Liposome-induced conformational changes of an epitopic peptide and its palmitoylated derivative of influenza virus hemagglutinin

Meningococcal sodC encodes periplasmic copper- and zinc-cofactored superoxide dismutase (Cu,Zn SOD) which catalyzes the conversion of the superoxide radical anion to hydrogen peroxide, preventing a sequence of reactions leading to production of toxic hydroxyl free radicals. From its periplasmic location, Cu,Zn SOD was inferred to acquire its substrate from outside the bacterial cell and was speculated to play a role in preserving meningococci from the action of microbicidal oxygen free radicals produced in the context of host defense. A sodC mutant was constructed by allelic exchange and was used to investigate the role of Cu,Zn SOD in pathogenicity. Wild-type and mutant meningococci grew at comparable rates and survived equally long in aerobic liquid culture. The mutant showed no increased sensitivity to paraquat, which generates superoxide within the cytosol, but was approximately 1,000-fold more sensitive to the toxicity of superoxide generated in solution by the xanthine/xanthine oxidase system. These data support a role for meningococcal Cu,Zn SOD in protection against exogenous superoxide. In experiments to translate this into a role in pathogenicity, wild-type and mutant organisms were used in an intraperitoneal mouse infection model. The sodC mutant was significantly less virulent. We conclude that periplasmic Cu,Zn SOD contributes to the virulence of Neisseria meningitidis, most likely by reducing the effectiveness of toxic oxygen host defenses.     

The isolation and characterisation of a gene encoding superoxide dismutase from Bradyrhizobium sp. (Parasponia) strain ANU289

Bradyrhizobium sp. (Parasponia) strain ANU289 expresses a single Mn-SOD in both the vegetative and symbiotic states. A 500 bp sod-homologous sequence was amplified from genomic DNA of strain ANU289 using PCR. A 1.3 kb SalI fragment was subsequently cloned which contained an ORF, sodA, encoding a 23 Kd protein. This putative SOD shares considerable homology with other Mn-SODs and analysis of the sodA sequence predicts that it is expressed. A lacZ-sodA fusion complemented the SOD-deficiency of E. coli QC779 and resulted in the expression of SOD activity in both mutant and wild type E. coli. We conclude that sodA encodes the Mn-SOD of strain ANU289.     

Prevalence of the "high-pathogenicity island" of Yersinia species among Escherichia coli strains that are pathogenic to humans

The fyuA-irp gene cluster contributes to the virulence of highly pathogenic Yersinia (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica 1B). The cluster encodes an iron uptake system mediated by the siderophore yersiniabactin and reveals features of a pathogenicity island. Two evolutionary lineages of this "high pathogenicity island" (HPI) can be distinguished on the basis of DNA sequence comparison: a Y. pestis group and a Y. enterocolitica group. In this study we demonstrate that the HPI of the Y. pestis evolutionary group is disseminated among species of the family Enterobacteriaceae which are pathogenic to humans. It prevails in enteroaggregative Escherichia coli and in E. coli blood culture isolates (93 and 80%, respectively), but is rarely found in enteropathogenic E. coli, enteroinvasive E. coli, and enterotoxigenic E. coli isolates. In contrast, the HPI was absent from enterohemorrhagic E. coli, Shigella, and Salmonella enterica strains investigated. Polypeptides encoded by the fyuA, irp1, and irp2 genes located on the HPI could be detected in E. coli strains pathogenic to humans. However, these E. coli strains showed a reduced sensitivity to the bacteriocin pesticin, whose uptake is mediated by the FyuA receptor. Escherichia strains do not possess the hms gene locus thought to be a part of the HPI of Y. pestis. Deletions of the juA-irp gene cluster affecting solely the fyuA part of the HPI were identified in 3% of the E. coli strains tested. These results suggest horizontal transfer of the HPI between Y. pestis and some pathogenic E. coli strains.     

