Assessing microbiome population dynamics using wild-type isogenic standardized hybrid (WISH)-tags.

Microbiomes feature recurrent compositional structures under given environmental conditions. However, these patterns may conceal diverse underlying population dynamics that require intrastrain resolution. Here we developed a genomic tagging system, termed wild-type isogenic standardized hybrid (WISH)-tags, that can be combined with quantitative polymerase chain reaction and next-generation sequencing for microbial strain enumeration. We experimentally validated the performance of 62 tags and showed that they can be differentiated with high precision. WISH-tags were introduced into model and non-model bacterial members of the mouse and plant microbiota. Intrastrain priority effects were tested using one species of isogenic barcoded bacteria in the murine gut and the Arabidopsis phyllosphere, both with and without microbiota context. We observed colonization resistance against late-arriving strains of Salmonella Typhimurium in the mouse gut, whereas the phyllosphere accommodated Sphingomonas latecomers in a manner proportional to their presence at the late inoculation timepoint. This demonstrates that WISH-tags are a resource for deciphering population dynamics underlying microbiome assembly across biological systems.

Salmonella T3SS-2 virulence enhances gut-luminal colonization by enabling chemotaxis-dependent exploitation of intestinal inflammation.

Salmonella Typhimurium (S.Tm) utilizes the chemotaxis receptor Tsr to exploit gut inflammation. However, the characteristics of this exploitation and the mechanism(s) employed by the pathogen to circumvent antimicrobial effects of inflammation are poorly defined. Here, using different naturally occurring S.Tm strains (SL1344 and 14028) and competitive infection experiments, we demonstrate that type-three secretion system (T3SS)-2 virulence is indispensable for the beneficial effects of Tsr-directed chemotaxis. The removal of the 14028-specific prophage Gifsy3, encoding virulence effectors, results in the loss of the Tsr-mediated fitness advantage in that strain. Surprisingly, without T3SS-2 effector secretion, chemotaxis toward the gut epithelium using Tsr becomes disadvantageous for either strain. Our findings reveal that luminal neutrophils recruited as a result of NLRC4 inflammasome activation locally counteract S.Tm cells exploiting the byproducts of the host immune response. This work highlights a mechanism by which S.Tm exploitation of gut inflammation for colonization relies on the coordinated effects of chemotaxis and T3SS activities.

mBARq: a versatile and user-friendly framework for the analysis of DNA barcodes from transposon insertion libraries, knockout mutants, and isogenic strain populations.

MOTIVATION: DNA barcoding has become a powerful tool for assessing the fitness of strains in a variety of studies, including random transposon mutagenesis screens, attenuation of site-directed mutants, and population dynamics of isogenic strain pools. However, the statistical analysis, visualization, and contextualization of the data resulting from such experiments can be complex and require bioinformatic skills. RESULTS: Here, we developed mBARq, a user-friendly tool designed to simplify these steps for diverse experimental setups. The tool is seamlessly integrated with an intuitive web app for interactive data exploration via the STRING and KEGG databases to accelerate scientific discovery. AVAILABILITY AND IMPLEMENTATION: The tool is implemented in Python. The source code is freely available (https://github.com/MicrobiologyETHZ/mbarq) and the web app can be accessed at: https://microbiomics.io/tools/mbarq-app.

From microbiome composition to functional engineering, one step at a time.

SUMMARYCommunities of microorganisms (microbiota) are present in all habitats on Earth and are relevant for agriculture, health, and climate. Deciphering the mechanisms that determine microbiota dynamics and functioning within the context of their respective environments or hosts (the microbiomes) is crucially important. However, the sheer taxonomic, metabolic, functional, and spatial complexity of most microbiomes poses substantial challenges to advancing our knowledge of these mechanisms. While nucleic acid sequencing technologies can chart microbiota composition with high precision, we mostly lack information about the functional roles and interactions of each strain present in a given microbiome. This limits our ability to predict microbiome function in natural habitats and, in the case of dysfunction or dysbiosis, to redirect microbiomes onto stable paths. Here, we will discuss a systematic approach (dubbed the N+1/N-1 concept) to enable step-by-step dissection of microbiome assembly and functioning, as well as intervention procedures to introduce or eliminate one particular microbial strain at a time. The N+1/N-1 concept is informed by natural invasion events and selects culturable, genetically accessible microbes with well-annotated genomes to chart their proliferation or decline within defined synthetic and/or complex natural microbiota. This approach enables harnessing classical microbiological and diversity approaches, as well as omics tools and mathematical modeling to decipher the mechanisms underlying N+1/N-1 microbiota outcomes. Application of this concept further provides stepping stones and benchmarks for microbiome structure and function analyses and more complex microbiome intervention strategies.

