Sequestration of a dual function DNA-binding protein by Vibrio cholerae CRP.

Although the mechanism by which the cyclic AMP receptor protein (CRP) regulates global gene transcription has been intensively studied for decades, new discoveries remain to be made. Here, we report that, during rapid growth, CRP associates with both the well-conserved, dual-function DNA-binding protein peptidase A (PepA) and the cell membrane. These interactions are not present under nutrient-limited growth conditions, due to post-translational modification of three lysines on a single face of CRP. Although coincident DNA binding is rare, dissociation from CRP results in increased PepA occupancy at many chromosomal binding sites and differential regulation of hundreds of genes, including several encoding cyclic dinucleotide phosphodiesterases. We show that PepA represses biofilm formation and activates motility/chemotaxis. We propose a model in which membrane-bound CRP interferes with PepA DNA binding. Under nutrient limitation, PepA is released. Together, CRP and free PepA activate a transcriptional response that impels the bacterium to seek a more hospitable environment. This work uncovers a function for CRP in the sequestration of a regulatory protein. More broadly, it describes a paradigm of bacterial transcriptome modulation through metabolically regulated association of transcription factors with the cell membrane.

Vibrio cholerae high cell density quorum sensing activates the host intestinal innate immune response.

Quorum sensing fundamentally alters the interaction of Vibrio cholerae with aquatic environments, environmental hosts, and the human intestine. At high cell density, the quorum-sensing regulator HapR represses not only expression of cholera toxin and the toxin co-regulated pilus, virulence factors essential in human infection, but also synthesis of the Vibrio polysaccharide (VPS) exopolysaccharide-based matrix required for abiotic and biotic surface attachment. Here, we describe a feature of V. cholerae quorum sensing that shifts the host-pathogen interaction toward commensalism. By repressing pathogen consumptive anabolic metabolism and, in particular, tryptophan uptake, V. cholerae HapR stimulates host intestinal serotonin production. This, in turn, activates host intestinal innate immune signaling to promote host survival.

Bioengineered 3D Tissue Model of Intestine Epithelium with Oxygen Gradients to Sustain Human Gut Microbiome.

The human gut microbiome is crucial to hosting physiology and health. Therefore, stable in vitro coculture of primary human intestinal cells with a microbiome community is essential for understanding intestinal disease progression and revealing novel therapeutic targets. Here, a three-dimensional scaffold system is presented to regenerate an in vitro human intestinal epithelium that recapitulates many functional characteristics of the native small intestines. The epithelium, derived from human intestinal enteroids, contains mature intestinal epithelial cells and possesses selectively permeable barrier functions. Importantly, by properly positioning the scaffolds cultured under normal atmospheric conditions, two physiologically relevant oxygen gradients, a proximal-to-distal oxygen gradient along the gastrointestinal (GI) tract, and a radial oxygen gradient across the epithelium, are distinguished in the tissues when the lumens are faced up and down in cultures, respectively. Furthermore, the presence of the low oxygen gradients supported the coculture of intestinal epithelium along with a complex living commensal gut microbiome (including obligate anaerobes) to simulate temporal microbiome dynamics in the native human gut. This unique silk scaffold platform may enable the exploration of microbiota-related mechanisms of disease pathogenesis and host-pathogen dynamics in infectious diseases including the potential to explore the human microbiome-gut-brain axis and potential novel microbiome-based therapeutics.

The Short-Chain Fatty Acids Propionate and Butyrate Augment Adherent-Invasive Escherichia coli Virulence but Repress Inflammation in a Human Intestinal Enteroid Model of Infection.

