Nucleator-driven assembly of curli organelles and their pathophysiological role in E. coli septic shock

Sammanfattning: The understanding of how polymeric surface structures are assembled in bacteria is one of general interests, which can provide insight into details of macromolecule interactions, and study how proteins fold into domains as assembly modules for building organelles. Protein-protein interactions in various subcellular compartments lead to the assembly of fimbriae at bacterial cell surfaces. Many clinical Salmonella and E. coli isolates as well as certain strains of E. coli K 12, express fibrillar surface structures, which are thin, irregular and highly aggregated, denoted curli. So far, six csg genes have been identified and their products are all involved in the assembly of curli. Among these, CsgA is a major subunit protein of curli. The co-transcribed CsgB protein shows high sequence homology to CsgA with 49% similarity and 30% identity. Part I of the present study demonstrates that curli are assembled on the bacterial cell surface following a distinct novel pathway, the extracellular nucleator/precipitation pathway, in which CsgB serves as a nucleator for the polymerization of secreted soluble CsgA at the microbial cell surface. CsgB is also present along the length of curli fibers as a minor component, to create branching of such fibrillar structures. The current data suggest that CsgB is translocated through the outer membrane, however, unlike CsgA (secreted in a soluble form), is insoluble on the bacterial surface. When overexpressed, CsgB is able to precipitate by itself resulting in short polymers. When both native proteins are expressed, CsgB is believed, by protein-protein interactions, to convey a conformational alteration upon CsgA, leading to heteropolymeric wild type curli fibers. The innate immune system interplays intimately with the adaptive immune system. The innate responses induced by microbial patterns, may at times be so dramatic that they are the major cause of the individual's symptoms. A good example is septic shock, which is the consequence of an over-activation of host cells by bacterial products, characterized by hypotension, by vasodilatation and increased vasopermeability. Bacterial extracellular proteins often play important roles in bacterial virulence, as they can interact with host factors directly. Curli are able to bind a series of host proteins, such as fibronectin, laminin, plasminogen, human contact phase proteins and MHC class I molecules. It has been shown that curliated E. coli and Salmonella are capable of activating the contact phase system in human plasma allowing for an anticoagulation effect and generation of the cellular mediator bradykinin. It has therefor, been speculated that curli may play a role during septic shock in man. Part 2 of the present work documents that more than 50% of E. coli isolates from blood cultures of patients with sepsis are able to express curli at 37O C in vitro. Curliated E. coli induce in vitro a massive expresstion of pro-inflammatory cytokines (IL-6, IL-8 and TNF-alpha) in human macrophages, and an overproduction of nitric oxide in vascular smooth muscle cells by inducing NOS2, as compared to the isogenic mutants bacteria-infected cells, lacking CsgA and/or CsgB proteins. In vivo, a higher level of plasma NO (nitrite/nitrate) is detected in mice after challenged intraperitoneally with 5x108 CFU viable E. coli expressing curli proteins (CsgA/CsgB). In contrast, no elevation of plasma NO was found in mice treated with an E. coli csgBA mutant. The overproduction of NO is closely associated with a dramatic fall in blood pressure in conscious wild type mice, so characteristic of septic shock. The blood pressure remains stable in infected NOS2-deficient mice, following an 8 h period of observation. An increased heart rate, a transient decrease in body temperature and a loss of gross activity are seen in all mice irrespective of curli expression. We therefore, conclude that curli as a conserved surface organelle on E. coli may serve as pattern recognition molecules that contribute to the pathophysiological symptoms and changes associated with E. coli septic shock in man.