Lynette Cegelski

The biology and future prospects of antivirulence therapies

The emergence and increasing prevalence of bacterial strains that are resistant to available antibiotics demand the discovery of new therapeutic approaches. Targeting bacterial virulence is an alternative approach to antimicrobial therapy that offers promising opportunities to inhibit pathogenesis and its consequences without placing immediate life-or-death pressure on the target bacterium. Certain virulence factors have been shown to be potential targets for drug design and therapeutic intervention, whereas new insights are crucial for exploiting others. Targeting virulence represents a new paradigm to empower the clinician to prevent and treat infectious diseases.

Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation

Curli are functional extracellular amyloid fibers produced by uropathogenic Escherichia coli (UPEC) and other Enterobacteriaceae. Ring-fused 2-pyridones, such as FN075 and BibC6, inhibited curli biogenesis in UPEC and prevented the in vitro polymerization of the major curli subunit protein CsgA. The curlicides FN075 and BibC6 share a common chemical lineage with other ring-fused 2-pyridones termed pilicides. Pilicides inhibit the assembly of type 1 pili, which are required for pathogenesis during urinary tract infection. Notably, the curlicides retained pilicide activities and inhibited both curli-dependent and type 1-dependent biofilms. Furthermore, pretreatment of UPEC with FN075 significantly attenuated virulence in a mouse model of urinary tract infection. Curli and type 1 pili exhibited exclusive and independent roles in promoting UPEC biofilms, and curli provided a fitness advantage in vivo. Thus, the ability of FN075 to block the biogenesis of both curli and type 1 pili endows unique anti-biofilm and anti-virulence activities on these compounds.

Oritavancin Exhibits Dual Mode of Action to Inhibit Cell-Wall Biosynthesis in Staphylococcus aureus

Solid-state NMR measurements performed on intact whole cells of Staphylococcus aureus labeled selectively in vivo have established that des-N-methylleucyl oritavancin (which has antimicrobial activity) binds to the cell-wall peptidoglycan, even though removal of the terminal N-methylleucyl residue destroys the D-Ala-D-Ala binding pocket. By contrast, the des-N-methylleucyl form of vancomycin (which has no antimicrobial activity) does not bind to the cell wall. Solid-state NMR has also determined that oritavancin and vancomycin are comparable inhibitors of transglycosylation, but that oritavancin is a more potent inhibitor of transpeptidation. This combination of effects on cell-wall binding and biosynthesis is interpreted in terms of a recent proposal that oritavancin-like glycopeptides have two cell-wall binding sites: the well-known peptidoglycan D-Ala-D-Ala pentapeptide stem terminus and the pentaglycyl bridging segment. The resulting dual mode of action provides a structural framework for coordinated cell-wall assembly that accounts for the enhanced potency of oritavancin and oritavancin-like analogues against vancomycin-resistant organisms.

Sum of the parts: composition and architecture of the bacterial extracellular matrix.

Bacterial biofilms are complex multicellular assemblies that exhibit resistance to antibiotics and contribute to the pathogenesis of serious and chronic infectious diseases. New approaches and quantitative data are needed to define the molecular composition of bacterial biofilms. Escherichia coli biofilms are known to contain polysaccharides and functional amyloid fibers termed curli, yet accurate determinations of biofilm composition at the molecular level have been elusive. The ability to define the composition of the extracellular matrix (ECM) is crucial for the elucidation of structure-function relationships that will aid the development of chemical strategies to disrupt biofilms. We have developed an approach that integrates non-perturbative preparation of the ECM with electron microscopy, biochemistry, and solid-state NMR spectroscopy to define the chemical composition of the intact and insoluble ECM of a clinically important pathogenic bacterium--uropathogenic E. coli. Our data permitted a sum-of-all-the-parts analysis. Electron microscopy revealed supramolecular shell-like structures that encapsulated single cells and enmeshed the bacterial community. Biochemical and solid-state NMR measurements of the matrix and constitutive parts established that the matrix is composed of two major components, curli and cellulose, each in a quantifiable amount. This approach to quantifying the matrix composition is widely applicable to other organisms and to examining the influence of biofilm inhibitors. Collectively, our NMR spectra and the electron micrographs of the purified ECM inspire us to consider the biofilm matrix not as an undefined slime, but as an assembly of polymers with a defined composition and architecture.

Nutrient-dependent structural changes in S. aureus peptidoglycan revealed by solid-state NMR spectroscopy.

The bacterial cell wall is essential to cell survival and is a major target of antibiotics. The main component of the bacterial cell wall is peptidoglycan, a cage-like macromolecule that preserves cellular integrity and maintains cell shape. The insolubility and heterogeneity of peptidoglycan pose a challenge to conventional structural analyses. Here we use solid-state NMR combined with specific isotopic labeling to probe a key structural feature of the Staphylococcus aureus peptidoglycan quantitatively and nondestructively. We observed that both the cell-wall morphology and the peptidoglycan structure are functions of growth stage in S. aureus synthetic medium (SASM). Specifically, S. aureus cells at stationary phase have thicker cell walls with nonuniformly thickened septa compared to cells in exponential phase, and remarkably, 12% (±2%) of the stems in their peptidoglycan do not have pentaglycine bridges attached. Mechanistically, we determined that these observations are triggered by the depletion of glycine in the nutrient medium, which is coincident with the start of the stationary phase, and that the production of the structurally altered peptidoglycan can be prevented by the addition of excess glycine. We also demonstrated that the structural changes primarily arise within newly synthesized peptidoglycan rather than through the modification of previously synthesized peptidoglycan. Collectively, our observations emphasize the plasticity in bacterial cell-wall assembly and the possibility to manipulate peptidoglycan structure with external stimuli.

