Secreted Proteases Control the Timing of Aggregative Community Formation in Vibrio cholerae

Bacteria orchestrate collective behaviors using the cell-cell communication process called quorum sensing (QS). QS relies on the synthesis, release, and group-wide detection of small molecules called autoinducers. In Vibrio cholerae, a multicellular community aggregation program occurs in liquid, during stationary phase, and in the high-cell-density QS state. Here, we demonstrate that this aggregation program consists of two subprograms. In one subprogram, which we call void formation, structures form that contain few cells but provide a scaffold within which cells can embed. The other subprogram relies on flagellar machinery and enables cells to enter voids. A genetic screen for factors contributing to void formation, coupled with companion molecular analyses, showed that four extracellular proteases, Vca0812, Vca0813, HapA, and PrtV control the onset timing of both void formation and aggregation, and moreover, proteolytic activity is required. These proteases, or their downstream products, can be shared between void-producing and non-void-forming cells and can elicit aggregation in a normally non-aggregating V. cholerae strain. Employing multiple proteases to control void formation and aggregation timing could provide a redundant and irreversible path to commitment to this community lifestyle.

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Secreted Proteases Control the Timing of Aggregative Community Formation in Vibrio cholerae

mBio ◽

10.1128/mbio.01518-21 ◽

2021 ◽

Author(s):

Matthew Jemielita ◽

Ameya A. Mashruwala ◽

Julie S. Valastyan ◽

Ned S. Wingreen ◽

Bonnie L. Bassler

Keyword(s):

Quorum Sensing ◽

Vibrio Cholerae ◽

Chemical Communication ◽

Communication Process ◽

Community Formation ◽

Aggregate Formation ◽

Content Type ◽

Secreted Proteases

Bacteria can work as collectives to form multicellular communities. Vibrio cholerae , the bacterium that causes the disease cholera in humans, forms aggregated communities in liquid. Aggregate formation relies on a chemical communication process called quorum sensing.

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The PqsE-RhlR Interaction Regulates RhlR DNA Binding to Control Virulence Factor Production in Pseudomonas aeruginosa

Microbiology Spectrum ◽

10.1128/spectrum.02108-21 ◽

2022 ◽

Author(s):

Kayla A. Simanek ◽

Isabelle R. Taylor ◽

Erica K. Richael ◽

Erica Lasek-Nesselquist ◽

Bonnie L. Bassler ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Dna Binding ◽

Quorum Sensing ◽

Virulence Factor ◽

Cell Communication ◽

Communication Process ◽

Collective Behaviors ◽

Factor Production ◽

A Cell ◽

Virulence Factor Production

Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of molecules called autoinducers (AI).

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Mechanism underlying autoinducer recognition in the Vibrio cholerae DPO-VqmA quorum-sensing pathway

10.1101/2019.12.19.881847 ◽

2019 ◽

Author(s):

Xiuliang Huang ◽

Olivia P. Duddy ◽

Justin E. Silpe ◽

Jon E. Paczkowski ◽

Jianping Cong ◽

...

Keyword(s):

Quorum Sensing ◽

Vibrio Cholerae ◽

Conformational Changes ◽

Site Directed Mutagenesis ◽

Communication Process ◽

Linear Molecule ◽

Gene Encoding ◽

Collective Behaviors ◽

Existing Structures ◽

Extracellular Signaling

ABSTRACTQuorum sensing is a bacterial communication process whereby bacteria produce, release and detect the accumulation of extracellular signaling molecules called autoinducers to coordinate collective behaviors. In Vibrio cholerae, the quorum-sensing autoinducer, DPO (3,5-dimethyl-pyrazin-2-ol), binds the receptor-transcription factor, VqmA. In response, the DPO-VqmA complex activates transcription of the vqmR gene encoding the VqmR small RNA. VqmR represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA has DPO-dependent and DPO-independent activity. We solved the DPO-VqmA crystal structure and compared it to existing structures to understand the conformational changes the protein undergoes upon DNA binding. Analysis of DPO analogs reveals that a hydroxyl or carbonyl group at the 2’ position is critical for binding. The proposed DPO precursor, a linear molecule, Ala-AA (N-alanyl-aminoacetone), also binds and activates VqmA. DPO and Ala-AA occupy the same binding site as judged by site-directed mutagenesis and competitive ligand binding analyses.

