Esculetin antagonizes iron-chelating agents and increases the virulence of Listeria monocytogenes

Iron is an essential compound for the growth and virulence of Listeria monocytogenes. In extracellular environments, iron often requires a siderophore to be acquired by microorganisms. Although it does not produce siderophores, L. monocytogenes can use some exogenous bacterial or fungal siderophores as well as a number of animal or plant o-diphenol compounds to overcome growth inhibition by the iron-chelating agents tropolone and 8-hydroxyquinoline. Esculin, a plant glycoside, can be hydrolysed by L. monocytogenes to the o-diphenol aglucon, esculetin. The latter neutralized in vitro growth inhibition induced by the iron-chelating agents. Furthermore, when injected into infected mice, esculetin enhanced mortality in a dose-dependent manner and increased bacterial counts in spleen induced by sublethal doses of L. monocytogenes. Esculetin apparently functioned as a siderophore for L. monocytogenes in murine tissues.     

Differential scanning calorimetry investigations on LPS and free lipids A of the bacterial cell wall

Differential scanning calorimetry (DSC) was used to investigate the thermal stability and behaviour of the lipopolysaccharides (LPS) of the outer membrane of Gram-negative bacteria and their lipid portion. DSC curves of LPS show thermal features between 200 and 129°C (depolymerization) and between −13 and −36°C (cooling phase transition). Both effects were related to the relative strength of the linkage types in the O-chain structure and to their capacity for intermolecular hydrogen bonding. DSC curves of lipids A show endotherm peaks between 40 and 24°C, around 15°C and between −23 and −4°C. Based on these effects, strong differences in thermal behaviour can be observed between that of Brucella and Vibrio cholerae on the one hand, and that of Escherichia coli and Shigella flexneri on the other. Fluidity of the acyl chains and lyotropism, which are important parameters in expression of biological activities, are discussed using the above data. To explain some properties, fluidity could be related to the temperature of the gel ↔ liquid crystalline (β ↔ α) phase transition, which occurs at physiological temperature. Nevertheless, fluidity could be related to temperature of the previous thermal effect (between 6 and 20°C), for which a softening or partial melting of the sample has been evidenced. The thermal effect measured between −23°C and −4°C indicates a greatly reduced water concentration of lipid A from Brucella, thus explaining its early fusion process and its activity by means of hydrophobic interactions.     

The winter ulcer bacterium Moritella viscosa demonstrates adhesion and cytotoxicity in a fish cell model

Moritella viscosa is considered the main aetiological agent of ‘winter ulcer’ disease in farmed salmonid fish. To further understand the pathogenesis of this disease, M. viscosa interaction with fish cells was studied using a Chinook salmon embryo cell line (CHSE-214). As winter ulcer appears exclusively at temperatures below 7–8 °C, we attempted to identify if this connection is explained by temperature regulated bacterial virulence. Therefore, infection studies were performed at a temperature range from 4 to 15 °C. At all temperatures, M. viscosa caused CHSE cells to retract and round up, lose their attachment abilities and finally disintegrate. The bacterium adhered to CHSE cells and caused changes to the cytoskeleton, however, it did not invade the cells. Increased adherence was demonstrated at 4 °C compared to adherence at higher temperatures. Extracellular proteins exerted rapid pore formation and lysis of CHSE cells at a temperature range from 4 to 22 °C. Furthermore, only small differences were found comparing extracellular proteomes of M. viscosa from 4 and 15 °C. We propose that the pathogenic mechanisms exerted by M. viscosa on CHSE cells are disruption of the cytoskeleton which affects cell rigidity and structure, followed by pore formation and lysis caused by secreted products from the bacterium. These processes can also occur at temperatures above those experienced from winter ulcer outbreaks. However, the adhesion mechanisms appear to be temperature regulated and may contribute to temperature dependent disease outbreaks.     

Characterization of non-virulence plasmids with homology to the virulence plasmid of Salmonella dublin

Six wild-type (wt) strains of Salmonella typhimurium, one wt strain of S. heidelberg and 12 wt strains of Escherichia coli were isolated based on both hybridization to a 6-kb HindIII fragment of the non-virulence coding part of the S. dublin serovar-specific virulence plasmid and the absence of hybridization to the virulence genes (spv genes) of the same plasmid. Such hybridization was shown to be caused by resident plasmids in all strains and to involve the same region of 30 to 37 kb of consecutive HindIII fragments on the S. dublin virulence plasmid, suggesting a common origin of this plasmid DNA. Nine of the plasmids were selected for detailed characterization and were shown not to be of the same plasmid species. They varied in size between 44 and 88 kb, they showed incompatibility with the plasmid K-MP10, or belonged to incompatibility group X, and with the exception of five plasmids from E. coli, they showed different HindIII restriction profile patterns.     

