killed by immune serum plus complement, immunity to the cyst stage depends on a cellular response. This dichotomous immune response is reminiscent of the extremes of the immune response associated with T helper 1 (Th1) and Th2 cytokine profiles. To characterize the cytokine response in cysticercosis, granulomas were removed from the peritoneal cavity of mice infected with Taenia crassiceps cysts and examined for cytokine message by in situ hybridization using 35S-labeled RNA probes. The granulomas were staged based on histologic appearance of the degenerating parasite. Message for gamma interferon (IFN-gamma) was identified by light microscopy in 11 of the 12 granulomas, and interleukin-2 (IL-2) message was identified in 9 of the 12. By laser scanning confocal microscopy, significantly increased IFN-gamma and IL-2 pixel intensity was identified in nearly all of the granulomas from early histologic stages. Message for IL-4 was seen in 6 of the 12 granulomas. Only granulomas with complete destruction of the parasite architecture displayed more than minimal amounts of IL-4 message by light microscopy, and only 2 of 12 granulomas had IL-4 pixel intensity significantly above background. Only minimal amounts of IL-10 message were detected in 4 of 11 granulomas. Thus, early granulomas in cysticercosis are predominantly associated with a Th1 response, whereas later granulomas, in which parasite destruction is complete, have a mixture of Th1 and IL-4. The Th1 response appears to play an important role both in the pathogenesis of disease as well as in the clearing of the parasites, with IL-4 involved in downregulation of the initial response.
Tyrosine phosphorylation is required for ehrlichial internalization and replication in P388D1 cells
Authors: Zhang Y , Rikihisa Y
Published: January 2020

Replication of Ehrlichia risticii was inhibited in P388D1 cells when a protein tyrosine kinase inhibitor (genistein or herbimycin A) was added after internalization of the organism at 3 h postinfection. Upon addition of genistein at day 1, 2, 3, or 4 postinfection, further proliferation of E. risticii was prevented. The inhibition was reversible, since regrowth of E. risticii occurred upon the removal of genistein. Genistein prevented spreading of E. risticii from P388D1 cells to THP-1 cells. Genistein did not prevent binding of [35S]methionine-labeled E. risticii to P388D1 cells but did prevent internalization of [35S]methionine-labeled E. risticii. 14CO2 production from L-[14C]glutamine in Percoll density gradient-purified E. risticii was not inhibited by genistein or herbimycin A, which suggests that these reagents did not directly inhibit ehrlichial energy metabolism. Double indirect immunofluorescence labeling with antiphosphotyrosine antibody and anti-E. risticii antibody revealed colocalization of tyrosine phosphoproteins with ehrlichial inclusions. There was, however, no colocalization of phosphotyrosine with phagosomes containing 0.5-microm-diameter fluorescent beads. Western immunoblot analysis revealed that 52- and 54-kDa proteins were tyrosine phosphorylated only in infected cells and that phosphorylation of these two proteins was reduced when infected cells were treated with genistein for 6 h. These results suggest that protein tyrosine phosphorylation is specific and essential for ehrlichial internalization, replication, and spreading in macrophages but not for binding.
Murine antibody responses to the verotoxin 1 B subunit: demonstration of major histocompatibility complex dependence and an immunodominant epitope involving phenylalanine 30
Authors: Bast D J , Sandhu J , Hozumi N , Barber B , Brunton J
Published: January 2020

Structurally conserved verotoxin 1 (VT1) mutant derivatives, showing reduced receptor binding and cytotoxicity, may serve as natural toxoids to protect against VT-mediated disease. In this study, the antibody responses to the wild-type VT1 B subunit, a B-subunit mutant (Phe30Ala B), and the corresponding holotoxin (Phe30Ala HT) were examined in three inbred mouse strains. BALB/c (H-2d) and CBA (H-2k) mice produced strong antibody responses to both wild-type and mutant B subunits. VT1 B-raised sera reacted more strongly with VT1 B than with Phe30Ala B in enzyme-linked immunosorbent assays, while Phe30Ala B-raised sera reacted equally with VT1 B and Phe30Ala B. C57BL/6 (H-2b) and congenic BALB/c (BALB x B [H-2b]) mice produced no detectable antibody response to either VT1 B or Phe30Ala B. However, an anti-VT1 B antibody response was detected in H-2b mice immunized with biologically active Phe30Ala HT. Based on these observations, we conclude that the VT1 B subunit possesses a B-cell immunodominant epitope formed partly by phenylalanine 30 and that the B-subunit antibody response is dependent on the H-2 haplotype of the mouse strain. Our results also support a potential role for the A subunit in providing the T-cell help necessary to overcome a deficient B-subunit antibody response in H-2b mice.
Induction and characterization of heat shock proteins of Salmonella typhi and their reactivity with sera from patients with typhoid fever
Authors: Tang S W , Abubakar S , Devi S , Puthucheary S , Pang T
Published: January 2020

