Biology subjects

Bridges, A. A.

Publications and source records attributed to Bridges, A. A..

11 recordsLinked to original sources

bioRxiv2026

A branching cell-fate decision in biofilm dispersal enables long-term surface persistence

Biofilms are the most ancient multicellular communities on Earth, representing a primitive developmental system that protects microbes from threats. Biofilm dispersal, whereby bacteria exit biofilms, is critical for the spread of pathogens to new infection sites. Here, using Vibrio cholerae, we show that dispersal events are accompanied by a branching cell-fate decision. While [~]90% of cells disperse, a viable subpopulation remains within a residual matrix. This post-dispersal biofilm community (PDBC) is established by the matrix protein RbmA and adopts a specialized anabolic program that enhances tolerance to antibiotics and bacteriophages. Our findings reveal that PDBCs act as a resilient "seed-bank" capable of rapidly re-populating the niche without requiring de novo matrix biosynthesis, providing a mechanistic basis for the recurrence and spread of chronic infections.

microbiology↗

bioRxiv2025

Label-free microscopy enables high-throughput identification of genes controlling biofilm development

The biofilm mode of growth plays a critical role in microbial ecology and in the persistence of human pathogens. Yet, much remains unknown regarding the molecular determinants of biofilms in human pathogens. In this study, we present label-free analysis of biofilms (LFAB), an imaging approach that combines time-lapse, low-magnification brightfield microscopy with regional optical density measurements to quantify biofilm biomass. Unlike other approaches to biofilm biomass quantification, LFAB enables real-time, non-perturbative, and high-throughput monitoring of biofilms. We validated LFAB in diverse microbes and found that our measurements strongly correlate with traditional biofilm assays. We then used LFAB to identify and characterize critical factors mediating biofilm formation in Streptococcus pneumoniae, a major human pathogen whose biofilm lifecycle is known to be intimately related to colonization and infection. Initial characterization revealed that S. pneumoniae microcolonies form by radial expansion of attached cells, displaying reproducible morphology and growth dynamics. Screening of a transposon mutant library revealed that genes spanning carbohydrate metabolism, signaling, surface binding, cell wall synthesis, and adhesion impinge on the biofilm lifecycle of S. pneumoniae. We performed follow-up investigations of choline binding protein A (CbpA) and its adjacently encoded two-component system regulator, which we find are critical for the dynamics of microcolony biofilms in S. pneumoniae. Overall, this work establishes LFAB as a powerful approach for identifying and characterizing biofilm determinants across bacteria and uncovers key regulators of the biofilm lifecycle in a major human pathogen.

microbiology↗

bioRxiv2025

A small periplasmic protein governs broad physiological adaptations in Vibrio cholerae via regulation of the DbfRS two-component system

Two-component signaling pathways allow bacteria to sense and respond to environmental changes, yet the sensory mechanisms of many remain poorly understood. In the pathogen Vibrio cholerae, the DbfRS two-component system controls the biofilm lifecycle, a critical process for environmental persistence and host colonization. Here, we identified DbfQ, a small periplasmic protein encoded adjacent to dbfRS, as a direct modulator of pathway activity. DbfQ directly binds the sensory domain of the histidine kinase DbfS, shifting it toward phosphatase activity and promoting biofilm dispersal. In contrast, outer membrane perturbations, caused by mutations in lipopolysaccharide biosynthesis genes or membrane-damaging antimicrobials, activate phosphorylation of the response regulator DbfR. Transcriptomic analyses reveal that DbfR phosphorylation leads to broad transcriptional changes spanning genes involved in biofilm formation, central metabolism, peptidoglycan synthesis, and cellular stress responses. Constitutive DbfR phosphorylation imposes severe fitness costs in an infection model, highlighting this pathway as a potential target for anti-infective therapeutics. We find that dbfQRS-like genetic modules are widely present across bacterial phyla, underscoring their broad relevance in bacterial physiology. Collectively, these findings establish DbfQ as a new class of periplasmic regulator that influences two-component signaling and bacterial adaptation.

