RESUMEN
Gonorrhea, which is caused by Neisseria gonorrhoeae, is the second most prevalent sexually transmitted infection worldwide. The increasing appearance of isolates that are resistant to approved therapeutics raises the concern that gonorrhea may become untreatable. Here, we serendipitously identified oxydifficidin as a potent N. gonorrhoeae antibiotic through the observation of a Bacillus amyloliquefaciens contaminant in a lawn of N. gonorrhoeae. Oxydifficidin is active against both wild-type and multidrug-resistant N. gonorrhoeae. It's potent activity results from a combination of DedA-assisted uptake into the cytoplasm and the presence of an oxydifficidin-sensitive ribosomal protein L7/L12 (RplL). Our data indicates that oxydifficidin binds to the ribosome at a site that is distinct from other antibiotics and that L7/L12 is uniquely associated with its mode of action. This study opens a potential new avenue for addressing antibiotic resistant gonorrhea and underscores the possibility of identifying overlooked natural products from cultured bacteria, particularly those with activity against previously understudied pathogens.
RESUMEN
Most biosynthetic gene clusters (BGCs) are functionally inaccessible by using fermentation methods. Bioinformatic-coupled total synthesis provides an alternative approach for accessing BGC-encoded bioactivities. To date, synthetic bioinformatic natural product (synBNP) methods have focused on lipopeptides containing simple lipids. Here we increase the bioinformatic and synthetic complexity of the synBNP approach by targeting BGCs that encode N-cinnamoyl lipids. This led to our synthesis of cinnamosyn, a 10-mer N-cinnamoyl-containing peptide that is cytotoxic to human cells.
Asunto(s)
Productos Biológicos , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/síntesis química , Humanos , Estructura Molecular , Biología Computacional , Familia de Multigenes , Lipopéptidos/química , Lipopéptidos/farmacología , Lipopéptidos/síntesis química , Cinamatos/química , Cinamatos/farmacología , Cinamatos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis químicaRESUMEN
Bacteria have adapted to phage predation by evolving a vast assortment of defence systems1. Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence data2. Here, to overcome this limitation, we infected Escherichia coli carrying a soil metagenomic DNA library3 with the lytic coliphage T4 to isolate clones carrying protective genes. Following this approach, we identified Brig1, a DNA glycosylase that excises α-glucosyl-hydroxymethylcytosine nucleobases from the bacteriophage T4 genome to generate abasic sites and inhibit viral replication. Brig1 homologues that provide immunity against T-even phages are present in multiple phage defence loci across distinct clades of bacteria. Our study highlights the benefits of screening unsequenced DNA and reveals prokaryotic DNA glycosylases as important players in the bacteria-phage arms race.
Asunto(s)
Bacterias , Bacteriófago T4 , ADN Glicosilasas , Bacterias/clasificación , Bacterias/enzimología , Bacterias/genética , Bacterias/inmunología , Bacterias/virología , Bacteriófago T4/crecimiento & desarrollo , Bacteriófago T4/inmunología , Bacteriófago T4/metabolismo , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Escherichia coli/genética , Escherichia coli/virología , Biblioteca de Genes , Metagenómica/métodos , Microbiología del Suelo , Replicación ViralRESUMEN
Cilagicin is a dual polyprenyl phosphate binding lipodepsipeptide antibiotic with strong activity against clinically relevant Gram-positive pathogens while evading antibiotic resistance. Cilagicin showed high serum binding that reduced its in vivo efficacy. Cilagicin-BP, which contains a biphenyl moiety in place of the N-terminal myristic acid found on cilagicin, showed reduced serum binding and increased in vivo efficacy but decreased potency against some pathogens. Here, we manipulated the acyl tail and the peptide core of cilagicin to identify an optimized collection of structural features that maintain potent antibiotic activity against a wide range of pathogens in the presence of serum. This led to the identification of the optimized antibiotic dodecacilagicin, which contains an N-terminal dodecanoic acid. Dodecacilagicin exhibits low MICs against clinically relevant pathogens in the presence of serum, retains polyprenyl phosphate binding, and evades resistance development even after long-term antibiotic exposure, making dodecacilagicin an appealing candidate for further therapeutic development.