Short-chain fatty acids (SCFAs), which consist of six or fewer carbons, are fermentation products of the bacterial community that inhabits the intestine. Due to an immunosuppressive effect on intestinal tissue, they have been touted as a therapeutic for inflammatory conditions of the bowel. Here, we study the impact of acetate, propionate, and butyrate, the three most abundant SCFAs in the intestine, on gene expression in the intestinal pathobiont adherent-invasive Escherichia coli. We pair this with adherence, invasion, and inflammation in Caco-2 and human intestinal enteroid (HIE)-derived monolayer models of the intestinal epithelium. We report that propionate and butyrate upregulate transcription of adherent-invasive Escherichia coli (AIEC) flagellar synthesis genes and decrease expression of capsule assembly and transport genes. These changes are predicted to augment AIEC invasiveness. In fact, SCFA supplementation increases AIEC adherence to and invasion of the Caco-2 monolayer but has no effect on these parameters in the HIE model. We attribute this to the anti-inflammatory effect of propionate and butyrate on HIEs but not on Caco-2 cells. We conclude that the potential of SCFAs to increase the virulence of intestinal pathogens should be considered in their use as anti-inflammatory agents. IMPORTANCE The human terminal ileum and colon are colonized by a community of microbes known as the microbiota. Short-chain fatty acids (SCFAs) excreted by bacterial members of the microbiota define the intestinal environment. These constitute an important line of communication within the microbiota and between the microbiota and the host epithelium. In inflammatory conditions of the bowel, SCFAs are often low and there is a preponderance of a conditionally virulent bacterium termed adherent-invasive Escherichia coli (AIEC). A connection between SCFA abundance and AIEC has been suggested. Here, we study AIEC in monoculture and in coculture with human intestinal enteroid-derived monolayers and show that the SCFAs propionate and butyrate increase expression of AIEC virulence genes while concurrently bolstering the intestinal epithelial barrier and reducing intestinal inflammation. While these SCFAs have been promoted as a therapy for inflammatory bowel conditions, our findings demonstrate that their effect on bacterial virulence must be considered.

Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex.

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, we asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals.

The Interplay of Sex Steroids, the Immune Response, and the Intestinal Microbiota.

The role of sex steroids in mammalian maturation is well established. Recently, it has been increasingly appreciated that sex steroids also play an important role in the propensity of adults to develop a myriad of diseases. The exposure and responsiveness of tissues to sex steroids varies among individuals and between the sexes, and this has been correlated with gender-specific differences in the composition of the intestinal microbiota and in susceptibility to metabolic, autoimmune, and neoplastic diseases. Here we focus on recent studies that demonstrate an interplay between sex steroids, the intestinal immune response, and the intestinal microbiota. While correlations between biological sex, the intestinal innate immune response, intestinal inflammation, and intestinal microbiota have been established, many gaps in our knowledge prevent the emergence of an overarching model for this complex interaction. Such a model could aid in the development of prebiotic, probiotic, or synthetic therapeutics that decrease the risk of autoimmune, metabolic, neoplastic, and infectious diseases of the intestine and mitigate the particular health risks faced by individuals receiving sex steroid treatment.

Methionine Availability in the Arthropod Intestine Is Elucidated through Identification of Vibrio cholerae Methionine Acquisition Systems.

While only a subset of Vibrio cholerae strains are human diarrheal pathogens, all are aquatic organisms. In this environment, they often persist in close association with arthropods. In the intestinal lumen of the model arthropod Drosophila melanogaster, methionine and methionine sulfoxide decrease susceptibility to V. cholerae infection. In addition to its structural role in proteins, methionine participates in the methionine cycle, which carries out synthetic and regulatory methylation reactions. It is, therefore, essential for the growth of both animals and bacteria. Methionine is scarce in some environments, and the facile conversion of free methionine to methionine sulfoxide in oxidizing environments interferes with its utilization. To ensure an adequate supply of methionine, the genomes of most organisms encode multiple high-affinity uptake pathways for methionine as well as multiple methionine sulfoxide reductases, which reduce free and protein-associated methionine sulfoxide to methionine. To explore the role of methionine uptake and reduction in V. cholerae colonization of the arthropod intestine, we mutagenized the two high-affinity methionine transporters and five methionine sulfoxide reductases encoded in the V. cholerae genome. We show that MsrC is the sole methionine sulfoxide reductase active on free methionine sulfoxide. Furthermore, in the absence of methionine synthesis, high-affinity methionine uptake but not reduction is essential for V. cholerae colonization of the Drosophila intestine. These findings allow us to place a lower limit of 0.05 mM and an upper limit of 0.5 mM on the methionine concentration in the Drosophila intestine.IMPORTANCE Methionine is an essential amino acid involved in both biosynthetic and regulatory processes in the bacterial cell. To ensure an adequate supply of methionine, bacteria have evolved multiple systems to synthesize, import, and recover this amino acid. To explore the importance of methionine synthesis, transport, and recovery in any environment, all of these systems must be identified and mutagenized. Here, we have mutagenized every high-affinity methionine uptake system and methionine sulfoxide reductase encoded in the genome of the diarrheal pathogen V. cholerae We use this information to determine that high-affinity methionine uptake systems are sufficient to acquire methionine in the intestine of the model arthropod Drosophila melanogaster but are not involved in virulence and that the intestinal concentration of methionine must be between 0.05 mM and 0.5 mM.

