Role of the Streptococcus mutans irvA Gene in GbpC-Independent, Dextran-Dependent Aggregation and Biofilm Formation

ABSTRACT Dextran-dependent aggregation (DDAG) of Streptococcus mutans is an in vitro phenomenon that is believed to represent a property of the organism that is beneficial for sucrose-dependent biofilm development. GbpC, a cell surface glucan-binding protein, is responsible for DDAG in S. mutans when cultured under defined stressful conditions. Recent reports have described a putative transcriptional regulator gene, irvA, located just upstream of gbpC, that is normally repressed by the product of an adjacent gene, irvR. When repression of irvA is relieved, there is a resulting increase in the expression of GbpC and decreases in competence and synthesis of the antibiotic mutacin I. This study examined the role of irvA in DDAG and biofilm formation by engineering strains that overexpressed irvA (IrvA+) on an extrachromosomal plasmid. The IrvA+ strain displayed large aggregation particles that did not require stressful growth conditions. A novel finding was that overexpression of irvA in a gbpC mutant background retained a measure of DDAG, albeit very small aggregation particles. Biofilms formed by the IrvA+ strain in the parental background possessed larger-than-normal microcolonies. In a gbpC mutant background, the overexpression of irvA reversed the fragile biofilm phenotype normally associated with loss of GbpC. Real-time PCR and Northern blot analyses found that expression of gbpC did not change significantly in the IrvA+ strain but expression of spaP, encoding the major surface adhesin P1, increased significantly. Inactivation of spaP eliminated the small-particle DDAG. The results suggest that IrvA promotes DDAG not only by GbpC, but also via an increase in P1.

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The cyclic di-GMP network is a global regulator of phase-transition and attachment-dependent host colonization in Erwinia amylovora

10.1101/2021.02.01.429191 ◽

2021 ◽

Author(s):

Roshni R. Kharadi ◽

Kayla Selbmann ◽

George W. Sundin

Keyword(s):

Signal Transduction ◽

Biofilm Formation ◽

Erwinia Amylovora ◽

Initial Surface ◽

Diguanylate Cyclase ◽

Wide Range ◽

Biofilm Development ◽

Bacterial Systems

AbstractCyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates the transition to biofilm formation in the phytopathogen Erwinia amylovora. The c-di-GMP system in E. amylovora is comprised of 12 diguanylate cyclase/Edc (dimerize cyclic-di-GMP) and phosphodiesterase/Pde (hydrolyze cyclic-di-GMP) proteins that are characterized by the presence of GGDEF and/or EAL motifs in their domain architecture. In order to study the global regulatory effect (without the inclusion of systemic regulatory impedance) of the c-di-GMP system in E. amylovora, we eliminated all 12 edc and pde genes in E. amylovora Ea1189Δ12. Comparisons between the representative transcriptomic profiles of Ea1189Δ12 and the combinatorial edc gene knockout mutant (Ea1189Δ5) revealed marked overall distinctions in expression levels for targets in a wide range of regulatory categories, including metabolic pathways involved in the utilization of methionine, isoleucine, histidine, etc. as well as critical signal transduction pathways including the Rcs phosphorelay and PhoPQ system. A complete loss of the cyclic-di-GMP signaling components resulted in the inability of Ea1189Δ12 cells to attach to and form biofilms in vitro and within the xylem vasculature in apple shoots. Using a flow-based in vitro biofilm system, we found that initial surface sensing was primarily dependent on the flagellar filament (FliC), following which the type IV pilus (HofC) was required to anchor cells to the surface to initialize biofilm development. A transcriptomic analysis of WT E. amylovora Ea1189 and Ea1189Δ12 cells in various stages of biofilm development revealed that cyclic-di-GMP based regulation had widespread effects on purine and pyrimidine biosynthesis pathways, amylovoran biosynthesis genes and the EnvZ/OmpR signal transduction system. Additionally, complementing individual eliminated genes back into Ea1189Δ12, and the collective evaluation of several virulence factors, enabled the correlative clustering of the functional effect rendered by each Edc and Pde enzyme in the system.SignificanceCyclic-di-GMP dependent regulation, in the context of biofilm formation, has been studied in several bacterial systems. However, the comprehensiveness of the studies exploring the role of individual genetic components related to cyclic-di-GMP is affected by the often large number of diguanylate cyclase and phosphodiesterase enzymes present within individual bacterial systems. To explore the evolutionary dependencies related to cyclic-di-GMP in E. amylovora, we used a collective elimination approach, whereby all of the enzymes involved in cyclic-di-GMP metabolism were eliminated from the system. This approach enabled us to highlight the critical importance of cyclic-di-GMP in plant xylem colonization due to its effect on surface attachment. Additionally, we highlight the global transcriptomic effect of cyclic-di-GMP dependent signaling at various stages of biofilm development. Our approach is aimed at exploring the regulatory role of individual cyclic-di-GMP related enzymes in a background that is free from any redundancy-based feedback.

