Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration

Hansen, Michael R.; Palmer, Robert; Udsen, Camilla; White, David C.; Molin, Søren

doi:10.1099/00221287-147-5-1383

Cited by 184 publications

(158 citation statements)

References 36 publications

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“…LB medium was supplemented with 200-g/ml erythromycin to maintain pCM18 (20) (Table 1), which contains the constitutive green fluorescent protein (GFP) vector and which allows visualization of the biofilm. The biofilm was formed at 37°C in a continuous flow cell that consists of a standard glass microscope slide on one side and a plastic coverslip on the other side with dimensions of 47.5 mm by 12.7 mm with a 1.6-mm gap between the surfaces (BST model FC81; Biosurface Technologies Corp., Bozeman, MT).…”

Section: Methodsmentioning

confidence: 99%

Autoinducer 2 Controls Biofilm Formation in Escherichia coli through a Novel Motility Quorum-Sensing Regulator (MqsR, B3022)

et al. 2006

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The cross-species bacterial communication signal autoinducer 2 (AI-2), produced by the purified enzymes Pfs (nucleosidase) and LuxS (terminal synthase) from S-adenosylhomocysteine, directly increased Escherichia coli biofilm mass 30-fold. Continuous-flow cells coupled with confocal microscopy corroborated these results by showing the addition of AI-2 significantly increased both biofilm mass and thickness and reduced the interstitial space between microcolonies. As expected, the addition of AI-2 to cells which lack the ability to transport AI-2 (lsr null mutant) failed to stimulate biofilm formation. Since the addition of AI-2 increased cell motility through enhanced transcription of five motility genes, we propose that AI-2 stimulates biofilm formation and alters its architecture by stimulating flagellar motion and motility. It was also found that the uncharacterized protein B3022 regulates this AI-2-mediated motility and biofilm phenotype through the two-component motility regulatory system QseBC. Deletion of b3022 abolished motility, which was restored by expressing b3022 in trans. Deletion of b3022 also decreased biofilm formation significantly, relative to the wild-type strain in three media (46 to 74%) in 96-well plates, as well as decreased biomass (8-fold) and substratum coverage (19-fold) in continuous-flow cells with minimal medium (growth rate not altered and biofilm restored by expressing b3022 in trans). Deleting b3022 changed the wild-type biofilm architecture from a thick (54-m) complex structure to one that contained only a few microcolonies. B3022 positively regulates expression of qseBC, flhD, fliA, and motA, since deleting b3022 decreased their transcription by 61-, 25-, 2.4-, and 18-fold, respectively. Transcriptome analysis also revealed that B3022 induces crl (26-fold) and flhCD (8-to 27-fold). Adding AI-2 (6.4 M) increased biofilm formation of wild-type K-12 MG1655 but not that of the isogenic b3022, qseBC, fliA, and motA mutants. Adding AI-2 also increased motA transcription for the wild-type strain but did not stimulate motA transcription for the b3022 and qseB mutants. Together, these results indicate AI-2 induces biofilm formation in E. coli through B3022, which then regulates QseBC and motility; hence, b3022 has been renamed the motility quorum-sensing regulator gene (the mqsR gene).There is an explosive amount of research on biofilms with the ultimate aim of their control (24); however, little is known about the regulation of this complex process of cell attachment leading to exquisite architecture (11). Since 65% of human bacterial infections involve biofilms (31), understanding the genetic basis of biofilm formation to find effective ways to prevent biofilms is important for combating disease and for engineering applications. To this end, we have studied the whole bacterial genome with DNA microarrays by two complementary approaches: studying biofilm gene expression relative to planktonic cells (34, 35) and studying plant-derived biofilm inhibitors that do not alter the bacterial g...

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Section: Methodsmentioning

confidence: 99%

Autoinducer 2 Controls Biofilm Formation in Escherichia coli through a Novel Motility Quorum-Sensing Regulator (MqsR, B3022)

et al. 2006

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“…5A). The fluorescence of endocytosed GFP-yeast (upper right quadrant) was lower than that of free yeast (upper left quadrant), possibly due to the acidic environment in the endosomes [40]. To discriminate between extracellular binding and phagocytosis, the tests were performed at 4 C and 37 C: 22.3 AE 1.6% of total splenic CD11c high DC showed GFP fluorescence at 37 C, as compared to 9.0 AE 2.4% at 4 C. Adding anti-dectin-1 antibodies inhibited phagocytosis of GFP-yeast by CD11c + DC cells to background levels (13.3 AE 1.5%) (Fig.…”