microbiology↗

bioRxiv2024

Biofilm dispersal patterns revealed using far-red fluorogenic probes

Bacteria frequently colonize niches by forming multicellular communities called biofilms. To explore new territories, cells exit biofilms through an active process called dispersal. Biofilm dispersal is essential for bacteria to spread between infection sites, yet how the process is executed at the single-cell level remains mysterious. Here, we characterize dispersal at unprecedented resolution for the global pathogen Vibrio cholerae. To do so, we first developed a far-red cell-labeling strategy that overcomes pitfalls of fluorescent protein-based approaches. We reveal that dispersal initiates at the biofilm periphery and [~]25% of cells never disperse. We define novel micro-scale patterns that occur during dispersal, including biofilm compression and the formation of dynamic channels. These patterns are attenuated in mutants that reduce overall dispersal or that increase dispersal at the cost of homogenizing local mechanical properties. Collectively, our findings provide fundamental insights into the mechanisms of biofilm dispersal, advancing our understanding of how pathogens disseminate.

microbiology↗

bioRxiv2023

Pneumococcal Extracellular Vesicles Mediate Horizontal Gene Transfer via the Transformation Machinery

Bacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogen Streptococcus pneumoniae (pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria, and in doing so, may promote the spread of drug resistance genes in the population. SignificanceThis work extends our understanding of horizontal gene transfer and the roles of extracellular vesicles in pneumococcus. This bacterium serves as the model for transformation, a process by which bacteria can take up naked DNA from the environment. Here we show that extracellular vesicles secreted by the pneumococcus have DNA on their surface, and that this DNA can be imported by the transformation machinery facilitating gene transfer. Understanding EV-mediated gene transfer may provide new avenues to manage the spread of antibiotic drug resistance.

microbiology↗

bioRxiv2022

Synergy between c-di-GMP and quorum-sensing signaling in Vibrio cholerae biofilm morphogenesis

Transitions between individual and communal lifestyles allow bacteria to adapt to changing environments. Bacteria must integrate information encoded in multiple sensory cues to appropriately undertake these transitions. Here, we investigate how two prevalent sensory inputs converge on biofilm morphogenesis: quorum sensing, which endows bacteria with the ability to communicate and coordinate group behaviors, and second messenger c-di-GMP signaling, which allows bacteria to detect and respond to environmental stimuli. We use Vibrio cholerae as our model system, the autoinducer AI-2 to modulate quorum sensing, and the polyamine norspermidine to modulate NspS-MbaA-mediated c-di-GMP production. Individually, AI-2 and norspermidine drive opposing biofilm phenotypes, with AI-2 repressing and norspermidine inducing biofilm formation. Surprisingly, however, when AI-2 and norspermidine are simultaneously detected, they act synergistically to elevate biofilm formation. We show that this effect is caused by quorum-sensing-mediated activation of nspS-mbaA expression, which increases the levels of NspS and MbaA, and in turn, c-di-GMP biosynthesis, in response to norspermidine. Increased MbaA-synthesized c-di-GMP activates the VpsR transcription factor, driving elevated expression of genes encoding key biofilm matrix components. Thus, in the context of biofilm formation in V. cholerae, quorum-sensing regulation of c-di-GMP-metabolizing receptor levels connects changes in cell population density to detection of environmental stimuli. ImportanceThe development of multicellular communities, known as biofilms, facilitates beneficial functions of gut microbiome bacteria and makes bacterial pathogens recalcitrant to treatment. Understanding how bacteria regulate the biofilm lifecycle is fundamental to biofilm control in industrial processes and in medicine. Here, we demonstrate how two major sensory inputs - quorum-sensing communication and second messenger c-di-GMP signaling - jointly regulate biofilm morphogenesis in the global pathogen Vibrio cholerae. We characterize the mechanism underlying a surprising synergy between quorum-sensing and c-di-GMP signaling in controlling biofilm development. Thus, the work connects changes in cell population density to detection of environmental stimuli in a pathogen of clinical significance.

