Adhesin Protein Research Articles

A Trimeric Autotransporter Enhances Biofilm Cohesiveness in Yersinia pseudotuberculosis but Not in Yersinia pestis.

Cohesion of biofilms made by Yersinia pestis and Yersinia pseudotuberculosis has been attributed solely to an extracellular polysaccharide matrix encoded by the hms genes (Hms-dependent extracellular matrix [Hms-ECM]). However, mutations in the Y. pseudotuberculosis BarA/UvrY/CsrB regulatory cascade enhance biofilm stability without dramatically increasing Hms-ECM production. We found that treatment with proteinase K enzyme effectively destabilized Y. pseudotuberculosiscsrB mutant biofilms, suggesting that cell-cell interactions might be mediated by protein adhesins or extracellular matrix proteins. We identified an uncharacterized trimeric autotransporter lipoprotein (YPTB2394), repressed by csrB, which has been referred to as YadE. Biofilms made by a ΔyadE mutant strain were extremely sensitive to mechanical disruption. Overexpression of yadE in wild-type Y. pseudotuberculosis increased biofilm cohesion, similar to biofilms made by csrB or uvrY mutants. We found that the Rcs signaling cascade, which represses Hms-ECM production, activated expression of yadE The yadE gene appears to be functional in Y. pseudotuberculosis but is a pseudogene in modern Y. pestis strains. Expression of functional yadE in Y. pestis KIM6+ weakened biofilms made by these bacteria. This suggests that although the YadE autotransporter protein increases Y. pseudotuberculosis biofilm stability, it may be incompatible with the Hms-ECM production that is essential for Y. pestis biofilm production in fleas. Inactivation of yadE in Y. pestis may be another instance of selective gene loss in the evolution of flea-borne transmission by this species.IMPORTANCE The evolution of Yersinia pestis from its Y. pseudotuberculosis ancestor involved gene acquisition and gene losses, leading to differences in biofilm production. Characterizing the unique biofilm features of both species may provide better understanding of how each adapts to its specific niches. This study identifies a trimeric autotransporter, YadE, that promotes biofilm stability of Y. pseudotuberculosis but which has been inactivated in Y. pestis, perhaps because it is not compatible with the Hms polysaccharide that is crucial for biofilms inside fleas. We also reveal that the Rcs signaling cascade, which represses Hms expression, activates YadE in Y. pseudotuberculosis The ability of Y. pseudotuberculosis to use polysaccharide or YadE protein for cell-cell adhesion may help it produce biofilms in different environments.

A strategy to control colonization of pathogens: embedding of lactic acid bacteria on the surface of urinary catheter.

Indwelling urinary catheterization is one of the major causes of urinary tract infection (UTI) in hospitalized patients worldwide. A catheter serves as a surface for the colonization and formation of biofilm by UTI-related pathogenic bacteria. To combat the biofilm formation on its surface, several strategies have already been employed such as coating it with antibiofilm and antimicrobial compounds. For instance, the application of lactic acid bacteria (LAB) offers a potential strategy for the treatment of biofilm formation on the surface of the urinary catheter due to its ability to kill the pathogenic bacteria. The killing of pathogenic bacteria by LAB occurs via the production of antimicrobial compounds such as lactic acid, bacteriocin, and hydrogen peroxide. LAB also displays a competitive exclusion mechanism to prevent the adhesion of pathogens on the surfaces. Hence, LAB has been extensively applied as a bacteriotherapy to combat infectious diseases. Several strategies have been employed to attach LAB to a surface, but its easy detachment during long time exposure becomes one of the drawbacks in its application. Here, we have proposed a novel strategy for its adhesion on the surface of the urinary catheter with the utilization of mannose-specific adhesin (Msa) protein in a way similar as uropathogenic bacteria interacts between Msa present on the tip of the type I fimbriae/pilus and the mannose moieties on the host epithelial cell surfaces. KEY POINTS: • Urinary tract infection (UTI) is one of the common hospital-acquired infections, which is associated with the application of an indwelling urinary catheter. • Based on the competitive exclusions properties of LAB, attachment of the LAB on the catheter surface would be a promising approach to control the formation of pathogenic biofilm. • The strategy employed for the adhesion of LAB is via a covalent interaction of its mannose-specific adhesin (Msa) protein to the mannose residues grafted on the catheter surface.

