scholarly journals Autoinducer-2 and QseC Control Biofilm Formation and In Vivo Virulence of Aggregatibacter actinomycetemcomitans

2010 ◽  
Vol 78 (7) ◽  
pp. 2919-2926 ◽  
Author(s):  
Elizabeth A. Novak ◽  
HanJuan Shao ◽  
Carlo Amorin Daep ◽  
Donald R. Demuth
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Biofilm Formation ◽  
Alveolar Bone ◽  
Wild Type ◽  
Two Component System ◽  
Component System ◽  
Autoinducer 2 ◽  
Two Component

ABSTRACT Biofilm formation by the periodontal pathogen Aggregatibacter actinomycetemcomitans is dependent upon autoinducer-2 (AI-2)-mediated quorum sensing. However, the components that link the detection of the AI-2 signal to downstream gene expression have not been determined. One potential regulator is the QseBC two-component system, which is part of the AI-2-dependent response pathway that controls biofilm formation in Escherichia coli. Here we show that the expression of QseBC in A. actinomycetemcomitans is induced by AI-2 and that induction requires the AI-2 receptors, LsrB and/or RbsB. Additionally, inactivation of qseC resulted in reduced biofilm growth. Since the ability to grow in biofilms is essential for A. actinomycetemcomitans virulence, strains that were deficient in QseC or the AI-2 receptors were examined in an in vivo mouse model of periodontitis. The ΔqseC mutant induced significantly less alveolar bone resorption than the wild-type strain (P < 0.02). Bone loss in animals infected with the ΔqseC strain was similar to that in sham-infected animals. The ΔlsrB, ΔrbsB, and ΔlsrB ΔrbsB strains also induced significantly less alveolar bone resorption than the wild type (P < 0.03, P < 0.02, and P < 0.01, respectively). However, bone loss induced by a ΔluxS strain was indistinguishable from that induced by the wild type, suggesting that AI-2 produced by indigenous microflora in the murine oral cavity may complement the ΔluxS mutation. Together, these results suggest that the QseBC two-component system is part of the AI-2 regulon and may link the detection of AI-2 to the regulation of downstream cellular processes that are involved in biofilm formation and virulence of A. actinomycetemcomitans.

scholarly journals Phosphorelay as the Sole Physiological Route of Signal Transmission by the Arc Two-Component System ofEscherichia coli

2000 ◽  
Vol 182 (13) ◽  
pp. 3858-3862 ◽  
Author(s):  
Ohsuk Kwon ◽  
Dimitris Georgellis ◽  
E. C. C. Lin
Keyword(s):  
Response Regulator ◽  
Signal Transmission ◽  
Phosphoryl Group ◽  
Sensor Kinase ◽  
Wild Type ◽  
Two Component System ◽  
Component System ◽  
Group Transfer ◽  
Two Component

ABSTRACT The Arc two-component system, comprising a tripartite sensor kinase (ArcB) and a response regulator (ArcA), modulates the expression of numerous genes involved in respiratory functions. In this study, the steps of phosphoryl group transfer from phosphorylated ArcB to ArcA were examined in vivo by using single copies of wild-type and mutantarcB alleles. The results indicate that the signal transmission occurs solely by His-Asp-His-Asp phosphorelay.

scholarly journals Identification of a Novel Two-Component System in Streptococcus gordonii V288 Involved in Biofilm Formation

2004 ◽  
Vol 72 (6) ◽  
pp. 3489-3494 ◽  
Author(s):  
Yongshu Zhang ◽  
Yu Lei ◽  
Ali Khammanivong ◽  
Mark C. Herzberg
Keyword(s):  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Streptococcus Gordonii ◽  
Wild Type ◽  
Two Component System ◽  
Component System ◽  
Two Component