Asunto(s)
Antibacterianos , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/química , Humanos , Farmacorresistencia Bacteriana/efectos de los fármacos , Depsipéptidos/farmacología , Depsipéptidos/química , Bacterias Grampositivas/efectos de los fármacosRESUMEN
DNA topoisomerases are attractive targets for anticancer agents. Dual topoisomeraseâ I/II inhibitors are particularly appealing due to their reduced rates of resistance. A number of therapeutically relevant topoisomerase inhibitors are bacterial natural products. Mining the untapped chemical diversity encoded by soil microbiomes presents an opportunity to identify additional natural topoisomerase inhibitors. Here we couple metagenome mining, bioinformatic structure prediction algorithms, and chemical synthesis to produce the dual topoisomerase inhibitor tapcin. Tapcin is a mixed p-aminobenzoic acid (PABA)-thiazole with a rare tri-thiazole substructure and picomolar antiproliferative activity. Tapcin reduced colorectal adenocarcinoma HT-29â cell proliferation and tumor volume in mouse hollow fiber and xenograft models, respectively. In both studies it showed similar activity to the clinically used topoisomeraseâ I inhibitor irinotecan. The study suggests that the interrogation of soil microbiomes using synthetic bioinformatic natural product methods has the potential to be a rewarding strategy for identifying potent, biomedically relevant, antiproliferative agents.
Asunto(s)
Antineoplásicos , Productos Biológicos , Humanos , Ratones , Animales , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Productos Biológicos/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Biología Computacional , Suelo , Tiazoles , Línea Celular TumoralRESUMEN
Cilagicin is a Gram-positive active antibiotic that has a dual polyprenyl phosphate binding mechanism that impedes resistance development. Here we bioinformatically screened predicted non-ribosomal polypeptide synthetase encoded structures to search for antibiotics that might similarly avoid resistance development. Synthesis and bioactivity screening of the predicted structures that we identified led to three antibiotics that are active against multidrug-resistant Gram-positive pathogens, two of which, paenilagicin and virgilagicin, did not lead to resistance even after prolonged antibiotic exposure.
Asunto(s)
Antibacterianos , Fosfatos de Poliisoprenilo , Antibacterianos/farmacología , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , FosfatosRESUMEN
Cyclic oligonucleotide-based antiphage signalling systems (CBASS) protect prokaryotes from viral (phage) attack through the production of cyclic oligonucleotides, which activate effector proteins that trigger the death of the infected host1,2. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a structured RNA transcribed from the terminase subunit genes, termed CBASS-activating bacteriophage RNA (cabRNA), which binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP to activate the CBASS immune response. Phages that escape the CBASS defence harbour mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. As the mammalian cyclase OAS1 also binds viral double-stranded RNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defence pathways.
Asunto(s)
Bacterias , Nucleotidiltransferasas , ARN Viral , Fagos de Staphylococcus , Animales , 2',5'-Oligoadenilato Sintetasa/metabolismo , Bacterias/enzimología , Bacterias/inmunología , Evolución Molecular , Inmunidad Innata , Nucleotidiltransferasas/metabolismo , Oligonucleótidos/inmunología , Oligonucleótidos/metabolismo , ARN Viral/inmunología , ARN Viral/metabolismo , Transducción de Señal/inmunología , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/inmunologíaRESUMEN
Novel antibiotics are in constant demand to combat a global increase in antibiotic-resistant infections. Bacterial natural products have been a long-standing source of antibiotic compounds, and metagenomic mining of environmental DNA (eDNA) has increasingly provided new antibiotic leads. The metagenomic small-molecule discovery pipeline can be divided into three main steps: surveying eDNA, retrieving a sequence of interest, and accessing the encoded natural product. Improvements in sequencing technology, bioinformatic algorithms, and methods for converting biosynthetic gene clusters into small molecules are steadily increasing our ability to discover metagenomically encoded antibiotics. We predict that, over the next decade, ongoing technological improvements will dramatically increase the rate at which antibiotics are discovered from metagenomes.
Asunto(s)
Productos Biológicos , ADN Ambiental , Antibacterianos/farmacología , Bacterias/genética , Metagenómica , Familia de MultigenesRESUMEN
The capture of metagenomic DNA in large clone libraries provides the opportunity to study microbial diversity that is inaccessible using culture-dependent methods. In this study, we harnessed nuclease-deficient Cas9 to establish a CRISPR counter-selection interruption circuit (CCIC) that can be used to retrieve target clones from complex libraries. Combining modern sequencing methods with CCIC cloning allows for rapid physical access to the genetic diversity present in natural ecosystems.
