Daniel R. Smith

Diversity, biogenesis and function of microbial amyloids

Amyloid is a distinct β-sheet-rich fold that many proteins can acquire. Frequently associated with neurodegenerative diseases in humans, including Alzheimers, Parkinsons and Huntingtons diseases, amyloids are traditionally considered the product of protein misfolding. However, the amyloid fold is now recognized as a ubiquitous part of normal cellular biology. Functional amyloids have been identified in nearly all facets of cellular life, with microbial functional amyloids leading the way. Unlike disease-associated amyloids, functional amyloids are assembled by dedicated, directed pathways and ultimately perform a physiological function that benefits the organism. The evolved amyloid assembly and disassembly pathways of microbes have provided novel insights into how cells have harnessed the amyloid assembly process for productive means. An understanding of functional amyloid biogenesis promises to provide a fresh perspective on the molecular events that underlie disease-associated amyloidogenesis. Here, we review functional microbial amyloids with an emphasis on curli fibers and their role in promoting biofilm formation and other community behaviors.

In vitro polymerization of a functional Escherichia coli amyloid protein.

Amyloid formation is characterized by the conversion of soluble proteins into biochemically and structurally distinct fibers. Although amyloid formation is traditionally associated with diseases such as Alzheimer disease, a number of biologically functional amyloids have recently been described. Curli are amyloid fibers produced by Escherichia coli that contribute to biofilm formation and other important physiological processes. We characterized the polymerization properties of the major curli subunit protein CsgA. CsgA polymerizes into an amyloid fiber in a sigmoidal kinetic fashion with a distinct lag, growth, and stationary phase. Adding sonicated preformed CsgA fibers to the polymerization reaction can significantly shorten the duration of the lag phase. We also demonstrate that the conversion of soluble CsgA into an insoluble fiber involves the transient formation of an intermediate similar to that characterized for several disease-associated amyloids. The CsgA core amyloid domain can be divided into five repeating units that share sequence and structural hallmarks. We show that peptides representing three of these repeating units are amyloidogenic in vitro. Although the defining characteristics of CsgA polymerization appear conserved with disease-associated amyloids, these proteins evolved in diverse systems and for different purposes. Therefore, amyloidogenesis appears to be an innate protein folding pathway that can be capitalized on to fulfill normal physiological tasks.

Spontaneous hemothorax. Report of 6 cases and review of the literature.

We present 6 cases of spontaneous hemothorax and comprehensively review the medical literature on this subject. We categorize the reported causes and offer a rational diagnostic approach to patients with nontraumatic hemothorax. We recommend specific treatments for specific etiologies, and emphasize the importance of well-established surgical principles for the treatment of hemothorax. Our suggestions should enable physicians to accurately diagnose and expeditiously treat patients with spontaneous hemothorax.

Economical Evolution: Microbes Reduce the Synthetic Cost of Extracellular Proteins