microbiology↗

bioRxiv2021

Quantitative input-output dynamics of a c-di-GMP signal-transduction cascade in Vibrio cholerae

Bacterial biofilms are multicellular communities that collectively overcome environmental threats and clinical treatments. To regulate the biofilm lifecycle, bacteria commonly transduce sensory information via the second-messenger molecule cyclic diguanylate (c-di-GMP). Using experimental and modeling approaches, we quantitatively capture c-di-GMP signal transmission via the bifunctional polyamine receptor NspS-MbaA, from ligand binding to output, in the pathogen Vibrio cholerae. Upon binding of norspermidine or spermidine, NspS-MbaA synthesizes or degrades c-di-GMP, respectively, which in turn, drives alterations specifically to biofilm gene expression. A longstanding question is how output specificity is achieved via c-di-GMP, a diffusible molecule that regulates dozens of effectors. We show that NspS-MbaA signals locally to specific effectors, sensitizing V. cholerae to polyamines. However, local signaling is not required for specificity, as changes to global cytoplasmic c-di-GMP levels can selectively regulate biofilm genes. This work establishes the input-output dynamics underlying c-di-GMP signaling, which could be useful for developing bacterial manipulation strategies.

microbiology↗

bioRxiv2020

Inverse regulation of Vibrio cholerae biofilm dispersal by polyamine signals

The global pathogen Vibrio cholerae undergoes cycles of biofilm formation and dispersal in the environment and the human host. Little is understood about biofilm dispersal. Here, we show that MbaA, a periplasmic polyamine sensor, and PotD1, a polyamine importer, regulate V. cholerae biofilm dispersal. Spermidine, a commonly produced polyamine, drives V. cholerae dispersal, whereas norspermidine, an uncommon polyamine produced by vibrios, inhibits dispersal. Spermidine and norspermidine differ by one methylene group. Both polyamines function to control dispersal via periplasmic detection by MbaA and subsequent signal relay. Biofilm dispersal fails in the absence of PotD1 because reuptake of endogenously produced norspermidine does not occur, so it accumulates in the periplasm where it stimulates MbaA. These results suggest that V. cholerae uses MbaA to monitor environmental polyamines, blends of which potentially provide information about numbers of self and other. This information is used to dictate whether or not to disperse from biofilms.

microbiology↗

bioRxiv2020

Identification of signaling pathways, matrix-digestion enzymes, and motility components controlling Vibrio cholerae biofilm dispersal

Bacteria alternate between being free-swimming and existing as members of sessile multicellular communities called biofilms. The biofilm lifecycle occurs in three stages: cell attachment, biofilm maturation, and biofilm dispersal. Vibrio cholerae biofilms are hyper-infectious and biofilm formation and dispersal are considered central to disease transmission. While biofilm formation is well-studied, almost nothing is known about biofilm dispersal. Here, we conduct an imaging screen for V. cholerae mutants that fail to disperse, revealing three classes of dispersal components: signal transduction proteins, matrix-degradation enzymes, and motility factors. Signaling proteins dominated the screen and among them, we focused on an uncharacterized two-component sensory system that we name DbfS/DbfR for Dispersal of Biofilm Sensor/Regulator. Phospho-DbfR represses biofilm dispersal. DbfS dephosphorylates and thereby inactivates DbfR, which permits dispersal. Matrix degradation requires two enzymes: LapG, which cleaves adhesins, and RbmB, which digests matrix polysaccharide. Reorientations in swimming direction, mediated by CheY3, are necessary for cells to escape from the porous biofilm matrix. We suggest that these components act sequentially: signaling launches dispersal by terminating matrix production and triggering matrix digestion and, subsequently, cell motility permits escape from biofilms. This study lays the groundwork for interventions that modulate V. cholerae biofilm dispersal to ameliorate disease. Significance statementThe pathogen Vibrio cholerae alternates between the free-swimming state and existing in sessile multicellular communities known as biofilms. Transitioning between these lifestyles is key for disease transmission. V. cholerae biofilm formation is well studied, however, almost nothing is known about how V. cholerae cells disperse from biofilms, precluding understanding of a central pathogenicity step. Here, we conducted a high-content imaging screen for V. cholerae mutants that failed to disperse. Our screen revealed three classes of components required for dispersal: signal transduction, matrix degradation, and motility factors. We characterized these components to reveal the sequence of molecular events that choreograph V. cholerae biofilm dispersal. Our report provides a framework for developing strategies to modulate biofilm dispersal to prevent or treat disease.