The role of ferredoxin-NADP+ reductase in the concerted cell defense against oxidative damage -- studies using Escherichia coli mutants and cloned plant genes

Ferredoxin-NADP+ reductases (FNR) participate in cellular defense against oxidative damage. Escherichia coli mutants deficient in FNR are abnormally sensitive to methyl viologen and hydrogen peroxide. Tolerance to these oxidants was regained by expression of plant FNR, superoxide dismutase, or catalase genes in the mutant cells. FNR contribution to the concerted defense against viologen toxicity under redox-cycling conditions was similar to that of the two major E. coli superoxide dismutases together, as judged by the phenotypes displayed by relevant mutant strains. However, FNR expression in sodA sodB strains failed to increase their tolerance to viologens, indicating that the FNR target is not the superoxide radical. Sensitivity of FNR-deficient cells to oxidants is related to extensive DNA damage. Incubation of the mutant bacteria with iron chelators or hydroxyl radical scavengers provided significant protection against viologens or peroxide, suggesting that oxidative injury in FNR-deficient cells was mediated by intracellular iron through the formation of hydroxyl radicals in situ. The NADP(H)-dependent activities of the reductase were necessary and sufficient for detoxification, without participation of either ferredoxin or flavodoxin in the process. Possible mechanisms by which FNR may exert its protective role are discussed.     

Superoxide dependence of the toxicity of short chain sugars

Erythrose inhibited the growth of a sodA sodB strain of Escherichia coli under aerobiosis; but did not inhibit anaerobic growth of the sodA sodB strain, or the aerobic growth of the superoxide dismutase (SOD)-competent parental strain. A SOD mimic protected the sodA sodB strain against the toxicity of erythrose as did the carbonyl-blocking reagents hydrazine and aminoguanidine. Three carbon sugars, such as glyceraldehyde and dihydroxy acetone, and the two carbon sugar glycolaldehyde, were similarly toxic in an O-2-dependent manner. An unidentified dialyzable component in E. coli extract augmented the oxidation of short chain sugars, and this was partially inhibitable by SOD. The toxicity of the short chain sugars appears to be because of an O-2-dependent oxidation to alpha, beta-dicarbonyl compounds. In keeping with this view was the O-2-independent toxicity of methylglyoxal.     

Reevaluation of the virulence phenotype of the inv yadA double mutants of Yersinia pseudotuberculosis

Yersinia pseudotuberculosis and Yersinia enterocolitica are closely related human pathogens causing gastroenteritis. Invasin and YadA are two of the most extensively studied virulence factors of the Yersinia genus. Invasin is the primary invasion factor encoded by the inv gene on the chromosome and is required for the penetration of the epithelial cells. YadA is encoded by the yadA gene on the 70-kb virulence plasmid and has multiple functions. Previous studies indicate that an inv yadA double mutant of Y. enterocolitica is avirulent while an inv yadA mutant of Y. pseudotuberculosis is hypervirulent. In this study, we investigated this unexpected difference. New constructs of the inv yadA mutants of Y. pseudotuberculosis were made and tested in mice. These new constructs were not hypervirulent; rather, they maintained the same virulence as the wild-type strain. Further examination of the inv mutant used for the previous study revealed that it carries an aberrant inv phenotype and has an altered outer membrane profile and an altered colony morphology. Therefore, the mutants used previously were not isogenic to the parental wild-type strain, which may in part account for the difference in the results obtained.     

Role of manganese superoxide dismutase in a mucoid isolate of Pseudomonas aeruginosa: adaptation to oxidative stress