The heat shock protein (HSP) response of Salmonella typhi following exposure to elevated growth temperatures was studied. Three major proteins with molecular sizes of 58, 68, and 88 kDa were abundantly expressed when S. typhi cells were shifted from 37 to 45 degrees C and to 55 degrees C. These proteins were also constitutively expressed at 37 degrees C. Western blotting and immunoprecipitation studies with anti-HSP monoclonal antibodies revealed that the 58- and 68-kDa proteins were analogous to the GroEL and DnaK proteins, respectively, of Escherichia coli. These HSPs are also abundantly present in the outer membrane fraction of disrupted cells and, to a lesser extent, in the cytosol. Immunoblotting experiments with sera from patients with a culture-positive diagnosis of typhoid fever showed the presence of antibodies to these HSPs. Nine of twelve sera reacted with the 58-, 68-, and 88-kDa proteins, while three sera reacted only with the 68- and 88-kDa proteins. All 10 sera from healthy individuals showed no binding to these HSPs. In light of the well-documented roles of HSPs in the pathogenesis of microbial infections and as immunodominant antigens, these findings may be relevant for a better understanding of disease processes and for the future development of diagnostic and preventive strategies.
Plasmodium falciparum AARP1, a giant protein containing repeated motifs rich in asparagine and aspartate residues, is associated with the infected erythrocyte membrane
Authors: Barale J C , Candelle D , Attal-Bonnefoy G , Dehoux P , Bonnefoy S , Ridley R , Pereira da Silva L , Langsley G
Published: January 2020

During Plasmodium falciparum asexual intraerythrocytic development, the host's cell plasma membrane is modified by the insertion of parasite proteins. One or more of these modifications mediate the cytoadherence of infected erythrocytes to host vascular endothelium. However, these surface antigens can be the target of cytophilic antibodies which promote phagocytosis of the infected erythrocyte. It has been proposed that antibodies directed to epitopes rich in asparagine play an important role in this process, which has promoted efforts to isolate the corresponding gene(s). We describe here P. falciparum asparagine- and aspartate-rich protein 1 (PfAARP1), a new giant (circa 700-kDa) protein associated with the infected erythrocyte membrane which is rich in asparagine and aspartate residues due to the presence of nine blocks of repeats. Topology analysis predicts that PfAARP1 has multiple transmembrane domains and at least five external loops. Human antibodies immunopurified against a sequence composed exclusively of asparagine and aspartate amino acids derived from PfAARP1 label the surface of the infected erythrocyte, demonstrating that such motifs are exposed. Interestingly, external loop 4 of PfAARP1 contains repetitions of these residues, and their possible role as a target of cytophilic antibodies is discussed.
Genetic control of antibody responses induced by recombinant Mycobacterium bovis BCG expressing a foreign antigen
Authors: Lagranderie M , Lo-Man R , Dériaud E , Gicquel B , Gheorghiu M , Leclerc C
Published: January 2020

Recombinant Mycobacterium bovis BCG expressing foreign antigens represents a promising candidate for the development of future vaccines and was shown in several experimental models to induce protective immunity against bacterial or parasitic infections. Innate resistance to BCG infection is under genetic control and could modify the immune responses induced against an antigen delivered by such engineered microorganisms. To investigate this question, we analyzed the immune responses of various inbred strains of mice to recombinant BCG expressing beta-galactosidase. These experiments demonstrated that BALB/c mice developed strong antibody responses against BCG expressing beta-galactosidase under the control of two different promoters. In contrast, C57BL/6, C3H, and CBA mice produced high anti-beta-galactosidase antibody titers only when immunized with recombinant BCG expressing beta-galactosidase under the control of the pblaF* promoter, which induced the production of high levels of this antigen. This difference in mouse responsiveness to recombinant BCG was not due to innate resistance to BCG infection, since similar immune responses were induced in Ity(r) and Ity(s) congenic strains of mice. In contrast, the analysis of anti-beta-galactosidase antibody responses of H-2 congenic mice in two different genetic backgrounds demonstrated that H-2 genes are involved in the immune responsiveness to beta-galactosidase delivered by recombinant BCG. Together, these results demonstrate that immune responses to an antigen delivered by recombinant BCG are under complex genetic influences which could play a crucial role in the efficiency of future recombinant BCG vaccines.
Gut intraepithelial lymphocytes induce immunity against Cryptosporidium infection through a mechanism involving gamma interferon production
Authors: Culshaw R J , Bancroft G J , McDonald V
Published: January 2020

