scholarly journals Molecular Strategy for Blocking Isopeptide Bond Formation in Nascent Pilin Proteins

2018 ◽  
Author(s):  
Jaime Andrés Rivas-Pardo ◽  
Carmen L. Badilla ◽  
Rafael Tapia-Rojo ◽  
Álvaro Alonso-Caballero ◽  
Julio M. Fernández
Keyword(s):  
Bacterial Adhesion ◽  
Oxygen Radicals ◽  
Rational Design ◽  
Mechanical Stability ◽  
Host Cells ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Isopeptide Bond ◽  
Isopeptide Bonds ◽  
Single Polypeptide

ABSTRACTBacteria anchor to their host cells through their adhesive pili, which must resist the large mechanical stresses induced by the host as it attempts to dislodge the pathogens. The pili of Gram-positive bacteria are constructed as a single polypeptide made of hundreds of pilin repeats, which contain intramolecular isopeptide bonds strategically located in the structure to prevent their unfolding under force, protecting the pilus from degradation by extant proteases and oxygen radicals. Here, we demonstrate the design of a short peptide that blocks the formation of the isopeptide bond present in the pilin Spy0128 from the human pathogen Streptococcus pyogenes, resulting in mechanically labile pilin domains. We use a combination of protein engineering and AFM force spectroscopy to demonstrate that the peptide blocks the formation of the native isopeptide bond and compromises the mechanics of the domain. While an intact Spy0128 is inextensible at any force, peptide-modified Spy0128 pilins readily unfold at very low forces, marking the abrogation of the intramolecular isopeptide bond as well as the absence of a stable pilin fold. We propose that isopeptide-blocking peptides could be further developed as a novel type of highly-specific anti-adhesive antibiotics to treat Gram-positive pathogens.SignificanceAt the onset of an infection, Gram-positive bacteria adhere to host cells through their pili, filamentous structures built by hundreds of repeats of pilin proteins. These proteins can withstand large mechanical challenges without unfolding, remaining anchored to the host and resisting cleavage by proteases and oxygen radicals present in the targeted tissues. The key structural component that gives pilins mechanical resilience are internal isopeptide bonds, strategically placed so that pilins become inextensible structures. We target this bond by designing a blocking peptide that interferes with its formation during folding. We demonstrate that peptide-modified pilins lack mechanical stability and extend at low forces. We propose this strategy as a rational design of mechanical antibiotics, targeting the Achilles’ Heel of bacterial adhesion.

scholarly journals Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins

2018 ◽  
Vol 115 (37) ◽  
pp. 9222-9227 ◽  
Author(s):  
Jaime Andrés Rivas-Pardo ◽  
Carmen L. Badilla ◽  
Rafael Tapia-Rojo ◽  
Álvaro Alonso-Caballero ◽  
Julio M. Fernández
Keyword(s):  
Bond Formation ◽  
Host Cells ◽  
Human Pathogen ◽  
Gram Positive ◽  
Force Microscopy ◽  
Gram Positive Bacteria ◽  
Isopeptide Bond ◽  
Atomic Force ◽  
Isopeptide Bonds ◽  
Single Polypeptide

Bacteria anchor to their host cells through their adhesive pili, which must resist the large mechanical stresses induced by the host as it attempts to dislodge the pathogens. The pili of gram-positive bacteria are constructed as a single polypeptide made of hundreds of pilin repeats, which contain intramolecular isopeptide bonds strategically located in the structure to prevent their unfolding under force, protecting the pilus from degradation by extant proteases and oxygen radicals. Here, we demonstrate the design of a short peptide that blocks the formation of the isopeptide bond present in the pilin Spy0128 from the human pathogen Streptococcus pyogenes, resulting in mechanically labile pilin domains. We use a combination of protein engineering and atomic-force microscopy force spectroscopy to demonstrate that the peptide blocks the formation of the native isopeptide bond and compromises the mechanics of the domain. While an intact Spy0128 is inextensible at any force, peptide-modified Spy0128 pilins readily unfold at very low forces, marking the abrogation of the intramolecular isopeptide bond as well as the absence of a stable pilin fold. We propose that isopeptide-blocking peptides could be further developed as a type of highly specific antiadhesive antibiotics to treat gram-positive pathogens.

scholarly journals Isopeptide Bonds Block the Mechanical Extension of Pilins in Gram-Positive Bacteria

2010 ◽  
Vol 98 (3) ◽  
pp. 596a
Author(s):  
Jorge Alegre-Cebollada ◽  
Carmen L. Badilla ◽  
Julio M. Fernandez
Keyword(s):  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Isopeptide Bonds

scholarly journals A slow-forming isopeptide bond in the structure of the major pilin SpaD fromCorynebacterium diphtheriaehas implications for pilus assembly

