Structural basis of ribosomal 30S subunit degradation by RNase R.

Protein synthesis is a major energy-consuming process of the cell that requires the controlled production1-3 and turnover4,5 of ribosomes. Although the past few years have seen major advances in our understanding of ribosome biogenesis, structural insight into the degradation of ribosomes has been lacking. Here we present native structures of two distinct small ribosomal 30S subunit degradation intermediates associated with the 3' to 5' exonuclease ribonuclease R (RNase R). The structures reveal that RNase R binds at first to the 30S platform to facilitate the degradation of the functionally important anti-Shine-Dalgarno sequence and the decoding-site helix 44. RNase R then encounters a roadblock when it reaches the neck region of the 30S subunit, and this is overcome by a major structural rearrangement of the 30S head, involving the loss of ribosomal proteins. RNase R parallels this movement and relocates to the decoding site by using its N-terminal helix-turn-helix domain as an anchor. In vitro degradation assays suggest that head rearrangement poses a major kinetic barrier for RNase R, but also indicate that the enzyme alone is sufficient for complete degradation of 30S subunits. Collectively, our results provide a mechanistic basis for the degradation of 30S mediated by RNase R, and reveal that RNase R targets orphaned 30S subunits using a dynamic mechanism involving an anchored switching of binding sites.

Uncovering new families and folds in the natural protein universe.

We are now entering a new era in protein sequence and structure annotation, with hundreds of millions of predicted protein structures made available through the AlphaFold database1. These models cover nearly all proteins that are known, including those challenging to annotate for function or putative biological role using standard homology-based approaches. In this study, we examine the extent to which the AlphaFold database has structurally illuminated this 'dark matter' of the natural protein universe at high predicted accuracy. We further describe the protein diversity that these models cover as an annotated interactive sequence similarity network, accessible at https://uniprot3d.org/atlas/AFDB90v4 . By searching for novelties from sequence, structure and semantic perspectives, we uncovered the ß-flower fold, added several protein families to Pfam database2 and experimentally demonstrated that one of these belongs to a new superfamily of translation-targeting toxin-antitoxin systems, TumE-TumA. This work underscores the value of large-scale efforts in identifying, annotating and prioritizing new protein families. By leveraging the recent deep learning revolution in protein bioinformatics, we can now shed light into uncharted areas of the protein universe at an unprecedented scale, paving the way to innovations in life sciences and biotechnology.

Structural Basis for Bacterial Ribosome-Associated Quality Control by RqcH and RqcP.

In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality control (RQC) pathways. RQC begins with the splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal "tails." How such tailing can occur without the regular suite of translational components is, however, unclear. Using single-particle cryo-electron microscopy (EM) of native complexes, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with RqcP, an Hsp15 family protein. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.

RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis.

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.

A field-wide assessment of differential expression profiling by high-throughput sequencing reveals widespread bias.

We assess inferential quality in the field of differential expression profiling by high-throughput sequencing (HT-seq) based on analysis of datasets submitted from 2008 to 2020 to the NCBI GEO data repository. We take advantage of the parallel differential expression testing over thousands of genes, whereby each experiment leads to a large set of p-values, the distribution of which can indicate the validity of assumptions behind the test. From a well-behaved p-value set π0, the fraction of genes that are not differentially expressed can be estimated. We found that only 25% of experiments resulted in theoretically expected p-value histogram shapes, although there is a marked improvement over time. Uniform p-value histogram shapes, indicative of <100 actual effects, were extremely few. Furthermore, although many HT-seq workflows assume that most genes are not differentially expressed, 37% of experiments have π0-s of less than 0.5, as if most genes changed their expression level. Most HT-seq experiments have very small sample sizes and are expected to be underpowered. Nevertheless, the estimated π0-s do not have the expected association with N, suggesting widespread problems of experiments with controlling false discovery rate (FDR). Both the fractions of different p-value histogram types and the π0 values are strongly associated with the differential expression analysis program used by the original authors. While we could double the proportion of theoretically expected p-value distributions by removing low-count features from the analysis, this treatment did not remove the association with the analysis program. Taken together, our results indicate widespread bias in the differential expression profiling field and the unreliability of statistical methods used to analyze HT-seq data.

A role for the S4-domain containing protein YlmH in ribosome-associated quality control in Bacillus subtilis.

