PotentIn VitroandIn VivoActivity of Plantibody Specific for Porphyromonas gingivalis FimA

ABSTRACTFimbrial protein fimbrillin (FimA), a major structural subunit ofPorphyromonas gingivalis, has been suggested as a vaccine candidate to controlP. gingivalis-induced periodontal disease. Previously, cDNAs encoding IgG monoclonal antibodies (MAbs) against purified FimA fromP. gingivalis2561 have been cloned, and the MAbs have been produced in rice cell suspension. Here we examined the biological activities of the plant-produced MAb specific for FimA (anti-FimA plantibody) ofP. gingivalisin vitroandin vivo. The anti-FimA plantibody recognized oligomeric/polymeric forms of native FimA in immunoblot analysis and showed high affinity for native FimA (KD= 0.11 nM). Binding ofP. gingivalis(108cells) to 2 mg of saliva-coated hydroxyapatite beads was reduced by 53.8% in the presence of 1 μg/ml plantibody. Anti-FimA plantibody (10 μg/ml) reduced invasion of periodontal ligament cells byP. gingivalis(multiplicity of infection, 100) by 68.3%. Intracellular killing ofP. gingivalisopsonized with the anti-FimA plantibody by mouse macrophages was significantly increased (77.1%) compared to killing of bacterial cells with irrelevant IgG (36.7%). In a mouse subcutaneous chamber model, the number of recoverableP. gingivaliscells from the chamber fluid was significantly reduced when the numbers of bacterial cells opsonized with anti-FimA plantibody were compared with the numbers of bacterial cells with irrelevant IgG, 66.7% and 37.1%, respectively. Thesein vitroandin vivoeffects of anti-FimA plantibody were comparable to those of the parental MAb. Further studies withP. gingivalisstrains with different types of fimbriae are needed to investigate the usefulness of anti-FimA plantibody for passive immunization to controlP. gingivalis-induced periodontal disease.

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Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

Infection and Immunity ◽

10.1128/iai.05773-11 ◽

2011 ◽

Vol 80 (1) ◽

pp. 3-13 ◽

Cited By ~ 27

Author(s):

Chen Li ◽

Kurniyati ◽

Bo Hu ◽

Jiang Bian ◽

Jianlan Sun ◽

...

Keyword(s):

Porphyromonas Gingivalis ◽

Biofilm Formation ◽

Adult Population ◽

Transcriptional Analysis ◽

Wild Type ◽

Content Type ◽

Multiple Organs ◽

Biochemical Analyses

ABSTRACTThe oral bacteriumPorphyromonas gingivalisis a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide.P. gingivalisexhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence ofP. gingivalisremain elusive. In this report, we found thatP. gingivalisencodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed thatPG0352is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPgis an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that thePG0352deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type,in vitrostudies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement.In vivostudies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPgis an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity ofP. gingivalis, and it can potentially serve as a new target for developing therapeutic agents againstP. gingivalisinfection.

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Assessment of Minocycline and Polymyxin B Combination against Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04110-14 ◽

2015 ◽

Vol 59 (5) ◽

pp. 2720-2725 ◽

Cited By ~ 34

Author(s):

Dana R. Bowers ◽

Henry Cao ◽

Jian Zhou ◽

Kimberly R. Ledesma ◽

Dongxu Sun ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Clinical Utility ◽

Efflux Pump ◽

Polymyxin B ◽

Intracellular Concentration ◽

Bacterial Cells ◽

Efflux Pump Inhibitor ◽

Content Type

ABSTRACTAntimicrobial resistance amongAcinetobacter baumanniiis increasing worldwide, often necessitating combination therapy. The clinical utility of using minocycline with polymyxin B is not well established. In this study, we investigated the activity of minocycline and polymyxin B against 1 laboratory isolate and 3 clinical isolates ofA. baumannii. Minocycline susceptibility testing was performed with and without an efflux pump inhibitor, phenylalanine-arginine β-naphthylamide (PAβN). The intracellular minocycline concentration was determined with and without polymyxin B (0.5 μg/ml). Time-kill studies were performed over 24 h using approximately 106CFU/ml of each strain with clinically relevant minocycline concentrations (2 μg/ml and 8 μg/ml), with and without polymyxin B (0.5 μg/ml). Thein vivoefficacy of the combination was assessed in a neutropenic murine pneumonia model. Infected animals were administered minocycline (50 mg/kg), polymyxin B (10 mg/kg), or both to achieve clinically equivalent exposures in humans. A reduction in the minocycline MIC (≥4×) was observed in the presence of PAβN. The intracellular concentration andin vitrobactericidal effect of minocycline were both enhanced by polymyxin B. With 2 minocycline-susceptible strains, the bacterial burden in lung tissue at 24 h was considerably reduced by the combination compared to monotherapy with minocycline or polymyxin B. In addition, the combination prolonged survival of animals infected with a minocycline-susceptible strain. Polymyxin B increased the intracellular concentration of minocycline in bacterial cells and enhanced the bactericidal activity of minocycline, presumably due to efflux pump disruption. The clinical utility of this combination should be further investigated.