Gasdermin D is the only Gasdermin that provides protection against acute Salmonella gut infection in mice.

Gasdermins (GSDMs) share a common functional domain structure and are best known for their capacity to form membrane pores. These pores are hallmarks of a specific form of cell death called pyroptosis and mediate the secretion of pro-inflammatory cytokines such as interleukin 1ß (IL1ß) and interleukin 18 (IL18). Thereby, Gasdermins have been implicated in various immune responses against cancer and infectious diseases such as acute Salmonella Typhimurium (S.Tm) gut infection. However, to date, we lack a comprehensive functional assessment of the different Gasdermins (GSDMA-E) during S.Tm infection in vivo. Here, we used epithelium-specific ablation, bone marrow chimeras, and mouse lines lacking individual Gasdermins, combinations of Gasdermins or even all Gasdermins (GSDMA1-3C1-4DE) at once and performed littermate-controlled oral S.Tm infections in streptomycin-pretreated mice to investigate the impact of all murine Gasdermins. While GSDMA, C, and E appear dispensable, we show that GSDMD i) restricts S.Tm loads in the gut tissue and systemic organs, ii) controls gut inflammation kinetics, and iii) prevents epithelium disruption by 72 h of the infection. Full protection requires GSDMD expression by both bone-marrow-derived lamina propria cells and intestinal epithelial cells (IECs). In vivo experiments as well as 3D-, 2D-, and chimeric enteroid infections further show that infected IEC extrusion proceeds also without GSDMD, but that GSDMD controls the permeabilization and morphology of the extruding IECs, affects extrusion kinetics, and promotes overall mucosal barrier capacity. As such, this work identifies a unique multipronged role of GSDMD among the Gasdermins for mucosal tissue defense against a common enteric pathogen.

The microbiota conditions a gut milieu that selects for wild-type Salmonella Typhimurium virulence.

Salmonella Typhimurium elicits gut inflammation by the costly expression of HilD-controlled virulence factors. This inflammation alleviates colonization resistance (CR) mediated by the microbiota and thereby promotes pathogen blooms. However, the inflamed gut-milieu can also select for hilD mutants, which cannot elicit or maintain inflammation, therefore causing a loss of the pathogen's virulence. This raises the question of which conditions support the maintenance of virulence in S. Typhimurium. Indeed, it remains unclear why the wild-type hilD allele is dominant among natural isolates. Here, we show that microbiota transfer from uninfected or recovered hosts leads to rapid clearance of hilD mutants that feature attenuated virulence, and thereby contributes to the preservation of the virulent S. Typhimurium genotype. Using mouse models featuring a range of microbiota compositions and antibiotic- or inflammation-inflicted microbiota disruptions, we found that irreversible disruption of the microbiota leads to the accumulation of hilD mutants. In contrast, in models with a transient microbiota disruption, selection for hilD mutants was prevented by the regrowing microbiota community dominated by Lachnospirales and Oscillospirales. Strikingly, even after an irreversible microbiota disruption, microbiota transfer from uninfected donors prevented the rise of hilD mutants. Our results establish that robust S. Typhimurium gut colonization hinges on optimizing its manipulation of the host: A transient and tempered microbiota perturbation is favorable for the pathogen to both flourish in the inflamed gut and also minimize loss of virulence. Moreover, besides conferring CR, the microbiota may have the additional consequence of maintaining costly enteropathogen virulence mechanisms.

Epithelial inflammasomes, gasdermins, and mucosal inflammation - Lessons from Salmonella and Shigella infected mice.