Optimization and qualification of an assay that demonstrates that a FimH vaccine induces functional antibody responses in women with histories of urinary tract infections

ABSTRACT Recurrent urinary tract infections (rUTI) are a serious disease associated with morbidities and mortality. Resistance to the standard of care antibiotics is now widespread because of the continued use of antibiotics among people who suffer from rUTI. We are therefore developing a vaccine to prevent recurrences among patients with rUTI. The antigen of the vaccine is FimH, a bacterial adhesin protein, and the vaccine is adjuvanted with a TLR-4 agonist. In a Phase 1 clinical study evaluating the vaccine, immunized individuals produced FimH-binding antibodies. Here we describe the optimization, qualification, and use of an assay to assess the functionality of these anti-FimH antibodies. The suitability of the assay for its intended purpose was demonstrated by selectivity, specificity, sensitivity, and intra-assay and inter-assay precision. The acceptance criteria were achieved for all parameters including intra-assay precision with ≤10% relative standard deviations and inter-assay precision with ≤25% relative standard deviations. The results presented herein suggest this functional assay will be important for supporting the vaccine’s efficacy in future human studies. Furthermore and of great significance, these results prove that vaccine-induced functional antibodies can be elicited in rUTI patients against an essential virulence factor, FimH.

The ever-expanding tcp conjugation locus of pCW3 from Clostridium perfringens

The spore-forming, anaerobic Gram positive pathogen Clostridium perfringens encodes many of its disease-causing toxins on closely related conjugative plasmids. Studies of the tetracycline resistance plasmid pCW3 have identified many of the genes involved in conjugative transfer, which are located in the tcp conjugation locus. Upstream of this locus is an uncharacterised region (the cnaC region) that is highly conserved. This study examined the importance in pCW3 conjugation of several highly conserved proteins encoded in the cnaC region. Conjugative mating studies suggested that the SrtD, TcpN and Dam proteins were required for efficient pCW3 transfer between C. perfringens cells from the same strain background. The requirement of these proteins for conjugation was amplified in matings between C. perfringens cells of different strain backgrounds. Additionally, the putative collagen adhesin protein, CnaC, was only required for the optimal transfer of pCW3 between cells of different strain backgrounds. Based on these studies we postulate that CnaC, SrtD, TcpN and Dam are involved in enhancing the transfer frequency of pCW3. These studies have led to a significant expansion of the tcp conjugation locus, which now encompasses a 19 kb region.

Legionella pneumophila Attachment to Biofilms of an Acidovorax Isolate from a Drinking Water-Consortium Requires the Lcl-Adhesin Protein.

Human infection by Legionella pneumophila (Lpn) only occurs via contaminated water from man-made sources, and eradication of these bacteria from man-made water systems is complicated by biofilm colonization. Using a continuously fed biofilm reactor model, we grew a biofilm consortium from potable water that was able to prolong recovery of Lpn CFU from biofilms. This effect was recreated using a subset of those species in a simplified consortium composed of eight bacterial isolates from the first biofilm reactor. In the reactor with the eight-species consortium, Lpn biofilm CFU was relatively stable over a 12-day trial. An isolate of Acidovorax from the consortium was, as a single species biofilm, able to promote Lpn surface attachment. Other isolates from the Pelomonas genus grew as equally robust biofilms alone, but did not promote surface attachment of Lpn. This attachment was disrupted by cationic polysaccharides and loss of the Lpn Lcl collagen-like adhesin protein. This work demonstrates that, while Lpn was fairly incompetent at attachment to surfaces to form a biofilm alone, pre-existing biofilms allowed attachment of Lpn as secondary colonizers. In addition, we demonstrate that initial attachment of Lpn to Acidovorax biofilms is likely via the Lcl-adhesin protein.

Effect of Adiantum philippense Extract on Biofilm Formation, Adhesion With Its Antibacterial Activities Against Foodborne Pathogens, and Characterization of Bioactive Metabolites: An in vitro-in silico Approach.