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is a leading cause of morbidity among cystic fibrosis (CF) patients. In the lungs of CF patients, the bacteria are exposed to activated oxygen species produced by the phagocytes of the host or resulting from the metabolism of oxygen. Two isoforms of superoxide dismutase are synthesized by P. aeruginosa; they differ by the metal present at their active site, which is either iron or manganese. To evaluate the role of manganese-containing superoxide dismutase (MnSOD), encoded by sodA, we have isolated a sodA mutant of the mucoid P. aeruginosa strain CHA isolated from the bronchopulmonary tract of a CF patient. The sodA mutant exhibited an increased sensitivity to oxidative stress generated by paraquat and was less resistant to oxidative stress in the stationary phase of growth compared with its parental strain. It was observed that MnSOD was expressed in the parental strain solely during the stationary phase of growth and that cells of the sodA mutant taken at the stationary phase resumed growth with a longer delay than the sodA+ cells when reinoculated in a new medium, especially in the presence of paraquat. These results suggest that MnSOD may participate in the adaptation of mucoid strains of P. aeruginosa to the stationary phase of growth in the lungs of CF patients.     

The fliA gene encoding sigma 28 in Yersinia enterocolitica

Yersinia enterocolitica is an enterobacterium responsible for gastrointestinal syndromes. Its pathogenicity depends on the presence of the 70-kb pYV plasmid, which directs Yop secretion. The Yop secretion machinery, consisting of the YscA-U and LcrD proteins, presents some structural similarity with the flagellum assembly machinery characterized in other bacteria. Flagellum assembly requires sigma 28, an alternative sigma factor. The region upstream of the lcrD gene resembles promoters recognized by sigma 28, suggesting that the similarity between Yop secretion and flagellum assembly could extend to their regulation. The chromosome of Y. enterocolitica also contains pathogenicity determinants such as myfA, which encodes the Myf antigen subunit. The promoter region of myfA also resembles promoters recognized by sigma 28. In an attempt to clarify the role of sigma 28 in the expression of lcrD, myfA, and flagellar genes, we cloned, sequenced, and mutagenized the fliA gene encoding the sigma 28 homolog in Y. enterocolitica. As is the case in other bacteria, fliA was required for motility. However, it was involved neither in fibrilla synthesis nor in Yop secretion. The fliA mutant allowed us to monitor the role of motility in pathogenesis. At least in the mouse model, motility seemed not to be required for Y. enterocolitica pathogenesis.     

The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5 degrees C)

The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent adaptations. To investigate Y. enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5 degrees C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy-defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp. Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp, including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5 degrees C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y. enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6-fold higher at 5 degrees C compared with 30 degrees C. A similarly enhanced level of PNPase at 5 degrees C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold-inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.     

Characterization of regulatory mutations causing anaerobic derepression of the sodA gene in Escherichia coli K12: cooperation between cis- and trans-acting regulatory loci

The genetic loci leading to anaerobic derepression of a sodA::lacZ protein fusion in a UV-generated mutant strain (UV14) of Escherichia coli were identified. The mutant (UV14) was found to harbour two altered loci: one is in the trans-regulatory gene fnr (fumarate nitrate reduction) where leucine-129 was changed to glutamine (fnr14), and the second (sodA14) is in the promoter region (cis) of the sodA gene apparently affecting the binding of the Fur (ferric uptake regulation) protein. Introduction of an fnr+ gene into UV14 restored anaerobic repression of sodA::lacZ and restored the ability of the cells to reduce nitrate. However, when either the fnr14 or the sodA14 mutation was introduced into an otherwise wild-type background, only slight anaerobic derepression of sodA was observed. When both the cis- and trans-acting mutations (i.e. sodA14 and fnr14) were combined simultaneously in an otherwise wild-type background, the specific activity of sodA::lacZ expression was comparable to that of the original mutant strain (UV14). Furthermore, a genetically confirmed fur fnr double mutant was also similarly derepressed in anaerobic sodA::lacZ expression. The data presented suggest that the cis-mutation in UV14 (sodA14) affects the Fur-binding site in the sodA promoter, while having no effect on Fnr or Arc mediated repression. Also, a second putative Fnr-binding site that straddles the ribosomal binding-site was identified in the sodA gene.     