ABSTRACT Streptococcus gordonii is a pioneer colonizer of the teeth, contributing to the initiation of the oral biofilm called dental plaque. To identify genes that may be important in biofilm formation, a plasmid integration library of S. gordonii V288 was used. After screening for in vitro biofilm formation on polystyrene, a putative biofilm-defective mutant was isolated. In this mutant, pAK36 was inserted into a locus encoding a novel two-component system (bfr [biofilm formation related]) with two cotranscribed genes that form an operon. bfrA encodes a putative response regulator, while bfrB encodes a receptor histidine kinase. The bfr mutant and wild-type strain V288 showed similar growth rates in Todd-Hewitt broth (THB). A bfr-cat fusion strain was constructed. During growth in THB, the reporter activity (chloramphenicol acetyltransferase) was first detected in mid-log phase and reached a maximum in stationary phase, suggesting that transcription of bfr was growth stage dependent. After being harvested from THB, the bfr mutant adhered less effectively than did wild-type strain V288 to saliva-coated hydroxyapatite (sHA). To simulate pioneer colonization of teeth, S. gordonii V288 was incubated with sHA for 4 h in THB with 10% saliva to develop biofilms. RNA was isolated, and expression of bfrAB was estimated. In comparison to that of cells grown in suspension (free-growing cells), bfr mRNA expression by sessile cells on sHA was 1.8-fold greater and that by surrounding planktonic cells was 3.5-fold greater. Therefore, bfrAB is a novel two-component system regulated in association with S. gordonii biofilm formation in vitro.

scholarly journals Enhancement of Osteoclastic Bone Resorption and Suppression of Osteoblastic Bone Formation in Response to Reduced Mechanical Stress Do Not Occur in the Absence of Osteopontin

2001 ◽  
Vol 193 (3) ◽  
pp. 399-404 ◽  
Author(s):  
Muneaki Ishijima ◽  
Susan R. Rittling ◽  
Teruhito Yamashita ◽  
Kunikazu Tsuji ◽  
Hisashi Kurosawa ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Bone Formation ◽  
Mechanical Stress ◽  
Bone Matrix ◽  
Osteoclastic Bone Resorption ◽  
Tail Suspension ◽  
Wild Type ◽  
Osteoblastic Bone Formation

Reduced mechanical stress to bone in bedridden patients and astronauts leads to bone loss and increase in fracture risk which is one of the major medical and health issues in modern aging society and space medicine. However, no molecule involved in the mechanisms underlying this phenomenon has been identified to date. Osteopontin (OPN) is one of the major noncollagenous proteins in bone matrix, but its function in mediating physical-force effects on bone in vivo has not been known. To investigate the possible requirement for OPN in the transduction of mechanical signaling in bone metabolism in vivo, we examined the effect of unloading on the bones of OPN−/− mice using a tail suspension model. In contrast to the tail suspension–induced bone loss in wild-type mice, OPN−/− mice did not lose bone. Elevation of urinary deoxypyridinoline levels due to unloading was observed in wild-type but not in OPN−/− mice. Analysis of the mechanisms of OPN deficiency–dependent reduction in bone on the cellular basis resulted in two unexpected findings. First, osteoclasts, which were increased by unloading in wild-type mice, were not increased by tail suspension in OPN−/− mice. Second, measures of osteoblastic bone formation, which were decreased in wild-type mice by unloading, were not altered in OPN−/− mice. These observations indicate that the presence of OPN is a prerequisite for the activation of osteoclastic bone resorption and for the reduction in osteoblastic bone formation in unloaded mice. Thus, OPN is a molecule required for the bone loss induced by mechanical stress that regulates the functions of osteoblasts and osteoclasts.

scholarly journals Site-Directed Mutagenesis Identifies a Molecular Switch Involved in Copper Sensing by the Histidine Kinase CinS in Pseudomonas putida KT2440

2009 ◽  
Vol 191 (16) ◽  
pp. 5304-5311 ◽  
Author(s):  
Davide Quaranta ◽  
Megan M. McEvoy ◽  
Christopher Rensing
Keyword(s):  
Pseudomonas Putida ◽  
Histidine Kinase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Copper Binding ◽  
Wild Type ◽  
Two Component System ◽  
Pseudomonas Putida Kt2440 ◽  
Component System ◽  
Two Component