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Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines againstStreptococcus pneumoniaeandSalmonella entericaSerovar Typhi

Infection and Immunity ◽

10.1128/iai.00211-18 ◽

2018 ◽

Vol 86 (9) ◽

Author(s):

Vivek Belde ◽

Matthew P. Cravens ◽

Dania Gulandijany ◽

Justin A. Walker ◽

Isabel Palomo-Caturla ◽

...

Keyword(s):

Antibody Response ◽

B Cell ◽

Salmonella Enterica ◽

Passive Immunization ◽

Terminal Deoxynucleotidyl Transferase ◽

Human Infants ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTB cell antigen receptor (BCR) diversity increases by several orders of magnitude due to the action of terminal deoxynucleotidyl transferase (TdT) during V(D)J recombination. Unlike adults, infants have limited BCR diversity, in part due to reduced expression of TdT. Since human infants and young mice respond poorly to polysaccharide vaccines, such as the pneumococcal polysaccharide vaccine Pneumovax23 and Vi polysaccharide (ViPS) ofSalmonella entericaserovar Typhi, we tested the contribution of TdT-mediated BCR diversity in response to these vaccines. We found that TdT+/−and TdT−/−mice generated comparable antibody responses to Pneumovax23 and survivedStreptococcus pneumoniaechallenge. Moreover, passive immunization of B cell-deficient mice with serum from Pneumovax23-immunized TdT+/−or TdT−/−mice conferred protection. TdT+/−and TdT−/−mice generated comparable levels of anti-ViPS antibodies and antibody-dependent, complement-mediated bactericidal activity againstS. Typhiin vitro. To test the protective immunity conferred by ViPS immunizationin vivo, TdT+/−and TdT−/−mice were challenged with a chimericSalmonella entericaserovar Typhimurium strain expressing ViPS, since mice are nonpermissive hosts forS. Typhi infection. Compared to their unimmunized counterparts, immunized TdT+/−and TdT−/−mice challenged with ViPS-expressingS. Typhimurium exhibited a significant reduction in the bacterial burden and liver pathology. These data suggest that the impaired antibody response to the Pneumovax23 and ViPS vaccines in the young is not due to limited TdT-mediated BCR diversification.

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Preclinical Experiments on the Release Behavior of Biodegradable Nanofibrous Multipharmaceutical Membranes in a Model of Four-Wall Intrabony Defect

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00506-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 9-14 ◽

Cited By ~ 19

Author(s):

Dave Wei-Chih Chen ◽

Fu-Ying Lee ◽

Jun-Yi Liao ◽

Shih-Jung Liu ◽

Chao-Ying Hsiao ◽

...

Keyword(s):

Periodontal Disease ◽

Periodontal Therapy ◽

Release Behavior ◽

Electrospinning Technique ◽

Content Type ◽

Intrabony Defect ◽

Regenerative Periodontal Therapy ◽

Collagen Membranes

ABSTRACTGuided tissue regeneration (GTR) therapy has been widely used to regenerate lost periodontium from periodontal disease. However, in terms of regenerative periodontal therapy, a multidrug-loaded biodegradable carrier can be even more promising in dealing with periodontal disease. In the current study, we fabricated biodegradable nanofibrous collagen membranes that were loaded with amoxicillin, metronidazole, and lidocaine by an electrospinning technique. Thein vitrorelease behavior and the cytotoxicity of the membranes were investigated. A four-wall intrabony defect was created in rabbits forin vivorelease analysis. The bioactivity of the released antibiotics was also examined. The experimental results showed that the drug-loaded collagen membranes could provide sustainable release of effective amoxicillin, metronidazole, and lidocaine for 28, 56, and 8 days, respectively,in vivo. Furthermore, the bioactivity of the released antibiotics remained high, with average bioactivities of 50.5% for amoxicillin againstStaphylococcus aureusand 58.6% for metronidazole againstEscherichia coli. The biodegradable nanofibrous multipharmaceutical membranes developed in this study may provide a promising solution for regenerative periodontal therapy.