Besides its crucial function in nutrient absorbance and as barrier against the microbiota, the gut epithelium is essential for sensing pathogenic insults and mounting of an appropriate early immune response. In mice, the activation of the canonical NAIP/NLRC4 inflammasome is critical for the defense against enterobacterial infections. Activation of the NAIP/NLRC4 inflammasome triggers the extrusion of infected intestinal epithelial cells (IEC) into the gut lumen, concomitant with inflammasome-mediated lytic cell death. The membrane permeabilization, a hallmark of pyroptosis, is caused by the pore-forming proteins called gasdermins (GSDMs). Recent work has revealed that NAIP/NLRC4-dependent extrusion of infected IECs can, however, also be executed in the absence of GSDMD. In fact, several reports highlighted that various cell death pathways (e.g., pyroptosis or apoptosis) and unique mechanisms specific to particular infection models and stages of gut infection are in action during epithelial inflammasome defense against intestinal pathogens. Here, we summarize the current knowledge regarding the underlying mechanisms and speculate on the putative functions of the epithelial inflammasome activation and cell death, with a particular emphasis on mouse infection models for two prominent enterobacterial pathogens, Salmonella Typhimurium and Shigella flexneri.

Intraluminal neutrophils limit epithelium damage by reducing pathogen assault on intestinal epithelial cells during Salmonella gut infection.

Recruitment of neutrophils into and across the gut mucosa is a cardinal feature of intestinal inflammation in response to enteric infections. Previous work using the model pathogen Salmonella enterica serovar Typhimurium (S.Tm) established that invasion of intestinal epithelial cells by S.Tm leads to recruitment of neutrophils into the gut lumen, where they can reduce pathogen loads transiently. Notably, a fraction of the pathogen population can survive this defense, re-grow to high density, and continue triggering enteropathy. However, the functions of intraluminal neutrophils in the defense against enteric pathogens and their effects on preventing or aggravating epithelial damage are still not fully understood. Here, we address this question via neutrophil depletion in different mouse models of Salmonella colitis, which differ in their degree of enteropathy. In an antibiotic pretreated mouse model, neutrophil depletion by an anti-Ly6G antibody exacerbated epithelial damage. This could be linked to compromised neutrophil-mediated elimination and reduced physical blocking of the gut-luminal S.Tm population, such that the pathogen density remained high near the epithelial surface throughout the infection. Control infections with a ssaV mutant and gentamicin-mediated elimination of gut-luminal pathogens further supported that neutrophils are protecting the luminal surface of the gut epithelium. Neutrophil depletion in germ-free and gnotobiotic mice hinted that the microbiota can modulate the infection kinetics and ameliorate epithelium-disruptive enteropathy even in the absence of neutrophil-protection. Together, our data indicate that the well-known protective effect of the microbiota is augmented by intraluminal neutrophils. After antibiotic-mediated microbiota disruption, neutrophils are central for maintaining epithelial barrier integrity during acute Salmonella-induced gut inflammation, by limiting the sustained pathogen assault on the epithelium in a critical window of the infection.

Differences in carbon metabolic capacity fuel co-existence and plasmid transfer between Salmonella strains in the mouse gut.

Antibiotic resistance plasmids can be disseminated between different Enterobacteriaceae in the gut. Here, we investigate how closely related Enterobacteriaceae populations with similar nutrient needs can co-bloom in the same gut and thereby facilitate plasmid transfer. Using different strains of Salmonella Typhimurium (S.Tm SL1344 and ATCC14028) and mouse models of Salmonellosis, we show that the bloom of one strain (i.e., recipient) from very low numbers in a gut pre-occupied by the other strain (i.e., donor) depends on strain-specific utilization of a distinct carbon source, galactitol or arabinose. Galactitol-dependent growth of the recipient S.Tm strain promotes plasmid transfer between non-isogenic strains and between E. coli and S.Tm. In mice stably colonized by a defined microbiota (OligoMM12), galactitol supplementation similarly facilitates co-existence of two S.Tm strains and promotes plasmid transfer. Our work reveals a metabolic strategy used by Enterobacteriaceae to expand in a pre-occupied gut and provides promising therapeutic targets for resistance plasmids spread.