microbiology↗

bioRxiv2019

Separating Functions of the Phage-Encoded Quorum-Sensing-Activated Antirepressor Qtip

Quorum sensing is a process of chemical communication that bacteria use to track cell density and coordinate gene expression across a population. Bacteria-infecting viruses, called phages, can encode quorum-sensing components that enable them to integrate host cell density information into the lysis-lysogeny decision. Vibriophage VP882 is one such phage, and activation of its quorum-sensing pathway leads to the production of an antirepressor called Qtip. Qtip interferes with the prophage repressor (cIVP882), leading to host-cell lysis. Here, we show that Qtip interacts with the N-terminus of cIVP882, inhibiting both cIVP882 DNA-binding and cIVP882 autoproteolysis. Qtip also sequesters cIVP882, localizing it to the poles. Qtip can localize to the poles independently of cIVP882. Alanine-scanning mutagenesis of Qtip shows that its localization and interference with cIVP882 activities are separable. Comparison of Qtip to a canonical phage antirepressor reveals that, despite both proteins interacting with their partner repressors, only Qtip drives polar localization.

microbiology↗

bioRxiv2019

The intra-genus and inter-species quorum-sensing autoinducers exert distinct control over Vibrio cholerae biofilm formation and dispersal

Vibrio cholerae possesses multiple quorum-sensing systems that control virulence and biofilm formation among other traits. At low cell densities, when quorum-sensing autoinducers are absent, V. cholerae forms biofilms. At high cell densities, when autoinducers have accumulated, biofilm formation is repressed and dispersal occurs. Here, we focus on the roles of two well-characterized quorum-sensing autoinducers that function in parallel. One autoinducer, called CAI-1, is used to measure vibrio abundance, and the other autoinducer, called AI-2, is a broadly-made universal autoinducer that is presumed to enable V. cholerae to assess the total bacterial cell density of the vicinal community. The two V. cholerae autoinducers funnel information into a shared signal relay pathway. This feature of the quorum-sensing system architecture has made it difficult to understand how specific information can be extracted from each autoinducer, how the autoinducers might drive distinct output behaviors, and in turn, how the bacteria use quorum sensing to distinguish self from other in bacterial communities. We develop a live-cell biofilm formation and dispersal assay that allows examination of the individual and combined roles of the two autoinducers in controlling V. cholerae behavior. We show that the quorum-sensing system works as a coincidence detector in which both autoinducers must be present simultaneously for repression of biofilm formation to occur. Within that context, the CAI-1 quorum-sensing pathway is activated when only a few V. cholerae cells are present, whereas the AI-2 pathway is activated only at much higher cell density. The consequence of this asymmetry is that exogenous sources of AI-2, but not CAI-1, contribute to satisfying the coincidence detector to repress biofilm formation and promote dispersal. We propose that V. cholerae uses CAI-1 to verify that some of its kin are present before committing to the high-cell-density quorum-sensing mode, but it is, in fact, the universal autoinducer AI-2, that sets the pace of the V. cholerae quorum-sensing program. This first report of unique roles for the different V. cholerae autoinducers suggests that detection of self fosters a distinct outcome from detection of other.

microbiology↗