2014 ◽  
Vol 70 (5) ◽  
pp. 1190-1201 ◽  
Author(s):  
Hae Joo Kang ◽  
Neil G. Paterson ◽  
Chae Un Kim ◽  
Martin Middleditch ◽  
Chungyu Chang ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Mechanical Stability ◽  
Crystallographic Analysis ◽  
Cross Link ◽  
Mass Spectral ◽  
Isopeptide Bond ◽  
Pilin Subunit ◽  
Isopeptide Bonds ◽  
Binding Loop ◽  
Pilus Assembly

The Gram-positive organismCorynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed byC. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys–Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys–Asn isopeptide-bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its isopeptide bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.

scholarly journals Sortase-assembled pili in Corynebacterium diphtheriae are built using a latch mechanism

2021 ◽  
Vol 118 (12) ◽  
pp. e2019649118
Author(s):  
Scott A. McConnell ◽  
Rachel A. McAllister ◽  
Brendan R. Amer ◽  
Brendan J. Mahoney ◽  
Christopher K. Sue ◽  
...  
Keyword(s):  
Solution Structure ◽  
Corynebacterium Diphtheriae ◽  
Crosslinking Reaction ◽  
Gram Positive ◽  
Kinetic Measurements ◽  
Sorting Signal ◽  
Gram Positive Bacteria ◽  
Isopeptide Bond ◽  
Assembly Mechanism ◽  
Binding Groove

Gram-positive bacteria assemble pili (fimbriae) on their surfaces to adhere to host tissues and to promote polymicrobial interactions. These hair-like structures, although very thin (1 to 5 nm), exhibit impressive tensile strengths because their protein components (pilins) are covalently crosslinked together via lysine–isopeptide bonds by pilus-specific sortase enzymes. While atomic structures of isolated pilins have been determined, how they are joined together by sortases and how these interpilin crosslinks stabilize pilus structure are poorly understood. Using a reconstituted pilus assembly system and hybrid structural biology methods, we elucidated the solution structure and dynamics of the crosslinked interface that is repeated to build the prototypical SpaA pilus from Corynebacterium diphtheriae. We show that sortase-catalyzed introduction of a K190-T494 isopeptide bond between adjacent SpaA pilins causes them to form a rigid interface in which the LPLTG sorting signal is inserted into a large binding groove. Cellular and quantitative kinetic measurements of the crosslinking reaction shed light onto the mechanism of pilus biogenesis. We propose that the pilus-specific sortase in C. diphtheriae uses a latch mechanism to select K190 on SpaA for crosslinking in which the sorting signal is partially transferred from the enzyme to a binding groove in SpaA in order to facilitate catalysis. This process is facilitated by a conserved loop in SpaA, which after crosslinking forms a stabilizing latch that covers the K190-T494 isopeptide bond. General features of the structure and sortase-catalyzed assembly mechanism of the SpaA pilus are likely conserved in Gram-positive bacteria.

scholarly journals Covalent host-targeting by thioester domains of Gram-positive pathogens

2014 ◽  
Vol 70 (a1) ◽  
pp. C848-C848
Author(s):  
Miriam Walden ◽  
John Edwards ◽  
Aleksandra Dziewulska ◽  
Uli Schwarz-Linek ◽  
Mark Banfield
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Molecular Mechanisms ◽  
Cell Attachment ◽  
Surface Proteins ◽  
Host Cells ◽  
Covalent Attachment ◽  
Gram Positive ◽  
Covalent Bonds ◽  
Isopeptide Bonds

Gram-positive pathogens are a major concern to global health, with increasing resistance to antimicrobials and the lack of preventative therapeutics. Understanding how these bacteria interact with host cells is vital for the development of novel strategies to combat disease. One of the most crucial steps in infection is adhesion to the host cell. The discovery of complex cell-surface associated proteins, such as pili, has advanced our knowledge of this interaction, however the precise molecular mechanisms underlying this process remain unclear. Structural studies of pili revealed the presence of highly unusual intramolecular covalent bonds between amino acid side chains. These include isopeptide bonds between Lys and Asp/Asn residues, conferring mechanical strength, thermal stability and resistance to proteases [1,2]. In Streptococcus pyogenes pili, the adhesin Spy0125 (or Cpa) interacts with the host cell. It comprises three domains, two of which contain stabilising isopeptide bonds [2,3]. Intriguingly, the third domain contains an extremely rare thioester bond, between a Cys and a Gln residue. A Cys to Ala mutation results in a 75% reduction in adhesion, suggesting that this internal linkage may mediate direct attachment [3]. We have now discovered putative thioester domains (TEDs) in cell-surface proteins of several clinically important pathogens. The only other example of an internal thioester is found in complement proteins, where the reactive bond enables the formation of covalent attachment to pathogens. The presence of these bonds in bacterial proteins suggests the possibility of an as-yet uncharacterised, conserved mechanism of covalent host cell attachment. For a selection of pathogens, we have used mass spectrometry and crystallography to confirm the presence of the covalent link between the Cys and Gln residues within the TEDs. Furthermore, we have identified putative host cell targets of TEDs and confirmed covalent linkages between the TED and the target.

