Project description:The formation of electroactive biofilms is a crucial process for the generation of bioelectricity and bioremediation. G. sulfurreducens is a dissimilatory metal-reducing microorganism that can couple oxidation of organic matter with extracellular electron transfer to different insoluble electron acceptors. It has the capability to form biofilms in insoluble metal oxides and electroconductive biofilms in electrodes in bioelectrochemical systems. The formation of electroactive biofilms in this microorganism is a process that has been studied from a physiological, genetic, physical, and electrochemical approach. In G. sulfurreducens, we found that the transcriptional regulator GSU1771 participates in the gene expression of essential genes involved in electron transfer and biofilm formation. Strains deficient in GSU1771 increases Fe(III) reduction, produces more c-type cytochromes and exopolysaccharides. Furthermore, the biofilms produced are thicker and more electroactive than wild-type. In this work, we investigate the global gene expression profile performing RNA-seq comparing Δgsu1771 mutant biofilm grown in non-conductive support (glass) and respiring-graphite electrode. RNA-seq analysis of Δgsu1771 biofilm grown in glass support revealed a total of 467 (167 upregulated and 300 downregulated) differentially expressed genes versus the wild-type biofilm. Meanwhile, in Δgsu1771 biofilm developed in respiring-electrode graphite, we detect 119 (79 upregulated and 40 downregulated) differentially expressed genes with respect to wild-type biofilm. Moreover, transcriptional changes of 67 (56 with the same regulation and in 11 counterregulation) genes were shared in Δgsu1771 biofilm developed in glass and graphite electrodes. We locate upregulated in Δgsu1771 biofilms potential target genes, involved in exopolysaccharide synthesis (gsu1961-63, gsu1959, gsu1972-73, gsu1976-77). We confirmed the upregulation of gsu1979, gsu0972, gsu0783, pgcA, omcM, aroG, panC gnfK, gsu2507, and the downregulation of asnA, ato-1, gsu0810, pilA, csrA, ppcD, and gsu3356 genes by RT-qPCR. DNA-protein binding assay shows direct binding of the GSU1771 regulator to the promoter region of pgcA, pulF, relA, and gsu3356. Also, heme-staining and western blotting revealed an increase of c-type cytochromes in Δgsu1771 biofilms such as OmcS and OmcZ. In general, our data shows that GSU1771 is a global regulator involved in controlling the extracellular electron transfer and exopolysaccharide synthesis, processes required for electroconductive biofilm development.

Project description:For Streptococcus pneumoniae, biofilms have been suggested to promote long-term colonization of the nasopharynx and contribute to the pathology of recurrent middle ear infections. To date numerous studies have investigated the contribution of specific genetic determinants for the development of pneumococcal biofilms, however, studies examining the global changes that occur during biofilm development and how they contribute to disease are lacking. Using Scanning and Transmission electron microscopy we examined development of a mature pneumococcal biofilm in a continuous flow through reactor. We determined that a mature biofilm is formed in discrete stages, is marked by the formation of complex 3-dimensional structures, and is primarily composed of dead pneumococci. Using genomic microarrays we determined that pneumococci in mature biofilms down regulate genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide production, and virulence. We confirmed these changes by testing bacterial resistance to antimicrobials, measuring capsule production by ELSIA, and immunoblotting for pneumolysin production. We determined that biofilm pneumococci are hyper-adhesive, binding to cell lines at levels 9 to 11-fold greater than planktonic counterparts. Using Western blot and ELISA, we determined that biofilm bacteria produce greater amounts of the adhesins PsrP, CbpA, and surface exposed phosphorylcholine. We subsequently determined that the hyper-adhesive phenotype was in part due to selection of the transparent phase variant during biofilm growth. Intranasal, intratracheal and intraperitoneal challenge of mice with biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Immunization of mice with ethanol-killed biofilm pneumococci of serotype 4 conferred protection against challenge with same isolate but not a serotype 3. ELISA for reactive IgG levels subsequently determined that biofilm pneumococci do not provide high levels of cross-reactive protein antigens. Together these studies suggest that biofilms do not directly contribute to disease but instead confer a protected mode of growth for the pneumococcus.

