Diversity of Virulence Phenotypes among Type III Secretion Negative Pseudomonas aeruginosa Clinical Isolates

Pseudomonas aeruginosa is a frequent cause of acute infections. The primary virulence factor that has been linked to clinical disease is the type III secretion system, a molecular syringe that delivers effector proteins directly into host cells. Despite the importance of type III secretion in dictating clinical outcomes and promoting disease in animal models of infections, clinical isolates often do not express the type III secretion system in vitro. Here we screened 81 clinical P. aeruginosa isolates for secretion of type III secretion system substrates by western blot. Non-expressing strains were also subjected to a functional test assaying the ability to intoxicate epithelial cells in vitro, and to survive and cause disease in a murine model of corneal infection. 26 of 81 clinical isolates were found to be type III secretion negative by western blot. 17 of these 26 non-expressing strains were tested for their ability to cause epithelial cell rounding. Of these, three isolates caused epithelial cell rounding in a type III secretion system dependent manner, and one strain was cytotoxic in a T3SS-independent manner. Five T3SS-negative isolates were also tested for their ability to cause disease in a murine model of corneal infection. Of these isolates, two strains caused severe corneal disease in a T3SS-independent manner. Interestingly, one of these strains caused significant disease (inflammation) despite being cleared. Our data therefore show that P. aeruginosa clinical isolates can cause disease in a T3SS-independent manner, demonstrating the existence of novel modifiers of clinical disease.     

Interleukin-3-mediated cell survival signals include phosphatidylinositol 3-kinase-dependent translocation of the glucose transporter GLUT1 to the cell surface

Maintenance of glucose uptake is a key component in the response of hematopoietic cells to survival factors. To investigate the mechanism of this response we employed the interleukin-3 (IL-3)-dependent murine mast cell line IC2.9. In these cells, hexose uptake decreased markedly upon withdrawal of IL-3, whereas its readdition led to rapid (t(1/2) approximately 10 min) stimulation of transport, associated with an approximately 4-fold increase in Vmax but no change in Km. Immunocytochemistry and photoaffinity labeling revealed that IL-3 caused translocation of intracellular GLUT1 transporters to the cell surface, whereas a second transporter isoform, GLUT3, remained predominantly intracellular. The inhibitory effects of latrunculin B and jasplakinolide, and of nocodazole and colchicine, respectively, revealed a requirement for both the actin and microtubule cytoskeletons in GLUT1 translocation and transport stimulation. Both IL-3 stimulation of transport and GLUT1 translocation were also prevented by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. The time courses for activation of phosphatidylinositol 3-kinase and its downstream target, protein kinase B, by IL-3 were consistent with a role in IL-3-induced transporter translocation and enhanced glucose uptake. We conclude that one component of the survival mechanisms elicited by IL-3 involves the subcellular redistribution of glucose transporters, thus ensuring the supply of a key metabolic substrate.     

The adhesive properties of salmonellae in the dynamics of the infectious process

In patients with toxic infections salmonellae were identified in 31% of cases. The patients were divided into two groups: the control group receiving treatment with infusion solutions and the test group treated, in addition to the usual scheme of therapy, with indomethacin in a daily dose of 150 mg. The study revealed that salmonellae isolated at the initial stages of the disease possessed highly pronounced adhesive properties. The adhesive properties of salmonellae isolated at the stage of convalescence from the patients of the test group were considerably less pronounced than those of salmonellae isolated from the same patients at the peak of the disease. In the control group no differences in the adhesive properties of salmonellae isolated from the same patients were established.     

Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

In the presence of 4 mM inorganic phosphate, differentiating chick limb-bud mesenchymal cells plated in micromass cultures form a mineralized matrix resembling that of chick calcified cartilage. To test the hypothesis that cartilage proteoglycans are inhibitors of cell mediated mineralization, the synthesis, content, and turnover of proteoglycans were altered in this system, and the extent of mineralization and properties of the mineral crystals examined. In all cases where the proteoglycan synthesis or proteoglycans present were modified to provide fewer or smaller molecules, mineralization was enhanced. Specifically, when proteoglycan synthesis was blocked by treatment with 10⁻¹⁰ M retinoic acid, extensive mineral deposition occurred on a matrix devoid of both proteoglycans and cartilage nodules. The crystals, which formed rapidly, were relatively large in size based on analysis by X-ray diffraction or FT-IR microspectroscopy, and were more abundant than in controls. When 2.5 or 5 mM xylosides were used to cause the synthesis of smaller proteoglycans, the extent of mineral accretion was also increased relative to controls; however, the matrix was less affected, and the extent of mineral deposition and the size of the crystals were not as markedly altered as in the case of retinoic acid. Modification of existing proteoglycans by either chondroinase ABC or hyaluronidase treatment similarly resulted in increased mineral accretion (based on ⁴⁵Ca uptake or total Ca uptake) relative to cultures in which the proteoglycan content was not manipulated. Crystals were more abundant and larger than in control mineralizing cultures. In contrast, when proteoglycan degradation by metalloproteases was inhibited by metal chelation with o-phenanthroline, the Ca accretion at early time points was increased, but as mineralization progressed, Ca accumulation decreased. These data provide evidence that in this culture system, proteoglycans are inhibitors of mineralization. J. Cell. Biochem. 64:632–643. © 1997 Wiley-Liss, Inc.     

The hemagglutinating properties of Pseudomonas aeruginosa strains isolated from patients with nonspecific lung diseases]

To detect d-mannose-sensitive (MS) pili in 31 P. aeruginosa strains isolated from the respiratory tract of patients with inflammatory and purulent destructive pulmonary diseases, the hemagglutination (HA) test was used. The isolated Pseudomonas under study differed in the degree of manifestation of their MS adhesins. Among them microorganisms with pronounced HA activity (high HA titer) occurred, as well as those whose HA activity was less pronounced (low HA titer). P. aeruginosa strains with pronounced HA activity were more frequently isolated from patients with purulent destructive processes in the lungs. A correlation between the state of the patient at the moment of bacteriological examination and the degree of manifestation of MS pili in the P. aeruginosa strain isolated from this patients was established. The value of HA titer in the presence of d-mannose is indicative of the presence of MS adhesins in a P. aeruginosa strain.