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Quorum-sensing systems LuxS/autoinducer 2 and Com regulate Streptococcus pneumoniae biofilms in a bioreactor with living cultures of human respiratory cells.

ABSTRACT: Streptococcus pneumoniae forms organized biofilms in the human upper respiratory tract that may play an essential role in both persistence and acute respiratory infection. However, the production and regulation of biofilms on human cells is not yet fully understood. In this work, we developed a bioreactor with living cultures of human respiratory epithelial cells (HREC) and a continuous flow of nutrients, mimicking the microenvironment of the human respiratory epithelium, to study the production and regulation of S. pneumoniae biofilms (SPB). SPB were also produced under static conditions on immobilized HREC. Our experiments demonstrated that the biomass of SPB increased significantly when grown on HREC compared to the amount on abiotic surfaces. Additionally, pneumococcal strains produced more early biofilms on lung cells than on pharyngeal cells. Utilizing the bioreactor or immobilized human cells, the production of early SPB was found to be regulated by two quorum-sensing systems, Com and LuxS/AI-2, since a mutation in either comC or luxS rendered the pneumococcus unable to produce early biofilms on HREC. Interestingly, while LuxS/autoinducer 2 (AI-2) regulated biofilms on both HREC and abiotic surfaces, Com control was specific for those structures produced on HREC. The biofilm phenotypes of strain D39-derivative ?comC and ?luxS QS mutants were reversed by genetic complementation. Of note, SPB formed on immobilized HREC and incubated under static conditions were completely lysed 24 h postinoculation. Biofilm lysis was also regulated by the Com and LuxS/AI-2 quorum-sensing systems.

SUBMITTER: Vidal JE

PROVIDER: S-EPMC3639605 | biostudies-other | 2013 Apr

REPOSITORIES: biostudies-other

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Quorum-sensing systems LuxS/autoinducer 2 and Com regulate Streptococcus pneumoniae biofilms in a bioreactor with living cultures of human respiratory cells.

Vidal Jorge E JE Howery Kristen E KE Ludewick Herbert P HP Nava Porfirio P Klugman Keith P KP

Infection and immunity 20130212 4

Streptococcus pneumoniae forms organized biofilms in the human upper respiratory tract that may play an essential role in both persistence and acute respiratory infection. However, the production and regulation of biofilms on human cells is not yet fully understood. In this work, we developed a bioreactor with living cultures of human respiratory epithelial cells (HREC) and a continuous flow of nutrients, mimicking the microenvironment of the human respiratory epithelium, to study the production ...[more]

PMID: 23403556

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Project description:UnlabelledThe use of pneumococcal capsular polysaccharide (PPS)-based vaccines has resulted in a substantial reduction in invasive pneumococcal disease. However, much remains to be learned about vaccine-mediated immunity, as seven-valent PPS-protein conjugate vaccine use in children has been associated with nonvaccine serotype replacement and 23-valent vaccine use in adults has not prevented pneumococcal pneumonia. In this report, we demonstrate that certain PPS-specific monoclonal antibodies (MAbs) enhance the transformation frequency of two different Streptococcus pneumoniae serotypes. This phenomenon was mediated by PPS-specific MAbs that agglutinate but do not promote opsonic effector cell killing of the homologous serotype in vitro. Compared to the autoinducer, competence-stimulating peptide (CSP) alone, transcriptional profiling of pneumococcal gene expression after incubation with CSP and one such MAb to the PPS of serotype 3 revealed changes in the expression of competence (com)-related and bacteriocin-like peptide (blp) genes involved in pneumococcal quorum sensing. This MAb was also found to induce a nearly 2-fold increase in CSP2-mediated bacterial killing or fratricide. These observations reveal a novel, direct effect of PPS-binding MAbs on pneumococcal biology that has important implications for antibody immunity to pneumococcus in the pneumococcal vaccine era. Taken together, our data suggest heretofore unsuspected mechanisms by which PPS-specific antibodies could affect genetic exchange and bacterial viability in the absence of host cells.ImportanceCurrent thought holds that pneumococcal capsular polysaccharide (PPS)-binding antibodies protect against pneumococcus by inducing effector cell opsonic killing of the homologous serotype. While such antibodies are an important part of how pneumococcal vaccines protect against pneumococcal disease, PPS-specific antibodies that do not exhibit this activity but are highly protective against pneumococcus in mice have been identified. This article examines the effect of nonopsonic PPS-specific monoclonal antibodies (MAbs) on the biology of Streptococcus pneumoniae. The results showed that in the presence of a competence-stimulating peptide (CSP), such MAbs increase the frequency of pneumococcal transformation. Further studies with one such MAb showed that it altered the expression of genes involved in quorum sensing and increased competence-induced killing or fratricide. These findings reveal a novel, previously unsuspected mechanism by which certain PPS-specific antibodies exert a direct effect on pneumococcal biology that has broad implications for bacterial clearance, genetic exchange, and antibody immunity to pneumococcus.

