Amyloidogenesis is the aggregation of soluble proteins into structurally conserved fibers. Amyloid fibers are distinguished by their resistance to proteinase K, tinctorial properties and β-sheet-rich secondary structure. Amyloid formation is a hallmark of many human diseases including Alzheimer's, Huntington's and the prion diseases. Therefore, understanding amyloidogenesis will provide insights into the development of therapeutics that target these debilitating diseases. A new class of 'functional' amyloids promises a unique glimpse at how nature has harnessed the amyloid fiber to accomplish important physiological tasks. Functional amyloids are produced by organisms spanning all aspects of cellular life. Herein we review amyloidogenesis, with special attention focused on the similarities and differences between the best characterized disease-associated amyloidogenic protein amyloid-β and the formation of several functional amyloids. The implications of studying functional amyloidogenesis and the strategies organisms employ to limit exposure to toxic intermediates will also be discussed.

Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β–strand-loop-β–strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB, and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the 1st, 2nd or 3rd repeating units were able to convert CsgA into fibers. However, mutants missing either the 4th or 5th repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane, but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2 and 4 self assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units.

Immune function is likely to be shaped by multiple infections over time. Infection with one pathogen can confer cross-protection against heterologous pathogens. We tested the hypothesis that latent murine gammaherpesvirus 68 (γHV68) infection modulates host inflammatory responses and susceptibility to mouse adenovirus type 1 (MAV-1). Mice were infected intranasally (i.n.) with γHV68. 21 days later, they were infected i.n. with MAV-1. We assessed cytokine and chemokine expression by quantitative reverse transcriptase real-time PCR, cellular inflammation by histology, and viral loads by quantitative real-time PCR. Previous γHV68 infection led to persistently upregulated IFN-γ in lungs and spleen and persistently upregulated CCL2 and CCL5 in the lungs. Previous γHV68 infection amplified MAV-1-induced CCL5 upregulation and cellular inflammation in the lungs. Previous γHV68 infection was associated with lower MAV-1 viral loads in the spleen but not the lung. There was no significant effect of previous γHV68 on IFN-γ expression or MAV-1 viral loads when the interval between infections was increased to 44 days. In summary, previous γHV68 infection modulated lung inflammatory responses and decreased susceptibility to a heterologous virus in an organ- and time-dependent manner.

Pseudomonas aeruginosa is capable of causing a variety of acute and chronic infections. Here, we provide evidence that sbrR (PA2895), a gene previously identified as required during chronic P. aeruginosa respiratory infection, encodes an anti-factor that inhibits the activity of its cognate extracytoplasmic-function factor, SbrI (PA2896). Bacterial two-hybrid analysis identified an N-terminal region of SbrR that interacts directly with SbrI and that was sufficient for inhibition of SbrI-dependent gene expression. We show that SbrI associates with RNA polymerase in vivo and identify the SbrIR regulon. In cells lacking SbrR, the SbrI-dependent expression of muiA was found to inhibit swarming motility and promote biofilm formation. Our findings reveal SbrR and SbrI as a novel set of regulators of swarming motility and biofilm formation in P. aeruginosa that mediate their effects through muiA, a gene not previously known to influence surface-associated behaviors in this organism. IMPORTANCEThis study characterizes a factor/anti-factor system that reciprocally regulates the surface-associated behaviors of swarming motility and biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. We present evidence that SbrR is an anti-factor specific for its cognate factor, SbrI, and identify the SbrIR regulon in P. aeruginosa. We find that cells lacking SbrR are severely defective in swarming motility and exhibit enhanced biofilm formation. Moreover, we identify muiA (PA1494) as the SbrI-dependent gene responsible for mediating these effects. SbrIR have been implicated in virulence and in responding to antimicrobial and cell envelope stress. SbrIR may therefore represent a stress response system that influences the surface behaviors of P. aeruginosa during infection.T he Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen notorious for being the principal cause of morbidity and mortality in cystic fibrosis (CF) patients (1). In patients with CF, chronic pulmonary colonization by P. aeruginosa leads to chronic inflammation, progressive loss of lung function, and eventually respiratory failure and death (1). P. aeruginosa is also the fifth leading cause of nosocomial infections overall in the United States and is the second most common cause of ventilator-associated pneumonia (VAP) and catheter-associated urinary tract infections (CAUTI) (2, 3). In patients with VAP or CAUTI, P. aeruginosa grows as a biofilm on endotracheal tubes and catheters, respectively (4-6). In addition, P. aeruginosa is thought to persist as a biofilm in the CF lung (7). P. aeruginosa biofilms are associated with chronic infection and exhibit increased antibiotic resistance and resistance to clearance by the immune system (8). Thus, the ability to form biofilms contributes significantly to the clinical burden of P. aeruginosa infection.In P. aeruginosa, growth as a biofilm is inversely regulated with a cooperative form of multicellular surface motility called swarming (9-12). Swarming motility is flagell...