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Function of BriC Peptide in the Pneumococcal Competence and Virulence Portfolio

10.1101/245902 ◽

2018 ◽

Cited By ~ 1

Author(s):

Surya D. Aggarwal ◽

Rory Eutsey ◽

Jacob West-Roberts ◽

Arnau Domenech ◽

Wenjie Xu ◽

...

Keyword(s):

Murine Model ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Nasopharyngeal Colonization ◽

Point Of Entry ◽

Abiotic Surfaces ◽

Biofilm Development

AbstractStreptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.

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Bovine-associated non-aureus staphylococci suppress Staphylococcus aureus biofilm dispersal in vitro yet not through agr regulation

Veterinary Research ◽

10.1186/s13567-021-00985-z ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Bruno Toledo-Silva ◽

Fernando N. de Souza ◽

Kristien Mertens ◽

Sofie Piepers ◽

Freddy Haesebrouck ◽

...

Keyword(s):

Biofilm Formation ◽

Communication Systems ◽

Bovine Milk ◽

Subclinical Mastitis ◽

Interspecies Interactions ◽

Environmental Signals ◽

Biofilm Dispersion ◽

Biofilm Development

AbstractBiofilm formation is a significant virulence factor in Staphylococcus (S.) aureus strains causing subclinical mastitis in dairy cows. A role of environmental signals and communication systems in biofilm development, such as the agr system in S. aureus, is suggested. In the context of multispecies biofilm communities, the presence of non-aureus staphylococci (NAS) might influence S. aureus colonization of the bovine mammary gland, yet, such interspecies interactions have been poorly studied. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices (TA) were able to affect biofilm formation and dispersion of S. aureus, and if so, how isolate traits such as the capacity to regulate the S. aureus agr quorum sensing system are determinants in this process. The capacity of an agr-positive S. aureus strain to form biofilm was increased more in the presence of S. chromogenes than in the presence of S. simulans and S. epidermidis isolates and in the presence of NAS isolates that do not harbor biofilm related genes. On the other hand, biofilm dispersion of this particular S. aureus strain was suppressed by NAS as a group, an effect that was more pronounced by isolates from TA. Furthermore, the observed effects on biofilm formation and dispersion of the agr-positive S. aureus strain as well as of an agr-negative S. aureus strain did not depend on the capacity of NAS to repress the agr system.

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Streptococcus mutans biofilm inhibition using antisense oligonucleotide to glucosyltransferases B and C

Acta medica Lituanica ◽

10.6001/actamedica.v22i2.3123 ◽

2015 ◽

Vol 22 (2) ◽

pp. 85-92

Author(s):

Povilas Kalesinskas ◽

Tomas Kačergius ◽

Arvydas Ambrozaitis ◽

Ryo Jimbo ◽

Dan Ericson

Keyword(s):

Streptococcus Mutans ◽

Biofilm Formation ◽

Roughness Parameter ◽

Preventive Measure ◽

Modern Society ◽

Biofilm Inhibition ◽

Dental Biofilm ◽

Glass Slides ◽

Biofilm Development

Background. Biofilm formation by Streptococcus mutans bacteria on teeth leads to dental caries, which still remains one of the most prevalent human diseases strongly related to increase of dietary sucrose consumption in modern society. In the biofilm, sucrose is metabolized by S. mutans to acids causing tooth decay. S. mutans also produces glucosyltransferases (Gtfs) for synthesis of sticky glucan polymers from sucrose that provides matrix for biofilm formation on teeth. For reducing biofilm build-up, one preventive measure could be blocking of Gtf synthesis. The aim of this study was to test antisense phosphorothioate oligodeoxyribonucleotide (PS-ODN) targeting simultaneously S. mutans gtfB and gtfC mRNAs in order to inhibit biofilm formation in vitro. Materials and methods. S. mutans bacteria were grown anaerobically on glass slides inserted vertically in 24-well cell culture plates containing Todd Hewitt broth with sucrose under exposure to antisense or missense PS-ODNs at the final concentration of 10 μM. Untreated bacteria served as controls. After 24 h of incubation, glass slides were removed, air-dried and further used for the quantitative evaluation of the streptococci biofilm applying an optical profilometry technique. Results. It was revealed that antisense PS-ODN considerably reduced the most critical biofilm surface roughness parameter Sa (average difference between the peak hight and valleys) inhibiting the biofilm development by 46% and 77% in comparison to untreated (p = 0.06) and missense PS-ODN-treated bacteria (p < 0.05), respectively. Conclusions. The results demonstrate that antisense PS-ODN considerably decreases streptococci-induced biofilm development on glass slides, and might therefore significantly suppress dental biofilm formation through simultaneous inactivation of S. mutans gtfB and gtfC mRNAs.