ABSTRACT In the presence of copper, Pseudomonas putida activates transcription of cinAQ via the two-component system CinS-CinR. The CinS-CinR TCS was responsive to 0.5 μM copper and was specifically activated only by copper and silver. Modeling studies of CinS identified a potential copper binding site containing H37 and H147. CinS mutants with H37R and H147R mutations had an almost 10-fold reduced copper-dependent induction of cinAQ compared to the wild type.

scholarly journals Quantitative Kinetic Analyses of Shutting Off a Two-Component System

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Rong Gao ◽  
Ann M. Stock
Keyword(s):  
Phosphatase Activity ◽  
Response Regulator ◽  
Two Component System ◽  
Component System ◽  
Signaling Systems ◽  
Cellular Environment ◽  
Two Component ◽  
The Impact

ABSTRACT Cells rely on accurate control of signaling systems to adapt to environmental perturbations. System deactivation upon stimulus removal is as important as activation of signaling pathways. The two-component system (TCS) is one of the major bacterial signaling schemes. In many TCSs, phosphatase activity of the histidine kinase (HK) is believed to play an essential role in shutting off the pathway and resetting the system to the prestimulus state. Two basic challenges are to understand the dynamic behavior of system deactivation and to quantitatively evaluate the role of phosphatase activity under natural cellular conditions. Here we report a kinetic analysis of the response to shutting off the archetype Escherichia coli PhoR-PhoB TCS pathway using both transcription reporter assays and in vivo phosphorylation analyses. Upon removal of the stimulus, the pathway is shut off by rapid dephosphorylation of the PhoB response regulator (RR) while PhoB-regulated gene products gradually reset to prestimulus levels through growth dilution. We developed an approach combining experimentation and modeling to assess in vivo kinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an estimation of the PhoR phosphatase activity in vivo , which is much stronger than the phosphatase activity of PhoR cytoplasmic domains analyzed in vitro . We quantitatively modeled how strong the phosphatase activity needs to be to suppress nonspecific phosphorylation in TCSs and discovered that strong phosphatase activity of PhoR is required for cross-phosphorylation suppression. IMPORTANCE Activation of TCSs has been extensively studied; however, the kinetics of shutting off TCS pathways is not well characterized. We present comprehensive analyses of the shutoff response for the PhoR-PhoB system that reveal the impact of phosphatase activity on shutoff kinetics. This allows development of a quantitative framework not only to characterize the phosphatase activity in the natural cellular environment but also to understand the requirement for specific strengths of phosphatase activity to suppress nonspecific phosphorylation. Our model suggests that the ratio of the phosphatase rate to the nonspecific phosphorylation rate correlates with TCS expression levels and the ratio of the RR to HK, which may contribute to the great diversity of enzyme levels and activities observed in different TCSs.

scholarly journals Regulation of Virulence by a Two-Component System in Group B Streptococcus

2005 ◽  
Vol 187 (3) ◽  
pp. 1105-1113 ◽  
Author(s):  
Sheng-Mei Jiang ◽  
Michael J. Cieslewicz ◽  
Dennis L. Kasper ◽  
Michael R. Wessels
Keyword(s):  
Response Regulator ◽  
Newborn Infants ◽  
Regulatory System ◽  
Virulence Determinants ◽  
Wild Type ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
Mutant Strains ◽  
Group B

ABSTRACT Group B Streptococcus (GBS) is frequently carried in the gastrointestinal or genitourinary tract as a commensal organism, yet it has the potential to cause life-threatening infection in newborn infants, pregnant women, and individuals with chronic illness. Regulation of virulence factor expression may affect whether GBS behaves as an asymptomatic colonizer or an invasive pathogen, but little is known about how such factors are controlled in GBS. We now report the characterization of a GBS locus that encodes a two-component regulatory system similar to CsrRS (or CovRS) in Streptococcus pyogenes. Inactivation of csrR, encoding the putative response regulator, in two unrelated wild-type strains of GBS resulted in a marked increase in production of beta-hemolysin/cytolysin and a striking decrease in production of CAMP factor, an unrelated cytolytic toxin. Quantitative RNA hybridization experiments revealed that these two phenotypes were associated with a marked increase and decrease in expression of the corresponding genes, cylE and cfb, respectively. The CsrR mutant strains also displayed increased expression of scpB encoding C5a peptidase. Similar, but less marked, changes in gene expression were observed in CsrS (putative sensor component) mutants, evidence that CsrR and CsrS constitute a functional two-component system. Experimental infection studies in mice demonstrated reduced virulence of both CsrR and CsrS mutant strains relative to the wild type. Together, these results indicate that CsrRS regulates expression of multiple GBS virulence determinants and is likely to play an important role in GBS pathogenesis.