Mouse models for bacterial enteropathogen infections: insights into the role of colonization resistance.

Globally, enteropathogenic bacteria are a major cause of morbidity and mortality.1-3 Campylobacter, Salmonella, Shiga-toxin-producing Escherichia coli, and Listeria are among the top five most commonly reported zoonotic pathogens in the European Union.4 However, not all individuals naturally exposed to enteropathogens go on to develop disease. This protection is attributable to colonization resistance (CR) conferred by the gut microbiota, as well as an array of physical, chemical, and immunological barriers that limit infection. Despite their importance for human health, a detailed understanding of gastrointestinal barriers to infection is lacking, and further research is required to investigate the mechanisms that underpin inter-individual differences in resistance to gastrointestinal infection. Here, we discuss the current mouse models available to study infections by non-typhoidal Salmonella strains, Citrobacter rodentium (as a model for enteropathogenic and enterohemorrhagic E. coli), Listeria monocytogenes, and Campylobacter jejuni. Clostridioides difficile is included as another important cause of enteric disease in which resistance is dependent upon CR. We outline which parameters of human infection are recapitulated in these mouse models, including the impact of CR, disease pathology, disease progression, and mucosal immune response. This will showcase common virulence strategies, highlight mechanistic differences, and help researchers from microbiology, infectiology, microbiome research, and mucosal immunology to select the optimal mouse model.

Fitness advantage of Bacteroides thetaiotaomicron capsular polysaccharide in the mouse gut depends on the resident microbiota.

Many microbiota-based therapeutics rely on our ability to introduce a microbe of choice into an already-colonized intestine. In this study, we used genetically barcoded Bacteroides thetaiotaomicron (B. theta) strains to quantify population bottlenecks experienced by a B. theta population during colonization of the mouse gut. As expected, this reveals an inverse relationship between microbiota complexity and the probability that an individual wildtype B. theta clone will colonize the gut. The polysaccharide capsule of B. theta is important for resistance against attacks from other bacteria, phage, and the host immune system, and correspondingly acapsular B. theta loses in competitive colonization against the wildtype strain. Surprisingly, the acapsular strain did not show a colonization defect in mice with a low-complexity microbiota, as we found that acapsular strains have an indistinguishable colonization probability to the wildtype strain on single-strain colonization. This discrepancy could be resolved by tracking in vivo growth dynamics of both strains: acapsular B.theta shows a longer lag phase in the gut lumen as well as a slightly slower net growth rate. Therefore, as long as there is no niche competitor for the acapsular strain, this has only a small influence on colonization probability. However, the presence of a strong niche competitor (i.e., wildtype B. theta, SPF microbiota) rapidly excludes the acapsular strain during competitive colonization. Correspondingly, the acapsular strain shows a similarly low colonization probability in the context of a co-colonization with the wildtype strain or a complete microbiota. In summary, neutral tagging and detailed analysis of bacterial growth kinetics can therefore quantify the mechanisms of colonization resistance in differently-colonized animals.

Chemotaxis and autoinducer-2 signalling mediate colonization and contribute to co-existence of Escherichia coli strains in the murine gut.

Bacteria communicate and coordinate their behaviour at the intra- and interspecies levels by producing and sensing diverse extracellular small molecules called autoinducers. Autoinducer 2 (AI-2) is produced and detected by a variety of bacteria and thus plays an important role in interspecies communication and chemotaxis. Although AI-2 is a major autoinducer molecule present in the mammalian gut and can influence the composition of the murine gut microbiota, its role in bacteria-bacteria and bacteria-host interactions during gut colonization remains unclear. Combining competitive infections in C57BL/6 mice with microscopy and bioinformatic approaches, we show that chemotaxis (cheY) and AI-2 signalling (via lsrB) promote gut colonization by Escherichia coli, which is in turn connected to the ability of the bacteria to utilize fructoselysine (frl operon). We further show that the genomic diversity of E. coli strains with respect to AI-2 signalling allows ecological niche segregation and stable co-existence of different E. coli strains in the mammalian gut.