Flow cytometric DNA analysis of abnormal endometrium

When tyrosine-Z of the D1-polypeptide of the photosystem II from Chlamydomonas reinhardtii was changed to phenylalanine, the rapid donor to P680+ was lost, and P680+ accumulated on illumination. The rapid donation from tyrosine-Z was replaced by a slow electron transfer from an endogenous donor. Spectrophotometric measurements showed that carotenoids and chlorophylls were bleached by the P680+ either directly or indirectly upon illumination. The carotenoid bleaching was inhibited in the presence of SOD or catalase, but the reaction did not require molecular oxygen as an electron acceptor. These observations led us to conclude that active oxygen radicals, possibly hydroxyl radicals, take part in the destruction of carotenoids in the Y161F mutant. Possible mechanisms for the destruction are discussed.     

Virulence and arsenic resistance in Yersiniae

The genus Yersinia contains three pathogenic species: Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica. Only a few biotypes and serotypes of Y. enterocolitica are pathogenic, and these form two distinct groups: some are of low virulence, and they are encountered worldwide; others, mainly encountered in North America, are markedly more virulent. All pathogenic yersiniae possess a 70-kb virulence plasmid called pYV which encodes secreted antihost proteins called Yops as well as a type III secretion machinery that is required for Yop secretion. Genes encoding Yop synthesis and secretion are tightly clustered in three quadrants of the pYV plasmid. We show here that in the low-virulence strains of Y. enterocolitica, the fourth quadrant of the plasmid contains a new class II transposon, Tn2502. This transposon encodes a defective transposase, but transposition can be complemented in trans by Tn2501, another class II transposon. Tn2502 was not detected in the pYV plasmids of the more virulent American strains of Y. enterocolitica, of Y. pseudotuberculosis, and of Y. pestis. Tn2502 confers arsenite and arsenate resistance. This resistance involves four genes; three are homologous to the arsRBC genes present on the Escherichia coli chromosome, but no homolog of the fourth one, arsH, has been found. The systematic presence of such a resistance operon on a virulence plasmid is unusual and could be related to the recent spread of low-virulence Y. enterocolitica strains. The presence of this ars operon also constitutes the first significant difference between the pYV plasmids from different Yersinia species.     

Experimental infection of axenic mice by "Yersinia enterocolitica" (author's transl)

Although most of Yersinia enterocolitica strains isolated from man have no pathogenicity for laboratory animals, it has been demonstrated that some strains are pathogenic for conventional mice and that most of the strains are probably pathogenic for Nude mice. The authors report the results of the infection of germ-free mice with a strain of Y. enterocolitica which is non pathogenic for holoxenic mice. It appears that C3H/He mice are sensitive to the infection by gavage or aerogenic and peritoneal routes. They all die within 8 to 12 days after injection of an inoculum of 5.10(5) viable cells. Germ-free NCS mice were also sensitive to the oral and aerogenic infection but not to the peritoneal infection; the difference between C3H/He and NCS sensitivity to this way of infection could be explained by a higher bactericidal activity of the peritoneal phagocytes of the latter. The C3H/He and NCS holoxenic control mice infected with the same inoculum of the same strain, did not show any symptoms and all attempts to isolate Y. enterocolitica failed three months after the challenge. Germ-free mice killed by the infection showed histopathological findings, i.e. abscesses involving intestinal wall. liver and spleen; they were similar to those described in experiments with pathogenic strains for conventional mice (holoxenic) and to those observed in infection of athymic Nude mice with strains non pathogenic for conventional mice. This infectious disease model is discussed in regards to the natural human infection.     

Direct detection and isolation of plasmid-bearing virulent serotypes of Yersinia enterocolitica from various foods

A procedure was developed for direct detection, isolation, and maintenance of plasmid-bearing virulent serotypes of Yersinia enterocolitica from different food sources. Plasmid-bearing virulent strains of Y. enterocolitica representing five serotypes were simultaneously detected and isolated from enriched swab samples of artificially contaminated pork chops, ground pork, cheese, and zucchini, using Congo red binding and low-calcium-response tests. The method was also effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Virulence of the strains isolated from these foods was confirmed by PCR, the expression of plasmid-associated phenotypes, and mouse pathogenicity.     