Ribosomes trapped on mRNAs during protein synthesis need to be rescued for the cell to survive. The most ubiquitous bacterial ribosome rescue pathway is trans-translation mediated by tmRNA and SmpB. Genetic inactivation of trans-translation can be lethal, unless ribosomes are rescued by ArfA or ArfB alternative rescue factors or the ribosome-associated quality control (RQC) system, which in Bacillus subtilis involves MutS2, RqcH, RqcP and Pth. Using transposon sequencing in a trans-translation-incompetent B. subtilis strain we identify a poorly characterized S4-domain-containing protein YlmH as a novel potential RQC factor. Cryo-EM structures reveal that YlmH binds peptidyl-tRNA-50S complexes in a position analogous to that of S4-domain-containing protein RqcP, and that, similarly to RqcP, YlmH can co-habit with RqcH. Consistently, we show that YlmH can assume the role of RqcP in RQC by facilitating the addition of poly-alanine tails to truncated nascent polypeptides. While in B. subtilis the function of YlmH is redundant with RqcP, our taxonomic analysis reveals that in multiple bacterial phyla RqcP is absent, while YlmH and RqcH are present, suggesting that in these species YlmH plays a central role in the RQC.

The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin-antitoxin and related phage defense systems.

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.

Involvement of Escherichia coli YbeX/CorC in ribosomal metabolism.

YbeX of Escherichia coli, a member of the CorC protein family, is encoded in the same operon with ribosome-associated proteins YbeY and YbeZ. Here, we report the involvement of YbeX in ribosomal metabolism. The ΔybeX cells accumulate distinct 16S rRNA degradation intermediates in the 30S particles and the 70S ribosomes. E. coli lacking ybeX has a lengthened lag phase upon outgrowth from the stationary phase. This growth phenotype is heterogeneous at the individual cell level and especially prominent under low extracellular magnesium levels. The ΔybeX strain is sensitive to elevated growth temperatures and to several ribosome-targeting antibiotics that have in common the ability to induce the cold shock response in E. coli. Although generally milder, the phenotypes of the ΔybeX mutant overlap with those caused by ybeY deletion. A genetic screen revealed partial compensation of the ΔybeX growth phenotype by the overexpression of YbeY. These findings indicate an interconnectedness among the ybeZYX operon genes, highlighting their roles in ribosomal assembly and/or degradation.

Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P.

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

A hyperpromiscuous antitoxin protein domain for the neutralization of diverse toxin domains.

Toxin-antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids as well as temperate bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analyzed the conservation of genomic neighborhoods using our computational tool FlaGs (for flanking genes), which allows high-throughput detection of TA-like operons. Focusing on the widespread but poorly experimentally characterized antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have named the domain Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralized TA pairs. While the majority of validated PanA-neutralized toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises transcription and translation as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralization mechanisms employed by Type II TA systems. Finally, through directed evolution, we have selected PanA variants that can neutralize noncognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain.

Structure of Escherichia coli heat shock protein Hsp15 in complex with the ribosomal 50S subunit bearing peptidyl-tRNA.

In Escherichia coli, the heat shock protein 15 (Hsp15) is part of the cellular response to elevated temperature. Hsp15 interacts with peptidyl-tRNA-50S complexes that arise upon dissociation of translating 70S ribosomes, and is proposed to facilitate their rescue and recycling. A previous structure of E. coli Hsp15 in complex with peptidyl-tRNA-50S complex reported a binding site located at the central protuberance of the 50S subunit. By contrast, recent structures of RqcP, the Hsp15 homolog in Bacillus subtilis, in complex with peptidyl-tRNA-50S complexes have revealed a distinct site positioned between the anticodon-stem-loop (ASL) of the P-site tRNA and H69 of the 23S rRNA. Here we demonstrate that exposure of E. coli cells to heat shock leads to a decrease in 70S ribosomes and accumulation of 50S subunits, thus identifying a natural substrate for Hsp15 binding. Additionally, we have determined a cryo-EM reconstruction of the Hsp15-50S-peptidyl-tRNA complex isolated from heat shocked E. coli cells, revealing that Hsp15 binds to the 50S-peptidyl-tRNA complex analogously to its B. subtilis homolog RqcP. Collectively, our findings support a model where Hsp15 stabilizes the peptidyl-tRNA in the P-site and thereby promotes access to the A-site for putative rescue factors to release the aberrant nascent polypeptide chain.

DrugVirus.info 2.0: an integrative data portal for broad-spectrum antivirals (BSA) and BSA-containing drug combinations (BCCs).