Adiantum philippense (A. philippense), an ethnomedicinally important fern, has become an interesting herb in the search for novel bioactive metabolites, which can also be used as therapeutic agents. Primarily, in this study, A. philippense crude extract was screened for its phytochemical constituents, antagonistic potential, and effect on bacterial adhesion and biofilm formation against common food pathogens. Phytochemical profiling of A. philippense was carried out by using High Resolution-Liquid Chromatography and Mass Spectroscopy (HR-LCMS) followed by antibacterial activity via agar cup/well diffusion, broth microdilution susceptibility methods, and growth curve analysis. Antibiofilm potency and efficacy were assessed on the development, formation, and texture of biofilms through light microscopy, fluorescent microscopy, scanning electron microscopy, and the assessment of exopolysaccharide production. Correspondingly, a checkerboard test was performed to evaluate the combinatorial effect of A. philippense and chloramphenicol. Lastly, molecular docking studies of identified phytochemicals with adhesin proteins of tested food pathogens, which helps the bacteria in surface attachment and leads to biofilm formation, were assessed. A. philippense crude extract was found to be active against all tested food pathogens, displaying the rapid time-dependent kinetics of bacterial killing. A. philippense crude extract also impedes the biofilm matrix by reducing the total content of exopolysaccharide, and, likewise, the microscopic images revealed a great extent of disruption in the architecture of biofilms. A synergy was observed between A. philippense crude extract and chloramphenicol for E. coli, S. aureus, and P. aeruginosa, whereas an additive effect was observed for S. flexneri. Various bioactive phytochemicals were categorized from A. philippense crude extract using HR-LCMS. The molecular docking of these identified phytochemicals was interrelated with the active site residues of adhesin proteins, IcsA, Sortase A, OprD, EspA, and FimH from S. flexneri, S. aureus, P. aeruginosa, and E. coli, respectively. Thus, our findings represent the bioactivity and potency of A. philippense crude extract against food pathogens not only in their planktonic forms but also against/in biofilms for the first time. We have also correlated these findings with the possible mechanism of biofilm inhibition via targeting adhesin proteins, which could be explored further to design new bioactive compounds against biofilm producing foodborne bacterial pathogens.

Diagnostic Value of Recombinant Heparin-binding Hemagglutinin Adhesin Protein in Spinal Tuberculosis.

Background and aimTo explore the diagnostic value of recombinant heparin-binding hemagglutinin adhesin (HBHA) protein antigen in spinal tuberculosis.Materials and methodsForty patients with spinal tuberculosis were included in the experimental group and 40 healthy people were included in the control group. Serum IgG antibody expression level was detected with recombinant HBHA protein as the antigen, using enzyme-linked immunosorbent assay (ELISA) detection.ResultsPatients with spinal tuberculosis and healthy volunteers were included in this study. A total of 40 eligible patients with spinal tuberculosis were included (24 males and 16 females, aged 18-72 years, with an average age of 41.24 ± 15.74 years). Forty healthy people were included (21 males and 19 females, aged 18-70 years, with an average age of 41.33 ± 12.36 years). On comparing the groups, no significant difference was found in the general data (P >0.05). IgG antibody level in the experimental group was higher than that in the control group, and the difference was significant (P < 0.00001).ConclusionsDetection of serum HBHA protein antibody is of great value in the auxiliary diagnosis of spinal tuberculosis, and high HBHA expression can be used as an indicator for diagnosis of spinal tuberculosis.

Characterization and potential mechanisms of highly antibiotic tolerant VBNC Escherichia coli induced by low level chlorination

Escherichia coli is an important pathogenic indicator in drinking water. Viable but non-culturable (VBNC) E. coli induced by low level chlorination was found to have higher antibiotic tolerance. The emerging of VBNC bacteria in drinking water systems is posing challenges to the control of bio-safety. It is necessary to study the underlying mechanisms of VBNC state E. coli under actual residual chlorine condition of drinking water pipe network. In this study, we investigated the changes of morphology and gene expressions that might present such state. The results indicated that the size of VBNC E. coli was not remarkably changed or recovered culturability under favorable environmental conditions. Results from transcriptomic analysis revealed that the regulated genes related to fimbrial-like adhesin protein, putative periplasmic pilin chaperone, regulators of the transcriptional regulation, antibiotic resistance genes and stress-induced genes, rendering VBNC cells more tolerant to adverse environmental conditions. In total of 16 genes were significantly up-regulated under the VBNC state, including three genes encoding toxic protein (ygeG, ibsD, shoB), indicating that VBNC E. coil was still a threat to human. The work is of great relevance in the context of better understanding this poorly understood physiological state.

