In vitro activity of rifabutin against 293 contemporary carbapenem-resistant Acinetobacter baumannii clinical isolates and characterization of rifabutin mode of action and resistance mechanisms.

BACKGROUND: Rifabutin, an oral drug approved to treat Mycobacterium avium infections, demonstrated potent activity against Acinetobacter baumannii in nutrient-limited medium enabled by rifabutin cellular uptake through the siderophore receptor FhuE. OBJECTIVES: To determine rifabutin in vitro activity and resistance mechanisms in a large panel of A. baumannii isolates. METHODS: Two hundred and ninety-three carbapenem-resistant A. baumannii clinical isolates collected from Europe, the USA and Asia during 2017-19 were used for MIC determination. Sequencing/genotyping of fhuE, rpoB and arr-2 genes in isolates with elevated rifabutin MIC combined with genetic engineering and gene expression quantification was used to characterize rifabutin's mode of action and resistance mechanisms. RESULTS: Rifabutin showed excellent activity on the strain panel, with an MIC50/90 of 0.008/1 mg/L, and was superior to all other antibiotics tested, including colistin, tigecycline and cefiderocol (MIC90 of 8 mg/L). Rifabutin remained active on resistant subpopulations, including strains resistant to the siderophore-drug conjugate cefiderocol (MIC90 of 2 mg/L, n = 23). At least two independent resistance mechanisms were required to abolish rifabutin activity, which is in line with the dose-dependent mutational resistance frequency reaching 10-9 at rifabutin concentrations at or above 2 mg/L. CONCLUSIONS: This study demonstrated the potent activity of rifabutin against carbapenem-resistant A. baumannii. We propose that FhuE-mediated active uptake of rifabutin enables activity against rifampicin-resistant isolates. To achieve clinically meaningful strain coverage and to avoid rapid resistance development, rifabutin concentrations ≥2 mg/L are required, something rifabutin oral formulations cannot deliver.

Alternative Evolutionary Paths to Bacterial Antibiotic Resistance Cause Distinct Collateral Effects.

When bacteria evolve resistance against a particular antibiotic, they may simultaneously gain increased sensitivity against a second one. Such collateral sensitivity may be exploited to develop novel, sustainable antibiotic treatment strategies aimed at containing the current, dramatic spread of drug resistance. To date, the presence and molecular basis of collateral sensitivity has only been studied in few bacterial species and is unknown for opportunistic human pathogens such as Pseudomonas aeruginosa. In the present study, we assessed patterns of collateral effects by experimentally evolving 160 independent populations of P. aeruginosa to high levels of resistance against eight commonly used antibiotics. The bacteria evolved resistance rapidly and expressed both collateral sensitivity and cross-resistance. The pattern of such collateral effects differed to those previously reported for other bacterial species, suggesting interspecific differences in the underlying evolutionary trade-offs. Intriguingly, we also identified contrasting patterns of collateral sensitivity and cross-resistance among the replicate populations adapted to the same drug. Whole-genome sequencing of 81 independently evolved populations revealed distinct evolutionary paths of resistance to the selective drug, which determined whether bacteria became cross-resistant or collaterally sensitive towards others. Based on genomic and functional genetic analysis, we demonstrate that collateral sensitivity can result from resistance mutations in regulatory genes such as nalC or mexZ, which mediate aminoglycoside sensitivity in ß-lactam-adapted populations, or the two-component regulatory system gene pmrB, which enhances penicillin sensitivity in gentamicin-resistant populations. Our findings highlight substantial variation in the evolved collateral effects among replicates, which in turn determine their potential in antibiotic therapy.

A Novel Genome-Editing Platform for Drug-Resistant Acinetobacter baumannii Reveals an AdeR-Unrelated Tigecycline Resistance Mechanism.

