Diversification of the AlpB Outer Membrane Protein of Helicobacter pylori Affects Biofilm Formation and Cellular Adhesion

ABSTRACT Helicobacter pylori is one of the most common causes of bacterial infection in humans, and it forms biofilms on human gastric mucosal epithelium as well as on in vitro abiotic surfaces. Bacterial biofilm is critical not only for environmental survival but also for successful infection. We previously demonstrated that strain TK1402, which was isolated from a Japanese patient with duodenal and gastric ulcers, has high biofilm-forming ability in vitro relative to other strains. In addition, we showed that outer membrane vesicles (OMV) play an important role in biofilm formation. The aim of this study was to analyze which protein(s) in the OMV contributes to biofilm formation in TK1402. We obtained a spontaneous mutant strain derived from TK1402 lacking biofilm-forming ability. The protein profiles of the OMV were compared between this mutant strain and the wild type, and it was found that AlpB, an outer membrane protein in the OMV of the mutant strain, was markedly decreased compared to that of the wild type. Restoration of TK1402 alpB to the mutant strain fully recovered the ability to form biofilm. However, restoration with alpB from other strains demonstrated incomplete recovery of biofilm-forming ability. We therefore inferred that the variable region of AlpB (amino acid positions 121 to 146) was involved in TK1402 biofilm formation. In addition, diversification of the AlpB sequence was shown to affect the ability to adhere to AGS cells. These results demonstrate a new insight into the molecular mechanisms of host colonization by H. pylori. IMPORTANCE Bacterial biofilm is critical not only for environmental survival but also for successful infection. The mechanism of Helicobacter pylori adherence to host cells mediated by cell surface adhesins has been the focus of many studies, but little is known regarding factors involved in H. pylori biofilm formation. Our study demonstrated that AlpB plays an important role in biofilm formation and that this property depends upon the specific sequence of alpB. This in turn was shown to be important in the ability to adhere to gastric cells. We anticipate that these results will provide new insight into the molecular mechanisms of H. pylori colonization.

Download Full-text

Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori

Journal of Bacteriology ◽

10.1128/jb.02533-14 ◽

2015 ◽

Vol 197 (11) ◽

pp. 1921-1930 ◽

Cited By ~ 5

Author(s):

Jennifer Tsang ◽

Timothy R. Hoover

Keyword(s):

Helicobacter Pylori ◽

Basal Body ◽

Protein Interactions ◽

Transcript Levels ◽

Content Type ◽

Genes Encoding ◽

H Pylori ◽

Flagellar Biogenesis ◽

Flagellar Genes

ABSTRACTFlagellar biogenesis inHelicobacter pyloriis regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ80), RpoN (σ54), and FliA (σ28), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaBandflgE) and FliA-dependent (flaA) genes. The MS ring (encoded byfliF) is one of the earliest flagellar structures assembled. Deletion offliFresulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease inflaAtranscript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions offliHand genes encoding C ring components (fliMandfliY) decreased transcript levels offlaBandflgEbut had little or no effect on transcript levels offlaA. Transcript levels offlaBandflgEwere elevated in mutants where genes encoding rod proteins (fliEandflgBC) were deleted, while transcript levels offlaAwas reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure.IMPORTANCEThe mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of theH. pyloriflagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of theH. pyloriRpoN regulon, a hypothesis that can be tested by examining protein-protein interactionsin vitro.

Download Full-text

The Helicobacter pylori Autotransporter ImaA (HP0289) Modulates the Immune Response and Contributes to Host Colonization

Infection and Immunity ◽

10.1128/iai.00312-12 ◽

2012 ◽

Vol 80 (7) ◽

pp. 2286-2296 ◽

Cited By ~ 13

Author(s):

William E. Sause ◽

Andrea R. Castillo ◽

Karen M. Ottemann

Keyword(s):

Immune Response ◽

Helicobacter Pylori ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Dependent Manner ◽