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Escherichia coliZipA Organizes FtsZ Polymers into Dynamic Ring-Like Protofilament Structures

mBio ◽

10.1128/mbio.01008-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 15

Author(s):

Marcin Krupka ◽

Marta Sobrinos-Sanguino ◽

Mercedes Jiménez ◽

Germán Rivas ◽

William Margolin

Keyword(s):

Cell Division ◽

High Surface ◽

Bacterial Cells ◽

Content Type ◽

E Coli ◽

Membrane Attachment ◽

Lipid Surface ◽

Confocal Fluorescence Imaging

ABSTRACTZipA is an essential cell division protein inEscherichia coli. Together with FtsA, ZipA tethers dynamic polymers of FtsZ to the cytoplasmic membrane, and these polymers are required to guide synthesis of the cell division septum. This dynamic behavior of FtsZ has been reconstituted on planar lipid surfacesin vitro, visible as GTP-dependent chiral vortices several hundred nanometers in diameter, when anchored by FtsA or when fused to an artificial membrane binding domain. However, these dynamics largely vanish when ZipA is used to tether FtsZ polymers to lipids at high surface densities. This, along with somein vitrostudies in solution, has led to the prevailing notion that ZipA reduces FtsZ dynamics by enhancing bundling of FtsZ filaments. Here, we show that this is not the case. When lower, more physiological levels of the soluble, cytoplasmic domain of ZipA (sZipA) were attached to lipids, FtsZ assembled into highly dynamic vortices similar to those assembled with FtsA or other membrane anchors. Notably, at either high or low surface densities, ZipA did not stimulate lateral interactions between FtsZ protofilaments. We also usedE. colimutants that are either deficient or proficient in FtsZ bundling to provide evidence that ZipA does not directly promote bundling of FtsZ filamentsin vivo. Together, our results suggest that ZipA does not dampen FtsZ dynamics as previously thought, and instead may act as a passive membrane attachment for FtsZ filaments as they treadmill.IMPORTANCEBacterial cells use a membrane-attached ring of proteins to mark and guide formation of a division septum at midcell that forms a wall separating the two daughter cells and allows cells to divide. The key protein in this ring is FtsZ, a homolog of tubulin that forms dynamic polymers. Here, we use electron microscopy and confocal fluorescence imaging to show that one of the proteins required to attach FtsZ polymers to the membrane duringE. colicell division, ZipA, can promote dynamic swirls of FtsZ on a lipid surfacein vitro. Importantly, these swirls are observed only when ZipA is present at low, physiologically relevant surface densities. Although ZipA has been thought to enhance bundling of FtsZ polymers, we find little evidence for bundlingin vitro. In addition, we present several lines ofin vivoevidence indicating that ZipA does not act to directly bundle FtsZ polymers.

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New Insight into Daptomycin Bioavailability and Localization in Staphylococcus aureus Biofilms by Dynamic Fluorescence Imaging

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00735-16 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4983-4990 ◽

Cited By ~ 22

Author(s):

Rym Boudjemaa ◽

Romain Briandet ◽

Matthieu Revest ◽

Cédric Jacqueline ◽

Jocelyne Caillon ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Fluorescence Imaging ◽

Diffusion Reaction ◽

Medical Community ◽

Bacterial Cells ◽

Content Type ◽

Dynamic Fluorescence ◽

Dynamic Fluorescence Imaging

ABSTRACTStaphylococcus aureusis one of the most frequent pathogens responsible for biofilm-associated infections (BAI), and the choice of antibiotics to treat these infections remains a challenge for the medical community. In particular, daptomycin has been reported to fail against implant-associatedS. aureusinfections in clinical practice, while its association with rifampin remains a good candidate for BAI treatment. To improve our understanding of such resistance/tolerance toward daptomycin, we took advantage of the dynamic fluorescence imaging tools (time-lapse imaging and fluorescence recovery after photobleaching [FRAP]) to locally and accurately assess the antibiotic diffusion reaction in methicillin-susceptible and methicillin-resistantS. aureusbiofilms. To provide a realistic representation of daptomycin action, we optimized anin vitromodel built on the basis of our recently publishedin vivomouse model of prosthetic vascular graft infections. We demonstrated that at therapeutic concentrations, daptomycin was inefficient in eradicating biofilms, while the matrix was not a shield to antibiotic diffusion and to its interaction with its bacterial target. In the presence of rifampin, daptomycin was still present in the vicinity of the bacterial cells, allowing prevention of the emergence of rifampin-resistant mutants. Conclusions derived from this study strongly suggest thatS. aureusbiofilm resistance/tolerance toward daptomycin may be more likely to be related to a physiological change involving structural modifications of the membrane, which is a strain-dependent process.