Infections with the Gram-negative coccobacillus Acinetobacter baumannii are a major threat in hospital settings. The progressing emergence of multidrug-resistant clinical strains significantly reduces the treatment options for clinicians to fight A. baumannii infections. The current lack of robust methods to genetically manipulate drug-resistant A. baumannii isolates impedes research on resistance and virulence mechanisms in clinically relevant strains. In this study, we developed a highly efficient and versatile genome-editing platform enabling the markerless modification of the genome of A. baumannii clinical and laboratory strains, regardless of their resistance profiles. We applied this method for the deletion of AdeR, a transcription factor that regulates the expression of the AdeABC efflux pump in tigecycline-resistant A. baumannii, to evaluate its function as a putative drug target. Loss of adeR reduced the MIC90 of tigecycline from 25 µg/ml in the parental strains to 3.1 µg/ml in the ΔadeR mutants, indicating its importance in the drug resistance phenotype. However, 60% of the clinical isolates remained nonsusceptible to tigecycline after adeR deletion. Evolution of artificial tigecycline resistance in two strains followed by whole-genome sequencing revealed loss-of-function mutations in trm, suggesting its role in an alternative AdeABC-independent tigecycline resistance mechanism. This finding was strengthened by the confirmation of trm disruption in the majority of the tigecycline-resistant clinical isolates. This study highlights the development and application of a powerful genome-editing platform for A. baumannii enabling future research on drug resistance and virulence pathways in clinically relevant strains.

The food additive vanillic acid controls transgene expression in mammalian cells and mice.

Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

Klebsiella pneumoniae OmpR facilitates lung infection through transcriptional regulation of key virulence factors.

IMPORTANCE: Bacteria use two-component regulatory systems (TCSs) to adapt to changes in their environment by changing their gene expression. In this study, we show that the EnvZ/OmpR TCS of the clinically relevant opportunistic pathogen Klebsiella pneumoniae plays an important role in successfully establishing lung infection and virulence. In addition, we elucidate the K. pneumoniae OmpR regulon within the host. This work suggests that K. pneumoniae OmpR might be a promising target for innovative anti-infectives.

A novel reporter system for bacterial and mammalian cells based on the non-ribosomal peptide indigoidine.

The biosynthesis of non-ribosomal peptides, many of which have pharmaceutical activities, is an evolutionary privilege of microorganisms. Capitalizing on the universal set of the Streptomyces lavendulae non-ribosomal peptide synthase BpsA and the Streptomyces verticillus 4'-phosphopantetheinyl transferase Svp, we have engineered Escherichia coli as well as mammalian cells, including human stem cells, to produce the blue 3,3'-bipyridyl pigment keto-indigoidine and the reduced colorless but fluorescent leuco-isoform. Detailed characterization of a tailored substrate-free chromogenic assay and FACS analysis showed that indigoidine's blue color and fluorescence could be reliably profiled in bacteria and mammalian cells using standard multiwell-compatible detection equipment. Besides serving as an inexpensive, reliable, versatile and easy-to-assay cross-kingdom reporter system, the potential of having mammalian cells produce non-ribosomal peptides, preferably ones with biopharmaceutical activities, may provide novel treatment opportunities in future gene- and cell-based therapies.

Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin.

Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida-mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies.

Targeting virulence regulation to disarm Acinetobacter baumannii pathogenesis.

The development of anti-virulence drug therapy against Acinetobacter baumannii infections would provide an alternative to traditional antibacterial therapy that are increasingly failing. Here, we demonstrate that the OmpR transcriptional regulator plays a pivotal role in the pathogenesis of diverse A. baumannii clinical strains in multiple murine and G. mellonella invertebrate infection models. We identified OmpR-regulated genes using RNA sequencing and further validated two genes whose expression can be used as robust biomarker to quantify OmpR inhibition in A. baumannii. Moreover, the determination of the structure of the OmpR DNA binding domain of A. baumannii and the development of in vitro protein-DNA binding assays enabled the identification of an OmpR small molecule inhibitor. We conclude that OmpR is a valid and unexplored target to fight A. baumannii infections and we believe that the described platform combining in silico methods, in vitro OmpR inhibitory assays and in vivo G. mellonella surrogate infection model will facilitate future drug discovery programs.

The small-molecule SMARt751 reverses Mycobacterium tuberculosis resistance to ethionamide in acute and chronic mouse models of tuberculosis.

The sensitivity of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis, which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.

Rifabutin for infusion (BV100) for the treatment of severe carbapenem-resistant Acinetobacter baumannii infections.

Rifamycin antibiotics were discovered during the 1950s, and their main representative, rifampicin, remains a cornerstone treatment for TB. The clinical use of rifamycin is restricted to mycobacteria and Gram-positive infections because of its poor ability to penetrate the Gram-negative outer membrane. Rifabutin, a rifamycin antibiotic approved for the prevention of Mycobacterium avium complex disease, makes an exception to this rule by hijacking the iron uptake system of Acinetobacter baumannii, resulting in potent activity against this important Gram-negative pathogen. Here, we describe recent findings on the specific activity of rifabutin and provide evidence of the need for the development of an intravenous formulation of rifabutin (BV100) for the treatment of difficult-to-treat carbapenem-resistant A.baumannii infections.