Wild Type ◽

Content Type ◽

H Pylori ◽

Murine Infections

ABSTRACTThe human pathogenHelicobacter pyloriemploys a diverse collection of outer membrane proteins to colonize, persist, and drive disease within the acidic gastric environment. In this study, we sought to elucidate the function of the host-induced geneHP0289, which encodes an uncharacterized outer membrane protein. We first generated an isogenicH. pylorimutant that lacksHP0289and found that the mutant has a colonization defect in single-strain infections and is greatly outcompeted in mouse coinfection experiments with wild-typeH. pylori. Furthermore, we used protease assays and biochemical fractionation coupled with an HP0289-targeted peptide antibody to verify that the HP0289 protein resides in the outer membrane. Our previous findings showed that theHP0289promoter is upregulated in the mouse stomach, and here we demonstrate thatHP0289expression is induced under acidic conditions in an ArsRS-dependent manner. Finally, we have shown that theHP0289mutant induces greater expression of the chemokine interleukin-8 (IL-8) and the cytokine tumor necrosis factor alpha (TNF-α) in gastric carcinoma cells (AGS). Similarly, transcription of the IL-8 homolog keratinocyte-derived chemokine (KC) is elevated in murine infections with the HP0289 mutant than in murine infections with wild-typeH. pylori. On the basis of this phenotype, we renamed HP0289 ImaA forimmunomodulatoryautotransporter protein. Our work has revealed that genes inducedin vivoplay an important role inH. pyloripathogenesis. Specifically, the outer membrane protein ImaA modulates a component of the host inflammatory response, and thus may allowH. pylorito fine tune the host immune response based on ImaA expression.

Download Full-text

Dynamics of Lewis b Binding and Sequence Variation of the babA Adhesin Gene during Chronic Helicobacter pylori Infection in Humans

mBio ◽

10.1128/mbio.02281-14 ◽

2014 ◽

Vol 5 (6) ◽

Cited By ~ 26

Author(s):

Sandra Nell ◽

Lynn Kennemann ◽

Sandra Schwarz ◽

Christine Josenhans ◽

Sebastian Suerbaum

Keyword(s):

Helicobacter Pylori ◽

Amino Acid ◽

Outer Membrane ◽

Chronic Infection ◽

Human Infection ◽

Complete Loss ◽

Blood Group Antigen ◽

Content Type ◽

Strain Pair ◽

H Pylori

ABSTRACTHelicobacter pyloriundergoes rapid microevolution during chronic infection, but very little is known about how this affects host interaction factors. The best-studied adhesin ofH. pyloriis BabA, which mediates binding to the blood group antigen Lewis b [Le(b)]. To study the dynamics of Le(b) adherence during human infection, we analyzed pairedH. pyloriisolates obtained sequentially from chronically infected individuals. A complete loss or significant reduction of Le(b) binding was observed in strains from 5 out of 23 individuals, indicating that the Le(b) binding phenotype is quite stable during chronic human infection. Sequence comparisons ofbabAidentified differences due to mutation and/or recombination in 12 out of 16 strain pairs analyzed. Most amino acid changes were found in the putative N-terminal extracellular adhesion domain. One strain pair that had changed from a Le(b) binding to a nonbinding phenotype was used to study the role of distinct sequence changes in Le(b) binding. By transformations of the nonbinding strain with ababAgene amplified from the binding strain,H. pyloristrains with mosaicbabAgenes were generated. Recombinants were enriched for a gain of Le(b) binding by biopanning or for BabA expression on the bacterial surface by pulldown assay. With this approach, we identified several amino acid residues affecting the strength of Le(b) binding. Additionally, the data showed that the C terminus of BabA, which is predicted to encode an outer membrane β-barrel domain, plays an essential role in the biogenesis of this protein.IMPORTANCEHelicobacter pyloricauses a chronic infection of the human stomach that can lead to ulcers and cancer. The bacterium can bind to gastric epithelial cells with specialized outer membrane proteins. The best-studied protein is the BabA adhesin which binds to the Lewis b blood group antigen. SinceH. pyloriis a bacterium with very high genetic variability, we asked whetherbabAevolves during chronic infection and how mutations or recombination inbabAaffect binding. We found that BabA-mediated adherence was stable in most individuals but observed a complete loss of binding or reduced binding in 22% of individuals. One strain pair in which binding was lost was used to generatebabAsequences that were mosaics of a functional allele and a nonfunctional allele, and the mosaic sequences were used to identify amino acids critically involved in binding of BabA to Lewis b.