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Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1500203112 ◽

2015 ◽

Vol 112 (7) ◽

pp. E766-E775 ◽

Cited By ~ 119

Author(s):

Kai Papenfort ◽

Konrad U. Förstner ◽

Jian-Ping Cong ◽

Cynthia M. Sharma ◽

Bonnie L. Bassler

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Posttranscriptional Regulation ◽

Cell Communication ◽

Rna Seq ◽

Gene Encoding ◽

Collective Behaviors ◽

Mrna Targets ◽

Transcriptional Start Sites ◽

Microarray Analyses

Quorum sensing (QS) is a process of cell-to-cell communication that enables bacteria to transition between individual and collective lifestyles. QS controls virulence and biofilm formation in Vibrio cholerae, the causative agent of cholera disease. Differential RNA sequencing (RNA-seq) of wild-type V. cholerae and a locked low-cell-density QS-mutant strain identified 7,240 transcriptional start sites with ∼47% initiated in the antisense direction. A total of 107 of the transcripts do not appear to encode proteins, suggesting they specify regulatory RNAs. We focused on one such transcript that we name VqmR. vqmR is located upstream of the vqmA gene encoding a DNA-binding transcription factor. Mutagenesis and microarray analyses demonstrate that VqmA activates vqmR transcription, that vqmR encodes a regulatory RNA, and VqmR directly controls at least eight mRNA targets including the rtx (repeats in toxin) toxin genes and the vpsT transcriptional regulator of biofilm production. We show that VqmR inhibits biofilm formation through repression of vpsT. Together, these data provide to our knowledege the first global annotation of the transcriptional start sites in V. cholerae and highlight the importance of posttranscriptional regulation for collective behaviors in this human pathogen.

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Mechanism underlying autoinducer recognition in the Vibrio cholerae DPO-VqmA quorum-sensing pathway

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012104 ◽

2020 ◽

Vol 295 (10) ◽

pp. 2916-2931 ◽

Cited By ~ 8

Author(s):

Xiuliang Huang ◽

Olivia P. Duddy ◽

Justin E. Silpe ◽

Jon E. Paczkowski ◽

Jianping Cong ◽

...

Keyword(s):

Dna Binding ◽

Quorum Sensing ◽

Vibrio Cholerae ◽

Conformational Changes ◽

Transcriptional Activity ◽

Function Analysis ◽

Site Directed Mutagenesis ◽

Communication Process ◽

Linear Molecule ◽

Collective Behaviors

Quorum sensing is a bacterial communication process whereby bacteria produce, release, and detect extracellular signaling molecules called autoinducers to coordinate collective behaviors. In the pathogen Vibrio cholerae, the quorum-sensing autoinducer 3,5-dimethyl-pyrazin-2-ol (DPO) binds the receptor and transcription factor VqmA. The DPO-VqmA complex activates transcription of vqmR, encoding the VqmR small RNA, which represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA is soluble and properly folded and activates basal-level transcription of its target vqmR in the absence of DPO. VqmA transcriptional activity is increased in response to increasing concentrations of DPO, allowing VqmA to drive the V. cholerae quorum-sensing transition at high cell densities. We solved the DPO-VqmA crystal structure to 2.0 Å resolution and compared it with existing structures to understand the conformational changes VqmA undergoes upon DNA binding. Analysis of DPO analogs showed that a hydroxyl or carbonyl group at the 2′-position is critical for binding to VqmA. The proposed DPO precursor, a linear molecule, N-alanyl-aminoacetone (Ala-AA), also bound and activated VqmA. Results from site-directed mutagenesis and competitive ligand-binding analyses revealed that DPO and Ala-AA occupy the same binding site. In summary, our structure-function analysis identifies key features required for VqmA activation and DNA binding and establishes that, whereas VqmA binds two different ligands, VqmA does not require a bound ligand for folding or basal transcriptional activity. However, bound ligand is required for maximal activity.