Pregnancy reduces the genetic resistance of C57BL/6 mice to Listeria monocytogenes infection by intragastric inoculation

In this study, we compared genetically resistant C57BL/6 and susceptible A/J mice for their resistance to Listeria monocytogenes infection during pregnancy. Intragastric infection with modest numbers of bacterial cells (10⁵ CFU) caused reproducible fetal infection and abortion in both mouse strains. Bioluminescence imaging demonstrated dissemination of L. monocytogenes cells from maternal to fetal organs within 3 days of intragastric infection. Although non-pregnant C57BL/6 mice were significantly more resistant to infection than non-pregnant A/J mice, C57BL/6 and A/J mice had similar microbial loads (CFU) in maternal and fetal tissues during pregnancy. Inflammation and necrosis, however, were more severe in A/J mice as evaluated by semi-quantitative histopathology. Although the microbial load in fetal tissues was similar for all fetuses within a single uterus, inflammation and necrosis varied among individual fetuses and placentas. We also noted that the uterus is a target for L. monocytogenes infection in non-pregnant mice.     

The role of the O-antigen lipopolysaccharide on the colonizationin vivoof the germfree chicken gut byAeromonas hydrophilaserogroup O:34

We compared the ability of differentAeromonas hydrophilastrains from serogroup O:34 grown at different temperatures to colonizein vivothe germfree chicken gut. We found a good colonization when the strains were grown at 20°C but not when they were grown at 37°C. We previously described that these strains were able to form the O-antigen lipopolysaccharide (LPS) when they grow at low temperature but not at high temperature. We also obtained by transposon mutagenesis mutants only devoid of the O-antigen LPS (rfbmutants), and showed that they were unable to colonize the germfree chicken gut. All these results prompted us to conclude that the O-antigen LPS, in these strains, is a main factor for colonization in this animal model system.     

Beta-naphthoflavone causes an AhR-independent inhibition of invasion and intracellular multiplication of Listeria monocytogenes in murine hepatocytes

We recently reported a heretofore unknown role for the aryl hydrocarbon receptor in host resistance to listeriosis in mice. Hepatocytes are an important site for Listeria monocytogenes multiplication in vivo. In this study, we investigated whether activation of AhR in TIB73 murine embryonic hepatocytes affects the ingestion and intracellular multiplication of L. monocytogenes. Treatment of TIB73 cells with the AhR agonist β-naphthoflavone (BNF) significantly inhibited the ingestion and intracellular growth of L. monocytogenes. The inhibitory effects of BNF were dose-dependent and correlated with up-regulation of CYP1A1. Surprisingly, pretreatment with AhR antagonists (3′-MNF or α-naphthoflavone) or knocking-down of AhR with siRNA did not abolish the inhibitory effects of BNF. Moreover, the inhibitory effects of BNF on invasion and intracellular growth of L. monocytogenes by BNF were observed in AhR-deficient (CRL-2710), or ARNT-dysfunctional (CRL-2717) Hepa cells. We also observed similar inhibitory effects of BNF treatment using primary hepatocytes recovered from AhR^+/− or AhR^−/− mice. Moreover, the prototypic AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) did not inhibit the invasion and intracellular growth of L. monocytogenes in TIB73 cells. Mechanistic studies demonstrated that ROS, but not TNF-α or iNOS, plays an important role in mediating BNF-induced inhibition. In conclusion, BNF caused an AhR-independent inhibition of ingestion and intracellular multiplication of L. monocytogenes in murine hepatocytes, mediated in part by production of ROS.     

Listeria monocytogenes as novel carrier system for the development of live vaccines

Listeria monocytogenes is a facultative intracellular bacterium that enters a variety of non-professional mammalian cells by triggered phagocytosis (“zipper mechanism”) and replicates in the cytosol of the infected host cells. Therefore, it is a promising vaccine vector for the presentation of passenger antigens to the MHC class II and especially class I pathways. Here, we review recent progress made in our laboratory on the development of novel attenuated L. monocytogenes carrier strains for the delivery of heterologous antigens or antigen-encoding DNA and RNA to eukaryotic host cells. Based on the deletion of the chromosomal copy of the tryptophanyl-tRNA synthetase gene (trpS) and plasmid-based in trans complementation of the same, we were able to establish a balanced-lethal plasmid system in L. monocytogenes. Safety concerns in the antigen delivery in vivo were addressed by chromosomal deletion of genes in the basic branch of the aromatic amino acid pathway, resulting in safe, attenuated L. monocytogenes carrier strains. Furthermore, plasmid-based expression of a cytosolically expressed phage lysin resulted in a self-destructing carrier strain that has been successfully used for the delivery of antigens as well as antigen-encoding plasmid DNA and particularly mRNA, therefore overcoming bottlenecks that have been shown to exist for bacteria-mediated DNA delivery.     