Download Full-text

Helicobacter pylori Infections in the Bronx, New York: Surveying Antibiotic Susceptibility and Strain Lineage by Whole-Genome Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.01591-19 ◽

2019 ◽

Vol 58 (3) ◽

Cited By ~ 2

Author(s):

Rajagopalan Saranathan ◽

Michael H. Levi ◽

Alice R. Wattam ◽

Adel Malek ◽

Emmanuel Asare ◽

...

Keyword(s):

Helicobacter Pylori ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Molecular Mechanisms ◽

Kappa Coefficient ◽

23S Rrna ◽

Whole Genome ◽

Content Type ◽

Phenotypic Resistance ◽

H Pylori

ABSTRACT The emergence of drug resistance in Helicobacter pylori has resulted in a greater need for susceptibility-guided treatment. While the alleles associated with resistance to clarithromycin and levofloxacin have been defined, there are limited data regarding the molecular mechanisms underlying resistance to other antimicrobials. Using H. pylori isolates from 42 clinical specimens, we compared phenotypic and whole-genome sequencing (WGS)-based detection of resistance. Phenotypic resistance correlated with the presence of alleles of 23S rRNA (A2142G/A2143G) for clarithromycin (kappa coefficient, 0.84; 95% confidence interval [CI], 0.67 to 1.0) and gyrA (N87I/N87K/D91Y/D91N/D91G/D99N) for levofloxacin (kappa coefficient, 0.90; 95% CI, 0.77 to 1.0). Phenotypic resistance to amoxicillin in three isolates correlated with mutations in pbp1, pbp2, and/or pbp3 within coding regions near known amoxicillin binding motifs. All isolates were phenotypically susceptible to tetracycline, although four bore a mutation in 16S rRNA (A926G). For metronidazole, nonsense mutations and R16H substitutions in rdxA correlated with phenotypic resistance (kappa coefficient, 0.76; 95% CI, 0.56 to 0.96). Previously identified mutations in the rpoB rifampin resistance-determining region (RRDR) were not present, but 14 novel mutations outside the RRDR were found in rifampin-resistant isolates. WGS also allowed for strain lineage determination, which may be important for future studies in associating precise MICs with specific resistance alleles. In summary, WGS allows for broad analyses of H. pylori isolates, and our findings support the use of WGS for the detection of clarithromycin and levofloxacin resistance. Additional studies are warranted to better define mutations conferring resistance to amoxicillin, tetracycline, and rifampin, but combinatorial analyses for rdxA gene truncations and R16H mutations have utility for determining metronidazole resistance.

Download Full-text

Helicobacter pylori-Induced Disruption of Monolayer Permeability and Proinflammatory Cytokine Secretion in Polarized Human Gastric Epithelial Cells

Infection and Immunity ◽

10.1128/iai.01406-12 ◽

2013 ◽

Vol 81 (3) ◽

pp. 876-883 ◽

Cited By ~ 29

Author(s):

Maria Fiorentino ◽

Hua Ding ◽

Thomas G. Blanchard ◽

Steven J. Czinn ◽

Marcelo B. Sztein ◽

...

Keyword(s):

