Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.Bacterial biofilms are communities of microorganisms residing within a polysaccharide matrix that have been imaged in dental plaques, medical prostheses, and contact lenses (7,31,33). It is well accepted that biofilms play important roles in bacterial persistence and antibiotic resistance in chronic infections, such as cystic fibrosis and otitis media (3,8,9,30). However, the existence and/or roles of biofilms in acute infections, which are defined by short time courses and high severity, have not been examined. The opportunistic gram-negative pathogen Pseudomonas aeruginosa causes both chronic and acute infections and is one of the leading causes of morbidity and mortality in thermally injured patients (27,37). In this study we examined the production of P. aeruginosa biofilms in the thermally injured mouse model of acute infections.The differentiation or maturation of P. aeruginosa biofilms in vitro depends on intercellular signaling systems or quorum sensing (QS) (5,22). QS systems in many gram-negative bacteria rely on acylated homoserine lactones (AHLs), which are produced at high levels when cell density is high and act as ligands for transcriptional regulators. The P. aeruginosa synthases LasI and RhlI synthesize two AHLs, N-3-oxododecanoyl homoserine lactone (3OC 12 -HSL) and N-butyryl-homoserine lactone (C 4 -HSL), which bind and modulate the activity of the transcriptional regulators LasR and RhlR, respectively (28). These transcriptional regulators then regulate the transcription of many genes whose products, including proteases, elastases, toxins, and hemolysins, are thought to be crucial for virulence (28). P. aeruginosa strains lacking functional QS systems are less virulent than wild-type strains (29) and form flat, undifferentiated biofilms on glass surfaces (5). These undifferentiated biofilms are less stable than the differentiated biofilms formed by wild-type P. aeruginosa as they can be easily disrupted by the detergent sodium dodecyl sulfate (5). However, the role of QS in biofilm formation has not previously been examined in vivo. Therefore, in this study we have also examined the role of QS in P. aeruginosa biofilm formation in the acute infection model. ...

Amyloids are aggregated proteins characterized by a specific cross-β-sheet structure and are typically associated with neurodegenerative diseases including Alzheimer's disease. Recently, however, several nonpathological amyloids have been found in intracellular organelles of normal mammalian tissues suggesting that amyloid may also carry out biological functions. We previously have shown that the epididymal cystatin CRES (cystatin-related epididymal spermatogenic), cst8, a reproductive-specific member of the cystatin superfamily of cysteine protease inhibitors, forms amyloid in vitro suggesting that CRES amyloid may also form in vivo within the epididymal lumen. Here we show that amyloid structures containing CRES are a component of the normal mouse epididymal lumen without any apparent cytotoxic effects on spermatozoa and that these structures change along the length of the tubule. These studies suggest the presence of a functional amyloid structure that may carry out roles in sperm maturation or maintenance of the luminal milieu and which itself may undergo maturational changes along the epididymis. In contrast to previous examples of functional amyloid which were intracellular, our studies now show that nonpathological/functional amyloid can also be extracellular. The presence of an extracellular and nonpathological amyloid in the epididymis suggests that similar amyloid structures may be present in other organ systems and may carry out distinctive tissue-specific functions.

STUDY QUESTION: Do the CRES (cystatin-related epididymal spermatogenic) subgroup members, including CRES2, CRES3 and cystatin E2, contribute to the formation of a nonpathological, functional amyloid matrix in the mouse epididymal lumen?SUMMARY ANSWER: CRES2, CRES3 and cystatin E2 self-assemble with different aggregation properties into amyloids in vitro, are part of a common amyloid matrix in the mouse epididymal lumen and are present in extracellular vesicles.WHAT IS KNOWN ALREADY: Although previously thought only to be pathological, accumulating evidence has established that amyloids, which are highly ordered protein aggregates, can also carry out functional roles in the absence of pathology. We previously demonstrated that nonpathological amyloids are present in the epididymis; specifically, that the reproductive cystatin CRES forms amyloid and is present in the mouse epididymal lumen in a film-like amyloid matrix that is intimately associated with spermatozoa. Because the related proteins CRES2, CRES3 and cystatin E2 are also expressed in the epididymis, the present studies were carried out to determine if these proteins are also amyloidogenic in vitro and in vivo and thus may coordinately function with CRES as an amyloid structure. STUDY DESIGN, SAMPLES/MATERIALS, METHODS:The epididymides from CD1 and Cst8 (CRES)129SvEv/B6 gene knockout (KO) and wild-type mice and antibodies that specifically recognize each CRES subgroup member were used for immunohistochemical and biochemical analyzes of CRES subgroup proteins. Methods classically used to identify amyloid, including the conformation-dependent dyes thioflavin S (ThS) and thioflavin T (ThT), conformation-dependent antibodies, protein aggregation disease ligand (which binds any amyloid independent of sequence) and negative stain electron microscopy (EM) were carried out to examine the amyloidogenic properties of CRES subgroup members. Immunofluorescence analysis and confocal microscopy were used for colocalization studies. MAIN RESULTS AND THE ROLE OF CHANCE:Immunoblot and immunofluorescence analyzes showed that CRES2, CRES3 and cystatin E2 were primarily found in the initial segment and intermediate zone of the epididymis and were profoundly downregulated in epididymides from CRES KO mice, suggesting integrated functions. Except for CRES3, which was only detected in a particulate form, proteins were present in the epididymal lumen in both soluble and particulate forms including in a film-like matrix and in extracellular vesicles. The use of amyloid-specific reagents determined that all CRES subgroup members were present as amyloids and colocalized to a common amyloid matrix present in the epididymal lumen. Negative stain EM, dot blot analysis and ThT plate assays showed that recombinant CRES2, CRES3 and cystatin E2 formed amyloid in vitro, albeit with different aggregation properties. Together, our studies demonstrate that a unique amyloid matrix composed of the CRES family of reproductive-specific cystatins and cystatin C is a normal component of the mouse ...