Epithelial invasion and cell lysis by virulent strains of Streptococcus suis is enhanced by the presence of suilysin

Streptococcus suis is an important pathogen of pigs causing arthritis, pneumonia and meningitis and is an occupational disease of farmers and those in the meat industry. As with other streptococci, both virulent and avirulent strains of S. suis are frequently carried asymptomatically in the tonsillar crypts and nasal cavities. Little is known about the process by which virulent strains cross the mucosal epithelia to generate systemic disease and whether this process requires expression of specific bacterial virulence factors. Although putative virulence factors have been postulated, no specific role in the disease process has yet been demonstrated for these factors. This study is the first demonstration that virulent strains of S. suis both invade and lyse HEp-2 cells, a continuous laryngeal epithelial cell line, and that at least one bacterial virulence factor, suilysin, is involved in this process.     

Degradation of collagen and proteoglycan by macrophages and fibroblasts Individual potentialities of each cell type and cooperative effects through the activation of fibroblasts by macrophages

Fibroblasts and macrophages of various sources (peritoneal, alveolar or bone marrow-derived), from either rabbit or mouse, were cultured, independently or together, at the surface of [³H]proteoglycan/[¹⁴C]collagen-coated plates to evaluate their capacities for proteoglycan and collagen degradation. The various macrophage populations differed widely in their potentialities for proteoglycan and particularly, for collagen degradation, native collagen being significantly degraded, in this model only by rabbit alveolar macrophages. Fibroblasts were as active in proteoglycan degradation as the most active macrophage preparations, but their potential for collagen degradation appeared much higher than that of macrophages. Moreover, all types of macrophages secreted a factor, a monokine, that activated collagen and proteoglycan degradation by fibroblasts. Thus, fibroblasts might well be a major effector cell, active in connective tissue degradations occurring under chronic inflammatory situations.     

Differential release of tumor necrosis factor-α from murine peritoneal macrophages stimulated with virulent and avirulent species of mycobacteria

Abstract The ability of Mycobacterium tuberculosis H37Rv and H37Ra, M. bovis BCG and M. smegmatis to induce the secretion of tumor necrosis factor-α (TNF-α) by cultured murine peritoneal macrophages is inversely related to their virulence. The avirulent species of mycobacteria which were unable to persist in macrophages were capable of inducing significant levels of TNF-α compared to that formed in cultures infected with the virulent M. tuberculosis H37Rv. This difference was also associated with an inherent toxicity by live H37Rv for macrophage cultures. Heat-killed H37Rv was non-toxic and induced significant levels of TNF-α; in contrast, live and heat-killed suspensions of avirulent mycobacteria had an equivalent ability to trigger TNF-α secretion. The TNF-α response was dose-dependent, related directly to the percentage of infected cells, and peaked 6–12 h post-infection. An early and vigorous TNF-α response appears to be a marker of macrophage resistance, while the downregulation of this response seems associated with macrophage toxicity and unrestricted mycobacterial growth.     

Grouping of 20 reference strains of Legionella species by the growth ability within mouse and guinea pig macrophages

20 Reference strains of Legionella species, isolated from human, were classified according to their ability to grow within thioglycolate-induced peritoneal macrophages of mice and guinea pigs. Inbred and congenic mice were used to study the effect of the natural resistance genes Lgn1 and Bcg that are expressed phenotypically in the mouse macrophages. The Lgn1 gene controlled the intracellular growth of Legionella pneumophila Philadelphia-1 and Legionella jordanis GIFU 12657, but the Bcg gene did not affect the intracellular growth of any organism examined. Based on these results and the growth ability in guinea pig macrophages, the 20 reference strains were divided into four groups. This grouping will help us to understand a variety of modes of interaction between Legionella species and macrophages.     

Unravelling the differences: comparative proteomic analysis of a clonal virulent and an attenuated Histomonas meleagridis strain

The current study focused on Histomonas meleagridis, a unicellular protozoan, responsible for histomonosis in poultry. Recently, the occurrence of the disease increased due to the ban of effective chemotherapeutic drugs. Basic questions regarding the molecular biology, virulence mechanisms or even life cycle of the flagellate are still puzzling. In order to address some of these issues, we conducted a comparative proteomic analysis of a virulent and an attenuated H. meleagridis strain traced back to a single cell and propagated in vitro as monoxenic mono-eukaryotic cultures. Using two-dimensional electrophoresis (2-DE) for proteome visualization with computational 2-DE gel image and statistical analysis, upregulated proteins in either of the two H. meleagridis strains were detected. Statistical analysis fulfilling two criteria (≥threefold upregulation and P?<?0.05) revealed 119 differentially expressed protein spots out of which 62 spots were noticed in gels with proteins from the virulent and 57 spots in gels with proteins from the attenuated culture. Mass spectrometric analysis of 32 protein spots upregulated in gels of the virulent strain identified 17 as H. meleagridis-specific. The identification revealed that these spots belonged to eight different proteins, with the majority related to cellular stress management. Two ubiquitous cellular proteins, actin and enolase, were upregulated in multiple gel positions in this strain, indicating either post-translational modification or truncation, or even both. Additionally, a known virulence factor named legumain cysteine peptidase was also detected. In contrast to this, mass spectrometric analysis of 49 protein spots, upregulated in gels of the attenuated strain, singled out 32 spots as specific for the flagellate. These spots were shown to correspond to 24 different proteins that reflect the increased metabolism, in vitro adaptation of the parasite, and amoeboid morphology. In addition to H. meleagridis proteins, the analysis identified differential expression of Escherichia coli DH5α proteins that could have been influenced by the co-cultivated H. meleagridis strain, indicating a reciprocal interaction of these two organisms during monoxenic cultivation.     