Kin discrimination and outer membrane exchange in Myxococcus xanthus: Experimental analysis of a natural population.

In some species of myxobacteria, adjacent cells sufficiently similar at the adhesin protein TraA can exchange components of their outer membranes. The primary benefits of such outer membrane exchange (OME) in natural populations are unclear, but in some OME interactions, transferred OM content can include SitA toxins that kill OME participants lacking an appropriate immunity gene. Such OME-dependent toxin transfer across Myxococcus xanthus strains that differ only in their sitBAI toxin/antitoxin cassette can mediate inter-strain killing and generate colony-merger incompatibilities (CMIs)–inter-colony border phenotypes between distinct genotypes that differ from respective self-self colony interfaces. Here we ask whether OME-dependent toxin transfer is a common cause of prevalent CMIs and antagonisms between M. xanthus natural isolates identical at TraA. We disrupted traA in eleven isolates from a cm-scale soil population and assayed whether traA disruption eliminated or reduced CMIs between swarming colonies or antagonisms between strains in mixed cultures. Among 33 isolate pairs identical at traA that form clear CMIs, in no case did functional disruption of traA in one partner detectably alter CMI phenotypes. Further, traA disruption did not alleviate strong antagonisms observed during starvation-induced fruiting-body development in seven pairs of strains identical at traA. Collectively, our results suggest that most mechanisms of interference competition and inter-colony kin discrimination in natural populations of myxobacteria do not require OME. Finally, our experiments also indicate that several closely related laboratory reference strains kill some natural isolates by toxins delivered by a shared, OME-independent type VI secretion system (T6SS), suggesting that some antagonisms between sympatric natural isolates may also involve T6SS toxins.

RMSD analysis of structures of the bacterial protein FimH identifies five conformations of its lectin domain.

FimH is a bacterial adhesin protein located at the tip of Escherichia coli fimbria that functions to adhere bacteria to host cells. Thus, FimH is a critical factor in bacterial infections such as urinary tract infections and is of interest in drug development. It is also involved in vaccine development and as a model for understanding shear-enhanced catch bond cell adhesion. To date, over 60 structures have been deposited in the Protein Data Bank showing interactions between FimH and mannose ligands, potential inhibitors, and other fimbrial proteins. In addition to providing insights about ligand recognition and fimbrial assembly, these structures provide insights into conformational changes in the two domains of FimH that are critical for its function. To gain further insights into these structural changes, we have superposed FimH's mannose binding lectin domain in all these structures and categorized the structures into five groups of lectin domain conformers using RMSD as a metric. Many structures also include the pilin domain, which anchors FimH to the fimbriae and regulates the conformation and function of the lectin domain. For these structures, we have also compared the relative orientations of the two domains. These structural analyses enhance our understanding of the conformational changes associated with FimH ligand binding and domain-domain interactions, including its catch bond behavior through allosteric action of force in bacterial adhesion.

Molecular Superglues: Discovery and Engineering Orthogonalization.

Molecular superglues covalently ligate two or more macromolecules together into super stable, covalently linked assemblies. The discovery of intramolecular isopeptide and ester bond crosslinks in bacterial adhesin proteins, inspired the development of two distinct protein ligating technologies based on split protein domains. These chemically distinct technologies could be combined as orthogonal (non-cross-reacting) technologies to make complex assemblies. Here we provide simple practical instructions in the discovery, characterisation, and application of orthogonal ester bond crosslinks as molecular superglues. A large toolkit of diverse, orthogonal molecular superglues will expand our assembly repertoire, and afford increasingly more complex one-, two-, and three-dimensional protein nanomaterials with exquisite control over the final molecular architecture.

The NDV-3A vaccine protects mice from multidrug resistant Candida auris infection.