Section: Phagocytosis Of Gfp-yeast By Both DC Subsets Is Mediated By mentioning

confidence: 96%

CD8^– dendritic cells preferentially cross‐present Saccharomyces cerevisiae antigens

et al. 2008

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Mouse splenic dendritic cell (DC) subsets possess distinct antigen-presentation abilities. CD8 + DC are specialized in cross-presentation of antigens to CD8 + T cells, whereas CD8 -DC are more efficient in antigen presentation to CD4 + T cells. In this study, we examined the capacity of CD8 + and CD8 -DC subsets to present fungal antigens in MHC class I and II molecules to CD8 + and CD4 + T cells, respectively. We used ovalbuminexpressing Saccharomyces cerevisiae (yeast-OVA) as a fungal model system. Both CD8 + and CD8 -DC subsets phagocytosed yeast in equal amounts and uptake was mediated via dectin-1. In addition, both DC subsets induced similar OVA-specific CD4 + T cell proliferation after incubation with yeast-OVA. However, the induction of OVA-specific CD8 + T cell activation was largely restricted to the CD8 -DC subset. Furthermore, only CD8 -DC produced cytokines such as IL-10 and TNF-a and increased IL-23p19 and IL-23p40 mRNA levels in response to yeast. Our results strongly suggest that DC subsets have different functions in the elicitation of adaptive immune responses in vivo. IntroductionFungal infections with pathogenic yeasts such as Candida albicans, Aspergillus fumigatus, Histoplasma capsulatum and Cryptococcus neoformans are a major problem in immunocompromised persons. These opportunistic pathogens usually do not pose problems in healthy individuals, but frequently become invasive in patients suffering from HIV, patients with malignancies or transplant recipients, resulting in life-threatening diseases [1][2][3].Both innate and adaptive immune responses are essential to induce protection against fungi. Dendritic cells (DC) play a central role in the adaptive immune response by capturing fungi and presenting their antigens in major histocompatibility complex (MHC) class I and MHC class II molecules to T cells [4, 5]. DC express a panel of microbial recognition receptors such as Toll-like receptors (TLR) and C-type lectin receptors that are essential for the elicitation of adaptive immune responses [6]. Of these receptors, TLR2 and the DCassociated C-type lectin (dectin-1) are specifically involved in recognition of fungal components [7][8][9]. Zymosan and fungal pathogens like C. albicans and A. fumigatus are sensed by TLR2 that signals via myeloid differentiation factor 88 (MyD88), resulting in NF-jB activation and the production of cytokines [10][11][12][13]. The importance of this signaling cascade can be inferred from the fact that MyD88-knockout mice show an increased susceptibility to fungal infections [14,15].Dectin-1 is expressed on myeloid cells like macrophages, neutrophils, monocytes, and even a subset of T cells [16]. Dectin-1 mediates fungal recognition and Revised 2/11/07 Accepted 11/12/07 [DOI 10.1002/eji.200737647] Key words: Antigen presentation Á Dendritic cell Á Fungal Á T cell Abbreviations: b2m: b2-microglobulin Á Dectin-1: DC-associated C-type lectin-1 Á CR3: complement receptor 3 Á MR: mannose receptor Á SIGNR1: ICAM-3-grabbing nonintegrin (DC-SIGN) homolog, SIGN-related...

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“…Probes that are fluorescent permit imaging by confocal laser microscopy and can be used to examine spatiotemporal relationships. Several types have been used with success, including green fluorescent protein (5,60), fluorescently labeled antibodies to surface antigens (5,75,120), and fluorescently labeled 16S rRNA-targeted probes (110).…”

Section: In Vitromentioning

confidence: 99%

Communication among Oral Bacteria

Kolenbrander

Andersen

Blehert

et al. 2002

Microbiol Mol Biol Rev

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853

745

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Human oral bacteria interact with their environment by attaching to surfaces and establishing mixed-species communities. As each bacterial cell attaches, it forms a new surface to which other cells can adhere. Adherence and community development are spatiotemporal; such order requires communication. The discovery of soluble signals, such as autoinducer-2, that may be exchanged within multispecies communities to convey information between organisms has emerged as a new research direction. Direct-contact signals, such as adhesins and receptors, that elicit changes in gene expression after cell-cell contact and biofilm growth are also an active research area. Considering that the majority of oral bacteria are organized in dense three-dimensional biofilms on teeth, confocal microscopy and fluorescently labeled probes provide valuable approaches for investigating the architecture of these organized communities in situ. Oral biofilms are readily accessible to microbiologists and are excellent model systems for studies of microbial communication. One attractive model system is a saliva-coated flowcell with oral bacterial biofilms growing on saliva as the sole nutrient source; an intergeneric mutualism is discussed. Several oral bacterial species are amenable to genetic manipulation for molecular characterization of communication both among bacteria and between bacteria and the host. A successful search for genes critical for mixed-species community organization will be accomplished only when it is conducted with mixed-species communities

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Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration

Cited by 184 publications

References 36 publications

Autoinducer 2 Controls Biofilm Formation in Escherichia coli through a Novel Motility Quorum-Sensing Regulator (MqsR, B3022)

Autoinducer 2 Controls Biofilm Formation in Escherichia coli through a Novel Motility Quorum-Sensing Regulator (MqsR, B3022)

CD8^– dendritic cells preferentially cross‐present Saccharomyces cerevisiae antigens

Communication among Oral Bacteria

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Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration

Cited by 184 publications

References 36 publications

Autoinducer 2 Controls Biofilm Formation in Escherichia coli through a Novel Motility Quorum-Sensing Regulator (MqsR, B3022)

Autoinducer 2 Controls Biofilm Formation in Escherichia coli through a Novel Motility Quorum-Sensing Regulator (MqsR, B3022)

CD8– dendritic cells preferentially cross‐present Saccharomyces cerevisiae antigens

Communication among Oral Bacteria

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Product

Resources

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CD8^– dendritic cells preferentially cross‐present Saccharomyces cerevisiae antigens