scholarly journals Bone Resorption Caused by Three Periodontal Pathogens In Vivo in Mice Is Mediated in Part by Prostaglandin

1998 ◽  
Vol 66 (9) ◽  
pp. 4158-4162 ◽  
Author(s):  
Yuval Zubery ◽  
Colin R. Dunstan ◽  
Beryl M. Story ◽  
Lakshmyya Kesavalu ◽  
Jeffrey L. Ebersole ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Alveolar Bone ◽  
Tissue Injury ◽  
Bone Destruction ◽  
Fusobacterium Nucleatum ◽  
Host Responses ◽  
Periodontal Pathogens ◽  
Tissue Destruction

ABSTRACT Gingival inflammation, bacterial infection, alveolar bone destruction, and subsequent tooth loss are characteristic features of periodontal disease, but the precise mechanisms of bone loss are poorly understood. Most animal models of the disease require injury to gingival tissues or teeth, and the effects of microorganisms are thus complicated by host responses to tissue destruction. To determine whether three putative periodontal pathogens, Porphyromonas gingivalis, Campylobacter rectus, andFusobacterium nucleatum, could cause localized bone resorption in vivo in the absence of tissue injury, we injected live or heat-killed preparations of these microorganisms into the subcutaneous tissues overlying the calvaria of normal mice once daily for 6 days and then examined the bones histologically. We found that all three microorganisms (both live and heat killed) stimulated bone resorption and that the strain of F. nucleatum used appeared to be the strongest inducer of osteoclast activity. Treatment of the mice concomitantly with indomethacin reduced but did not completely inhibit bone resorption by these microorganisms, suggesting that their effects were mediated, in part, by arachidonic acid metabolites (e.g., prostaglandins). Our findings indicate that these potential pathogens can stimulate bone resorption locally when placed beside a bone surface in vivo in the absence of prior tissue injury and support a role for them in the pathogenesis of bone loss around teeth in periodontitis.

scholarly journals The NarX-NarL two-component system is a global regulator of biofilm formation, natural product biosynthesis, and host-associated survival in Burkholderia pseudomallei

2020 ◽  
Author(s):  
Mihnea R. Mangalea ◽  
Bradley R. Borlee
Keyword(s):  
Gene Expression ◽  
Surface Waters ◽  
Biofilm Formation ◽  
Burkholderia Pseudomallei ◽  
Nitrosative Stress ◽  
Two Component System ◽  
Component System ◽  
Sensing System ◽  
Two Component ◽  
Nitrate And Nitrite