Vibrio cholerae Sheds Its Coat to Make Itself Comfortable in the Gut.

Invertebrates molt, furry mammals shed, and human skin exfoliates. In this issue of Cell Host & Microbe, Zingl et al. describe a virulence mechanism in which the bacterial pathogen Vibrio cholerae jettisons outer membrane proteins and lipids in vesicles as it enters the mammalian intestine.

Microbial Control of Intestinal Homeostasis via Enteroendocrine Cell Innate Immune Signaling.

A community of commensal microbes, known as the intestinal microbiota, resides within the gastrointestinal tract of animals and plays a role in maintenance of host metabolic homeostasis and resistance to pathogen invasion. Enteroendocrine cells, which are relatively rare in the intestinal epithelium, have evolved to sense and respond to these commensal microbes. Specifically, they express G-protein-coupled receptors and functional innate immune signaling pathways that recognize products of microbial metabolism and microbe-associated molecular patterns, respectively. Here we review recent evidence from Drosophila melanogaster that microbial cues recruit antimicrobial, mechanical, and metabolic branches of the enteroendocrine innate immune system and argue that this response may play a role not only in maintaining host metabolic homeostasis but also in intestinal resistance to invasion by bacterial, viral, and parasitic pathogens.

A high-throughput, whole cell assay to identify compounds active against carbapenem-resistant Klebsiella pneumoniae.

Enteric Gram-negative rods (GNR), which are frequent causes of community-acquired and nosocomial infections, are increasingly resistant to the antibiotics in our current armamentarium. One solution to this medical dilemma is the development of novel classes of antimicrobial compounds. Here we report the development of a robust, whole cell-based, high-throughput metabolic assay that detects compounds with activity against carbapenem-resistant Klebsiella pneumoniae. We have used this assay to screen approximately 8,000 fungal extracts and 50,000 synthetic compounds with the goal of identifying extracts and compounds active against a highly resistant strain of Klebsiella pneumoniae. The primary screen identified 43 active fungal extracts and 144 active synthetic compounds. Patulin, a known fungal metabolite and inhibitor of bacterial quorum sensing and alanine racemase, was identified as the active component in the most potent fungal extracts. We did not study patulin further due to previously published evidence of toxicity. Three synthetic compounds termed O06, C17, and N08 were chosen for further study. Compound O06 did not have significant antibacterial activity but rather interfered with sugar metabolism, while compound C17 had only moderate activity against GNRs. Compound N08 was active against several resistant GNRs and showed minimal toxicity to mammalian cells. Preliminary studies suggested that it interferes with protein expression. However, its direct application may be limited by susceptibility to efflux and a tendency to form aggregates in aqueous media. Rapid screening of 58,000 test samples with identification of several compounds that act on CR-K. pneumoniae demonstrates the utility of this screen for the discovery of drugs active against this highly resistant GNR.

Removal of a Membrane Anchor Reveals the Opposing Regulatory Functions of Vibrio cholerae Glucose-Specific Enzyme IIA in Biofilms and the Mammalian Intestine.