Studying antibiotic persistence in vivo using the model organism Salmonella Typhimurium.

Antibiotic persistence permits a subpopulation of susceptible bacteria to survive lethal concentrations of bactericidal antibiotics. This prolongs antibiotic therapy, promotes the evolution of antibiotic-resistant pathogen strains and can select for pathogen virulence within infected hosts. Here, we review the literature exploring antibiotic persistence in vivo, and describe the consequences of recalcitrant subpopulations, with a focus on studies using the model pathogen Salmonella Typhimurium. In vitro studies have established a concise set of features distinguishing true persisters from other forms of bacterial recalcitrance to bactericidal antibiotics. We discuss how animal infection models are useful for exploring these features in vivo, and describe how technical challenges can sometimes prevent the conclusive identification of true antibiotic persistence within infected hosts. We propose using two complementary working definitions for studying antibiotic persistence in vivo: the strict definition for studying the mechanisms of persister formation, and an operative definition for functional studies assessing the links between invasive virulence and persistence as well as the consequences for horizontal gene transfer, or the emergence of antibiotic-resistant mutants. This operative definition will enable further study of how antibiotic persisters arise in vivo, and of how surviving populations contribute to diverse downstream effects such as pathogen transmission, horizontal gene transfer and the evolution of virulence and antibiotic resistance. Ultimately, such studies will help to improve therapeutic control of antibiotic- recalcitrant populations.

The Mobilizable Plasmid P3 of Salmonella enterica Serovar Typhimurium SL1344 Depends on the P2 Plasmid for Conjugative Transfer into a Broad Range of Bacteria In Vitro and In Vivo.

The global rise of drug-resistant bacteria is of great concern. Conjugative transfer of antibiotic resistance plasmids contributes to the emerging resistance crisis. Despite substantial progress in understanding the molecular basis of conjugation in vitro, the in vivo dynamics of intra- and interspecies conjugative plasmid transfer are much less understood. In this study, we focused on the streptomycin resistance-encoding mobilizable plasmid pRSF1010SL1344 (P3) of Salmonella enterica serovar Typhimurium strain SL1344. We show that P3 is mobilized by interacting with the conjugation machinery of the conjugative plasmid pCol1B9SL1344 (P2) of SL1344. Thereby, P3 can be transferred into a broad range of relevant environmental and clinical bacterial isolates in vitro and in vivo. Our data suggest that S. Typhimurium persisters in host tissues can serve as P3 reservoirs and foster transfer of both P2 and P3 once they reseed the gut lumen. This adds to our understanding of resistance plasmid transfer in ecologically relevant niches, including the mammalian gut. IMPORTANCE S. Typhimurium is a globally abundant bacterial species that rapidly occupies new niches and survives unstable environmental conditions. As an enteric pathogen, S. Typhimurium interacts with a broad range of bacterial species residing in the mammalian gut. High abundance of bacteria in the gut lumen facilitates conjugation and spread of plasmid-carried antibiotic resistance genes. By studying the transfer dynamics of the P3 plasmid in vitro and in vivo, we illustrate the impact of S. Typhimurium-mediated antibiotic resistance spread via conjugation to relevant environmental and clinical bacterial isolates. Plasmids are among the most critical vehicles driving antibiotic resistance spread. Further understanding of the dynamics and drivers of antibiotic resistance transfer is needed to develop effective solutions for slowing down the emerging threat of multidrug-resistant bacterial pathogens.

Results from Canton Grisons of Switzerland suggest repetitive testing reduces SARS-CoV-2 incidence (February-March 2021).

In February 2021, in response to emergence of more transmissible SARS-CoV-2 virus variants, the Canton Grisons launched a unique RNA mass testing program targeting the labour force in local businesses. Employees were offered weekly tests free of charge and on a voluntary basis. If tested positive, they were required to self-isolate for ten days and their contacts were subjected to daily testing at work. Thereby, the quarantine of contact persons could be waved.Here, we evaluate the effects of the testing program on the tested cohorts. We examined 121,364 test results from 27,514 participants during February-March 2021. By distinguishing different cohorts of employees, we observe a noticeable decrease in the test positivity rate and a statistically significant reduction in the associated incidence rate over the considered period. The reduction in the latter ranges between 18 and 50%. The variability is partly explained by different exposures to exogenous infection sources (e.g., contacts with visiting tourists or cross-border commuters). Our analysis provides the first empirical evidence that applying repetitive mass testing to a real population over an extended period of time can prevent spread of COVID-19 pandemic. However, to overcome logistic, uptake, and adherence challenges it is important that the program is carefully designed and that disease incursion from the population outside of the program is considered and controlled.