Persistent production of interferon-gamma (IFN-γ) and IL-12 is essential for the generation of protective immunity against Listeria monocytogenes

IFN-γ and IL-12 are believed to be important in the host defence against Listeria infection in mice. However, the relationship between these two cytokines and generation of protective immunity remains poorly understood. In the present study, it was found that at least 4 days of immunizing infection were required for the generation of protective immunity against L. monocytogenes. Protective immunity was generated only by immunizing infection with virulent strain. Even repeated injections of avirulent strain failed to induce protective immunity. When the immunizing infection was terminated with antibiotics, generation of protective immunity and IFN-γ-producing ability was impaired, while expression of IFN-γ and IL-12 was also impaired. The mutual relationship between IFN-γ and IL-12 in L. monocytogenes infection was analysed in vitro. After neutralization of IL-12, IFN-γ production was completely blocked and IFN-γ expression was also inhibited. In contrast, there was no change of IL-12 expression after neutralization of IFN-γ. Taking all facts into consideration, it may be concluded that persistent production of IFN-γ induced by persistent production of IL-12 during immunizing infection is essential for the generation of protective immunity against L. monocytogenes.     

Prevalence,identification by a DNA microarray-based assay of human and food isolates Listeria spp. from Tunisia

Objectives

We aimed at evaluating the prevalence of Listeria species isolated from food samples and characterizing food and human cases isolates.

Material and methods

Between 2005 and 2007, one hundred food samples collected in the markets of Tunis were analysed in our study. Five strains of Listeria monocytogenes responsible for human listeriosis isolated in hospital of Tunis were included. Multiplex PCR serogrouping and pulsed field gel electrophoresis (PFGE) applying the enzyme AscI and ApaI were used for the characterization of isolates of L. monocytogenes. We have developed a rapid microarray-based assay to a reliable discrimination of species within the Listeria genus.

Results

The prevalence of Listeria spp. in food samples was estimated at 14% by using classical biochemical identification. Two samples were assigned to L. monocytogenes and 12 to L. innocua. DNA microarray allowed unambiguous identification of Listeria species. Our results obtained by microarray-based assay were in accordance with the biochemical identification. The two food L. monocytogenes isolates were assigned to the PCR serogroup IIa (serovar 1/2a). Whereas human L. monocytogenes isolates were of PCR serogroup IVb, (serovars 4b). These isolates present a high similarity in PFGE. Food L. monocytogenes isolates were classified into two different pulsotypes. These pulsotypes were different from that of the five strains responsible for the human cases.

Conclusion

We confirmed the presence of Listeria spp. in variety of food samples in Tunis. Increased food and clinical surveillance must be taken into consideration in Tunisia to identify putative infections sources.     

Rat dorsal root ganglia neurons as a model for Listeria monocytogenes infections in culture

Neurotropism of Listeria monocytogenes was studied in rat dorsal root ganglia (DRG) and hippocampal neurons in culture. Using a system in which the DRG neurons can grow relatively free from other cells, it was observed that such DRG neurons, in contrast to hippocampal neurons, can be effectively infected by L. monocytogenes. The bacteria aligned along DRG axons, but not along hippocampal neurites. A mutant deficient in internalin, a protein required for entry into E-cadherin-expressing cells, did not interact with DRG neurons. Axonal migration of bacteria was studied in the DRG neurons grown in a double-chamber system, where either the neurites or the nerve cell bodies were exposed to the bacteria. The data suggest that L. monocytogenes can infect both axons and DRG nerve cell bodies, and that the bacteria can migrate in a retrograde as well as anterograde direction. These results support the notion that L. monocytogenes can spread via primary sensory neurons to the central nervous system. Infection of DRG primary sensory neurons, as employed in the present study, provides a model for analysis of bacterial and neuronal factors of importance for neurovirulence of L. monocytogenes. Received: 3 April 1999     

Interaction of Leishmania mexicana promastigotes with the plasminogen–plasmin system