Helicobacter Pylori ◽

Tight Junction ◽

Proinflammatory Cytokines ◽

Barrier Function ◽

Cytokine Secretion ◽

Gastric Epithelial Cells ◽

Content Type ◽

Monolayer Permeability ◽

H Pylori

ABSTRACTHelicobacter pyloriinfection of the stomach is related to the development of diverse gastric pathologies. The ability ofH. pylorito compromise epithelial junctional complexes and to induce proinflammatory cytokines is believed to contribute to pathogenesis. The purpose of this study was to use anin vitrohuman gastric epithelial model to investigate the ability ofH. pylorito affect permeability and the extent and polarity of the host inflammatory response. NCI-N87 monolayers were cocultured with live or heat-killedH. pylorior culture supernatants. Epithelial barrier function was measured by transepithelial electric resistance (TEER) analysis, diffusion of fluorescein isothiocyanate (FITC)-labeled markers, and immunostaining for tight junction proteins. Supernatants from both apical and basolateral chambers were tested for cytokine production by multiplex analysis.H. pyloricaused a significant decrease in TEER, an increased passage of markers through the infected monolayer, and severe disruption and mislocalization of ZO-1 and claudin-1 proteins. Cell viability was not altered byH. pylori, indicating that loss of barrier function could be attributed to a breakdown of tight junction integrity. Significantly high levels of cytokine secretion were induced by either viable or heat-killedH. pylori.H. pyloriaffects monolayer permeability of polarized human gastric epithelial cells. Proinflammatory cytokines were secreted in a polarized manner, mostly basolaterally. Live bacteria are required for disruption of tight junctions but not for the induction of cytokine secretion. The NCI-N87 cell line provides an excellent model for thein vitrostudy ofH. pyloripathogenesis and the epithelial cell host response to infection.

Download Full-text

The Helicobacter pylori CZB Cytoplasmic Chemoreceptor TlpD Forms an Autonomous Polar Chemotaxis Signaling Complex That Mediates a Tactic Response to Oxidative Stress

Journal of Bacteriology ◽

10.1128/jb.00071-16 ◽

2016 ◽

Vol 198 (11) ◽

pp. 1563-1575 ◽

Cited By ~ 23

Author(s):

Kieran D. Collins ◽

Tessa M. Andermann ◽

Jenny Draper ◽

Lisa Sanders ◽

Susan M. Williams ◽

...

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Helicobacter Pylori ◽

Molecular Mechanisms ◽

Reactive Oxygen ◽

Host Cells ◽

Oxygen Species ◽

Content Type ◽

Signaling Complex ◽

H Pylori

ABSTRACTCytoplasmic chemoreceptors are widespread among prokaryotes but are far less understood than transmembrane chemoreceptors, despite being implicated in many processes. One such cytoplasmic chemoreceptor isHelicobacter pyloriTlpD, which is required for stomach colonization and drives a chemotaxis response to cellular energy levels. Neither the signals sensed by TlpD nor its molecular mechanisms of action are known. We report here that TlpD functions independently of the other chemoreceptors. When TlpD is the sole chemoreceptor, it is able to localize to the pole and recruits CheW, CheA, and at least two CheV proteins to this location. It loses the normal membrane association that appears to be driven by interactions with other chemoreceptors and with CheW, CheV1, and CheA. These results suggest that TlpD can form an autonomous signaling unit. We further determined that TlpD mediates a repellent chemotaxis response to conditions that promote oxidative stress, including being in the presence of iron, hydrogen peroxide, paraquat, and metronidazole. Last, we found that all testedH. pyloristrains express TlpD, whereas other chemoreceptors were present to various degrees. Our data suggest a model in which TlpD coordinates a signaling complex that responds to oxidative stress and may allowH. pylorito avoid areas of the stomach with high concentrations of reactive oxygen species.IMPORTANCEHelicobacter pylorisenses its environment with proteins called chemoreceptors. Chemoreceptors integrate this sensory information to affect flagellum-based motility in a process called chemotaxis. Chemotaxis is employed during infection and presumably aidsH. pyloriin encountering and colonizing preferred niches. A cytoplasmic chemoreceptor named TlpD is particularly important in this process, and we report here that this chemoreceptor is able to operate independently of other chemoreceptors to organize a chemotaxis signaling complex and mediate a repellent response to oxidative stress conditions.H. pyloriencounters and must cope with oxidative stress during infection due to oxygen and reactive oxygen species produced by host cells. TlpD's repellent response may allow the bacteria to escape niches experiencing inflammation and elevated reactive oxygen species (ROS) production.

Download Full-text

Tissue Plasminogen Activator Coating on Implant Surfaces Reduces Staphylococcus aureus Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.02803-15 ◽

2015 ◽

Vol 82 (1) ◽

pp. 394-401 ◽

Cited By ~ 14

Author(s):

Jakub Kwiecinski ◽

Manli Na ◽

Anders Jarneborn ◽

Gunnar Jacobsson ◽

Marijke Peetermans ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Medical Devices ◽

Biofilm Formation ◽

Bacterial Biofilm ◽

Content Type ◽

Tissue Plasminogen

ABSTRACTStaphylococcus aureusbiofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role inS. aureusbiofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating onS. aureusbiofilm formation was tested within vitromicroplate biofilm assays and anin vivomouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by variousS. aureusstrains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics.In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduceS. aureusbiofilm formation bothin vitroandin vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices.