The Role of OmpR in Bile Tolerance and Pathogenesis of Adherent-Invasive Escherichia coli.

Gut microbiota dysbiosis toward adherent-invasive Escherichia coli (AIEC) plays an important role in Crohn's disease (CD). The OmpR transcriptional regulator is required for the AIEC LF82 prototype strain to adhere and invade intestinal epithelial cells. In this study, we explored the role of OmpR in AIEC pathogenesis using a panel of eight Escherichia coli strains isolated from CD patients and identified as AIEC. The deletion of ompR together with the implementation of two cell-based assays revealed that the role of OmpR in adhesion in vitro was not conserved in AIEC clinical strains. Nevertheless, we showed that OmpR was required for robust gut colonization of transgenic mice expressing human CEACAM receptors, suggesting that OmpR is involved in alternative virulence mechanisms in AIEC strains. We found that deletion of ompR compromised the ability of AIEC strains to cope with the stress induced by bile salts, which may be key for AIEC pathogenesis. More specifically, we demonstrated that OmpR was involved in a tolerance mechanism toward sodium deoxycholate (DOC), one of bile salts main component. We showed that the misregulation of OmpF or the loss of outer membrane integrity are not the drivers of OmpR-mediated DOC tolerance, suggesting that OmpR regulates a specific mechanism enhancing AIEC survival in the presence of DOC. In conclusion, the newly discovered role of OmpR in AIEC bile tolerance suggests that OmpR inhibition would interfere with different aspects of AIEC virulence arsenal and could be an alternative strategy for CD-treatment.

Compounds targeting OSBPL7 increase ABCA1-dependent cholesterol efflux preserving kidney function in two models of kidney disease.

Impaired cellular cholesterol efflux is a key factor in the progression of renal, cardiovascular, and autoimmune diseases. Here we describe a class of 5-arylnicotinamide compounds, identified through phenotypic drug discovery, that upregulate ABCA1-dependent cholesterol efflux by targeting Oxysterol Binding Protein Like 7 (OSBPL7). OSBPL7 was identified as the molecular target of these compounds through a chemical biology approach, employing a photoactivatable 5-arylnicotinamide derivative in a cellular cross-linking/immunoprecipitation assay. Further evaluation of two compounds (Cpd A and Cpd G) showed that they induced ABCA1 and cholesterol efflux from podocytes in vitro and normalized proteinuria and prevented renal function decline in mouse models of proteinuric kidney disease: Adriamycin-induced nephropathy and Alport Syndrome. In conclusion, we show that small molecule drugs targeting OSBPL7 reveal an alternative mechanism to upregulate ABCA1, and may represent a promising new therapeutic strategy for the treatment of renal diseases and other disorders of cellular cholesterol homeostasis.

A nutrient-limited screen unmasks rifabutin hyperactivity for extensively drug-resistant Acinetobacter baumannii.

Industry screens of large chemical libraries have traditionally relied on rich media to ensure rapid bacterial growth in high-throughput testing. We used eukaryotic, nutrient-limited growth media in a compound screen that unmasked a previously unknown hyperactivity of the old antibiotic, rifabutin (RBT), against highly resistant Acinetobacter baumannii. In nutrient-limited, but not rich, media, RBT was 200-fold more potent than rifampin. RBT was also substantially more effective in vivo. The mechanism of enhanced efficacy was a Trojan horse-like import of RBT, but not rifampin, through fhuE, only in nutrient-limited conditions. These results are of fundamental importance to efforts to discover antibacterial agents.

Dissecting Colistin Resistance Mechanisms in Extensively Drug-Resistant Acinetobacter baumannii Clinical Isolates.