Candida auris is an emerging, multi-drug resistant, health care-associated fungal pathogen. Its predominant prevalence in hospitals and nursing homes indicates its ability to adhere to and colonize the skin, or persist in an environment outside the host—a trait unique from other Candida species. Besides being associated globally with life-threatening disseminated infections, C. auris also poses significant clinical challenges due to its ability to adhere to polymeric surfaces and form highly drug-resistant biofilms. Here, we performed bioinformatic studies to identify the presence of adhesin proteins in C. auris, with sequence as well as 3-D structural homologies to the major adhesin/invasin of C. albicans, Als3. Anti-Als3p antibodies generated by vaccinating mice with NDV-3A (a vaccine based on the N-terminus of Als3 protein formulated with alum) recognized C. auris in vitro, blocked its ability to form biofilms and enhanced macrophage-mediated killing of the fungus. Furthermore, NDV-3A vaccination induced significant levels of C. auris cross-reactive humoral and cellular immune responses, and protected immunosuppressed mice from lethal C. auris disseminated infection, compared to the control alum-vaccinated mice. The mechanism of protection is attributed to anti-Als3p antibodies and CD4+ T helper cells activating tissue macrophages. Finally, NDV-3A potentiated the protective efficacy of the antifungal drug micafungin, against C. auris candidemia. Identification of Als3-like adhesins in C. auris makes it a target for immunotherapeutic strategies using NDV-3A, a vaccine with known efficacy against other Candida species and safety as well as efficacy in clinical trials. Considering that C. auris can be resistant to almost all classes of antifungal drugs, such an approach has profound clinical relevance.

The Role of Pili Protein 38,6 kDa Klebsiella pneumoniae as a Hemagglutinin and Adhesin Protein which Serves as a Virulence Factor

Klebsiella pneumoniae is an infectious bacteria in various parts of the body. The presence of proteins in pili that will bind to cell surface receptors (adhesin proteins) and cell membrane sugar molecules (hemagglutinin proteins) is a very influential factor in the ability of K. pneumoniae to enter the human body. The purpose of this study was to determine the role of pili 38.6 kDa K. pneumoniae protein as a hemagglutinin and adhesin protein which functions as a virulence factor. This type of research is pure experimental research using the electrophoresis method (SDS-PAGE) to obtain protein. The protein obtained was then tested for hemagglutination and adhesion test using erythrocyte cells and enterocytes of BALB / C mice to determine their role as hemagglutinin and adhesin proteins. Conclusion of this study pili protein 38.6 kDa Klebsiella pneumoniae acts as a hemagglutinin and adhesion protein which functions as a virulence factor.&#x0D; Keywords: Klebsiella pneumonia, 38,6 kDa Protein, Hemagglutinin, Adhesin

Pathogenic interactions between Helicobacter pylori adhesion protein HopQ and human cell surface adhesion molecules CEACAMs in gastric epithelial cells.

Objective(s):The present paper aims to review the studies describing the interactions between HopQ and CEACAMs along with possible mechanisms responsible for pathogenicity of Helicobacter pylori.Materials and Methods:The literature was searched on “PubMed” using different key words including Helicobacter pylori, CEACAM and gastric.Results:HopQ is one of the outer membrane proteins of H. pylori and belongs to the family of adhesin proteins. In contrast to other adhesins, HopQ interacts with host cell surface molecules in a glycan independent manner. Human CEACAMs are the cell surface adhesion molecules mainly present on the epithelial cells, endothelial cells and leukocytes. The overexpression of these molecules may contribute to cancer progression and relapse. Recent studies have shown that HopQ may interact with human CEACAMs, particularly CEACAM1, CEACAM3, CEACAM5 and CEACAM6, but not CEACAM8. HopQ interacts with GFCC’C” interaction surface of IgV domain of N- terminal region of CEACAM1. Moreover, binding of HopQ to CEACAM1 prevent its trans-dimerization and stabilizes it in monomeric form. H. pylori may use these HopQ-CEACAM interactions to transfer its CagA oncoprotein into host gastric epithelial cells, which is followed by its phosphorylation and release of interleukin-8. HopQ-CEACAM interactions may also utilize T4SS, instead of CagA, to activate NF-κB signaling and trigger inflammation. Conclusion:HopQ of H. pylori may interact with CEACAMs of the human gastric cells to induce the development of gastric ulcers and cancers by transferring CagA oncoprotein or inducing activation of T4SS to initiate and maintain inflammatory reactions.