The Vibrio cholerae phosphoenolpyruvate phosphotransferase system (PTS) is a well-conserved, multicomponent phosphotransfer cascade that coordinates the bacterial response to carbohydrate availability through direct interactions of its components with protein targets. One such component, glucose-specific enzyme IIA (EIIAGlc), is a master regulator that coordinates bacterial metabolism, nutrient uptake, and behavior by direct interactions with cytoplasmic and membrane-associated protein partners. Here, we show that an amphipathic helix (AH) at the N terminus of V. cholerae EIIAGlc serves as a membrane association domain that is dispensable for interactions with cytoplasmic partners but essential for regulation of integral membrane protein partners. By deleting this AH, we reveal previously unappreciated opposing regulatory functions for EIIAGlc at the membrane and in the cytoplasm and show that these opposing functions are active in the laboratory biofilm and the mammalian intestine. Phosphotransfer through the PTS proceeds in the absence of the EIIAGlc AH, while PTS-dependent sugar transport is blocked. This demonstrates that the AH couples phosphotransfer to sugar transport and refutes the paradigm of EIIAGlc as a simple phosphotransfer component in PTS-dependent transport. Our findings show that Vibrio cholerae EIIAGlc, a central regulator of pathogen metabolism, contributes to optimization of bacterial physiology by integrating metabolic cues arising from the cytoplasm with nutritional cues arising from the environment. Because pathogen carbon metabolism alters the intestinal environment, we propose that it may be manipulated to minimize the metabolic cost of intestinal infection.IMPORTANCE The V. cholerae phosphoenolpyruvate phosphotransferase system (PTS) is a well-conserved, multicomponent phosphotransfer cascade that regulates cellular physiology and virulence in response to nutritional signals. Glucose-specific enzyme IIA (EIIAGlc), a component of the PTS, is a master regulator that coordinates bacterial metabolism, nutrient uptake, and behavior by direct interactions with protein partners. We show that an amphipathic helix (AH) at the N terminus of V. cholerae EIIAGlc serves as a membrane association domain that is dispensable for interactions with cytoplasmic partners but essential for regulation of integral membrane protein partners. By removing this amphipathic helix, hidden, opposing roles for cytoplasmic partners of EIIAGlc in both biofilm formation and metabolism within the mammalian intestine are revealed. This study defines a novel paradigm for AH function in integrating opposing regulatory functions in the cytoplasm and at the bacterial cell membrane and highlights the PTS as a target for metabolic modulation of the intestinal environment.

The Drosophila Immune Deficiency Pathway Modulates Enteroendocrine Function and Host Metabolism.

Enteroendocrine cells (EEs) are interspersed between enterocytes and stem cells in the Drosophila intestinal epithelium. Like enterocytes, EEs express components of the immune deficiency (IMD) innate immune pathway, which activates transcription of genes encoding antimicrobial peptides. The discovery of large lipid droplets in intestines of IMD pathway mutants prompted us to investigate the role of the IMD pathway in the host metabolic response to its intestinal microbiota. Here we provide evidence that the short-chain fatty acid acetate is a microbial metabolic signal that activates signaling through the enteroendocrine IMD pathway in a PGRP-LC-dependent manner. This, in turn, increases transcription of the gene encoding the endocrine peptide Tachykinin (Tk), which is essential for timely larval development and optimal lipid metabolism and insulin signaling. Our findings suggest innate immune pathways not only provide the first line of defense against infection but also afford the intestinal microbiota control over host development and metabolism.

Sublingual Adjuvant Delivery by a Live Attenuated Vibrio cholerae-Based Antigen Presentation Platform.

A sublingually delivered heterologous antigen presentation platform that does not depend on antigen or adjuvant purification would be of great benefit in protection against diarrheal disease. In proof-of-concept studies, we previously showed that when a fusion protein comprised of the Vibrio cholerae biofilm matrix protein RbmA and the B subunit of cholera toxin (R-CTB) is expressed from a plasmid within V. cholerae, R-CTB is sequestered in the biofilm matrix, leading to decoration of the cell surface. Sublingual delivery of live attenuated R-CTB-decorated cells results in a mucosal immune response to CTB. To improve the immune response to diarrheal antigens presented by this platform, we have engineered our live attenuated vaccine to express the mucosal adjuvant mmCT (i.e., multiply mutated CT). Here we report that delivery of this adjuvant via sublingual administration of our vaccine enhances the mucosal immune response to V. cholerae LPS and elicits a systemic and mucosal immune response to CTB. However, provision of R-CTB with mmCT selectively blunts the mucosal immune response to CTB. We propose that mmCT delivered by this live attenuated Vibrio cholerae vaccine platform may serve as a mucosal adjuvant for heterologous antigens, provided they are not too similar to mmCT.IMPORTANCE Diarrheal disease is the most common infectious disease of children in the developing world. Our goal is to develop a diarrheal antigen presentation platform based on whole Vibrio cholerae cells that does not depend on protein purification. We have previously shown the feasibility of genetically fusing antigens to the V. cholerae biofilm matrix protein RbmA for presentation on the cell surface. A mucosal adjuvant could improve immunogenicity of such a vaccine at the mucosal surface. Here we engineer a live attenuated V. cholerae vaccine to constitutively synthesize mmCT, a nontoxic form of cholera toxin. When this vaccine is delivered sublingually, in vivo-synthesized mmCT acts as both an adjuvant and antigen. This could greatly increase the magnitude and duration of the immune response elicited by codelivered heterologous antigens.