Metabolic reconstitution of germ-free mice by a gnotobiotic microbiota varies over the circadian cycle.

The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiota to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat mass than germ-free (GF) or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure, and respiratory quotient using a novel isolator-housed metabolic cage system, which allows long-term measurements without contamination risk. This demonstrated that microbiota-released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and GF mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well integrated into appetite control. We also observed no significant difference in energy expenditure after normalization by lean mass between the different microbiota groups, suggesting that cumulative small differences in energy balance, or altered energy storage, must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: In GF mice, the respiratory exchange ratio (RER) was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly, the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied GF mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a GF-like drop in liver glycogen storage during the light phase and both liver and plasma metabolomes of OligoMM12 mice clustered closely with GF mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle and which are absent in the OligoMM12 consortium.

Pathogenic and Commensal Gut Bacteria Harboring Glycerol/Diol Dehydratase Metabolize Glycerol and Produce DNA-Reactive Acrolein.

Bacteria harboring glycerol/diol dehydratase (GDH) encoded by the genes pduCDE metabolize glycerol and release acrolein during growth. Acrolein has antimicrobial activity, and exposure of human cells to acrolein gives rise to toxic and mutagenic responses. These biological responses are related to acrolein's high reactivity as a chemical electrophile that can covalently bind to cellular nucleophiles including DNA and proteins. Various food microbes and gut commensals transform glycerol to acrolein, but there is no direct evidence available for bacterial glycerol metabolism giving rise to DNA adducts. Moreover, it is unknown whether pathogens, such as Salmonella Typhymurium, catalyze this transformation. We assessed, therefore, acrolein formation by four GDH-competent strains of S. Typhymurium grown under either aerobic or anaerobic conditions in the presence of 50 mM glycerol. On the basis of analytical derivatization with a heterocyclic amine, all wild-type strains were observed to produce acrolein, but to different extents, and acrolein production was not detected in fermentations of a pduC-deficient mutant strain. Furthermore, we found that, in the presence of calf thymus DNA, acrolein-DNA adducts were formed as a result of bacterial glycerol metabolism by two strains of Limosilactobacillus reuteri, but not a pduCDE mutant strain. The quantification of the resulting adducts with increasing levels of glycerol up to 600 mM led to the production of up to 1.5 mM acrolein and 3600 acrolein-DNA adducts per 108 nucleosides in a model system. These results suggest that GDH-competent food microbes, gut commensals, and pathogens alike have the capacity to produce acrolein from glycerol. Further, the acrolein production can lead to DNA adduct formation, but requires high glycerol concentrations that are not available in the human gut.

Transient cell-in-cell formation underlies tumor relapse and resistance to immunotherapy.

Despite the remarkable successes of cancer immunotherapies, the majority of patients will experience only partial response followed by relapse of resistant tumors. While treatment resistance has frequently been attributed to clonal selection and immunoediting, comparisons of paired primary and relapsed tumors in melanoma and breast cancers indicate that they share the majority of clones. Here, we demonstrate in both mouse models and clinical human samples that tumor cells evade immunotherapy by generating unique transient cell-in-cell structures, which are resistant to killing by T cells and chemotherapies. While the outer cells in this cell-in-cell formation are often killed by reactive T cells, the inner cells remain intact and disseminate into single tumor cells once T cells are no longer present. This formation is mediated predominantly by IFNγ-activated T cells, which subsequently induce phosphorylation of the transcription factors signal transducer and activator of transcription 3 (STAT3) and early growth response-1 (EGR-1) in tumor cells. Indeed, inhibiting these factors prior to immunotherapy significantly improves its therapeutic efficacy. Overall, this work highlights a currently insurmountable limitation of immunotherapy and reveals a previously unknown resistance mechanism which enables tumor cells to survive immune-mediated killing without altering their immunogenicity.