Role of Evolutionary Selection Acting on Vaccine Antigens in the Re-Emergence of Bordetella Pertussis.

Pertussis (“whooping cough”) is a re-emerging disease with increasing incidence among fully vaccinated individuals. We explored the genetic diversity of five Bordetella pertussis proteins used to generate the subunit vaccine across ancestral and newly emergent strains using immunoinformatics and evolutionary selection measurements. The five subunits of pertussis toxin (Ptx1–Ptx5) were highly conserved with regard to sequence, predicted structure, predicted antigenicity, and were under purifying selection. In contrast, the adhesin proteins pertactin (Prn) and filamentous hemagglutinin (FHA) were under statistically significant (p < 0.01) diversifying selection. Most heavily diversified sites of each protein fell within antigenic epitopes, and the functional adhesin motifs were conserved. Protein secondary structure was conserved despite sequence diversity for FHA but was changeable in Prn. These findings suggest that subunit vaccine-derived immunity does not impact Ptx1–Ptx5 but may apply evolutionary pressure to Prn and FHA to undergo diversifying selection. These findings offer further insight into the emergence of vaccine-resistant strains of B. pertussis.

Deposition of Host Matrix Proteins on Breast Implant Surfaces Facilitates Staphylococcus Epidermidis Biofilm Formation: In Vitro Analysis.

Staphylococcus epidermidis is a primary cause of breast implant-associated infection. S epidermidis possesses several virulence factors that enable it to bind both abiotic surfaces and host factors to form a biofilm. In addition S epidermidis colocalizes with matrix proteins coating explanted human breast implants. The authors sought to identify matrix proteins that S epidermidis may exploit to infect various breast implant surfaces in vitro. A combination of in vitro assays was used to characterize S epidermidis strains isolated from human breast implants to gain a better understanding of how these bacteria colonize breast implant surfaces. These included determining the (1) minimum inhibitory and bactericidal concentrations for irrigation solutions commonly used to prevent breast implant contamination; (2) expression and carriage of polysaccharide intercellular adhesin and serine-aspartate repeat proteins, which bind fibrinogen (SdrG) and collagen (SdrF), respectively; and (3) biofilm formation on varying implant surface characteristics, in different growth media, and supplemented with fibrinogen and Types I and III collagen. Scanning electron microscopy and immunofluorescence staining analyses were performed to corroborate findings from these assays. Textured breast implant surfaces support greater bacterial biofilm formation at baseline, and the addition of collagen significantly increases biomass on all surfaces tested. We found that S epidermidis isolated from breast implants all encoded SdrF. Consistent with this finding, these strains had a clear affinity for Type I collagen, forming dense, highly structured biofilms in its presence. The authors found that S epidermidis may utilize SdrF to interact with Type I collagen to form biofilm on breast implant surfaces.

Gene Duplication Associated with Increased Fluconazole Tolerance in Candida auris cells of Advanced Generational Age

Candida auris is an emerging multi-drug resistant yeast that causes systemic infections. Here we show that C. auris undergoes replicative aging (RA) that results from asymmetric cell division and causes phenotypic differences between mother and daughter cells similar to other pathogenic yeasts. Importantly, older C. auris cells (10 generations) exhibited higher tolerance to fluconazole (FLC), micafungin, 5- flucytosine and amphotericin B compared to younger (0–3 generation) cells. Increased FLC tolerance was associated with increased Rhodamine 6G (R6G) efflux and therapeutic failure of FLC in a Galleria infection model. The higher efflux in the older cells correlated with overexpression of the efflux pump encoding gene CDR1 (4-fold). In addition, 8-fold upregulation of the azole target encoding gene ERG11 was noted in the older cells. Analysis of genomic DNA from older cells by qPCR indicates that transient gene duplication of CDR1 and ERG11 causes the observed age-dependent enhanced FLC tolerance in C. auris strains. Furthermore, older cells exhibited a thickened cell wall, decreased neutrophil killing (24% vs 50%), increased epithelial cell adhesion (31.6% vs 17.8%) and upregulation of adhesin protein Als5p. Thus, this study demonstrates that transient gene duplication can occur during RA, causing increased FLC tolerance in old C. auris cells.