Project description:Background: Air-pollutants containing toxic particulate matters (PM) deposit in the respiratory tract and increases microbial infections. However, the mechanism underline is not well understood. In this study, we evaluated the effect of urban particles (UP) on S. pneumoniae in-vitro biofilm formation, colonization on Human middle ear epithelium cells (HMEECs) and in mouse nasal cavity and transition to middle ear and lungs. Methods: S. pneumoniae in vitro biofilms and planktonic growth was evaluated in metal ion free medium in presence of UP, and biofilms were quantified by CV-microplate assay, cfu counts and resazurin staining. Biofilm structures were analyzed using scanning electron microscope (SEM) and confocal microscopy (CM). Gene expressions of biofilms were evaluated using real time RT-PCR. Effects of UP exposure on S. pneumoniae colonization to HMEECs was evaluated using fluorescent in-situ hybridization (FISH), cell viability was detected by EZcyto kit, apoptosis in HMEECs were evaluated using Annexin-V/PI based cytometry analysis and reactive oxygen species (ROS) production were evaluated using Oxiselect kit. Alteration of HMEECs gene expressions on UP exposure or pneumococci colonization were evaluated using microarray. In vivo colonization of pneumococci in presence of UP and transition to middle ear and lungs were evaluated using intranasal mice colonization model. Results: UP exposure significantly (*p< 0.05) increased pneumococcal in vitro biofilms and planktonic growth. In presence of UP pneumococci formed organized biofilms with matrix, while in absence of UP bacteria was unable to form biofilms. The luxS, ply, lytA, comA, comB and ciaR genes involved in bacterial pathogenesis, biofilms formation and quorum sensing were up-regulated in pneumococci biofilms grown in presence of UP. The HMEECs viability was significantly (p<0.05) decreased and bacteria colonization was significantly (p<0.05) elevated in co-treatment (UP+S. pneumoniae) in compare to single treatment. Similarly, increased apoptosis and ROS produce were detected in HMEECs treated with UP+ pneumococci. The microarray analysis of HMEECs revealed that the genes involve in apoptosis and cell death, inflammation, immune response were up-regulated in co-treatment, those genes were unchanged or expressed in less fold in single treatments of UP or S. pneumoniae. In vivo study showed increased pneumococcal colonization to nasal cavity in presence of UP and higher transition of bacteria to middle ear and lungs in presence of UP.

Project description:For Streptococcus pneumoniae, biofilms have been suggested to promote long-term colonization of the nasopharynx and contribute to the pathology of recurrent middle ear infections. To date numerous studies have investigated the contribution of specific genetic determinants for the development of pneumococcal biofilms, however, studies examining the global changes that occur during biofilm development and how they contribute to disease are lacking. Using Scanning and Transmission electron microscopy we examined development of a mature pneumococcal biofilm in a continuous flow through reactor. We determined that a mature biofilm is formed in discrete stages, is marked by the formation of complex 3-dimensional structures, and is primarily composed of dead pneumococci. Using genomic microarrays we determined that pneumococci in mature biofilms down regulate genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide production, and virulence. We confirmed these changes by testing bacterial resistance to antimicrobials, measuring capsule production by ELSIA, and immunoblotting for pneumolysin production. We determined that biofilm pneumococci are hyper-adhesive, binding to cell lines at levels 9 to 11-fold greater than planktonic counterparts. Using Western blot and ELISA, we determined that biofilm bacteria produce greater amounts of the adhesins PsrP, CbpA, and surface exposed phosphorylcholine. We subsequently determined that the hyper-adhesive phenotype was in part due to selection of the transparent phase variant during biofilm growth. Intranasal, intratracheal and intraperitoneal challenge of mice with biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Immunization of mice with ethanol-killed biofilm pneumococci of serotype 4 conferred protection against challenge with same isolate but not a serotype 3. ELISA for reactive IgG levels subsequently determined that biofilm pneumococci do not provide high levels of cross-reactive protein antigens. Together these studies suggest that biofilms do not directly contribute to disease but instead confer a protected mode of growth for the pneumococcus. Pneumococcal biofilms compared to planktonic control at 4, 12, 24, 48 hours. 3 biological replicates each of 4 and 12 hour time points, and 2 biological replicates each of 24 and 48 hour time points. Flip dye (technical replicates) performed for 4, 12, and 24 hour time points; no technical replicate performed for 48 hour time point due to limiting material. Ratios were determined by averaging across technical and biological replicates. The following hybridizations made up each biological replicate: 14090167.tav.annot and 14090190.tav.annot (4hr biol rep 1); 14090169.tav.annot and 14090176.tav.annot (4hr biol rep 2); 14090192.tav.annot and 14090188.tav.annot (4hr biol rep 3); 14087688.tav.annot and 14090180.tav.annot (12hr biol rep 1); 14090185.tav.annot and 14090168.tav.annot (12hr biol rep 2); 14090191.tav.annot and 14090174.tav.annot (12hr biol rep 3); 14090170.tav.annot and 14090175.tav.annot (24hr biol rep 2); 14090193.tav.annot and 14087687.tav.annot (24hr biol rep 3); 14090181.tav.annot (48hr biol rep 1); 14090187.tav.annot (48hr biol rep 2)

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Pyrimidine-diones structure containing small molecule inhibits Streptococcus pneumoniae in-vitro biofilms growth

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