AbstractIn the environment, Burkholderia pseudomallei exists as a saprophyte inhabiting soils and surface waters where denitrification is important for anaerobic respiration. As an opportunistic pathogen, B. pseudomallei transitions from the environment to infect human and animal hosts where respiratory nitrate reduction enables replication in anoxic conditions. We have previously shown that B. pseudomallei responds to nitrate and nitrite in part by inhibiting biofilm formation and altering cyclic di-GMP signaling. Here, we describe the global transcriptomic response to nitrate and nitrite to characterize the nitrosative stress response relative to biofilm inhibition. To better understand the roles of nitrate-sensing in the biofilm inhibitory phenotype of B. pseudomallei, we created in-frame deletions of narX (Bp1026b_I1014) and narL (Bp1026b_I1013), which are adjacent components of the conserved nitrate-sensing two-component system. Through differential expression analysis of RNA-seq data, we observed that key components of the biofilm matrix are downregulated in response to nitrate and nitrite. In addition, several gene loci associated with the stringent response, central metabolism dysregulation, antibiotic tolerance, and pathogenicity determinants were significantly altered in their expression. Some of the most differentially expressed genes were nonribosomal peptide synthases (NRPS) and/or polyketide synthases (PKS) encoding the proteins for the biosynthesis of bactobolin, malleilactone, and syrbactin, in addition to an uncharacterized cryptic NRPS biosynthetic cluster. We also observed reduced expression of ribosomal structural and biogenesis loci, and gene clusters associated with translation and DNA replication, indicating modulation of growth rate and metabolism under nitrosative stress conditions. The differences in expression observed under nitrosative stress were reversed in narX and narL mutants, suggesting that nitrate sensing is an important checkpoint for regulating the diverse metabolic changes occurring in the biofilm inhibitory phenotype. Moreover, in a macrophage model of infection, narX and narL mutants were attenuated in intracellular replication, suggesting that nitrate sensing is important for host survival.Author SummaryBurkholderia pseudomallei is a saprophytic bacterium inhabiting soils and surface waters throughout the tropics causing severe disease in humans and animals. Environmental signals such as the accumulation of inorganic ions mediates the biofilm forming capabilities and survival of B. pseudomallei. In particular, nitrate metabolism inhibits B. pseudomallei biofilm formation through complex regulatory cascades that relay environmental cues to intracellular second messengers that modulate bacterial physiology. Nitrates are common environmental contaminants derived from artificial fertilizers and byproducts of animal wastes that can be readily reduced by bacteria capable of denitrification. In B. pseudomallei 1026b, biofilm dynamics are in part regulated by a gene pathway involved in nitrate sensing, metabolism, and transport. This study investigated the role of a two-component nitrate sensing system, NarX-NarL, in regulating gene expression, biofilm formation, and cellular invasion. Global gene expression analyses in the wild type, as compared to Δ narX and Δ narL mutant strains with nitrate or nitrite implicate the NarX-NarL system in the regulation of biofilm components as well as B. pseudomallei host-associated survival. This study characterizes a conserved nitrate sensing system that is important in environmental and host-associated contexts and aims to bridge a gap between these two important B. pseudomallei lifestyles.

scholarly journals The NarX-NarL two-component system regulates biofilm formation, natural product biosynthesis, and host-associated survival in Burkholderia pseudomallei

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Mihnea R. Mangalea ◽  
Bradley R. Borlee
Keyword(s):  
Biofilm Formation ◽  
Burkholderia Pseudomallei ◽  
Severe Disease ◽  
Expression Patterns ◽  
Inorganic Ions ◽  
Two Component System ◽  
Component System ◽  
Environmental Signals ◽  
Two Component ◽  
Nitrate And Nitrite

AbstractBurkholderia pseudomallei is a saprophytic bacterium endemic throughout the tropics causing severe disease in humans and animals. Environmental signals such as the accumulation of inorganic ions mediates the biofilm forming capabilities and survival of B. pseudomallei. We have previously shown that B. pseudomallei responds to nitrate and nitrite by inhibiting biofilm formation and altering cyclic di-GMP signaling. To better understand the roles of nitrate-sensing in the biofilm inhibitory phenotype of B. pseudomallei, we created in-frame deletions of narX (Bp1026b_I1014) and narL (Bp1026b_I1013), which are adjacent components of a conserved nitrate-sensing two-component system. We observed transcriptional downregulation in key components of the biofilm matrix in response to nitrate and nitrite. Some of the most differentially expressed genes were nonribosomal peptide synthases (NRPS) and/or polyketide synthases (PKS) encoding the proteins for the biosynthesis of bactobolin, malleilactone, and syrbactin, and an uncharacterized cryptic NRPS biosynthetic cluster. RNA expression patterns were reversed in ∆narX and ∆narL mutants, suggesting that nitrate sensing is an important checkpoint for regulating the diverse metabolic changes occurring in the biofilm inhibitory phenotype. Moreover, in a macrophage model of infection, ∆narX and ∆narL mutants were attenuated in intracellular replication, suggesting that nitrate sensing contributes to survival in the host.

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