Immunological control of infection with cryptosporidia in mice is dependent on CD4+ T cells and the production of gamma interferon (IFN-gamma), but to date, the mucosal T cells which produce IFN-gamma local to the infection have not been characterized. We previously showed that immunity against the gastric parasite Cryptosporidium muris could be adoptively transferred to adult SCID (severe combined immunodeficiency) mice with small intestinal intraepithelial lymphocytes (IEL) from previously infected immunocompetent mice, but only if the donor CD4+ T cells were intact. The present investigation examined whether IFN-gamma was important in the effector mechanisms mediated by immune IEL in SCID mice. The development of resistance against C. muris infection in SCID mice given immune IEL was prevented by treatment with a hamster anti-mouse IFN-gamma-neutralizing monoclonal antibody, but following cessation of antibody treatment, the mice recovered from infection. In further experiments, an enzyme-linked immunospot (ELISPOT) technique was used to compare frequencies of IFN-gamma-producing cells in activated T-cell populations from C. muris-immune and naive donor mice. Stimulation with concanavalin A or a rat anti-mouse CD3 monoclonal antibody resulted in detection of greater numbers of cells producing IFN-gamma from immune than naive IEL populations. Small numbers of IEL from C. muris-immune mice, but not from naive mice, also produced IFN-gamma when cultured with soluble oocyst antigen, but this occurred only if gamma-irradiated spleen cells were cocultured with the immune IEL. These results suggested that IEL were important in the generation of immunity to Cryptosporidium and that one of their crucial functions was to produce IFN-gamma at the site of infection.
Cloning, characterization, and chromosomal mapping of a phospholipase (lecithinase) produced by Vibrio cholerae
Authors: Fiore A E , Michalski J M , Russell R G , Sears C L , Kaper J B
Published: January 2020

Phospholipases are associated with virulence in bacterial diseases. Vibrio cholerae produces a phospholipase (lecithinase), with enzyme production visualized as a zone of clearing around colonies plated on egg yolk agar. The role of phospholipase in gut colonization or disease pathogenesis is unknown. We used the egg yolk agar assay to clone and characterize a gene encoding a phospholipase from V. cholerae El Tor strain E7946. Sequence analysis revealed a 1,254-bp open reading frame (lec) encoding a 418-amino-acid protein with a predicted molecular weight of 47,600. The predicted sequence exhibits DNA homology to other Vibrionaceae phospholipases. A potential signal sequence exists in the predicted amino acid sequence, as does a lipid binding motif found in prokaryotic and eukaryotic phospholipases and lipases. Polyacrylamide gel electrophoresis combined with an egg yolk agarose overlay demonstrated phospholipase activity migrating at a relative molecular weight of 45,000 in preparations of V. cholerae and the Escherichia coli clone. Restriction mapping and Southern blot analysis revealed that lec, hlyA (hemolysin), and hlyC (lipase) are adjacent on the V. cholerae chromosome, and chromosomal digests of several El Tor, classical, and O139 (Bengal) strains demonstrated conservation of this gene arrangement. An in-frame internal deletion of the lec gene was constructed and recombined into the chromosome of attenuated V. cholerae El Tor strain CVD 110. The resulting mutant lacked lecithinase activity on egg yolk agar but had undiminished reactivity in rabbit ligated ileal loop assays.
Inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus
Authors: Cahill R J , Foltz C J , Fox J G , Dangler C A , Powrie F , Schauer D B
Published: January 2020

Inflammatory bowel disease (IBD) is thought to result from either an abnormal immunological response to enteric flora or a normal immunological response to a specific pathogen. No study to date has combined both factors. The present studies were carried out with an immunologically manipulated mouse model of IBD. Mice homozygous for the severe combined immunodeficiency (scid) mutation develop IBD with adoptive transfer of CD4+ T cells expressing high levels of CD45RB (CD45RB(high) CD4+ T cells). These mice do not develop IBD in germfree conditions, implicating undefined intestinal flora in the pathogenesis of lesions. In controlled duplicate studies, the influence of a single murine pathogen, Helicobacter hepaticus, in combination with the abnormal immunological response on the development of IBD was assessed. The combination of H. hepaticus infection and CD45RB(high) CD4+ T-cell reconstitution resulted in severe disease expression similar to that observed in human IBD. This study demonstrates that IBD develops in mice as a consequence of an abnormal immune response in the presence of a single murine pathogen, H. hepaticus. The interaction of host immunity and a single pathogen in this murine system provides a novel model of human IBD, an immunity-mediated condition triggered by bacterial infection.