A Self-Assembling Whole-Cell Vaccine Antigen Presentation Platform.

Diarrhea is the most common infection in children under the age of 5 years worldwide. In spite of this, only a few vaccines to treat infectious diarrhea exist, and many of the available vaccines are sparingly and sporadically administered. Major obstacles to the development and widespread implementation of vaccination include the ease and cost of production, distribution, and delivery. Here we present a novel, customizable, and self-assembling vaccine platform that exploits the Vibrio cholerae bacterial biofilm matrix for antigen presentation. We use this technology to create a proof-of-concept, live-attenuated whole-cell vaccine that is boosted by spontaneous association of a secreted protein antigen with the cell surface. Sublingual administration of this live-attenuated vaccine to mice confers protection against V. cholerae challenge and elicits the production of antigen-specific IgA in stool. The platform presented here enables the development of antigen-boosted vaccines that are simple to produce and deliver, addressing many of the obstacles to vaccination against diarrheal diseases. This may also serve as a paradigm for the development of broadly protective biofilm-based vaccines against other mucosal infections.IMPORTANCE Diarrheal disease is the most common infection afflicting children worldwide. In resource-poor settings, these infections are correlated with cognitive delay, stunted growth, and premature death. With the development of efficacious, affordable, and easily administered vaccines, such infections could be prevented. While a major focus of research on biofilms has been their elimination, here we harness the bacterial biofilm to create a customizable platform for cost-effective, whole-cell mucosal vaccines that self-incorporate secreted protein antigens. We use this platform to develop a sublingually administered live-attenuated prototype vaccine based on Vibrio cholerae This serves not only as a proof of concept for a multivalent vaccine against common bacterial enteric pathogens but also as a paradigm for vaccines utilizing other bacterial biofilms to target mucosal infections.

Activation of Vibrio cholerae quorum sensing promotes survival of an arthropod host.

Vibrio cholerae colonizes the human terminal ileum to cause cholera, and the arthropod intestine and exoskeleton to persist in the aquatic environment. Attachment to these surfaces is regulated by the bacterial quorum-sensing signal transduction cascade, which allows bacteria to assess the density of microbial neighbours. Intestinal colonization with V. cholerae results in expenditure of host lipid stores in the model arthropod Drosophila melanogaster. Here we report that activation of quorum sensing in the Drosophila intestine retards this process by repressing V. cholerae succinate uptake. Increased host access to intestinal succinate mitigates infection-induced lipid wasting to extend survival of V. cholerae-infected flies. Therefore, quorum sensing promotes a more favourable interaction between V. cholerae and an arthropod host by reducing the nutritional burden of intestinal colonization.

Vibrio cholerae ensures function of host proteins required for virulence through consumption of luminal methionine sulfoxide.

Vibrio cholerae is a diarrheal pathogen that induces accumulation of lipid droplets in enterocytes, leading to lethal infection of the model host Drosophila melanogaster. Through untargeted lipidomics, we provide evidence that this process is the product of a host phospholipid degradation cascade that induces lipid droplet coalescence in enterocytes. This infection-induced cascade is inhibited by mutation of the V. cholerae glycine cleavage system due to intestinal accumulation of methionine sulfoxide (MetO), and both dietary supplementation with MetO and enterocyte knock-down of host methionine sulfoxide reductase A (MsrA) yield increased resistance to infection. MsrA converts both free and protein-associated MetO to methionine. These findings support a model in which dietary MetO competitively inhibits repair of host proteins by MsrA. Bacterial virulence strategies depend on functional host proteins. We propose a novel virulence paradigm in which an intestinal pathogen ensures the repair of host proteins essential for pathogenesis through consumption of dietary MetO.

Erysipelothrix rhusiopathiae Suppurative Arthritis in a 12-year-old Boy After an Unusual Fresh Water Exposure.