scholarly journals Structure-Activity Relationships of Antimicrobial and Lipoteichoic Acid-Sequestering Properties in Polyamine Sulfonamides

2008 ◽  
Vol 53 (1) ◽  
pp. 57-62 ◽  
Author(s):  
Hemamali J. Warshakoon ◽  
Mark R. Burns ◽  
Sunil A. David
Keyword(s):  
Antimicrobial Activity ◽  
Rational Design ◽  
Acyl Chain ◽  
Lipoteichoic Acid ◽  
Major Constituent ◽  
Dependent Manner ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Structure Activity

ABSTRACTWe have recently confirmed that lipoteichoic acid (LTA), a major constituent of the gram-positive bacterial surface, is the endotoxin of gram-positive bacteria that induces proinflammatory molecules in a Toll-like receptor 2 (TLR2)-dependent manner. LTA is an anionic amphipath whose physicochemical properties are similar to those of lipopolysaccharide (LPS), which is found on the outer leaflet of the outer membranes of gram-negative organisms. Hypothesizing that compounds that sequester LPS could also bind to and inhibit LTA-induced cellular activation, we screened congeneric series of polyamine sulfonamides which we had previously shown effectively neutralized LPS both in vitro and in animal models of endotoxemia. We observed that these compounds do bind to and neutralize LTA, as reflected by the inhibition of TLR2-mediated NF-κB induction in reporter gene assays. Structure-activity studies showed a clear dependence of the acyl chain length on activity against LTA in compounds with spermine and homospermine scaffolds. We then sought to examine possible correlations between the neutralizing potency toward LTA and antimicrobial activity inStaphylococcus aureus. A linear relationship between LTA sequestration activity and antimicrobial activity for compounds with a spermine backbone was observed, while all compounds with a homospermine backbone were equally active againstS. aureus, regardless of their neutralizing potency toward LTA. These results suggest that the number of protonatable charges is a key determinant of the activity toward the membranes of gram-positive bacteria. The development of resistance to membrane-active antibiotics has been relatively slower than that to conventional antibiotics, and it is possible that compounds such as the acylpolyamines may be useful clinically, provided that they have an acceptable safety profile and margin of safety. A more detailed understanding of the mechanisms of interactions of these compounds with LPS and LTA, as well as the gram-negative and -positive bacterial cell surfaces, will be instructive and should allow the rational design of analogues which combine antisepsis and antibacterial properties.

Specific Inhibitors of Bacterial Adhesion: Observations From the Study of Gram-Positive Bacteria that Initiate Biofilm Formation on the Tooth Surface

1997 ◽  
Vol 11 (1) ◽  
pp. 168-175 ◽  
Author(s):  
J.O. Cisar ◽  
Y. Takahashi ◽  
S. Ruhl ◽  
J.A. Donkersloot ◽  
A.L. Sandberg
Keyword(s):  
Structural Design ◽  
Bacterial Adhesion ◽  
Biofilm Formation ◽  
Tooth Surface ◽  
Viridans Streptococci ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Bacterial Adhesins ◽  
Potential Inhibitors ◽  
Secretory Antibodies

Oral surfaces are bathed in secretory antibodies and other salivary macromolecules that are potential inhibitors of specific microbial adhesion. Indigenous Gram-positive bacteria that colonize teeth, including viridans streptococci and actinomyces, may avoid inhibition of adhesion by host secretory molecules through various strategies that involve the structural design and binding properties of bacterial adhesins and receptors. Further studies to define the interactions of these molecules within the host environment may suggest novel approaches for the control of oral biofilm formation.

scholarly journals Listeria monocytogenes virulence factors are secreted in biologically active Extracellular Vesicles