Asunto(s)
Ecosistema , Metagenómica , Células ClonalesRESUMEN
Bacterial genomes contain large reservoirs of biosynthetic gene clusters (BGCs) that are predicted to encode unexplored natural products. Heterologous expression of previously unstudied BGCs should facilitate the discovery of additional therapeutically relevant bioactive molecules from bacterial culture collections, but the large-scale manipulation of BGCs remains cumbersome. Here, we describe a method to parallelize the identification, mobilization and heterologous expression of BGCs. Our solution simultaneously captures large numbers of BGCs by cloning the genomes of a strain collection in a large-insert library and uses the CONKAT-seq (co-occurrence network analysis of targeted sequences) sequencing pipeline to efficiently localize clones carrying intact BGCs which represent candidates for heterologous expression. Our discovery of several natural products, including an antibiotic that is active against multi-drug resistant Staphylococcus aureus, demonstrates the potential of leveraging economies of scale with this approach to systematically interrogate cryptic BGCs contained in strain collections.
Asunto(s)
Productos Biológicos , Staphylococcus aureus Resistente a Meticilina , Antibacterianos , Productos Biológicos/metabolismo , Vías Biosintéticas/genética , Genoma Bacteriano/genética , Staphylococcus aureus Resistente a Meticilina/genética , Familia de MultigenesRESUMEN
Cationic nonribosomal lipopeptides (CNRLPs) from Paenibacillus spp. have been a rewarding source of Gram-negative-active antibiotics. Here we systematically screened sequenced bacterial genomes for CNRLP biosynthetic gene clusters (BGCs) that we predicted might encode additional Gram-negative-active antibiotics. Total chemical synthesis of the bioinformatically predicted products of seven such BGCs led to our identification of new laterocidine, tridecaptin, and paenibacterin-like antibiotics with potent activity against both multiple-drug-resistant Gram-negative and Gram-positive pathogens.
Asunto(s)
Antibacterianos , Paenibacillus , Antibacterianos/farmacología , Genoma Bacteriano , Lipopéptidos/farmacología , Familia de Multigenes , Paenibacillus/genéticaRESUMEN
The mechanisms by which commensal organisms affect human physiology remain poorly understood. Lectins are non-enzymatic carbohydrate binding proteins that all organisms employ as part of establishing a niche, evading host-defenses and protecting against pathogens. Although lectins have been extensively studied in plants, bacterial pathogens and human immune cells for their role in disease pathophysiology and as therapeutics, the role of bacterial lectins in the human microbiome is largely unexplored. Here we report on the characterization of a lectin produced by a common human associated bacterium that interacts with myeloid cells in the blood and intestine. In mouse and cell-based models, we demonstrate that this lectin induces distinct immunologic responses in peripheral and intestinal leukocytes and that these responses are specific to monocytes, macrophages and dendritic cells. Our analysis of human microbiota sequencing data reveal thousands of unique sequences that are predicted to encode lectins, many of which are highly prevalent in the human microbiome yet completely uncharacterized. Based on the varied domain architectures of these lectins we predict they will have diverse effects on the human host. The systematic investigation of lectins in the human microbiome should improve our understanding of human health and provide new therapeutic opportunities.
Asunto(s)
Lectinas , Microbiota , Animales , Bacterias/metabolismo , Humanos , Lectinas/metabolismo , Ratones , Plantas/metabolismoRESUMEN
Emerging resistance to currently used antibiotics is a global public health crisis. Because most of the biosynthetic capacity within the bacterial kingdom has remained silent in previous antibiotic discovery efforts, uncharacterized biosynthetic gene clusters found in bacterial genome-sequencing studies remain an appealing source of antibiotics with distinctive modes of action. Here, we report the discovery of a naturally inspired lipopeptide antibiotic called cilagicin, which we chemically synthesized on the basis of a detailed bioinformatic analysis of the cil biosynthetic gene cluster. Cilagicin's ability to sequester two distinct, indispensable undecaprenyl phosphates used in cell wall biosynthesis, together with the absence of detectable resistance in laboratory tests and among multidrug-resistant clinical isolates, makes it an appealing candidate for combating antibiotic-resistant pathogens.
Asunto(s)
Antibacterianos , Lipopéptidos , Antibacterianos/farmacología , Biología Computacional , Lipopéptidos/farmacología , Pruebas de Sensibilidad Microbiana , Familia de MultigenesRESUMEN
In natural product discovery programs, the power of synthetic chemistry is often leveraged for the total synthesis and diversification of characterized metabolites. The synthesis of structures that are bioinformatically predicted to arise from uncharacterized biosynthetic gene clusters (BGCs) provides a means for synthetic chemistry to enter this process at an early stage. The recent identification of non-ribosomal peptides (NRPs) containing multiple ρ-aminobenzoic acids (PABAs) led us to search soil metagenomes for BGCs that polymerize PABA. Here, we use PABA-specific adenylation-domain sequences to guide the cloning of the lap BGC directly from soil. This BGC was predicted to encode a unique N-acylated PABA and thiazole containing structure. Chemical synthesis of this structure gave lapcin, a dual topoisomerase I/II inhibitor with nM to pM IC50s against diverse cancer cell lines. The discovery of lapcin highlights the power of coupling metagenomics, bioinformatics and total chemical synthesis to unlock the biosynthetic potential contained in even complex uncharacterized BGCs.