Nosocomial infections with Acinetobacter baumannii are a global problem in intensive care units with high mortality rates. Increasing resistance to first- and second-line antibiotics has forced the use of colistin as last-resort treatment, and increasing development of colistin resistance in A. baumannii has been reported. We evaluated the transcriptional regulator PmrA as potential drug target to restore colistin efficacy in A. baumannii Deletion of pmrA restored colistin susceptibility in 10 of the 12 extensively drug-resistant A. baumannii clinical isolates studied, indicating the importance of PmrA in the drug resistance phenotype. However, two strains remained highly resistant, indicating that PmrA-mediated overexpression of the phosphoethanolamine (PetN) transferase PmrC is not the exclusive colistin resistance mechanism in A. baumannii A detailed genetic characterization revealed a new colistin resistance mechanism mediated by genetic integration of the insertion element ISAbaI upstream of the PmrC homolog EptA (93% identity), leading to its overexpression. We found that eptA was ubiquitously present in clinical strains belonging to the international clone 2, and ISAbaI integration upstream of eptA was required to mediate the colistin-resistant phenotype. In addition, we found a duplicated ISAbaI-eptA cassette in one isolate, indicating that this colistin resistance determinant may be embedded in a mobile genetic element. Our data disprove PmrA as a drug target for adjuvant therapy but highlight the importance of PetN transferase-mediated colistin resistance in clinical strains. We suggest that direct targeting of the homologous PetN transferases PmrC/EptA may have the potential to overcome colistin resistance in A. baumanniiIMPORTANCE The discovery of antibiotics revolutionized modern medicine and enabled us to cure previously deadly bacterial infections. However, a progressive increase in antibiotic resistance rates is a major and global threat for our health care system. Colistin represents one of our last-resort antibiotics that is still active against most Gram-negative bacterial pathogens, but increasing resistance is reported worldwide, in particular due to the plasmid-encoded protein MCR-1 present in pathogens such as Escherichia coli and Klebsiella pneumoniae Here, we showed that colistin resistance in A. baumannii, a top-priority pathogen causing deadly nosocomial infections, is mediated through different avenues that result in increased activity of homologous phosphoethanolamine (PetN) transferases. Considering that MCR-1 is also a PetN transferase, our findings indicate that PetN transferases might be the Achilles heel of superbugs and that direct targeting of them may have the potential to preserve the activity of polymyxin antibiotics.

Quantitative contribution of efflux to multi-drug resistance of clinical Escherichia coli and Pseudomonas aeruginosa strains.

BACKGROUND: Efflux pumps mediate antimicrobial resistance in several WHO critical priority bacterial pathogens. However, most available data come from laboratory strains. The quantitative relevance of efflux in more relevant clinical isolates remains largely unknown. METHODS: We developed a versatile method for genetic engineering in multi-drug resistant (MDR) bacteria, and used this method to delete tolC and specific antibiotic-resistance genes in 18 representative MDR clinical E. coli isolates. We determined efflux activity and minimal inhibitory concentrations for a diverse set of clinically relevant antibiotics in these mutants. We also deleted oprM in MDR P. aeruginosa strains and determined the impact on antibiotic susceptibility. FINDINGS: tolC deletion abolished detectable efflux activity in 15 out of 18 tested E. coli strains, and modulated antibiotic susceptibility in many strains. However, all mutant strains retained MDR status, primarily because of other, antibiotic-specific resistance genes. Deletion of oprM altered antibiotic susceptibility in a fraction of clinical P. aeruginosa isolates. INTERPRETATION: Efflux modulates antibiotic resistance in clinical MDR isolates of E. coli and P. aeruginosa. However, when other antimicrobial-resistance mechanisms are present, inhibition of MDR efflux pumps alone is often not sufficient to restore full susceptibility even for antibiotics with a dramatic impact of efflux in laboratory strains. We propose that development of novel antibiotics should include target validation in clinical MDR isolates. FUND: Innovative Medicines Initiative of European Union and EFPIA, Schweizerischer Nationalfonds, Swiss National Research Program 72, EU Marie Sklodowska-Curie program. The funders played no role in design, data collection, data analysis, interpretation, writing of the report, and in the decision to submit the paper for publication.

A fragment-based approach towards the discovery of N-substituted tropinones as inhibitors of Mycobacterium tuberculosis transcriptional regulator EthR2.

Tuberculosis (TB) caused by the pathogen Mycobacterium tuberculosis, represents one of the most challenging threat to public health worldwide, and with the increasing resistance to approved TB drugs, it is needed to develop new strategies to address this issue. Ethionamide is one of the most widely used drugs for the treatment of multidrug-resistant TB. It is a prodrug that requires activation by mycobacterial monooxygenases to inhibit the enoyl-ACP reductase InhA, which is involved in mycolic acid biosynthesis. Very recently, we identified that inhibition of a transcriptional repressor, termed EthR2, derepresses a new bioactivation pathway that results in the boosting of ethionamide activation. Herein, we describe the identification of potent EthR2 inhibitors using fragment-based screening and structure-based optimization. A target-based screening of a fragment library using thermal shift assay followed by X-ray crystallography identified 5 hits. Rapid optimization of the tropinone chemical series led to compounds with improved in vitro potency.

Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties.

A monomeric variant of triosephosphate isomerase (TIM) with a new engineered binding groove has been characterized further. In this variant (ml8bTIM), the phosphate binding loop had been shortened, causing the binding site to be much more extended. Here, it is reported that in the V233A variant of ml8bTIM (A-TIM), three important properties of the wild-type TIM active site have been restored: (i) the structural properties of loop-7, (ii) the binding site of a conserved water molecule between loop-7 and loop-8 and (iii) the binding site of the phosphate moiety. It is shown that the active site of A-TIM can bind TIM transition state analogs and suicide inhibitors competently. It is found that the active site geometry of the A-TIM complexes is less compact and more solvent exposed, as in wild-type TIM. This correlates with the observation that the catalytic efficiency of A-TIM for interconverting the TIM substrates is too low to be detected. It is also shown that the A-TIM active site can bind compounds which do not bind to wild-type TIM and which are completely different from the normal TIM substrate, like a citrate molecule. The binding of this citrate molecule is stabilized by hydrogen bonding interactions with the new binding groove.

Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420.

Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis have now spread worldwide. Some key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in M. tuberculosis, circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.

Antisense RNA based down-regulation of RNaseE in E. coli.

BACKGROUND: Messenger RNA decay is an important mechanism for controlling gene expression in all organisms. The rate of the mRNA degradation directly affects the steady state concentration of mRNAs and therefore influences the protein synthesis. RNaseE has a key importance for the general mRNA decay in E. coli. While RNaseE initiates the degradation of most mRNAs in E. coli, it is likely that the enzyme is also responsible for the degradation of recombinant RNAs. As RNaseE is essential for cell viability and knockout mutants cannot be cultured, we investigated the possibility for a down-regulation of the intracellular level of RNaseE by antisense RNAs. During this study, an antisense RNA based approach could be established which revealed a strong reduction of the intracellular level of RNaseE in E. coli. RESULTS: Despite the autoregulation of rne mRNA by its gene product, significant antisense downregulation of RNaseE is possible. The expression of antisense RNAs did not effect the cell growth negatively. The amount of antisense RNA was monitored quantitatively by a fluorescence based sandwich hybridisation assay. Induction by anhydrotetracycline was followed by a 25-fold increase of the detectable antisense RNA molecules per cell. The antisense RNA level was maintained above 400 molecules per cell until the stationary phase, which caused the level of expressed antisense RNAs to decrease markedly. Western blot experiments revealed the strongest reduction in the RNaseE protein level 90 min after antisense RNA induction. The cellular level of RNaseE could be decreased to 35% of the wild type level. When the growth entered the stationary phase, the RNaseE level was maintained still at 50 to 60% of the wild type level. CONCLUSION: In difference to eukaryotic cells, where the RNAi technology is widely used, this technology is rather unexplored in bacteria, although different natural systems use antisense RNA-based silencing, and a few studies have earlier indicated the potential of this technology also in prokaryotes. Our results show that even complicated self-regulatory systems such as RNaseE may be controlled by antisense RNA technology, indicating that systems based on antisense RNA expression may have a potential for controlling detrimental factors with plasmid-based constructs in arbitrary strains while maintaining their beneficial characteristics. The study also proved that the RNA sandwich hybridisation technique is directly applicable to quantify small RNA molecules in crude cell extracts, which may have a broader application potential as a monitoring tool in RNA inhibition applications.

A designer cell-based histamine-specific human allergy profiler.

Allergic disorders are markedly increasing in industrialized countries. The identification of compounds that trigger the immunoglobulin E-dependent allergic reaction remains the key to limit patients' exposure to critical allergens and improve their quality of life. Here we use synthetic biology principles to design a mammalian cell-based allergy profiler that scores the allergen-triggered release of histamine from whole-blood-derived human basophils. A synthetic signalling cascade engineered within the allergy profiler rewires histamine input to the production of reporter protein, thereby integrating histamine levels in whole-blood samples with remarkable sensitivity and a wide dynamic range, allowing for rapid results or long-term storage of output, respectively. This approach provides non-intrusive allergy profiles for the personalized medicine era.