The binding of human plasminogen and plasmin to the promastigote form of Leishmania mexicana was investigated. L. mexicana was capable to bind both molecules, the binding being inhibited by -aminocaproic acid. Scatchard plot analysis revealed a dissociation constant (K_d) value of 2.4±0.8 μM and 0.9±0.1×10⁴ binding sites per cell for plasminogen and a K_d value of 1.2±0.4 μM and 1.6±0.2×10⁵ binding sites per cell for plasmin. C-terminal lysine residues are involved in plasminogen binding to cells, since carboxypeptidase B treatment reduced this binding by 34%. Ligand blotting analysis showed a group of proteins, with molecular masses between 105 and 115 kDa, capable to interact with plasminogen. Zymogram analysis showed that the protease activity acquired by L. mexicana, due to the interaction with either plasminogen or plasmin, comprises an important fraction of the total protease activity at pH 7.7. Plasminogen activation by tissue-type plasminogen activator (t-PA) was enhanced by the presence of L. mexicana promastigotes. These results raise the question whether the interaction of L. mexicana with components of the fibrinolytic system is involved in the virulence of the parasite.     

Temperature dependence of mycosubtilin homologue production in Bacillus subtilis ATCC6633

Bacillus subtilis ATCC6633 produces mycosubtilin, a non-ribosomally synthesized lipopeptide of the iturin family which presents antagonistic activities toward various phytopathogens. Different homologues with fatty acid moiety varying from C₁₅ to C₁₇ are usually co-produced, with their biological activities increasing with the number of carbons in the fatty acid chain. In the present report, we highlight that growth temperature modulates both the extent of mycosubtilin production and the relative abundance of the different homologues. A 30-fold increase in mycosubtilin production was observed when the temperature was decreased from 37 °C to 25 °C for both strain ATCC6633 and its derivative BBG100, a constitutive mycosubtilin overproducer. However, no significant difference in either the expression of the mycosubtilin synthetase encoding genes or in the intracellular synthetase concentration could be found, suggesting that the observed phenotype originated from a higher mycosubtilin synthetase turnover at lower temperature. We also point out that lower growth temperature leads to an increased proportion of odd-numbered fatty acid homologues as a consequence of de novo synthesis of C₁₇ anteiso fatty acid following cell adaptation to low temperatures.     

Effect of rush immunotherapy on airway inflammation and airway hyperresponsiveness after bronchoprovocation with allergen in asthma

Background: Rush immunotherapy (RIT) has been shown to be effective in allergic asthma. Objective: We investigated the mechanisms of RIT on the basis of cytokine production by T-cell lines and airway inflammation and responsiveness. Methods: Subjects were 8 patients with house dust mite–allergic asthma treated with dust mite extract RIT for 6 months and 6 RIT-untreated control patients. IL-5 production by Dermatophagoides farinae –specific T-cell lines, eosinophil percentages, and eosinophil cationic protein (ECP) in induced sputum and airway responsiveness to allergen and histamine were evaluated before and after treatment. Changes in eosinophil percentages and ECP in induced sputum and responsiveness to histamine 24 hours after allergen inhalation were also studied. Results: After 6 months of RIT, percentages of total eosinophils (43.0% ± 6.90% to 16.8% ± 2.48%; P < .01), percentages of EG2⁺ eosinophils (32.6% ± 6.39% to 19.7% ± 4.68%; P < .01) and ECP (362.7 ± 125.3 ng/mL to 26.2 ± 5.15 ng/mL; P < .05) decreased in induced sputum, and IL-5 production by T-cell lines decreased (617 ± 93.2 pg/mL to 200.0 ± 34.1 pg/mL; P < .01). RIT decreased both early- and late-phase bronchoconstriction (early phase: 33.2% ± 3.46% to 25.4% ± 1.42%; P < .03; late phase: 16.2% ± 3.52% to 6.2% ± 1.96%; P < .03) and suppressed increases in the percentages of total (61.8% ± 4.89% to 42.0% ± 4.67%; P < .01) and EG2-positive eosinophils (55.54% ± 7.21% to 36.5% ± 6.43%; P < .01) and ECP (685.6 ± 217.0 ng/mL to 85.4 ± 23.4 ng/mL; P < .05) in induced sputum after allergen inhalation. RIT also decreased airway responsiveness to dust mite (1:303.7 ± 123.7 wt/vol to 1:65.0 ± 13.2 wt/vol; P < .03) and to histamine before (397.1 ± 206.9 μg/mL to 1391.3 ± 283.3 μg/mL; P < .03) and after allergen inhalation (139.2 ± 36.5 μg/mL to 629.1 ± 196.3 μg/mL; P < .03). Conclusion: RIT decreases airway inflammation and airway hyperresponsiveness before and after bronchial provocation with allergen, possibly by inhibiting both allergen-specific T-cell– and mast cell-dependent pathways. RIT is an effective antiinflammatory treatment in allergic asthma. (J Allergy Clin Immunol 1998;102:927-34.)     