Asunto(s)
Productos Biológicos/farmacología , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Metagenoma , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Vías Biosintéticas/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Biología Computacional , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Metagenoma/genética , Metagenómica , Familia de Multigenes , SueloRESUMEN
Gram-negative bacteria are responsible for an increasing number of deaths caused by antibiotic-resistant infections1,2. The bacterial natural product colistin is considered the last line of defence against a number of Gram-negative pathogens. The recent global spread of the plasmid-borne mobilized colistin-resistance gene mcr-1 (phosphoethanolamine transferase) threatens the usefulness of colistin3. Bacteria-derived antibiotics often appear in nature as collections of similar structures that are encoded by evolutionarily related biosynthetic gene clusters. This structural diversity is, at least in part, expected to be a response to the development of natural resistance, which often mechanistically mimics clinical resistance. Here we propose that a solution to mcr-1-mediated resistance might have evolved among naturally occurring colistin congeners. Bioinformatic analysis of sequenced bacterial genomes identified a biosynthetic gene cluster that was predicted to encode a structurally divergent colistin congener. Chemical synthesis of this structure produced macolacin, which is active against Gram-negative pathogens expressing mcr-1 and intrinsically resistant pathogens with chromosomally encoded phosphoethanolamine transferase genes. These Gram-negative bacteria include extensively drug-resistant Acinetobacter baumannii and intrinsically colistin-resistant Neisseria gonorrhoeae, which, owing to a lack of effective treatment options, are considered among the highest level threat pathogens4. In a mouse neutropenic infection model, a biphenyl analogue of macolacin proved to be effective against extensively drug-resistant A. baumannii with colistin-resistance, thus providing a naturally inspired and easily produced therapeutic lead for overcoming colistin-resistant pathogens.
Asunto(s)
Antibacterianos , Colistina , Farmacorresistencia Bacteriana , Bacterias Gramnegativas , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/enzimología , Acinetobacter baumannii/genética , Animales , Antibacterianos/farmacología , Vías Biosintéticas/genética , Colistina/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Etanolaminas , Genes Bacterianos , Genoma Bacteriano , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/genética , Ratones , Pruebas de Sensibilidad Microbiana , Familia de Multigenes , Neutropenia/tratamiento farmacológico , Neutropenia/microbiología , Plásmidos , Transferasas (Grupos de Otros Fosfatos Sustitutos)RESUMEN
Bacterial resistance threatens the utility of currently available antibiotics. Rifampicin, a cornerstone in the treatment of persistent Gram-positive infections, is prone to the development of resistance resulting from single point mutations in the antibiotic's target, RNA polymerase. One strategy to circumvent resistance is the use of 'hybrid' antibiotics consisting of two covalently linked antibiotic entities. These compounds generally have two distinct cellular targets, reducing the probability of resistance development and potentially providing simplified pharmacological properties compared to combination therapies using the individual antibiotics. Here we evaluate a series of semi-synthetic hybrid antibiotics formed by linking kanglemycin A (Kang A), a rifampicin analog, and a collection of fluoroquinolones. Kang A is a natural product antibiotic which contains a novel dimethyl succinic acid moiety that offers a new attachment point for the synthesis of hybrid antibiotics. We compare the activity of the Kang A hybrids generated via the acid attachment point to a series of hybrids linked at the compound's naphthoquinone ring system. Several hybrids exhibit activity against bacteria resistant to Kang A via the action of the partnered antibiotic, suggesting that the Kang scaffold may provide new avenues for generating antibiotics effective against drug-resistant infections.