Detection of virulent Shigella and enteroinvasive Escherichia coli by induction of the 43 kDa invasion plasmid antigen, ipaC

The invasion plasmid antigen, ipaC (43 kDa) of Shigella spp. and enteroinvasive Escherichia coli (EIEC) could be induced in vitro by growing them in the presence of Congo red. An enzyme-linked immunosorbent assay (ELISA) using antibodies to the 43 kDa protein of Shigella has been developed for specific detection of virulent Shigella spp and EIEC. The test is independent of initial isolation of individual colonies. As few as 10² CFU/ml of virulent Shigella present in mixed cultures could be detected and concurrently their susceptibility to antibiotics could be analysed after an initial growth of 8–16 h in Congo red-containing medium. The test may prove useful in the diagnosis and treatment of bacillary dysentery caused either by Shigella or EIEC through their rapid identification and proper antimicrobial therapy.     

Comparison of the oxidation of glutamine,glucose, ketone bodies and fatty acids by human diploid fibroblasts

The contribution of glutamine, glucose, ketone bodies and fatty acids to the oxidative energy metabolism of human diploid fibroblasts was studied. The rate of glutamine oxidation by fibroblasts was 98 nmol/h per mg cell protein compared to 2 nmol/h per mg cell protein or less for glucose, acetoacetate, d-3-hydroxybutyrate, octanoic acid and palmitic acid. Glucose inhibited glutamine oxidation by 85%, while the other substrates had no effect. Therefore, these cells meet their energy requirement almost solely by anaerobic glycolysis and glutamine oxidation.     

Interactions of liposomes with the reticuloendothelial system: II. Nonspecific and receptor-mediated uptake of liposomes by mouse peritoneal macrophages

Liposomes are taken up as intact vesicles by mouse peritoneal macrophages in a process which is temperature sensitive and is affected by inhibitors of glycolytic metabolism and of microfilament activity. Macrophages take up negatively charged vesicles more readily than positively charged vesicles (2-fold) or neutral vesicles (4-fold). Macrophages take up similar amounts of multilamellar liposomes, reversed phase liposomes and small unilamellar liposomes in terms of lipid, however this corresponds to vastly different numbers of particles and amounts of trapped volume. Coating the liposomes with macromolecular ligands capable of interacting with macrophage surface receptors can markedly promote liposome uptake. Thus, formation of an IgG-antigen complex on the liposome surface results in a 10²-fold enhancement of liposome uptake, while coating the vesicles with fibronectin results in a 10-fold augmentation of uptake. Uptake via IgG-mediated and fibronectin-mediated processes seem to be independent since excess unlabelled, IgG-coated liposomes will inhibit the uptake of radioactively-labelled IgG-coated liposomes much more effectively than the uptake of radioactively-labelled fibronectin-coated liposomes. Cell-bound liposomes can readily be visualized on and inside of the macrophages using fluorescence microscopy techniques.     

Island colonization and founder effects: the invasion of the Guadeloupe islands by ship rats (Rattus rattus)

The stepwise colonization of islands within an archipelago is typically punctuated by successive founder effects, with each newly founded population being a subsample of the gene pool of the source island. Thus, the genetic signature of successive bottlenecks should be detected when analysing the genetic structure between islands of an archipelago. To test this prediction, we investigated introduced ship rat populations, Rattus rattus (Linnaeus, 1758), in the Guadeloupe Archipelago. Three different methods, commonly named the heterozygosity excess, the mode-shift indicator and the M ratio method, were used to detect bottlenecks from genetic data obtained with eight microsatellite markers on Guadeloupe and two neighbouring islands, Petite-Terre and Fajou. Moreover, a recent eradication failure on Fajou allowed us to test the accuracy of the methods in an 'experimental-like' situation. The results indicate that rats were introduced on Guadeloupe first, which then became the source population for independent secondary colonization of Fajou and Petite-Terre. Moreover, the heterozygosity excess and the mode-shift indicator only detected bottlenecks for the recent colonization of Petite-Terre and the eradication failure on Fajou. However, bottlenecks were detected for all the populations using the M ratio method. This could be interpreted as the remaining signature of the early introduction of the ship rat in the archipelago.     