Download Full-text

Phosphodiesterase Genes Regulate Amylovoran Production, Biofilm Formation, and Virulence inErwinia amylovora

Applied and Environmental Microbiology ◽

10.1128/aem.02233-18 ◽

2018 ◽

Vol 85 (1) ◽

Cited By ~ 5

Author(s):

Roshni R. Kharadi ◽

Luisa F. Castiblanco ◽

Christopher M. Waters ◽

George W. Sundin

Keyword(s):

Biofilm Formation ◽

Erwinia Amylovora ◽

Virulence Gene ◽

Virulence Determinants ◽

Content Type ◽

Precise Control ◽

Intracellular Degradation ◽

Successful Infection ◽

And Function

ABSTRACTCyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger molecule that is an important virulence regulator in the plant pathogenErwinia amylovora. Intracellular levels of c-di-GMP are modulated by diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP and by phosphodiesterase (PDE) enzymes that degrade c-di-GMP. The regulatory role of the PDE enzymes inE. amylovorahas not been determined. Using a combination of single, double, and triple deletion mutants, we determined the effects of each of the four putative PDE-encoding genes (pdeA,pdeB,pdeC, andedcA) inE. amylovoraon cellular processes related to virulence. Our results indicate thatpdeAandpdeCare the two phosphodiesterases most active in virulence regulation inE. amylovoraEa1189. The deletion ofpdeCresulted in a measurably significant increase in the intracellular pool of c-di-GMP, and the highest intracellular concentrations of c-di-GMP were observed in the Ea1189 ΔpdeACand Ea1189 ΔpdeABCmutants. The regulation of virulence traits due to the deletion of thepdegenes showed two patterns. A stronger regulatory effect was observed on amylovoran production and biofilm formation, where both Ea1189 ΔpdeAand Ea1189 ΔpdeCmutants exhibited significant increases in these two phenotypesin vitro. In contrast, the deletion of two or morepdegenes was required to affect motility and virulence phenotypes. Our results indicate a functional redundancy among thepdegenes inE. amylovorafor certain traits and indicate that the intracellular degradation of c-di-GMP is mainly regulated bypdeAandpdeC, but they also suggest a role forpdeBin regulating motility and virulence.IMPORTANCEPrecise control of the expression of virulence genes is essential for successful infection of apple hosts by the fire blight pathogen,Erwinia amylovora. The presence and buildup of a signaling molecule called cyclic di-GMP enables the expression and function of some virulence determinants inE. amylovora, such as amylovoran production and biofilm formation. However, other determinants, such as those for motility and the type III secretion system, are expressed and functional when cyclic di-GMP is absent. Here, we report studies of enzymes called phosphodiesterases, which function in the degradation of cyclic di-GMP. We show the importance of these enzymes in virulence gene regulation and the ability ofE. amylovorato cause plant disease.

Download Full-text

Multiple Drug-Induced Stress Responses Inhibit Formation of Escherichia coli Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.01113-20 ◽

2020 ◽

Vol 86 (21) ◽

Cited By ~ 1

Author(s):

Nataliya A. Teteneva ◽

Sergey V. Mart’yanov ◽

María Esteban-López ◽

Jörg Kahnt ◽

Timo Glatter ◽

...

Keyword(s):