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Role of Multicellular Aggregates in Biofilm Formation

mBio ◽

10.1128/mbio.00237-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 133

Author(s):

Kasper N. Kragh ◽

Jaime B. Hutchison ◽

Gavin Melaugh ◽

Chris Rodesney ◽

Aled E. L. Roberts ◽

...

Keyword(s):

Biofilm Formation ◽

Single Cells ◽

Subsequent Development ◽

Relative Fitness ◽

Bacterial Cells ◽

Ecological Benefit ◽

Multicellular Aggregates ◽

Biofilm Development

ABSTRACT In traditional models of in vitro biofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development of Pseudomonas aeruginosa biofilms. We find that the relative fitness of aggregates depends markedly on the density of surrounding single cells, i.e., the level of competition for growth resources. When competition between aggregates and single cells is low, an aggregate has a growth disadvantage because the aggregate interior has poor access to growth resources. However, if competition is high, aggregates exhibit higher fitness, because extending vertically above the surface gives cells at the top of aggregates better access to growth resources. Other advantages of seeding by aggregates, such as earlier switching to a biofilm-like phenotype and enhanced resilience toward antibiotics and immune response, may add to this ecological benefit. Our findings suggest that current models of biofilm formation should be reconsidered to incorporate the role of aggregates in biofilm initiation. IMPORTANCE During the past decades, there has been a consensus around the model of development of a biofilm, involving attachment of single planktonic bacterial cells to a surface and the subsequent development of a mature biofilm. This study presents results that call for a modification of this rigorous model. We show how free floating biofilm aggregates can have a profound local effect on biofilm development when attaching to a surface. Our findings show that an aggregate landing on a surface will eventually outcompete the biofilm population arising from single cells attached around the aggregate and dominate the local biofilm development. These results point to a regime where preformed biofilm aggregates may have a fitness advantage over planktonic cells when it comes to accessing nutrients. Our findings add to the increasingly prominent comprehension that biofilm lifestyle is the default for bacteria and that planktonic single cells may be only a transition state at the most.

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Reduction of Streptococcus mutans Adherence and Dental Biofilm Formation by Surface Treatment with Phosphorylated Polyethylene Glycol

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00380-07 ◽

2007 ◽

Vol 51 (10) ◽

pp. 3634-3641 ◽

Cited By ~ 27

Author(s):

Akira Shimotoyodome ◽

Takashi Koudate ◽

Hisataka Kobayashi ◽

Junji Nakamura ◽

Ichiro Tokimitsu ◽

...

Keyword(s):

Polyethylene Glycol ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

De Novo ◽

Bacterial Attachment ◽

Salivary Proteins ◽

Dental Biofilm ◽

Biofilm Development

ABSTRACT Initial attachment of the cariogenic Streptococcus mutans onto dental enamel is largely promoted by the adsorption of specific salivary proteins on enamel surface. Some phosphorylated salivary proteins were found to reduce S. mutans adhesion by competitively inhibiting the adsorption of S. mutans-binding salivary glycoproteins to hydroxyapatite (HA). The aim of this study was to develop antiadherence compounds for preventing dental biofilm development. We synthesized phosphorylated polyethylene glycol (PEG) derivatives and examined the possibility of surface pretreatment with them for preventing S. mutans adhesion in vitro and dental biofilm formation in vivo. Pretreatment of the HA surface with methacryloyloxydecyl phosphate (MDP)-PEG prior to saliva incubation hydrophilized the surface and thereby reduced salivary protein adsorption and saliva-promoted bacterial attachment to HA. However, when MDP-PEG was added to the saliva-pretreated HA (S-HA) surface, its inhibitory effect on bacterial binding was completely diminished. S. mutans adhesion onto S-HA was successfully reduced by treatment of the surface with pyrophosphate (PP), which desorbs salivary components from S-HA. Treatment of S-HA surfaces with MDP-PEG plus PP completely inhibited saliva-promoted S. mutans adhesion even when followed by additional saliva treatment. Finally, mouthwash with MDP-PEG plus PP prevented de novo biofilm development after thorough teeth cleaning in humans compared to either water or PP alone. We conclude that MDP-PEG plus PP has the potential for use as an antiadherence agent that prevents dental biofilm development.

