Roger D. Klein

Bacterial Amyloid Formation: Structural Insights into Curli Biogensis

Curli are functional amyloid fibers assembled by many Gram-negative bacteria as part of an extracellular matrix that encapsulates the bacteria within a biofilm. A multicomponent secretion system ensures the safe transport of the aggregation-prone curli subunits across the periplasm and outer membrane, and coordinates subunit self-assembly into surface-attached fibers. To avoid the build-up of potentially toxic intracellular protein aggregates, the timing and location of the interactions of the different curli proteins are of paramount importance. Here we review the structural and molecular biology of curli biogenesis, with a focus on the recent breakthroughs in our understanding of subunit chaperoning and secretion. The mechanistic insight into the curli assembly pathway will provide tools for new biotechnological applications and inform the design of targeted inhibitors of amyloid polymerization and biofilm formation.

Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30–50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.

Antivirulence Isoquinolone Mannosides: Optimization of the Biaryl Aglycone for FimH Lectin Binding Affinity and Efficacy in the Treatment of Chronic UTI

Uropathogenic E. coli (UPEC) employ the mannose‐binding adhesin FimH to colonize the bladder epithelium during urinary tract infection (UTI). Previously reported FimH antagonists exhibit good potency and efficacy, but low bioavailability and a short half‐life in vivo. In a rational design strategy, we obtained an X‐ray structure of lead mannosides and then designed mannosides with improved drug‐like properties. We show that cyclizing the carboxamide onto the biphenyl B‐ring aglycone of biphenyl mannosides into a fused heterocyclic ring, generates new biaryl mannosides such as isoquinolone 22 (2‐methyl‐4‐(1‐oxo‐1,2‐dihydroisoquinolin‐7‐yl)phenyl α‐d‐mannopyranoside) with enhanced potency and in vivo efficacy resulting from increased oral bioavailability. N‐Substitution of the isoquinolone aglycone with various functionalities produced a new potent subseries of FimH antagonists. All analogues of the subseries have higher FimH binding affinity than unsubstituted lead 22, as determined by thermal shift differential scanning fluorimetry assay. Mannosides with pyridyl substitution on the isoquinolone group inhibit bacteria‐mediated hemagglutination and prevent biofilm formation by UPEC with single‐digit nanomolar potency, which is unprecedented for any FimH antagonists or any other antivirulence compounds reported to date.

Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation

Significance Curli are functional amyloids produced on the surface of many gram-negative bacteria. These amyloids, consisting primarily of CsgA, are involved in cell adhesion, colonization, and biofilm formation. CsgE is a periplasmic accessory protein that plays a central role in curli biogenesis by its interaction with CsgA and with the pore protein CsgG. To understand the mechanism of curli formation, it is critical to determine the structure of the proteins that are required for their formation. Here, we report the atomic solution structure of a double mutant of CsgE, as determined by NMR. The study reveals unique structural features of CsgE and provides insights into the assembly of the secretion channel and the regulation of curli biogenesis. Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on the surface of Escherichia coli, as well as many other enteric bacteria, and are involved in cell colonization and biofilm formation. CsgE is a periplasmic accessory protein that plays a crucial role in curli biogenesis. CsgE binds to both CsgA and the nonameric pore protein CsgG. The CsgG–CsgE complex is the curli secretion channel and is essential for the formation of the curli fibril in vivo. To better understand the role of CsgE in curli formation, we have determined the solution NMR structure of a double mutant of CsgE (W48A/F79A) that appears to be similar to the wild-type (WT) protein in overall structure and function but does not form mixed oligomers at NMR concentrations similar to the WT. The well-converged structure of this mutant has a core scaffold composed of a layer of two α-helices and a layer of three-stranded antiparallel β-sheet with flexible N and C termini. The structure of CsgE fits well into the cryoelectron microscopy density map of the CsgG–CsgE complex. We highlight a striking feature of the electrostatic potential surface in CsgE structure and present an assembly model of the CsgG–CsgE complex. We suggest a structural mechanism of the interaction between CsgE and CsgA. Understanding curli formation can provide the information necessary to develop treatments and therapeutic agents for biofilm-related infections and may benefit the prevention and treatment of amyloid diseases. CsgE could establish a paradigm for the regulation of amyloidogenesis because of its unique role in curli formation.

Precision antimicrobial therapeutics: the path of least resistance?

The emergence of drug-resistant pathogens has led to a decline in the efficacy of traditional antimicrobial therapy. The rise in resistance has been driven by widespread use, and in some cases misuse, of antibacterial agents in treating a variety of infections. A growing body of research has begun to elucidate the harmful effects of broad-spectrum antibiotic therapy on the beneficial host microbiota. To combat these threats, increasing effort is being directed toward the development of precision antimicrobial therapeutics that target key virulence determinants of specific pathogens while leaving the remainder of the host microbiota undisturbed. This includes the recent development of small molecules termed “mannosides” that specifically target uropathogenic E. coli (UPEC). Mannosides are glycomimetics of the natural mannosylated host receptor for type 1 pili, extracellular appendages that promotes UPEC colonization in the intestine. Type 1 pili are also critical for colonization and infection in the bladder. In both cases, mannosides act as molecular decoys which potently prevent bacteria from binding to host tissues. In mice, oral treatment with mannosides simultaneously clears active bladder infection and removes intestinal UPEC while leaving the gut microbiota structure relatively unchanged. Similar treatment strategies successfully target other pathogens, like adherent-invasive E. coli (AIEC), an organism associated with Crohn’s disease (CD), in mouse models. While not without its challenges, antibiotic-sparing therapeutic approaches hold great promise in a variety of disease systems, including UTI, CD, otitis media (OM), and others. In this perspective we highlight the benefits, progress, and roadblocks to the development of precision antimicrobial therapeutics.