Escherichia Coli ◽

Stress Responses ◽

Biofilm Formation ◽

Molecular Mechanisms ◽

Drug Repurposing ◽

Bacterial Attachment ◽

Bacterial Biofilm ◽

Multiple Drug ◽

Content Type ◽

Approved Drugs

ABSTRACT In most ecosystems, bacteria exist primarily as structured surface-associated biofilms that can be highly tolerant to antibiotics and thus represent an important health issue. Here, we explored drug repurposing as a strategy to identify new antibiofilm compounds, screening over 1,000 compounds from the Prestwick Chemical Library of approved drugs for specific activities that prevent biofilm formation by Escherichia coli. Most growth-inhibiting compounds, which include known antibacterial but also antiviral and other drugs, also reduced biofilm formation. However, we also identified several drugs that were biofilm inhibitory at doses where only a weak effect or no effect on planktonic growth could be observed. The activities of the most specific antibiofilm compounds were further characterized using gene expression analysis, proteomics, and microscopy. We observed that most of these drugs acted by repressing genes responsible for the production of curli, a major component of the E. coli biofilm matrix. This repression apparently occurred through the induction of several different stress responses, including DNA and cell wall damage, and homeostasis of divalent cations, demonstrating that biofilm formation can be inhibited through a variety of molecular mechanisms. One tested drug, tyloxapol, did not affect curli expression or cell growth but instead inhibited biofilm formation by suppressing bacterial attachment to the surface. IMPORTANCE The prevention of bacterial biofilm formation is one of the major current challenges in microbiology. Here, by systematically screening a large number of approved drugs for their ability to suppress biofilm formation by Escherichia coli, we identified a number of prospective antibiofilm compounds. We further demonstrated different mechanisms of action for individual compounds, from induction of replicative stress to disbalance of cation homeostasis to inhibition of bacterial attachment to the surface. Our work demonstrates the potential of drug repurposing for the prevention of bacterial biofilm formation and suggests that also for other bacteria, the activity spectrum of antibiofilm compounds is likely to be broad.

Download Full-text

In SilicoDiscovery andIn VitroValidation of Catechol-Containing Sulfonohydrazide Compounds as Potent Inhibitors of the Diguanylate Cyclase PleD

Journal of Bacteriology ◽

10.1128/jb.00742-15 ◽

2015 ◽

Vol 198 (1) ◽

pp. 147-156 ◽

Cited By ~ 14

Author(s):

Silvia Fernicola ◽

Alessandro Paiardini ◽

Giorgio Giardina ◽

Giordano Rampioni ◽

Livia Leoni ◽

...

Keyword(s):

Biofilm Formation ◽

Pathogenic Bacteria ◽

Caulobacter Crescentus ◽

Pharmacophore Model ◽

Second Messenger ◽

Binding Mode ◽

Structural Features ◽

Bacterial Biofilm ◽

Content Type

ABSTRACTBiofilm formation is responsible for increased antibiotic tolerance in pathogenic bacteria. Cyclic di-GMP (c-di-GMP) is a widely used second-messenger signal that plays a key role in bacterial biofilm formation. c-di-GMP is synthesized by diguanylate cyclases (DGCs), a conserved class of enzymes absent in mammals and hence considered attractive molecular targets for the development of antibiofilm agents. Here, the results of a virtual screening approach aimed at identifying small-molecule inhibitors of the DGC PleD fromCaulobacter crescentusare described. A three-dimensional (3D) pharmacophore model, derived from the mode of binding of GTP to the active site of PleD, was exploited to screen the ZINC database of compounds. Seven virtual hits were testedin vitrofor their ability to inhibit the activity of purified PleD by using circular dichroism spectroscopy. Two drug-like molecules with a catechol moiety and a sulfonohydrazide scaffold were shown to competitively inhibit PleD at the low-micromolar range (50% inhibitory concentration [IC50] of ∼11 μM). Their predicted binding mode highlighted key structural features presumably responsible for the efficient inhibition of PleD by both hits. These molecules represent the most potentin vitroinhibitors of PleD identified so far and could therefore result in useful leads for the development of novel classes of antimicrobials able to hamper biofilm formation.IMPORTANCEBiofilm-mediated infections are difficult to eradicate, posing a threatening health issue worldwide. The capability of bacteria to form biofilms is almost universally stimulated by the second messenger c-di-GMP. This evidence has boosted research in the last decade for the development of new antibiofilm strategies interfering with c-di-GMP metabolism. Here, two potent inhibitors of c-di-GMP synthesis have been identifiedin silicoand characterizedin vitroby using the well-characterized DGC enzyme PleD fromC. crescentusas a structural template and molecular target. Given that the protein residues implied as crucial for enzyme inhibition are found to be highly conserved among DGCs, the outcome of this study could pave the way for the future development of broad-spectrum antibiofilm compounds.

Download Full-text