Project description:Streptococcus pneumoniae 5'-methylthioadenosine/S-adenosylhomocysteine hydrolase (MTAN) catalyzes the hydrolytic deadenylation of its substrates to form adenine and 5-methylthioribose or S-ribosylhomocysteine (SRH). MTAN is not found in mammals but is involved in bacterial quorum sensing. MTAN gene disruption affects the growth and pathogenicity of bacteria, making it a target for antibiotic design. Kinetic isotope effects and computational studies have established a dissociative S(N)1 transition state for Escherichia coli MTAN, and transition state analogues resembling the transition state are powerful inhibitors of the enzyme [Singh, V., Lee, J. L., Núñez, S., Howell, P. L., and Schramm, V. L. (2005) Biochemistry 44, 11647-11659]. The sequence of MTAN from S. pneumoniae is 40% identical to that of E. coli MTAN, but S. pneumoniae MTAN exhibits remarkably distinct kinetic and inhibitory properties. 5'-Methylthio-Immucillin-A (MT-ImmA) is a transition state analogue resembling an early S(N)1 transition state. It is a weak inhibitor of S. pneumoniae MTAN with a K(i) of 1.0 microM. The X-ray structure of S. pneumoniae MTAN with MT-ImmA indicates a dimer with the methylthio group in a flexible hydrophobic pocket. Replacing the methyl group with phenyl (PhT-ImmA), tolyl (p-TolT-ImmA), or ethyl (EtT-ImmA) groups increases the affinity to give K(i) values of 335, 60, and 40 nM, respectively. DADMe-Immucillins are geometric and electrostatic mimics of a fully dissociated transition state and bind more tightly than Immucillins. MT-DADMe-Immucillin-A inhibits with a K(i) value of 24 nM, and replacing the 5'-methyl group with p-Cl-phenyl (p-Cl-PhT-DADMe-ImmA) gave a K(i) value of 0.36 nM. The inhibitory potential of DADMe-Immucillins relative to the Immucillins supports a fully dissociated transition state structure for S. pneumoniae MTAN. Comparison of active site contacts in the X-ray crystal structures of E. coli and S. pneumoniae MTAN with MT-ImmA would predict equal binding, yet most analogues bind 10(3)-10(4)-fold more tightly to the E. coli enzyme. Catalytic site efficiency is primarily responsible for this difference since k(cat)/K(m) for S. pneumoniae MTAN is decreased 845-fold relative to that of E. coli MTAN.

Project description:BACKGROUND:Quorum sensing is a mechanism of cell to cell communication that requires the production and detection of signaling molecules called autoinducers. Although mesophilic bacteria is known to utilize this for synchronization of physiological processes such as bioluminescence, virulence, biofilm formation, motility and cell competency through signaling molecules (acyl homoserine lactones, AI-1; oligopeptides, peptide based system and furanosyl borate diester, AI-2), the phenomenon of quorum sensing in thermophiles is largely unknown. RESULTS:In this study, proteomes of 106 thermophilic eubacteria and 21 thermophilic archaea have been investigated for the above three major quorum sensing systems to find the existence of quorum sensing in these thermophiles as there are evidences for the formation of biofilms in hot environments. Our investigation demonstrated that AI-1 system is absent in thermophiles. Further, complete peptide based two component systems for quorum sensing was also not found in any thermophile however the traces for the presence of response regulators for peptide based system were found in some of them. BLASTp search using LuxS (AI-2 synthase) protein sequence of Escherichia coli str. K-12 substr. MG1655 and autoinducer-2 receptors (LuxP of Vibrio harveyi, LsrB of E. coli str. K-12 substr. MG1655 and RbsB of Aggregatibacter actinomycetemcomitans) as queries revealed that 17 thermophilic bacteria from phyla Deinococcus- Thermus and Firmicutes possess complete AI-2 system (LuxS and LsrB and/or RbsB). Out of 106 thermophilic eubacteria 18 from phyla Deinococcus- Thermus, Proteobacteria and Firmicutes have only LuxS that might function as AI-2 synthesizing protein whereas, 16 are having only LsrB and/or RbsB which may function as AI-2 receptor in biofilms. CONCLUSIONS:We anticipate that thermophilic bacteria may use elements of LsrB and RbsB operon for AI-2 signal transduction and they may use quorum sensing for purposes like biofilm formation. Nevertheless, thermophiles in which no known quorum sensing system was found may use some unknown mechanisms as the mode of communication. Further information regarding quorum sensing will be explored to develop strategies to disrupt the biofilms of thermophiles.

Dataset Information

Quorum-sensing systems LuxS/autoinducer 2 and Com regulate Streptococcus pneumoniae biofilms in a bioreactor with living cultures of human respiratory cells.

Publications

Quorum-sensing systems LuxS/autoinducer 2 and Com regulate Streptococcus pneumoniae biofilms in a bioreactor with living cultures of human respiratory cells.

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