Role of a fluid-phase PRR in fighting an intracellular pathogen: PTX3 in Shigella infection.

Shigella spp. are pathogenic bacteria that cause bacillary dysentery in humans by invading the colonic and rectal mucosa where they induce dramatic inflammation. Here, we have analyzed the role of the soluble PRR Pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity. Mice that had been intranasally infected with S. flexneri were rescued from death by treatment with recombinant PTX3. In vitro PTX3 exerts the antibacterial activity against Shigella, impairing epithelial cell invasion and contributing to the bactericidal activity of serum. PTX3 is produced upon LPS-TLR4 stimulation in accordance with the lipid A structure of Shigella. In the plasma of infected patients, the level of PTX3 amount only correlates strongly with symptom severity. These results signal PTX3 as a novel player in Shigella pathogenesis and its potential role in fighting shigellosis. Finally, we suggest that the plasma level of PTX3 in shigellosis patients could act as a biomarker for infection severity.

Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation.

LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1ß in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection.

Microscopical and microbiologic characterization of customized titanium abutments after different cleaning procedures.

AIM: To assess and characterize pollution micro-particles and bacterial growth on customized titanium abutments after steaming, ultrasonic and plasma cleaning treatments. MATERIALS AND METHODS: Thirty commercially available implant abutments, after customization, were randomly divided into 3 groups of 10 and cleansed by steam (considered as control group), ultrasonic cleaning (test group 1) and plasma of Argon (test group 2). For all specimens, SEM analysis and EDAX microanalysis were performed to count and characterize pollution micro-particles, both on the abutment surface and implant-abutment connection. For the control and test groups, mean values and standard deviations were calculated for number and density of micro-particles. Statistical differences were determined by one-way ANOVA with Scheffe multiple comparison test. The level of statistical significance was set at P ≤ 0.05. Additional microbiologic analysis was performed to detect bacterial contamination on the abutment surface. RESULTS: In the control group, the number of micro-particles on average was 117.5, and 14.1, respectively, on the abutment surface and connection. In the test groups, no pollution was revealed on the abutment (average of 1.09 and 1.13 spots, respectively, in test group 1 and test group 2) and connection (1.28 and 1.41, respectively, in test group 1 and test group 2). The analysis of variance (ANOVA) showed a statistically significant difference for all the variables examined. For each variable, at least one of the groups differs from the others. Scheffe multiple comparison test showed that all comparisons for every variables between the control group and both groups are significant, while there were some comparisons between test group 1 and test group 2 that were not significant. EDAX microanalysis identified micro-particles as residual of lubricant mixed with traces of Titanium and other metals. Microbiologic analysis demonstrated the presence of bacterial growth on the abutment surface only in the control group (111.5 ± 11.43 CFU/ml/implant-abutment as mean value). In the test groups, absence of growing microorganisms was found. CONCLUSIONS: This study confirmed that both plasma and ultrasonic treatments can be beneficially adopted for abutment cleaning process after laboratory technical stages, to supposedly favor soft tissue healing and implant-prosthetic connection stability.

Chemistry and biology of the potent endotoxin from a Burkholderia dolosa clinical isolate from a cystic fibrosis patient.

This is the first report of the chemical and biological properties of the lipooligosaccharide (LOS) endotoxin isolated from Burkholderia dolosa IST4208, an isolate recovered from a cystic fibrosis (CF) patient in a Portuguese CF center. B. dolosa is a member of the Burkholderia cepacia complex, a group of closely related species that are highly problematic and opportunistic pathogens in CF. B. dolosa infection leads to accelerated loss of lung function and decreased survival. The structural determination of its endotoxin was achieved using a combination of chemistry and spectroscopy, and has revealed a novel endotoxin structure. The purified LOS was tested for its immunostimulatory activity on human HEK 293 cells expressing TLR-4, MD-2, and CD-14. In these assays, the LOS showed strong proinflammatory activity.

Intracellular bacteriolysis triggers a massive apoptotic cell death in Shigella-infected epithelial cells.

Infected epithelial cells, which act as a first barrier against pathogens, seldom undergo apoptosis. Rather, infected epithelial cells undergo a slow cell death that displays hallmarks of necrosis. Here, we demonstrate that rapid intracellular lysis of Shigella flexneri, provoked by either the use of a diaminopimelic acid auxotroph mutant or treatment of infected cells with antibiotics of the beta-lactam family, resulted in a massive and rapid induction of apoptotic cell death. This intracellular bacteriolysis-mediated apoptotic death (IBAD) was characterized by the specific involvement of the mitochondrial-dependent cytochrome c/Apaf-1 axis that resulted in the activation of caspases-3, -6 and -9. Importantly, Bcl-2 family members and the NF-kappaB pathway seemed to be critical modulators of IBAD. Finally, we identified that IBAD was also triggered by Salmonella enterica serovar Typhimurium but not by the Gram-positive bacteria, Listeria monocytogenes. Together, our results demonstrate that, contrary to previous findings, epithelial cells are intrinsically able to mount an efficient apoptotic cell death response following infection. Indeed, apoptosis in normal circumstances is masked by powerful anti-apoptotic mechanisms, which are overcome in IBAD. Our results also uncover an unexpected consequence of the treatment of infected cells with certain classes of antibiotics.

Bradyrhizobium Lipid A: Immunological Properties and Molecular Basis of Its Binding to the Myeloid Differentiation Protein-2/Toll-Like Receptor 4 Complex.

Lipopolysaccharides (LPS) are potent activator of the innate immune response through the binding to the myeloid differentiation protein-2 (MD-2)/toll-like receptor 4 (TLR4) receptor complexes. Although a variety of LPSs have been characterized so far, a detailed molecular description of the structure-activity relationship of the lipid A part has yet to be clarified. Photosynthetic Bradyrhizobium strains, symbiont of Aeschynomene legumes, express distinctive LPSs bearing very long-chain fatty acids with a hopanoid moiety covalently linked to the lipid A region. Here, we investigated the immunological properties of LPSs isolated from Bradyrhizobium strains on both murine and human immune systems. We found that they exhibit a weak agonistic activity and, more interestingly, a potent inhibitory effect on MD-2/TLR4 activation exerted by toxic enterobacterial LPSs. By applying computational modeling techniques, we also furnished a plausible explanation for the Bradyrhizobium LPS inhibitory activity at atomic level, revealing that its uncommon lipid A chemical features could impair the proper formation of the receptorial complex, and/or has a destabilizing effect on the pre-assembled complex itself.