We report the case of a 12-year-old boy who experienced recurrent suppurative arthritis of the distal interphalangeal joint after near simultaneous exposure to a frog, a dog bite and lake water. Anaerobic cultures of synovial fluid obtained during operative debridement grew small round gray colonies ultimately identified as Erysipelothrix rhusiopathiae by routine laboratory tests and mass spectrometry.

The interplay between intestinal bacteria and host metabolism in health and disease: lessons from Drosophila melanogaster.

All higher organisms negotiate a truce with their commensal microbes and battle pathogenic microbes on a daily basis. Much attention has been given to the role of the innate immune system in controlling intestinal microbes and to the strategies used by intestinal microbes to overcome the host immune response. However, it is becoming increasingly clear that the metabolisms of intestinal microbes and their hosts are linked and that this interaction is equally important for host health and well-being. For instance, an individual's array of commensal microbes can influence their predisposition to chronic metabolic diseases such as diabetes and obesity. A better understanding of host-microbe metabolic interactions is important in defining the molecular bases of these disorders and could potentially lead to new therapeutic avenues. Key advances in this area have been made using Drosophila melanogaster. Here, we review studies that have explored the impact of both commensal and pathogenic intestinal microbes on Drosophila carbohydrate and lipid metabolism. These studies have helped to elucidate the metabolites produced by intestinal microbes, the intestinal receptors that sense these metabolites, and the signaling pathways through which these metabolites manipulate host metabolism. Furthermore, they suggest that targeting microbial metabolism could represent an effective therapeutic strategy for human metabolic diseases and intestinal infection.

Regulation of CsrB/C sRNA decay by EIIA(Glc) of the phosphoenolpyruvate: carbohydrate phosphotransferase system.

Csr is a conserved global regulatory system, which uses the sequence-specific RNA-binding protein CsrA to activate or repress gene expression by binding to mRNA and altering translation, stability and/or transcript elongation. In Escherichia coli, CsrA activity is regulated by two sRNAs, CsrB and CsrC, which bind to multiple CsrA dimers, thereby sequestering this protein away from its mRNA targets. Turnover of CsrB/C sRNAs is tightly regulated by a GGDEF-EAL domain protein, CsrD, which targets them for cleavage by RNase E. Here, we show that EIIA(Glc) of the glucose-specific PTS system is also required for the normal decay of these sRNAs and that it acts by binding to the EAL domain of CsrD. Only the unphosphorylated form of EIIA(Glc) bound to CsrD in vitro and was capable of activating CsrB/C turnover in vivo. Genetic studies confirmed that this mechanism couples CsrB/C sRNA decay to the availability of a preferred carbon source. These findings reveal a new physiological influence on the workings of the Csr system, a novel function for the EAL domain, and an important new way in which EIIA(Glc) shapes global regulatory circuitry in response to nutritional status.

In situ proteolysis of the Vibrio cholerae matrix protein RbmA promotes biofilm recruitment.

The estuarine gram-negative rod and human diarrheal pathogen Vibrio cholerae synthesizes a VPS exopolysaccharide-dependent biofilm matrix that allows it to form a 3D structure on surfaces. Proteins associated with the matrix include, RbmA, RbmC, and Bap1. RbmA, a protein whose crystallographic structure suggests two binding surfaces, associates with cells by means of a VPS-dependent mechanism and promotes biofilm cohesiveness and recruitment of cells to the biofilm. Here, we show that RbmA undergoes limited proteolysis within the biofilm. This proteolysis, which is carried out by the hemagglutinin/protease and accessory proteases, yields the 22-kDa C-terminal polypeptide RbmA*. RbmA* remains biofilm-associated. Unlike full-length RbmA, the association of RbmA* with cells is no longer VPS-dependent, likely due to an electropositive surface revealed by proteolysis. We provide evidence that this proteolysis event plays a role in recruitment of VPS(-) cells to the biofilm surface. Based on our findings, we propose that association of RbmA with the matrix reinforces the biofilm structure and leads to limited proteolysis of RbmA to RbmA*. RbmA*, in turn, promotes recruitment of cells that have not yet initiated VPS synthesis to the biofilm surface. The assignment of two functions to RbmA, separated by a proteolytic event that depends on matrix association, dictates an iterative cycle in which reinforcement of recently added biofilm layers precedes the recruitment of new VPS(-) cells to the biofilm.