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The Vibrio cholerae quorum-sensing protein VqmA integrates cell density, environmental, and host-derived cues into the control of virulence

10.1101/2020.05.04.076810 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ameya A. Mashruwala ◽

Bonnie L. Bassler

Keyword(s):

Small Intestine ◽

Quorum Sensing ◽

Vibrio Cholerae ◽

Cell Density ◽

Chemical Communication ◽

Disulfide Bonds ◽

Redox Status ◽

Communication Process ◽

Signal Molecules ◽

Collective Behaviors

Scientific AbstractQuorum sensing is a chemical communication process in which bacteria use the production, release, and detection of signal molecules called autoinducers to orchestrate collective behaviors. The human pathogen Vibrio cholerae requires quorum sensing to infect the small intestine. There, V. cholerae encounters the absence of oxygen and the presence of bile. We show that these two stimuli differentially affect quorum sensing function and, in turn, V. cholerae pathogenicity. The quorum-sensing receptor-transcription factor called VqmA, that detects the autoinducer called DPO, also detects the lack of oxygen and the presence of bile. Detection occurs via DPO-, oxygen-, bile-, and redox-responsive disulfide bonds that alter VqmA DNA binding activity. We propose that VqmA serves as an information processing hub that integrates quorum- sensing information, redox status, the presence or absence of oxygen, and host cues. In response to the information acquired through this mechanism, V. cholerae appropriately modulates its virulence output.Lay AbstractQuorum sensing (QS) is a process of chemical communication bacteria use to orchestrate collective behaviors. QS communication relies on chemical signal molecules called autoinducers. QS regulates virulence in Vibrio cholerae, the causative agent of the disease cholera. Transit into the human small intestine, the site of cholera infection, exposes V. cholerae to the host environment. In this study, we show that the combination of two stimuli encountered in the small intestine, the absence of oxygen and the presence of host-produced bile, impinge on V. cholerae QS function and, in turn, pathogenicity. We suggest that possessing a QS system that is responsive to multiple environmental, host, and cell density cues enables V. cholerae to fine-tune its virulence capacity in the human intestine.

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Gap junctions in the prothoracic glands of the tobacco hornworm, Manduca sexta

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123933 ◽

1992 ◽

Vol 50 (1) ◽

pp. 706-707

Author(s):

Ji-da Dai ◽

M. Joseph Costello ◽

Lawrence I. Gilbert

Keyword(s):

Gap Junctions ◽

Small Molecules ◽

Manduca Sexta ◽

Freeze Fracture ◽

Cell Communication ◽

Cellular Level ◽

Thin Sections ◽

Prothoracic Glands ◽

Polyhydroxylated Steroids ◽

Stimulation Of

Insect molting and metamorphosis are elicited by a class of polyhydroxylated steroids, ecdysteroids, that originate in the prothoracic glands (PGs). Prothoracicotropic hormone stimulation of steroidogenesis by the PGs at the cellular level involves both calcium and cAMP. Cell-to-cell communication mediated by gap junctions may play a key role in regulating signal transduction by controlling the transmission of small molecules and ions between adjacent cells. This is the first report of gap junctions in the PGs, the evidence obtained by means of SEM, thin sections and freeze-fracture replicas.

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A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators

International Journal of Molecular Sciences ◽

10.3390/ijms22031417 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1417

Author(s):

Azeem Danish ◽

Robin Gedschold ◽

Sonja Hinz ◽

Anke C. Schiedel ◽

Dominik Thimm ◽

...

Keyword(s):

Gap Junctions ◽

Small Molecules ◽

High Throughput Screening ◽

Cell Communication ◽

Receptor Activation ◽

Adenosine A2a Receptor ◽

Intracellular Camp ◽

Donor Cells ◽

A2a Receptor ◽

Adenosine A2a

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

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