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Bacterial adhesion to cerebrospinal fluid shunts

Journal of Neurosurgery ◽

10.3171/jns.1987.67.3.0438 ◽

1987 ◽

Vol 67 (3) ◽

pp. 438-445 ◽

Cited By ~ 40

Author(s):

Juan A. Guevara ◽

Graciela Zúccaro ◽

Alejandro Trevisán ◽

Claudio D. Denoya

Keyword(s):

Cerebrospinal Fluid ◽

Microbial Pathogens ◽

Scanning Electron Micrographs ◽

Bacterial Cells ◽

Csf Shunts ◽

Content Type ◽

Surface Irregularities ◽

Fine Print

✓ Bacterial adherence to cerebrospinal fluid (CSF) shunts was analyzed in vivo and in vitro. Scanning electron micrographs (SEM's) of catheters removed from pediatric patients with shunts infected by Staphylococcus aureus or Klebsiella pneumoniae revealed numerous bacterial cells and microcolonies, leukocytes, and erythrocytes attached to the CSF catheters' inner walls, as well as the existence of surface irregularities, such as fissures, rugosities, and holes. Permeability analyses and SEM's demonstrated that catheters develop physical alterations over the period of implantation. Different bacterial strains presented a different in vitro adherence to CSF shunts, suggesting that this attachment may be affected by specific properties of the outer structures of each strain. The attachment of microbial pathogens to CSF shunts seems to contribute to the persistence of bacterial cells within a catheter and the onset of recurrent shunt infection. This study demonstrated that some bacteria can remain attached within shunts in vitro despite a CSF flow at rates up to 200 times higher than those normally demonstrated in vivo. Furthermore, surface irregularities found throughout this study may help to anchor and hide bacterial microcolonies. Based on these findings, it seems advisable to remove an infected shunt and to replace it with a new one after proper antimicrobial therapy, in order to prevent recurrent infections.

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Identification of Small-Molecule Inhibitors Targeting Porphyromonas gingivalis Interspecies Adherence and Determination of Their In Vitro and In Vivo Efficacies

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00884-20 ◽

2020 ◽

Vol 64 (11) ◽

Cited By ~ 1

Author(s):

Mohammad Roky ◽

Jinlian Tan ◽

Maryta N. Sztukowska ◽

John O. Trent ◽

Donald R. Demuth

Keyword(s):

Oral Cavity ◽

Cell Lines ◽

Porphyromonas Gingivalis ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Alveolar Bone ◽

Structural Similarity ◽

Content Type

ABSTRACT Porphyromonas gingivalis is one of the primary causative agents of periodontal disease and initially colonizes the oral cavity by adhering to commensal streptococci. Adherence requires the interaction of a minor fimbrial protein (Mfa1) of P. gingivalis with streptococcal antigen I/II (AgI/II). Our previous work identified an AgI/II peptide that potently inhibited adherence and significantly reduced P. gingivalis virulence in vivo, suggesting that this interaction represents a potential target for drug discovery. To develop targeted small-molecule inhibitors of this protein-protein interaction, we performed a virtual screen of the ZINC databases to identify compounds that exhibit structural similarity with the two functional motifs (NITVK and VQDLL) of the AgI/II peptide. Thirty three compounds were tested for in vitro inhibition of P. gingivalis adherence and the three most potent compounds, namely, N7, N17, and V8, were selected for further analysis. The in vivo efficacy of these compounds was evaluated in a murine model of periodontitis. Treatment of mice with each of the compounds significantly reduced maxillary alveolar bone resorption in infected animals. Finally, a series of cytotoxicity tests were performed against human and murine cell lines. Compounds N17 and V8 exhibited no significant cytotoxic activity toward any of the cell lines, whereas compound N7 was cytotoxic at the highest concentrations that were tested (20 and 40 μM). These results identify compounds N17 and V8 as potential lead compounds that will facilitate the design of more potent therapeutic agents that may function to limit or prevent P. gingivalis colonization of the oral cavity.