Citrullination mediated by PPAD constrains biofilm formation in P. gingivalis strain 381

Porphyromonas gingivalis is the only known human-associated prokaryote that produces a peptidylarginine deiminase (PPAD), a protein-modifying enzyme that is secreted along with a number of virulence factors via a type IX secretion system (T9SS). While the function of PPAD in P. gingivalis physiology is not clear, human peptidylarginine deiminases are known to convert positively charged arginine residues within proteins to neutral citrulline and, thereby, impact protein conformation and function. Here, we report that the lack of citrullination in a PPAD deletion mutant (Δ8820) enhances biofilm formation. More Δ8820 cells attached to the surface than the parent strain during the early stages of biofilm development and, ultimately, mature Δ8820 biofilms were comprised of significantly more cell–cell aggregates and extracellular matrix. Imaging by electron microscopy discovered that Δ8820 biofilm cells secrete copious amounts of protein aggregates. Furthermore, gingipain-derived adhesin proteins, which are also secreted by the T9SS were predicted by mass spectrometry to be citrullinated and citrullination of these targets by wild-type strain 381 in vitro was confirmed. Lastly, Δ8820 biofilms contained more gingipain-derived adhesin proteins and more gingipain activity than 381 biofilms. Overall, our findings support the model that citrullination of T9SS cargo proteins known to play a key role in colonization, such as gingipain-derived adhesin proteins, is an underlying mechanism that modulates P. gingivalis biofilm development.

Sequence analysis and confirmation of the type IV pili-associated proteins PilY1, PilW and PilV in Acidithiobacillus thiooxidans.

Acidithiobacillus thiooxidans is an acidophilic chemolithoautotrophic bacterium widely used in the mining industry due to its metabolic sulfur-oxidizing capability. The biooxidation of sulfide minerals is enhanced through the attachment of At. thiooxidans cells to the mineral surface. The Type IV pili (TfP) of At. thiooxidans may play an important role in the bacteria attachment since TfP play a key adhesive role in the attachment and colonization of different surfaces. In this work, we report for the first time the mRNA sequence of three TfP proteins from At. thiooxidans, the adhesin protein PilY1 and the TfP pilins PilW and PilV. The nucleotide sequences of these TfP proteins show changes in some nucleotide positions with respect to the corresponding annotated sequences. The bioinformatic analyses and 3D-modeling of protein structures sustain their classification as TfP proteins, as structural homologs of the corresponding proteins of Ps. aeruginosa, results that sustain the role of PilY1, PilW and PilV in pili assembly. Also, that PilY1 comprises the conserved Neisseria-PilC (superfamily) domain of the tip-associated adhesin, while PilW of the superfamily of putative TfP assembly proteins and PilV belongs to the superfamily of TfP assembly protein. In addition, the analyses suggested the presence of specific functional domains involved in adhesion, energy transduction and signaling functions. The phylogenetic analysis indicated that the PilY1 of Acidithiobacillus genus forms a cohesive group linked with iron- and/or sulfur-oxidizing microorganisms from acid mine drainage or mine tailings.

Designer artificial membrane binding proteins to direct stem cells to the myocardium.

We present a new cell membrane modification methodology where the inherent heart tissue homing properties of the infectious bacteria Streptococcus gordonii are transferred to human stem cells. This is achieved via the rational design of a chimeric protein-polymer surfactant cell membrane binding construct, comprising the cardiac fibronectin (Fn) binding domain of the bacterial adhesin protein CshA fused to a supercharged protein. Significantly, the protein-polymer surfactant hybrid spontaneously inserts into the plasma membrane of stem cells without cytotoxicity, instilling the cells with a high affinity for immobilized fibronectin. Moreover, we show that this cell membrane reengineering approach significantly improves retention and homing of stem cells delivered either intracardially or intravenously to the myocardium in a mouse model.