ABSTRACT Protein evolution is not simply a race toward improved function. Because organisms compete for limited resources, fitness is also affected by the relative economy of an organism’s proteome. Indeed, many abundant proteins contain relatively high percentages of amino acids that are metabolically less taxing for the cell to make, thus reducing cellular cost. However, not all abundant proteins are economical, and many economical proteins are not particularly abundant. Here we examined protein composition and found that the relative synthetic cost of amino acids constrains the composition of microbial extracellular proteins. In Escherichia coli, extracellular proteins contain, on average, fewer energetically expensive amino acids independent of their abundance, length, function, or structure. Economic pressures have strategically shaped the amino acid composition of multicomponent surface appendages, such as flagella, curli, and type I pili, and extracellular enzymes, including type III effector proteins and secreted serine proteases. Furthermore, in silico analysis of Pseudomonas syringae, Mycobacterium tuberculosis, Saccharomyces cerevisiae, and over 25 other microbes spanning a wide range of GC content revealed a broad bias toward more economical amino acids in extracellular proteins. The synthesis of any protein, especially those rich in expensive aromatic amino acids, represents a significant investment. Because extracellular proteins are lost to the environment and not recycled like other cellular proteins, they present a greater burden on the cell, as their amino acids cannot be reutilized during translation. We hypothesize that evolution has optimized extracellular proteins to reduce their synthetic burden on the cell. IMPORTANCE Microbes secrete proteins to perform essential interactions with their environment, such as motility, pathogenesis, biofilm formation, and resource acquisition. However, because microbes generally lack protein import systems, secretion is often a one-way street. Consequently, secreted proteins are less likely to be recycled by the cell due to environmental loss. We demonstrate that evolution has in turn selected these extracellular proteins for increased economy at the level of their amino acid composition. Compared to their cellular counterparts, extracellular proteins have fewer synthetically expensive amino acids and more inexpensive amino acids. The resulting bias lessens the loss of cellular resources due to secretion. Furthermore, this economical bias was observed regardless of the abundance, length, structure, or function of extracellular proteins. Thus, it appears that economy may address the compositional bias seen in many extracellular proteins and deliver further insight into the forces driving their evolution. Microbes secrete proteins to perform essential interactions with their environment, such as motility, pathogenesis, biofilm formation, and resource acquisition. However, because microbes generally lack protein import systems, secretion is often a one-way street. Consequently, secreted proteins are less likely to be recycled by the cell due to environmental loss. We demonstrate that evolution has in turn selected these extracellular proteins for increased economy at the level of their amino acid composition. Compared to their cellular counterparts, extracellular proteins have fewer synthetically expensive amino acids and more inexpensive amino acids. The resulting bias lessens the loss of cellular resources due to secretion. Furthermore, this economical bias was observed regardless of the abundance, length, structure, or function of extracellular proteins. Thus, it appears that economy may address the compositional bias seen in many extracellular proteins and deliver further insight into the forces driving their evolution.

Experimental Manipulation of the Microbial Functional Amyloid Called Curli

Curli are proteinaceous fibrous structures produced on the surface of many gram-negative bacteria. As a major constituent of the extracellular matrix, curli mediate interactions between the bacteria and its environment, and as such, curli play a critical role in biofilm formation. Curli fibers share biophysical properties with a growing number of remarkably stable and ordered protein aggregates called amyloid. Here we describe experimental methods to study the biogenesis and assembly of curli by exploiting their amyloid properties. We also present methods to analyze curli-mediated biofilm formation. These approaches are straightforward and can easily be adapted to study other bacterially produced amyloids.

The production of curli amyloid fibers is deeply integrated into the biology of escherichia coli

Curli amyloid fibers are the major protein component of the extracellular matrix produced by Enterobacteriaceae during biofilm formation. Curli are required for proper biofilm development and environmental persistence by Escherichia coli. Here, we present a complete and vetted genetic analysis of functional amyloid fiber biogenesis. The Keio collection of single gene deletions was screened on Congo red indicator plates to identify E. coli mutants that had defective amyloid production. We discovered that more than three hundred gene products modulated curli production. These genes were involved in fundamental cellular processes such as regulation, environmental sensing, respiration, metabolism, cell envelope biogenesis, transport, and protein turnover. The alternative sigma factors, σS and σE, had opposing roles in curli production. Mutations that induced the σE or Cpx stress response systems had reduced curli production, while mutant strains with increased σS levels had increased curli production. Mutations in metabolic pathways, including gluconeogenesis and the biosynthesis of lipopolysaccharide (LPS), produced less curli. Regulation of the master biofilm regulator, CsgD, was diverse, and the screen revealed several proteins and small RNAs (sRNA) that regulate csgD messenger RNA (mRNA) levels. Using previously published studies, we found minimal overlap between the genes affecting curli biogenesis and genes known to impact swimming or swarming motility, underlying the distinction between motile and sessile lifestyles. Collectively, the diversity and number of elements required suggest curli production is part of a highly regulated and complex developmental pathway in E. coli.

Cytokines and Lung Injury

Pulmonary inflammation represents a consequence of local tissue responses to a variety of direct or indirect stimuli. Many clinical entities, including trauma, infection, ischemia-reperfusion injury, as well as the syndrome of acute respiratory distress in adults (ARDS), are characterized by varying degrees of pulmonary insult and the resulting impairment of normal gas exchange. These inflammatory responses involve coordinated interactions between immune and non-immune cells and are specifically initiated, maintained and finally resolved. A variety of mediators are involved in the coordination of these activities and include lipids, such as prostaglandins; a group of small peptides, including bradykinin; and numerous polypeptides, of which a group has been classified as cytokines.