Suppression of the granulocyte colony-stimulating factor response to Escherichia coli challenge by alcohol intoxication

Alcohol's suppressive effects on polymorphonuclear leukocyte (PMN) production and function increases host susceptibility to a wide variety of infections and impairs the ability of these effector cells to seek and destroy invading pathogens. Granulocyte colony-stimulating factor (G-CSF), an important regulator of PMN production and function, is known to be increased in the plasma during infectious episodes. In previous studies we found acute alcohol intoxication to suppress the tumor necrosis factor-alpha (TNF alpha) response to in vivo challenges with bacteria or lipopolysaccharide. The present study was initiated to determine the impact of alcohol intoxication on the plasma G-CSF response to gram-negative infection. For this purpose, rats received an intravenous challenge of Escherichia coli (10(6) CFU) 30 min after an intraperitoneal injection of ethanol (5.5 g/kg) or an equivalent volume of saline (control). Ethanol-intoxicated rats had a greater 48 hr mortality to live E. coli injection than did unintoxicated animals (45% vs. 8%). Despite an increased bacterial burden in both the lung and liver at 24 hr after initiating E. coli infection in alcohol-intoxicated animals, PMN tissue recruitment, indexed as myeloperoxidase activity, did not differ between control and alcohol-treated rats. Moreover, alcohol suppressed blood PMN phagocytic capacity to a greater extent in animals given alcohol than controls at 5 and 24 hr after initiating infection. In control animals after intravenous E. coli injection, bioactive G-CSF increased in plasma and peaked near 300 ng/ml at 8 hr. In rats pretreated with alcohol, the plasma G-CSF response was markedly suppressed in response to intravenous E. coli (p < 0.05). In a second experiment, neutralization of the E. coli-induced plasma TNF alpha response by pretreatment with anti-TNF alpha antibody similarly inhibited the plasma G-CSF response. These results support the postulate that alcohol-induced inhibition of TNF alpha directly contributes to the adverse effects of alcohol on PMN function by suppressing the normal autocrine amplification pathway responsible for G-CSF production.     

Penetration of M cells and destruction of Peyer's patches by Yersinia enterocolitica: an ultrastructural and histological study

Yersinia enterocolitica is enteropathogenic for man and rodents. Previous studies provided evidence that Y. enterocolitica invades the lymphoid follicles of the Peyer's patches (PP) of the small intestine. In this study Y. enterocolitica-induced tissue alterations of the follicle-associated epithelium (FAE) and the underlying PP tissue were analysed by scanning (SEM) and transmission electron microscopy (TEM) as well as by conventional histological examination. For this purpose, an experimental mouse infection model including orogastric infections as well as ileal loop experiments were used. A rapid and selective colonisation of the FAE after orogastric yersinia infection was observed by SEM. TEM studies confirmed that Y. enterocolitica adhered closely to the FAE including M cells and enterocytes. Histological studies and TEM revealed that Y. enterocolitica selectively invaded the PP via M cells but not via other cells of the FAE. One day after Y. enterocolitica infection the FAE was altered and small micro-abscesses comprising yersiniae expressing the major outer-membrane protein YadA were observed immediately beneath the FAE. Adjacent villi were dilated from lymphangiectasis and transmigrating polymorphonuclear leucocytes (PMNL) were found within the epithelium. At 5-7 days after infection the FAE and parts of PP were destroyed. Profound alterations of the cyto-architecture of the PP were due to the enormous recruitment of PMNL. By day 5 after infection, abscesses were found in the mesenteric lymph nodes. However, TEM studies revealed evidence that Y. enterocolitica may disseminate from the PP not only via the lymphatics but also by invasion of blood vessels. Taken together, the results of this study demonstrate that the FAE is the primary site of host-pathogen interaction in Y. enterocolitica infection and that this pathogen penetrates M cells and subsequently induces destruction of the PP.