Cancer immunotherapies use the body's own immune system to fight off cancer. But, despite some remarkable success stories, many patients only see a temporary improvement before the immunotherapy stops being effective and the tumours regrow. It is unclear why this occurs, but it may have to do with how the immune system attacks cancer cells. Immunotherapies aim to activate a special group of cells known as killer T-cells, which are responsible for the immune response to tumours. These cells can identify cancer cells and inject toxic granules through their membranes, killing them. However, killer T-cells are not always effective. This is because cancer cells are naturally good at avoiding detection, and during treatment, their genes can mutate, giving them new ways to evade the immune system. Interestingly, when scientists analysed the genes of tumour cells before and after immunotherapy, they found that many of the genes that code for proteins recognized by T-cells do not change significantly. This suggests that tumours' resistance to immune attack may be physical, rather than genetic. To investigate this hypothesis, Gutwillig et al. developed several mouse tumour models that stop responding to immunotherapy after initial treatment. Examining cells from these tumours revealed that when the immune system attacks, they reorganise by getting inside one another. This allows some cancer cells to hide under many layers of cell membrane. At this point killer T-cells can identify and inject the outer cell with toxic granules, but it cannot reach the cells inside. This ability of cancer cells to hide within one another relies on them recognising when the immune system is attacking. This happens because the cancer cells can detect certain signals released by the killer T-cells, allowing them to hide. Gutwillig et al. identified some of these signals, and showed that blocking them stopped cancer cells from hiding inside each other, making immunotherapy more effective. This new explanation for how cancer cells escape the immune system could guide future research and lead to new cancer treatments, or approaches to boost existing treatments. Understanding the process in more detail could allow scientists to prevent it from happening, by revealing which signals to block, and when, for best results.

KappaBle fluorescent reporter mice enable low-background single-cell detection of NF-κB transcriptional activity in vivo.

Nuclear factor-κB (NF-κB) is a transcription factor with a key role in a great variety of cellular processes from embryonic development to immunity, the outcome of which depends on the fine-tuning of NF-κB activity. The development of sensitive and faithful reporter systems to accurately monitor the activation status of this transcription factor is therefore desirable. To address this need, over the years a number of different approaches have been used to generate NF-κB reporter mice, which can be broadly subdivided into bioluminescence- and fluorescence-based systems. While the former enables whole-body visualization of the activation status of NF-κB, the latter have the potential to allow the analysis of NF-κB activity at single-cell level. However, fluorescence-based reporters frequently show poor sensitivity and excessive background or are incompatible with high-throughput flow cytometric analysis. In this work we describe the generation and analysis of ROSA26 knock-in NF-κB reporter (KappaBle) mice containing a destabilized EGFP, which showed sensitive, dynamic, and faithful monitoring of NF-κB transcriptional activity at the single-cell level of various cell types during inflammatory and infectious diseases.

Impact of horizontal gene transfer on emergence and stability of cooperative virulence in Salmonella Typhimurium.

Intestinal inflammation fuels the transmission of Salmonella Typhimurium (S.Tm). However, a substantial fitness cost is associated with virulence expression. Mutations inactivating transcriptional virulence regulators generate attenuated variants profiting from inflammation without enduring virulence cost. Such variants interfere with the transmission of fully virulent clones. Horizontal transfer of functional regulatory genes (HGT) into attenuated variants could nevertheless favor virulence evolution. To address this hypothesis, we cloned hilD, coding for the master regulator of virulence, into a conjugative plasmid that is highly transferrable during intestinal colonization. The resulting mobile hilD allele allows virulence to emerge from avirulent populations, and to be restored in attenuated mutants competing against virulent clones within-host. However, mutations inactivating the mobile hilD allele quickly arise. The stability of virulence mediated by HGT is strongly limited by its cost, which depends on the hilD expression level, and by the timing of transmission. We conclude that robust evolution of costly virulence expression requires additional selective forces such as narrow population bottlenecks during transmission.