C-di-GMP Regulates Motile to Sessile Transition by Modulating MshA Pili Biogenesis and Near-Surface Motility Behavior in Vibrio cholerae

In many bacteria, including Vibrio cholerae, cyclic dimeric guanosine monophosphate (c-di-GMP) controls the motile to biofilm life style switch. Yet, little is known about how this occurs. In this study, we report that changes in c-di-GMP concentration impact the biosynthesis of the MshA pili, resulting in altered motility and biofilm phenotypes in V. cholerae. Previously, we reported that cdgJ encodes a c-di-GMP phosphodiesterase and a ΔcdgJ mutant has reduced motility and enhanced biofilm formation. Here we show that loss of the genes required for the mannose-sensitive hemagglutinin (MshA) pilus biogenesis restores motility in the ΔcdgJ mutant. Mutations of the predicted ATPase proteins mshE or pilT, responsible for polymerizing and depolymerizing MshA pili, impair near surface motility behavior and initial surface attachment dynamics. A ΔcdgJ mutant has enhanced surface attachment, while the ΔcdgJmshA mutant phenocopies the high motility and low attachment phenotypes observed in a ΔmshA strain. Elevated concentrations of c-di-GMP enhance surface MshA pilus production. MshE, but not PilT binds c-di-GMP directly, establishing a mechanism for c-di-GMP signaling input in MshA pilus production. Collectively, our results suggest that the dynamic nature of the MshA pilus established by the assembly and disassembly of pilin subunits is essential for transition from the motile to sessile lifestyle and that c-di-GMP affects MshA pilus assembly and function through direct interactions with the MshE ATPase.

Dimethyl Sulfoxide and Ethanol Elicit Increased Amyloid Biogenesis and Amyloid-Integrated Biofilm Formation in Escherichia coli

ABSTRACT Escherichia coli directs the assembly of functional amyloid fibers termed “curli” that mediate adhesion and biofilm formation. We discovered that E. coli exhibits a tunable and selective increase in curli protein expression and fiber assembly in response to moderate concentrations of dimethyl sulfoxide (DMSO) and ethanol. Furthermore, the molecular alterations resulted in dramatic functional phenotypes associated with community behavior, including (i) cellular agglutination in broth, (ii) altered colony morphology, and (iii) increased biofilm formation. Solid-state nuclear magnetic resonance (NMR) spectra of intact pellicles formed in the presence of [13C2]DMSO confirmed that DMSO was not being transformed and utilized directly for metabolism. Collectively, the chemically induced phenotypes emphasize the plasticity of E. colis response to environmental stimuli to enhance amyloid production and amyloid-integrated biofilm formation. The data also support our developing model of the extracellular matrix as an organized assembly of polymeric components, including amyloid fibers, in which composition relates to bacterial physiology and community function.

Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy.

ABSTRACT Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The 13C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional 15N and 31P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions.

Solid-state NMR for bacterial biofilms

Bacteria associate with surfaces and one another by elaborating an extracellular matrix to encapsulate cells, creating communities termed biofilms. Biofilms are beneficial in some ecological niches, but also contribute to the pathogenesis of serious and chronic infectious diseases. New approaches and quantitative measurements are needed to define the composition and architecture of bacterial biofilms to help drive the development of strategies to interfere with biofilm assembly. Solid-state nuclear magnetic resonance (NMR) is uniquely suited to the examination of insoluble and complex macromolecular and whole-cell systems. This article highlights three examples that implement solid-state NMR to deliver insights into bacterial biofilm composition and changes in cell-wall composition as cells transition to the biofilm lifestyle. Most recently, solid-state NMR measurements provided a total accounting of the protein and polysaccharide components in the extracellular matrix of an Escherichia coli biofilm and transformed our qualitative descriptions of matrix composition into chemical parameters that permit quantitative comparisons among samples. We present additional data for whole biofilm samples (cells plus the extracellular matrix) that complement matrix-only analyses. The study of bacterial biofilms by solid-state NMR is an exciting avenue ripe with many opportunities and we close the article by articulating some outstanding questions and future directions in this area.

Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose

A naturally modified cellulose Cellulose is the most abundant biopolymer on Earth and an important component of bacterial biofilms. Thongsomboon et al. used solid-state nuclear magnetic resonance spectroscopy to identify a naturally derived, chemically modified cellulose, phosphoethanolamine cellulose (see the Perspective by Galperin and Shalaeva). They went on to identify the genetic basis and molecular signaling involved in introducing this modification in bacteria, which regulates biofilm matrix architecture and function. This discovery has implications for understanding bacterial biofilms and for the generation of new cellulosic materials. Science, this issue p. 334; see also p. 276 Solid-state nuclear magnetic resonance spectroscopy identifies naturally produced, chemically modified cellulose crucial for bacterial biofilm architecture. Cellulose is a major contributor to the chemical and mechanical properties of plants and assumes structural roles in bacterial communities termed biofilms. We find that Escherichia coli produces chemically modified cellulose that is required for extracellular matrix assembly and biofilm architecture. Solid-state nuclear magnetic resonance spectroscopy of the intact and insoluble material elucidates the zwitterionic phosphoethanolamine modification that had evaded detection by conventional methods. Installation of the phosphoethanolamine group requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling pathway. The bcsEFG operon is present in many bacteria, including Salmonella species, that also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and the genetic and molecular basis for its production offers opportunities to modulate its production in bacteria and inspires efforts to biosynthetically engineer alternatively modified cellulosic materials.