A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.

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.

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.

Divide and conquer: the Pseudomonas aeruginosa two-component hybrid SagS enables biofilm formation and recalcitrance of biofilm cells to antimicrobial agents via distinct regulatory circuits.

The opportunistic pathogen Pseudomonas aeruginosa forms antimicrobial resistant biofilms through sequential steps requiring several two-component regulatory systems. The sensor-regulator hybrid SagS plays a central role in biofilm development by enabling the switch from the planktonic to the biofilm mode of growth, and by facilitating the transition of biofilm cells to a highly tolerant state. However, the mechanism by which SagS accomplishes both functions is unknown. SagS harbours a periplasmic sensory HmsP, and phosphorelay HisKA and Rec domains. SagS domain was used as constructs and site-directed mutagenesis to elucidate how SagS performs its dual functions. It was demonstrated that HisKA-Rec and the phospho-signalling between SagS and BfiS contribute to the switch to the biofilm mode of growth, but not to the tolerant state. Instead, expression of SagS domain constructs harbouring HmsP rendered ΔsagS biofilm cells as recalcitrant to antimicrobial agents as wild-type biofilms, likely by restoring BrlR production and cellular c-di-GMP levels to wild-type levels. Restoration of biofilm tolerance by HmsP was independent of biofilm biomass accumulation, RsmA, RsmYZ, HptB and BfiSR-downstream targets. Our findings thus suggest that SagS likely makes use of a "divide-and-conquer" mechanism to regulate its dual switch function, by activating two distinct regulatory networks via its individual domains.

BrlR from Pseudomonas aeruginosa is a c-di-GMP-responsive transcription factor.

The transcriptional regulator BrlR is a member of the MerR family of multidrug transport activators that contributes to the high-level drug tolerance of Pseudomonas aeruginosa biofilms. While MerR regulators are known to activate both the expression of multidrug efflux pump genes and their own transcription upon inducer binding, little is known about BrlR activation. We demonstrate using promoter reporter strains, in vivo and in vitro DNA-binding assays combined with 5'RACE, that BrlR binds to its own promoter, likely via a MerR-like palindromic sequence. Unlike known MerR multidrug transport activators, BrlR and brlR expression are not activated by multidrug transporter substrates. Instead, BrlR-DNA binding was enhanced by the secondary messenger c-di-GMP. In addition to enhanced BrlR-DNA binding, c-di-GMP levels contributed to PbrlR promoter activity in initial attached cells with elevated c-di-GMP levels correlating with increased expression of brlR. While not harbouring amino acid motifs resembling previously defined c-di-GMP-binding domains, BrlR was found to bind c-di-GMP in vitro at a ratio of one c-di-GMP per two BrlR. Cross-linking assays confirmed dimer formation to be enhanced in the presence of elevated c-di-GMP levels. Our findings demonstrate BrlR to be an unusual MerR-family member in that BrlR function and expression require the secondary messenger c-di-GMP.

Elevated levels of the second messenger c-di-GMP contribute to antimicrobial resistance of Pseudomonas aeruginosa.

Biofilms are highly structured, surface-associated communities. A hallmark of biofilms is their extraordinary resistance to antimicrobial agents that is activated during early biofilm development of Pseudomonas aeruginosa and requires the regulatory hybrid SagS and BrlR, a member of the MerR family of multidrug efflux pump activators. However, little is known about the mechanism by which SagS contributes to BrlR activation or drug resistance. Here, we demonstrate that ΔsagS biofilm cells harbour the secondary messenger c-di-GMP at reduced levels similar to those observed in wild-type cells grown planktonically rather than as biofilms. Restoring c-di-GMP levels to wild-type biofilm-like levels restored brlR expression, DNA binding by BrlR, and recalcitrance to killing by antimicrobial agents of ΔsagS biofilm cells. We likewise found that increasing c-di-GMP levels present in planktonic cells to biofilm-like levels (≥ 55 pmol mg(-1) ) resulted in planktonic cells being significantly more resistant to antimicrobial agents, with increased resistance correlating with increased brlR, mexA, and mexE expression and BrlR production. In contrast, reducing cellular c-di-GMP levels of biofilm cells to ≤ 40 pmol mg(-1) correlated with increased susceptibility and reduced brlR expression. Our findings suggest that a signalling pathway involving a specific c-di-GMP pool regulated by SagS contributes to the resistance of P. aeruginosa biofilms.

The MerR-like regulator BrlR confers biofilm tolerance by activating multidrug efflux pumps in Pseudomonas aeruginosa biofilms.

A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In Pseudomonas aeruginosa, biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR. Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a ΔbrlR strain partly restored tolerance of ΔbrlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents.

The MerR-like transcriptional regulator BrlR contributes to Pseudomonas aeruginosa biofilm tolerance.

Biofilms are composed of surface-attached microbial communities. A hallmark of biofilms is their profound tolerance of antimicrobial agents. While biofilm drug tolerance has been considered to be multifactorial, our findings indicate, instead, that bacteria within biofilms employ a classical regulatory mechanism to resist the action of antimicrobial agents. Here we report that the transcriptional regulator BrlR, a member of the MerR family of multidrug transport activators, plays a role in the high-level drug tolerance of biofilms formed by Pseudomonas aeruginosa. Expression of brlR was found to be biofilm specific, with brlR inactivation not affecting biofilm formation, motility, or pslA expression but increasing ndvB expression. Inactivation of brlR rendered biofilms but not planktonic cells grown to exponential or stationary phase significantly more susceptible to hydrogen peroxide and five different classes of antibiotics by affecting the MICs and the recalcitrance of biofilms to killing by microbicidal antimicrobial agents. In contrast, overexpression of brlR rendered both biofilms and planktonic cells more tolerant to the same compounds. brlR expression in three cystic fibrosis (CF) isolates was elevated regardless of the mode of growth, suggesting a selection for constitutive brlR expression upon in vivo biofilm formation associated with chronic infections. Despite increased brlR expression, however, isolate CF1-8 was as susceptible to tobramycin as was a ΔbrlR mutant because of a nonsense mutation in brlR. Our results indicate for the first time that biofilms employ a specific regulatory mechanism to resist the action of antimicrobial agents in a BrlR-dependent manner which affects MIC and recalcitrance to killing by microbicidal antimicrobial agents.