2017 ◽  
Author(s):  
Carolina Coelho ◽  
Lisa Brown ◽  
Maria Maryam ◽  
Meagan C. Burnet ◽  
Jennifer E. Kyle ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Extracellular Vesicles ◽  
Outer Membrane Vesicles ◽  
Biologically Active ◽  
Host Cells ◽  
Immunogold Electron Microscopy ◽  
Dependent Manner ◽  
Listeriolysin O ◽  
Gram Positive ◽  
Gram Positive Bacteria

ABSTRACTOuter membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secreted extracellular vesicles (EVs) was not pursued due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteriaare implicated in virulence, toxin release and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that is the etiological agent of listeriosis. Here we report that L. monocytogenes produces EVs with diameter ranging from 20-200 nm, containing the pore-forming toxin listeriolysin O(LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multi-omics we characterized protein, lipid and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Cell-free EV preparations were toxic to the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC can restrain LLO activity. Using immunogold electron microscopy we detect LLO localization at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes that colocalize with LLO during infection. Our findings demonstrate that L. monocytogenes utilize EVs for toxin release and implicate these structures in mammalian cytotoxicity.

scholarly journals Role of Extracellular DNA in Initial Bacterial Adhesion and Surface Aggregation

2010 ◽  
Vol 76 (10) ◽  
pp. 3405-3408 ◽  
Author(s):  
Theerthankar Das ◽  
Prashant K. Sharma ◽  
Henk J. Busscher ◽  
Henny C. van der Mei ◽  
Bastiaan P. Krom
Keyword(s):  
Bacterial Adhesion ◽  
Extracellular Dna ◽  
Acid Base ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Initial Adhesion ◽  
Biofilm Matrix

ABSTRACT Extracellular DNA (eDNA) is an important component of the biofilm matrix. We show that removal of eDNA from Gram-positive bacteria reduces initial adhesion to and aggregation of bacteria on surfaces. Thermodynamic analyses indicated that eDNA introduces favorable acid-base interactions, explaining the effect of eDNA on aggregation and adhesion to the surface.

scholarly journals Increasing the Antimicrobial Activity of Nisin-Based Lantibiotics against Gram-Negative Pathogens

2018 ◽  
Vol 84 (12) ◽  
Author(s):  
Qian Li ◽  
Manuel Montalban-Lopez ◽  
Oscar P. Kuipers
Keyword(s):  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Rational Design ◽  
Bacterial Species ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Lipid Ii

ABSTRACTLantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial compounds containing lanthionine and methyl-lanthionine residues. Nisin, one of the most extensively studied and used lantibiotics, has been shown to display very potent activity against Gram-positive bacteria, and stable resistance is rarely observed. By binding to lipid II and forming pores in the membrane, nisin can cause the efflux of cellular constituents and inhibit cell wall biosynthesis. However, the activity of nisin against Gram-negative bacteria is much lower than that against Gram-positive bacteria, mainly because lipid II is located at the inner membrane, and the rather impermeable outer membrane in Gram-negative bacteria prevents nisin from reaching lipid II. Thus, if the outer membrane-traversing efficiency of nisin could be increased, the activity against Gram-negative bacteria could, in principle, be enhanced. In this work, several relatively short peptides with activity against Gram-negative bacteria were selected from literature data to be fused as tails to the C terminus of either full or truncated nisin species. Among these, we found that one of three tails (tail 2 [T2; DKYLPRPRPV], T6 [NGVQPKY], and T8 [KIAKVALKAL]) attached to a part of nisin displayed improved activity against Gram-negative microorganisms. Next, we rationally designed and reengineered the most promising fusion peptides. Several mutants whose activity significantly outperformed that of nisin against Gram-negative pathogens were obtained. The activity of the tail 16 mutant 2 (T16m2) construct against several important Gram-negative pathogens (i.e.,Escherichia coli,Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa,Enterobacter aerogenes) was increased 4- to 12-fold compared to that of nisin. This study indicates that the rational design of nisin can selectively and significantly improve its outer membrane-permeating capacity as well as its activity against Gram-negative pathogens.IMPORTANCELantibiotics are antimicrobial peptides that are highly active against Gram-positive bacteria but that have relatively poor activity against most Gram-negative bacteria. Here, we modified the model lantibiotic nisin by fusing parts of it to antimicrobial peptides with known activity against Gram-negative bacteria. The appropriate selection of peptidic moieties that could be attached to (parts of) nisin could lead to a significant increase in its inhibitory activity against Gram-negative bacteria. Using this strategy, hybrids that outperformed nisin by displaying 4- to 12-fold higher levels of activity against relevant Gram-negative bacterial species were produced. This study shows the power of modified peptide engineering to alter target specificity in a desired direction.

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