Synergistic induction of endothelial tissue factor by tumor necrosis factor and vascular endothelial growth factor: functional analysis of the tumor necrosis factor receptors

Tissue factor expression on the surface of endothelial cells can be induced by tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) in a synergistic manner. We have investigated the role of the two different TNF receptors for this synergy. Firstly, stimulation of the 60 kDa TNF receptor (TNFR60) by a mutant of TNF specific for TNFR60 induced responses comparable to wild-type TNF. In contrast, stimulation of TNFR80 by a TNFR80-specific TNF mutein did not result in enhancement of tissue factor expression even in the presence of a suboptimal TNFR60 triggering. Secondly, we tested neutralizing TNF receptor antibodies for inhibition of tissue factor synthesis induced by VEGF and TNF. A TNFR60-specific antibody inhibited tissue factor production over a broad range of TNF concentrations, indicating an essential role of TNFR60 in the TNF/VEGF synergy. In contrast, blocking of TNF binding to TNFR80 strongly inhibited TNF-induced tissue factor expression at low, but less pronounced at high, TNF concentrations. In conclusion, these data are in agreement with a model in which TNFR80 participates in the synergy between VEGF and low concentrations of soluble TNF by passing the ligand to the signalling TNFR60.     

Macrophage depletion in the rat after intraperitoneal administration of liposome-encapsulated clodronate: Depletion kinetics and accelerated repopulation of peritoneal and omental macrophages by administration of freund's adjuvant

The purpose of this study was to develop a method for the depletion of macrophages from the peritoneal cavity and the omentum of the rat. Rats received two intraperitoneal injections (at days 0 and 3) with liposome-encapsulated clodronate (dichloromethylene bisphosphonate: Cl₂MBP-liposomes). This treatment resulted in complete elimination of mature tissue macrophages (ED2-positive macrophages) from the peritoneal cavity and the omentum within 2 days. The elimination included the strongly ED2-positive spindle-shaped cells of the omental membrane. Repopulation of the omental ED2-positive macrophages was not seen within the next 23 days. Whereas ED2-positive macrophages were completely depleted, few ED1-positive cells remained and repopulation of ED1-positive cells was faster. The treatment further depleted macrophages from the spleen, especially from the red pulp, parathymic lymph nodes and liver. Freund's incomplete adjuvant administered one day after the last injection of Cl₂MBP-liposomes considerably accelerated repopulation in the omentum. The protocol described might be used to investigate the contribution of mature tissue macrophages to the induction of immune responses, drug metabolism and the elimination of intestinal tumours.     

The role of flagella and motility in the adherence and invasion to fish cell lines by Aeromonas hydrophila serogroup O:34 strains

We compared the ability of Aeromonas hydrophila wild-type strains of serogroup O:34, non-motile Tn5 aflagellar mutants and the same mutants harboring a recombinant cosmid DNA from a library of A. hydrophila AH-3 (O:34, wild-type) that allows these mutants to make flagella and to be motile, to adhere and invade two fish cell lines. We found that motility is essential in these strains for adhesion, and also that possession of flagella is essential for the ability to invade the fish cell lines. We cannot rule out that flagella may be an adhesin, or that motility may also be involved in A. hydrophila serogroup O:34 bacterial invasion of both fish cell lines.     

Insight into the routes of Wolbachia invasion: high levels of horizontal transfer in the spider genus Agelenopsis revealed by Wolbachia strain and mitochondrial DNA diversity

The pandemic distribution of Wolbachia (alpha-proteobacteria) across arthropods is largely due to the ability of these maternally inherited endosymbionts to successfully shift hosts across species boundaries. Yet it remains unclear whether Wolbachia has preferential routes of transfer among species. Here, we examined populations of eight species of the North American funnel-web spider genus Agelenopsis to evaluate whether Wolbachia show evidence for host specificity and the relative contribution of horizontal vs. vertical transmission of strains within and among related host species. Wolbachia strains were characterized by multilocus sequence typing (MLST) and Wolbachia surface protein (WSP) sequences, and analysed in relation to host phylogeny, mitochondrial diversity and geographical range. Results indicate that at least three sets of divergent Wolbachia strains invaded the genus Agelenopsis. After each invasion, the Wolbachia strains preferentially shuffled across species of this host genus by horizontal transfer rather than cospeciation. Decoupling of Wolbachia and host mitochondrial haplotype (mitotypes) evolutionary histories within single species reveals an extensive contribution of horizontal transfer also in the rapid dispersal of Wolbachia among conspecific host populations. These findings provide some of the strongest evidence to support the association of related Wolbachia strains with related hosts by means of both vertical and horizontal strain transmission. Similar analyses across a broader range of invertebrate taxa are needed, using sensitive methods for strain typing such as MLST, to determine if this pattern of Wolbachia dispersal is peculiar to Agelenopsis (or spiders), or is in fact a general pattern in arthropods.