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Multifunctional Role of Human SPLUNC1 in Pseudomonas aeruginosa Infection

Infection and Immunity ◽

10.1128/iai.00500-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 285-291 ◽

Cited By ~ 41

Author(s):

Sameera Sayeed ◽

Laura Nistico ◽

Claudette St Croix ◽

Y. Peter Di

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Cell ◽

Cell Injury ◽

Respiratory Infections ◽

Structural Similarity ◽

Cell Permeability ◽

Bacterial Cells ◽

Content Type

The human short PLUNC1 (SPLUNC1) protein has been identified as a component of the pulmonary antimicrobial response based on its structural similarity to the bactericidal/permeability-increasing (BPI) protein. Using a genetically modified mouse model, we recently verified the antimicrobial activity of SPLUNC1 againstPseudomonas aeruginosain vivo. To further define the mechanism of epithelial SPLUNC1-mediated antibacterial action, we carried out studies to determine how SPLUNC1 protects the host from acute respiratory infections.P. aeruginosatreated with recombinant human SPLUNC1 protein showed decreased growthin vitro. This antibacterial activity was due to growth inhibition, as a consequence of a SPLUNC1-induced increase in bacterial cell permeability. Removal of SPLUNC1 allowed the recovery ofP. aeruginosaand suggested no permanent cell injury or direct killing of bacteria. Further investigation showed coating of bacterial cells by SPLUNC1. We suggest that this “bacterial cell coating” is necessary for the bacteriostatic function of SPLUNC1. Additionally, we demonstrated a novel role for SPLUNC1 as a chemoattractant that facilitated migration of macrophages and neutrophils. Taking the findings together, we propose synergistic roles for human SPLUNC1 as an antibacterial agent with bacteriostatic and chemotactic activities.

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Vitamin E Increases Antimicrobial Sensitivity by Inhibiting Bacterial Lipocalin Antibiotic Binding

mSphere ◽

10.1128/msphere.00564-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 7

Author(s):

Marwa M. Naguib ◽

Miguel A. Valvano

Keyword(s):

Cystic Fibrosis ◽

Vitamin E ◽

Water Soluble ◽

Burkholderia Cenocepacia ◽

Bacterial Cells ◽

Adjuvant Effect ◽

Bacterial Killing ◽

Content Type

ABSTRACT Burkholderia cenocepacia is an opportunistic Gram-negative bacterium that causes serious respiratory infections in patients with cystic fibrosis. Recently, we discovered that B. cenocepacia produces the extracellular bacterial lipocalin protein BcnA upon exposure to sublethal concentrations of bactericidal antibiotics. BcnA captures a range of antibiotics outside bacterial cells, providing a global extracellular mechanism of antimicrobial resistance. In this study, we investigated water-soluble and liposoluble forms of vitamin E as inhibitors of antibiotic binding by BcnA. Our results demonstrate that in vitro, both vitamin E forms bind strongly to BcnA and contribute to reduce the MICs of norfloxacin (a fluoroquinolone) and ceftazidime (a β-lactam), both of them used as model molecules representing two different chemical classes of antibiotics. Expression of BcnA was required for the adjuvant effect of vitamin E. These results were replicated in vivo using the Galleria mellonella larva infection model whereby vitamin E treatment, in combination with norfloxacin, significantly increased larva survival upon infection in a BcnA-dependent manner. Together, our data suggest that vitamin E can be used to increase killing by bactericidal antibiotics through interference with lipocalin binding. IMPORTANCE Bacteria exposed to stress mediated by sublethal antibiotic concentrations respond by adaptive mechanisms leading to an overall increase of antibiotic resistance. One of these mechanisms involves the release of bacterial proteins called lipocalins, which have the ability to sequester antibiotics in the extracellular space before they reach bacterial cells. We speculated that interfering with lipocalin-mediated antibiotic binding could enhance the efficacy of antibiotics to kill bacteria. In this work, we report that when combined with bactericidal antibiotics, vitamin E contributes to enhance bacterial killing both in vitro and in vivo. This adjuvant effect of vitamin E requires the presence of BcnA, a bacterial lipocalin produced by the cystic fibrosis pathogen Burkholderia cenocepacia. Since most bacteria produce lipocalins like BcnA, we propose that our findings could be translated into making novel antibiotic adjuvants to potentiate bacterial killing by existing antibiotics.

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