Study of the haemolytic process and receptors of thermostable direct haemolysin from Vibrio parahaemolyticus

The haemolytic action of ¹²⁵I-labelled thermostable direct haemolysin from Vibrio parahaemolyticus was studied on human and equine erythrocytes. In the first step, the haemolysin bound to the membranes of both erythrocyte species. This binding seemed temperature-independent. Then, for human erythrocytes, haemolysin produced cell disruption, and haemoglobin was released. Following this step, haemolysin was also released in a temperature-dependent manner. In contrast, equine erythrocytes were not disrupted, and no release of haemolysin occurred. The receptors of labelled haemolysin were analysed by assaying the lipid/toxin interaction on a nylon membrane and by binding on thin-layer chromatograms. The ganglioside asialo-G_M2 was found to be the most potent receptor, but asialo-G_M1 and lactocerebroside may also have been involved.     

Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: Relationship with expression of adhesins and production of toxins

A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesinencoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap⁺ and/or sfa⁺ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing a-haemolysin (Hly⁺) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesinencoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.La PCR a permis de détecter les opérons pap, sfa et afa chez 243 souches de Escherichia coli isolées d'infections de l'arbre urinaire. On observe que respectivement 54, 53 et 2% des souches sont du génotype pap, sfa et afa. Les souches pap⁺ et/ou sfa⁺ sont plus fréquentes dans les pyelonephritis aiguës (94%) que dans les cystites (67%) (P < 0,001) et dans les bactériuries asymptomatiques (57%) (P < 0,001). Les opérons pap et/ou sfa sont décelés chez 90% des souches MRHA⁺ (hémagglutination mannoserésistante) et 37% des souches MRHA⁻ (P < 0,001). La présence des opérons pap et sfa est spécialement significative chez les souches appartenant au type III de MRHA (100%) sans adhésines P, et au type IVa (97 %) exprimant la liaison Gai-Gal typique des adhésines P. Les opérons pap et sfa sont tous deux étroitement associés aux souches toxigéniques produisant l'α-hémolysine (Hly⁺) et le facteur cytotoxique nécrotique de type 1. Une corrélation apparaît entre les opérons pap et sfa et le sérogroupe: 93 % des souches appartenant aux groupes O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 et O83 possèdent ces opérons, et seulement 37 % des souches appartenant à d'autres sérogroupes (P < 0,001) les possèdent. Ces résultats confirment l'utilité de notre typage MRHA pour l'identification (présomptive) des souches pathogènes de E. coli porteuses de divers facteurs de virulence. Ainsi, 85 % des souches possédant à la fois les opérons pap et sfa codant les adhésines sont du type III ou IVa (de MRHA) en relation avec la virulence des souches responsables d'infections urinaires et de bactériémies.     

Antibodies to Campylobacter flagellin recognize epitopes common to phase 1 and phase 2 flagella

We used a simple method to obtain purified flagellin from Campylobacter, suitable for an immunization procedure in mice. Western blot analysis of cross-reacting antibodies showed that there were epitopes common to phase 1 and 2 flagellins. Analysis by ELISA suggested that certain common flagellar epitopes are conformational, and antibody immobilization tests confirmed that common surface-exposed epitopes exist in a region of flagella necessary for conferring motility to the bacteria.     

A Sch9 protein kinase homologue controlling virulence independently of the cAMP pathway in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

The polysaccharide capsule is one of the established virulence factors in Cryptococcus neoformans that provides a barrier against the host-mediated immune response. Mutation of the gene encoding the Saccharomyces cerevisiae Sch9 protein kinase homologue resulted in cells with enlarged capsules in C. neoformans. Capsule production was abrogated in sch9 pka1 double mutants, indicating that protein kinase A (PKA) signaling is still necessary for capsule formation in sch9 mutants. The sch9 mutant also exhibited increased thermal tolerance, a phenotype similar to sch9 mutant strains of S. cerevisiae. In addition, the sch9 mutant was attenuated in mating and the highly encapsulated cells were attenuated in virulence, in contrast to the pkr1 mutant, lacking the regulatory subunit of protein kinase A, that produced similarly enlarged capsules yet was increased in virulence. Interestingly, the virulence for the sch9 mutant strain could be restored by introduction of a pkr1 mutation; and the sch9 pkr1 mutant strain was dramatically increased in size and capsule thickness, suggesting that Sch9 and PKA function via different targets involved in virulence. Our findings support a model in which Sch9 modulates capsule formation and contributes to the virulence of C. neoformans both independently of and in conjunction with the cAMP–PKA pathway.