Asunto(s)
Antibacterianos/farmacología , Fluoroquinolonas/farmacología , Rifamicinas/farmacología , Antibacterianos/síntesis química , Antibacterianos/toxicidad , Farmacorresistencia Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Fluoroquinolonas/síntesis química , Fluoroquinolonas/toxicidad , Células HEK293 , Humanos , Pruebas de Sensibilidad Microbiana , Rifamicinas/síntesis química , Rifamicinas/toxicidad , Staphylococcus aureus/efectos de los fármacosRESUMEN
The emergence of multidrug-resistant bacteria poses a threat to global health and necessitates the development of additional in vivo active antibiotics with diverse modes of action. Directly targeting menaquinone (MK), which plays an important role in bacterial electron transport, is an appealing, yet underexplored, mode of action due to a dearth of MK-binding molecules. Here we combine sequence-based metagenomic mining with a motif search of bioinformatically predicted natural product structures to identify six biosynthetic gene clusters that we predicted encode MK-binding antibiotics (MBAs). Their predicted products (MBA1-6) were rapidly accessed using a synthetic bioinformatic natural product approach, which relies on bioinformatic structure prediction followed by chemical synthesis. Among these six structurally diverse MBAs, four make up two new MBA structural families. The most potent member of each new family (MBA3, MBA6) proved effective at treating methicillin-resistant Staphylococcus aureus infection in a murine peritonitis-sepsis model. The only conserved feature present in all MBAs is the sequence 'GXLXXXW', which we propose represents a minimum MK-binding motif. Notably, we found that a subset of MBAs were active against Mycobacterium tuberculosis both in vitro and in macrophages. Our findings suggest that naturally occurring MBAs are a structurally diverse and untapped class of mechanistically interesting, in vivo active antibiotics.
Asunto(s)
Antibacterianos/metabolismo , Antibacterianos/uso terapéutico , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Vitamina K 2/metabolismo , Animales , Farmacorresistencia Bacteriana Múltiple , Femenino , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Metagenómica/métodos , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Peritonitis/tratamiento farmacológico , Peritonitis/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Vitamina K 2/aislamiento & purificaciónRESUMEN
The COVID-19 pandemic has highlighted the need to identify additional antiviral small molecules to complement existing therapies. Although increasing evidence suggests that metabolites produced by the human microbiome have diverse biological activities, their antiviral properties remain poorly explored. Using a cell-based SARS-CoV-2 infection assay, we screened culture broth extracts from a collection of phylogenetically diverse human-associated bacteria for the production of small molecules with antiviral activity. Bioassay-guided fractionation uncovered three bacterial metabolites capable of inhibiting SARS-CoV-2 infection. This included the nucleoside analogue N6-(Δ2-isopentenyl)adenosine, the 5-hydroxytryptamine receptor agonist tryptamine, and the pyrazine 2,5-bis(3-indolylmethyl)pyrazine. The most potent of these, N6-(Δ2-isopentenyl)adenosine, had a 50% inhibitory concentration (IC50) of 2 µM. These natural antiviral compounds exhibit structural and functional similarities to synthetic drugs that have been clinically examined for use against COVID-19. Our discovery of structurally diverse metabolites with anti-SARS-CoV-2 activity from screening a small fraction of the bacteria reported to be associated with the human microbiome suggests that continued exploration of phylogenetically diverse human-associated bacteria is likely to uncover additional small molecules that inhibit SARS-CoV-2 as well as other viral infections. IMPORTANCE The continued prevalence of COVID-19 and the emergence of new variants has once again put the spotlight on the need for the identification of SARS-CoV-2 antivirals. The human microbiome produces an array of small molecules with bioactivities (e.g., host receptor ligands), but its ability to produce antiviral small molecules is relatively underexplored. Here, using a cell-based screening platform, we describe the isolation of three microbiome-derived metabolites that are able to prevent SARS-CoV-2 infection in vitro. These molecules display structural similarities to synthetic drugs that have been explored for the treatment of COVID-19, and these results suggest that the microbiome may be a fruitful source of the discovery of small molecules with antiviral activities.
Asunto(s)
Antivirales/farmacología , Bacterias/metabolismo , Medios de Cultivo/química , Redes y Vías Metabólicas , Microbiota/fisiología , SARS-CoV-2/efectos de los fármacos , Simbiosis/fisiología , Bacterias/química , Bacterias/clasificación , Bacterias/crecimiento & desarrollo , Bioensayo , Línea Celular Tumoral , Medios de Cultivo/farmacología , Humanos , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/farmacología , Unión ProteicaRESUMEN
The human microbiota plays a critical role in host health. Proper development of the infant microbiome is particularly important. Its dysbiosis leads to both short-term health issues and long-term disorders lasting into adulthood. A central way in which the microbiome interacts with the host is through the production of effector molecules, such as proteins and small molecules. Here, a metagenomic library constructed from 14 infant stool microbiomes is analyzed for the production of effectors that modulate three distinct host pathways: immune response (nuclear factor κB [NF-κB] activation), autophagy (LC3-B puncta formation), and redox potential (NADH:NAD ratio). We identify microbiome-encoded bioactive metabolites, including commendamide and hydrogen sulfide and their associated biosynthetic genes, as well as a previously uncharacterized autophagy-inducing operon from Klebsiella spp. This work extends our understanding of microbial effector molecules that are known to influence host pathways. Parallel functional screening of metagenomic libraries can be easily expanded to investigate additional host processes.