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Functional Genomics Approach to Identifying Genes Required for Biofilm Development by Streptococcus mutans

Applied and Environmental Microbiology ◽

10.1128/aem.68.3.1196-1203.2002 ◽

2002 ◽

Vol 68 (3) ◽

pp. 1196-1203 ◽

Cited By ~ 159

Author(s):

Zezhang T. Wen ◽

Robert A. Burne

Keyword(s):

Streptococcus Mutans ◽

Biofilm Formation ◽

Regulatory Protein ◽

Amino Acid Residues ◽

Global Regulators ◽

Insertional Inactivation ◽

Bacterial Quorum Sensing ◽

Bacterial Quorum ◽

Biofilm Development

ABSTRACT Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS Sm) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.

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The Role of Candida albicans Secreted Polysaccharides in Augmenting Streptococcus mutans Adherence and Mixed Biofilm Formation: In vitro and in vivo Studies

Frontiers in Microbiology ◽

10.3389/fmicb.2020.00307 ◽

2020 ◽

Vol 11 ◽

Cited By ~ 3

Author(s):

Zaid H. Khoury ◽

Taissa Vila ◽

Taanya R. Puthran ◽

Ahmed S. Sultan ◽

Daniel Montelongo-Jauregui ◽

...

Keyword(s):

Candida Albicans ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

In Vivo Studies

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Phase Variation of LPS and Capsule Is Responsible for Stochastic Biofilm Formation in Francisella tularensis

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.808550 ◽

2022 ◽

Vol 11 ◽

Author(s):

Kevin D. Mlynek ◽

Christopher T. Lopez ◽

David P. Fetterer ◽

Janice A. Williams ◽

Joel A. Bozue

Keyword(s):

Francisella Tularensis ◽

Biofilm Formation ◽

Potential Role ◽

Phase Variation ◽

Type A ◽

Biofilm Development ◽

Initial Biofilm ◽

Reproducible Manner

Biofilms have been established as an important lifestyle for bacteria in nature as these structured communities often enable survivability and persistence in a multitude of environments. Francisella tularensis is a facultative intracellular Gram-negative bacterium found throughout much of the northern hemisphere. However, biofilm formation remains understudied and poorly understood in F. tularensis as non-substantial biofilms are typically observed in vitro by the clinically relevant subspecies F. tularensis subsp. tularensis and F. tularensis subsp. holarctica (Type A and B, respectively). Herein, we report conditions under which robust biofilm development was observed in a stochastic, but reproducible manner in Type A and B isolates. The frequency at which biofilm was observed increased temporally and appeared switch-like as progeny from the initial biofilm quickly formed biofilm in a predictable manner regardless of time or propagation with fresh media. The Type B isolates used for this study were found to more readily switch on biofilm formation than Type A isolates. Additionally, pH was found to function as an environmental checkpoint for biofilm initiation independently of the heritable cellular switch. Multiple colony morphologies were observed in biofilm positive cultures leading to the identification of a particular subset of grey variants that constitutively produce biofilm. Further, we found that constitutive biofilm forming isolates delay the onset of a viable non-culturable state. In this study, we demonstrate that a robust biofilm can be developed by clinically relevant F. tularensis isolates, provide a mechanism for biofilm initiation and examine the potential role of biofilm formation.

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PapRIV, a BV-2 microglial cell activating quorum sensing peptide

Scientific Reports ◽

10.1038/s41598-021-90030-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yorick Janssens ◽

Nathan Debunne ◽

Anton De Spiegeleer ◽

Evelien Wynendaele ◽

Marta Planas ◽

...

Keyword(s):

Quorum Sensing ◽

Cell Model ◽

Dependent Manner ◽

Communication Signals ◽

Gram Positive Bacteria ◽

A Cell ◽

Gastro Intestinal Tract

AbstractQuorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood–brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction.

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