Viruses can cross species barriers and cause unpredictable outbreaks in man with substantial economic and public health burdens. Broad-spectrum antivirals, (BSAs, compounds inhibiting several human viruses), and BSA-containing drug combinations (BCCs) are deemed as immediate therapeutic options that fill the void between virus identification and vaccine development. Here, we present DrugVirus.info 2.0 (https://drugvirus.info), an integrative interactive portal for exploration and analysis of BSAs and BCCs, that greatly expands the database and functionality of DrugVirus.info 1.0 webserver. Through the data portal that now expands the spectrum of BSAs and provides information on BCCs, we developed two modules for (i) interactive analysis of users' own antiviral drug and combination screening data and their comparison with published datasets, and (ii) exploration of the structure-activity relationship between various BSAs. The updated portal provides an essential toolbox for antiviral drug development and repurposing applications aiming to identify existing and novel treatments of emerging and re-emerging viral threats.

Structure of Gcn1 bound to stalled and colliding 80S ribosomes.

The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.

Development of In Vitro and Ex Vivo Biofilm Models for the Assessment of Antibacterial Fibrous Electrospun Wound Dressings.

Increasing evidence suggests that the chronicity of wounds is associated with the presence of bacterial biofilms. Therefore, novel wound care products are being developed, which can inhibit biofilm formation and/or treat already formed biofilms. A lack of standardized assays for the analysis of such novel antibacterial drug delivery systems enhances the need for appropriate tools and models for their characterization. Herein, we demonstrate that optimized and biorelevant in vitro and ex vivo wound infection and biofilm models offer a convenient approach for the testing of novel antibacterial wound dressings for their antibacterial and antibiofilm properties, allowing one to obtain qualitative and quantitative results. The in vitro model was developed using an electrospun (ES) thermally crosslinked gelatin-glucose (GEL-Glu) matrix and an ex vivo wound infection model using pig ear skin. Wound pathogens were used for colonization and biofilm development on the GEL-Glu matrix or pig skin with superficial burn wounds. The in vitro model allowed us to obtain more reproducible results compared with the ex vivo model, whereas the ex vivo model had the advantage that several pathogens preferred to form a biofilm on pig skin compared with the GEL-Glu matrix. The in vitro model functioned poorly for Staphylococcus epidermidis biofilm formation, but it worked well for Escherichia coli and Staphylococcus aureus, which were able to use the GEL-Glu matrix as a nutrient source and not only as a surface for biofilm growth. On the other hand, all tested pathogens were equally able to produce a biofilm on the surface of pig skin. The developed biofilm models enabled us to compare different ES dressings [pristine and chloramphenicol-loaded polycaprolactone (PCL) and PCL-poly(ethylene oxide) (PEO) (PCL/PEO) dressings] and understand their biofilm inhibition and treatment properties on various pathogens. Furthermore, we show that biofilms were formed on the wound surface as well as on a wound dressing, indicating that the demonstrated methods mimic well the in vivo situation. Colony forming unit (CFU) counting and live biofilm matrix as well as bacterial DNA staining together with microscopic imaging were performed for biofilm quantification and visualization, respectively. The results showed that both wound biofilm models (in vitro and ex vivo) enabled the evaluation of the desired antibiofilm properties, thus facilitating the design and development of more effective wound care products and screening of various formulations and active substances.

Seven classes of antiviral agents.

The viral epidemics and pandemics have stimulated the development of known and the discovery of novel antiviral agents. About a hundred mono- and combination antiviral drugs have been already approved, whereas thousands are in development. Here, we briefly reviewed 7 classes of antiviral agents: neutralizing antibodies, neutralizing recombinant soluble human receptors, antiviral CRISPR/Cas systems, interferons, antiviral peptides, antiviral nucleic acid polymers, and antiviral small molecules. Interferons and some small molecules alone or in combinations possess broad-spectrum antiviral activity, which could be beneficial for treatment of emerging and re-emerging viral infections.

A widespread toxin-antitoxin system exploiting growth control via alarmone signaling.

Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.

Investigation of 29 Antimicrobial Compounds in Soil Using Newly Developed UHPLC-MS/MS Method.