Chaos Controlled: Discovery of a Powerful Amyloid Inhibitor

In this issue of Molecular Cell, Evans et al. (2015) report that the hitherto largely unstudied CsgC protein is responsible for the suppression of premature amyloidogenesis within the cellular periplasm, preventing early aggregation and cellular toxicity.

Structure-Function Analysis of the Curli Accessory Protein CsgE Defines Surfaces Essential for Coordinating Amyloid Fiber Formation

ABSTRACT Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA. The CsgE chaperone facilitates the secretion of CsgA through CsgG by forming a cap at the base of the nonameric CsgG outer membrane pore. We elucidated a series of finely tuned nonpolar and charge-charge interactions that facilitate the oligomerization of CsgE and its ability to transport unfolded CsgA to CsgG for translocation. CsgE oligomerization in vitro is temperature dependent and is disrupted by mutations in the W48 and F79 residues. Using nuclear magnetic resonance (NMR), we identified two regions of CsgE involved in the CsgE-CsgA interaction: a head comprising a positively charged patch centered around R47 and a stem comprising a negatively charged patch containing E31 and E85. Negatively charged residues in the intrinsically disordered N- and C-terminal “tails” were not implicated in this interaction. Head and stem residues were mutated and interrogated using in vivo measurements of curli production and in vitro amyloid polymerization assays. The R47 head residue of CsgE is required for stabilization of CsgA- and CsgE-mediated curli fiber formation. Mutation of the E31 and E85 stem residues to positively charged side chains decreased CsgE-mediated curli fiber formation but increased CsgE-mediated stabilization of CsgA. No single-amino-acid substitutions in the head, stem, or tail regions affected the ability of CsgE to cap the CsgG pore as determined by a bile salt sensitivity assay. These mechanistic insights into the directed assembly of functional amyloids in extracellular biofilms elucidate possible targets for biofilm-associated bacterial infections. IMPORTANCE Curli represent a class of functional amyloid fibers produced by Escherichia coli and other Gram-negative bacteria that serve as protein scaffolds in the extracellular biofilm matrix. Despite the lack of sequence conservation among different amyloidogenic proteins, the structural and biophysical properties of functional amyloids such as curli closely resemble those of amyloids associated with several common neurodegenerative diseases. These parallels are underscored by the observation that certain proteins and chemicals can prevent amyloid formation by the major curli subunit CsgA and by alpha-synuclein, the amyloid-forming protein found in Lewy bodies during Parkinson’s disease. CsgA subunits are targeted to the CsgG outer membrane pore by CsgE prior to secretion and assembly into fibers. Here, we use biophysical, biochemical, and genetic approaches to elucidate a mechanistic understanding of CsgE function in curli biogenesis. IMPORTANCE Curli represent a class of functional amyloid fibers produced by Escherichia coli and other Gram-negative bacteria that serve as protein scaffolds in the extracellular biofilm matrix. Despite the lack of sequence conservation among different amyloidogenic proteins, the structural and biophysical properties of functional amyloids such as curli closely resemble those of amyloids associated with several common neurodegenerative diseases. These parallels are underscored by the observation that certain proteins and chemicals can prevent amyloid formation by the major curli subunit CsgA and by alpha-synuclein, the amyloid-forming protein found in Lewy bodies during Parkinson’s disease. CsgA subunits are targeted to the CsgG outer membrane pore by CsgE prior to secretion and assembly into fibers. Here, we use biophysical, biochemical, and genetic approaches to elucidate a mechanistic understanding of CsgE function in curli biogenesis.

Innovative Solutions to Sticky Situations: Antiadhesive Strategies for Treating Bacterial Infections.

Bacterial adherence to host tissue is an essential process in pathogenesis, necessary for invasion and colonization and often required for the efficient delivery of toxins and other bacterial effectors. As existing treatment options for common bacterial infections dwindle, we find ourselves rapidly approaching a tipping point in our confrontation with antibiotic-resistant strains and in desperate need of new treatment options. Bacterial strains defective in adherence are typically avirulent and unable to cause infection in animal models. The importance of this initial binding event in the pathogenic cascade highlights its potential as a novel therapeutic target. This article seeks to highlight a variety of strategies being employed to treat and prevent infection by targeting the mechanisms of bacterial adhesion. Advancements in this area include the development of novel antivirulence therapies using small molecules, vaccines, and peptides to target a variety of bacterial infections. These therapies target bacterial adhesion through a number of mechanisms, including inhibition of pathogen receptor biogenesis, competition-based strategies with receptor and adhesin analogs, and the inhibition of binding through neutralizing antibodies. While this article is not an exhaustive description of every advancement in the field, we hope it will highlight several promising examples of the therapeutic potential of antiadhesive strategies.