While the prudent and reasonable use of veterinary antimicrobial agents in food-producing animals is necessary, researchers over the decades have shown that these antimicrobial agents can spread into the environment through livestock manure and wastewater. The analysis of the occurrence of antimicrobial compounds in soil samples is of a great importance to determine potential impacts on human and animal health and the environment. In this study, an affordable, rugged and simple analytical method has been developed for the determination of twenty-nine antimicrobial compounds from five different classes (tetracyclines, fluoro(quinolones), macrolides, sulfonamides and diaminopirimidines). Liquid-liquid extraction (LLE) with extract filtration combined with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was the best strategy for the simultaneous determination of all analytes. The developed method was validated according to the Commission Implementing Regulation (EU) 2021/808. The limit of detections (LODs) ranged from 0.5 to 2.0 µg/kg, while the limit of quantitation (LOQ) was established at 1.0 to 20.0 µg/kg. The developed method was successfully applied for the determination of antimicrobial residues in one hundred and eighteen soil samples obtained from four European countries (Austria, Czech Republic, Estonia and Portugal). Doxycycline in the concentration levels of 9.07 µg/kg-20.6 µg/kg was detected in eight of the analysed samples. Samples were collected from areas where natural fertilizers (swine or cow manure) were applied. Our method can be efficiently used to monitor anti-microbial compounds in soil samples.

Making Antimicrobial Susceptibility Testing More Physiologically Relevant with Bicarbonate?

Azithromycin is a clinically important drug for treating invasive salmonellosis despite poor activity in laboratory assays for MIC. Addition of the main buffer in blood, bicarbonate, has been proposed for more physiologically relevant and more predictive testing conditions. However, we show here that bicarbonate-triggered lowering of azithromycin MIC is entirely due to alkalization of insufficiently buffered media. In addition, bicarbonate is unlikely to be altering efflux pump activity.

Uropathogenic Escherichia coli Shows Antibiotic Tolerance and Growth Heterogeneity in an In Vitro Model of Intracellular Infection.

Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections, can invade different types of host cells. To compare the pharmacodynamic properties of antibiotics against intra- and extracellular UPEC, an in vitro model of intracellular infection was established in J774 mouse macrophages infected by the UPEC strain CFT073. We tested antibiotics commonly prescribed against urinary tract infections (gentamicin, ampicillin, nitrofurantoin, trimethoprim, sulfamethoxazole, and ciprofloxacin) and the investigational fluoroquinolone finafloxacin. The metabolic activity of individual bacteria was assessed by expressing the fluorescent reporter protein TIMERbac within CFT073. Concentration-response experiments revealed that all tested antibiotics were much less effective against intracellular bacteria than extracellular ones. Most antibiotics, except fluoroquinolones, were unable to reach a bactericidal effect intracellularly at clinically achievable concentrations. Ciprofloxacin and finafloxacin killed 99.9% of extracellular bacteria at concentrations around the MIC, while for intracellular bacteria, concentrations more than 100× over the MIC were required to achieve a bactericidal effect. Time-kill curves showed that finafloxacin was more rapidly bactericidal in acidic medium than at neutral pH, while the reverse observation was made for ciprofloxacin. Intracellularly, kill curves showed biphasic kinetics for both fluoroquinolones, suggesting the presence of drug-tolerant subpopulations. Flow cytometry analysis of TIMERbac fluorescence revealed a marked heterogeneity in intracellular growth of individual bacteria, suggesting that the presence of subpopulations reaching a state of metabolic dormancy was the main reason for increased antibiotic tolerance of intracellular UPEC.

Gluten-degrading bacteria: availability and applications.

Gluten is a mixture of storage proteins in wheat and occurs in smaller amounts in other cereal grains. It provides favorable structure to bakery products but unfortunately causes disease conditions with increasing prevalence. In the human gastrointestinal tract, gluten is cleaved into proline and gluten rich peptides that are not degraded further. These peptides trigger immune responses that might lead to celiac disease, wheat allergy, and non-celiac gluten sensitivity. The main treatment option is a gluten-free diet. Alternatively, using enzymes or microorganisms with gluten-degrading properties might alleviate the disease. These components can be used during food production or could be introduced into the digestive tract as food supplements. In addition, natural food from the environment is known to enrich the microbial communities in gut and natural environmental microbial communities have high potential to degrade gluten. It remains to be investigated if food and environment-induced changes in the gut microbiome could contribute to the triggering of gluten-related diseases. KEY POINTS: • Wheat proteins, gluten, are incompletely digested in human digestive tract leading to gluten intolerance. • The only efficient treatment of gluten intolerance is life-long gluten-free diet. • Environmental bacteria acquired together with food could be source of gluten-degrading bacteria detoxifying undigested gluten peptides.