Publications by authors named "Wolf-Dietrich Hardt"

164 Publications

Silicon Nitride, a Bioceramic for Bone Tissue Engineering: A Reinforced Cryogel System With Antibiofilm and Osteogenic Effects.

Front Bioeng Biotechnol 2021 15;9:794586. Epub 2021 Dec 15.

Department of Health Sciences and Technology, Institute for Biomechanics, ETH Zurich, Zurich, Switzerland.

Silicon nitride (SiN [SiN]) is a promising bioceramic for use in a wide variety of orthopedic applications. Over the past decades, it has been mainly used in industrial applications, such as space shuttle engines, but not in the medical field due to scarce data on the biological effects of SiN. More recently, it has been increasingly identified as an emerging material for dental and orthopedic implant applications. Although a few reports about the antibacterial properties and osteoconductivity of SiN have been published to date, there have been limited studies of SiN-based scaffolds for bone tissue engineering. Here, we developed a silicon nitride reinforced gelatin/chitosan cryogel system (SiN-GC) by loading silicon nitride microparticles into a gelatin/chitosan cryogel (GC), with the aim of producing a biomimetic scaffold with antibiofilm and osteogenic properties. In this scaffold system, the GC component provides a hydrophilic and macroporous environment for cells, while the SiN component not only provides antibacterial properties and osteoconductivity but also increases the mechanical stiffness of the scaffold. This provides enhanced mechanical support for the defect area and a better osteogenic environment. First, we analyzed the scaffold characteristics of SiN-GC with different SiN concentrations, followed by evaluation of its apatite-forming capacity in simulated body fluid and protein adsorption capacity. We further confirmed an antibiofilm effect of SiN-GC against () and () as well as enhanced cell proliferation, mineralization, and osteogenic gene upregulation for MC3T3-E1 pre-osteoblast cells. Finally, we developed a bioreactor to culture cell-laden scaffolds under cyclic compressive loading to mimic physiological conditions and were able to demonstrate improved mineralization and osteogenesis from SiN-GC. Overall, we confirmed the antibiofilm and osteogenic effect of a silicon nitride reinforced cryogel system, and the results indicate that silicon nitride as a biomaterial system component has a promising potential to be developed further for bone tissue engineering applications.
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http://dx.doi.org/10.3389/fbioe.2021.794586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8714913PMC
December 2021

Pathogen invasion-dependent tissue reservoirs and plasmid-encoded antibiotic degradation boost plasmid spread in the gut.

Elife 2021 12 7;10. Epub 2021 Dec 7.

Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland.

Many plasmids encode antibiotic resistance genes. Through conjugation, plasmids can be rapidly disseminated. Previous work identified gut luminal donor/recipient blooms and tissue-lodged plasmid-bearing persister cells of the enteric pathogen serovar Typhimurium (.Tm) that survive antibiotic therapy in host tissues, as factors promoting plasmid dissemination among Enterobacteriaceae. However, the buildup of tissue reservoirs and their contribution to plasmid spread await experimental demonstration. Here, we asked if re-seeding-plasmid acquisition-invasion cycles by .Tm could serve to diversify tissue-lodged plasmid reservoirs, and thereby promote plasmid spread. Starting with intraperitoneal mouse infections, we demonstrate that .Tm cells re-seeding the gut lumen initiate clonal expansion. Extended spectrum beta-lactamase (ESBL) plasmid-encoded gut luminal antibiotic degradation by donors can foster recipient survival under beta-lactam antibiotic treatment, enhancing transconjugant formation upon re-seeding. .Tm transconjugants can subsequently re-enter host tissues introducing the new plasmid into the tissue-lodged reservoir. Population dynamics analyses pinpoint recipient migration into the gut lumen as rate-limiting for plasmid transfer dynamics in our model. Priority effects may be a limiting factor for reservoir formation in host tissues. Overall, our proof-of-principle data indicates that luminal antibiotic degradation and shuttling between the gut lumen and tissue-resident reservoirs can promote the accumulation and spread of plasmids within a host over time.
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http://dx.doi.org/10.7554/eLife.69744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8651294PMC
December 2021

Smart investment of virus RNA testing resources to enhance Covid-19 mitigation.

PLoS One 2021 30;16(11):e0259018. Epub 2021 Nov 30.

Department of Mechanical and Process Engineering, Swiss Federal Institute of Technology, IFD, Zürich, Switzerland.

A variety of mitigation strategies have been employed against the Covid-19 pandemic. Social distancing is still one of the main methods to reduce spread, but it entails a high toll on personal freedom and economic life. Alternative mitigation strategies that do not come with the same problems but are effective at preventing disease spread are therefore needed. Repetitive mass-testing using PCR assays for viral RNA has been suggested, but as a stand-alone strategy this would be prohibitively resource intensive. Here, we suggest a strategy that aims at targeting the limited resources available for viral RNA testing to subgroups that are more likely than the average population to yield a positive test result. Importantly, these pre-selected subgroups include symptom-free people. By testing everyone in these subgroups, in addition to symptomatic cases, large fractions of pre- and asymptomatic people can be identified, which is only possible by testing-based mitigation. We call this strategy smart testing (ST). In principle, pre-selected subgroups can be found in different ways, but for the purpose of this study we analyze a pre-selection procedure based on cheap and fast virus antigen tests. We quantify the potential reduction of the epidemic reproduction number by such a two-stage ST strategy. In addition to a scenario where such a strategy is available to the whole population, we analyze local applications, e.g. in a country, company, or school, where the tested subgroups are also in exchange with the untested population. Our results suggest that a two-stage ST strategy can be effective to curb pandemic spread, at costs that are clearly outweighed by the economic benefit. It is technically and logistically feasible to employ such a strategy, and our model predicts that it is even effective when applied only within local groups. We therefore recommend adding two-stage ST to the portfolio of available mitigation strategies, which allow easing social distancing measures without compromising public health.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0259018PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8631684PMC
December 2021

The Polar Icm/Dot T4SS Establishes Distinct Contact Sites with the Pathogen Vacuole Membrane.

mBio 2021 10 12;12(5):e0218021. Epub 2021 Oct 12.

Institute of Medical Microbiology, Faculty of Medicine, University of Zürich, Zürich, Switzerland.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen that survives inside phagocytic host cells by establishing a protected replication niche, termed the "-containing vacuole" (LCV). To form an LCV and subvert pivotal host pathways, L. pneumophila employs a type IV secretion system (T4SS), which translocates more than 300 different effector proteins into the host cell. The L. pneumophila T4SS complex has been shown to span the bacterial cell envelope at the bacterial poles. However, the interactions between the T4SS and the LCV membrane are not understood. Using cryo-focused ion beam milling, cryo-electron tomography, and confocal laser scanning fluorescence microscopy, we show that up to half of the intravacuolar L. pneumophila bacteria tether their cell pole to the LCV membrane. Tethering coincides with the presence and function of T4SSs and likely promotes the establishment of distinct contact sites between T4SSs and the LCV membrane. Contact sites are characterized by indentations in the limiting LCV membrane and localize juxtaposed to T4SS machineries. The data are in agreement with the notion that effector translocation occurs by close membrane contact rather than by an extended pilus. Our findings provide novel insights into the interactions of the L. pneumophila T4SS with the LCV membrane . Legionnaires' disease is a life-threatening pneumonia, which is characterized by high fever, coughing, shortness of breath, muscle pain, and headache. The disease is caused by the amoeba-resistant bacterium L. pneumophila found in various soil and aquatic environments and is transmitted to humans via the inhalation of small bacteria-containing droplets. An essential virulence factor of L. pneumophila is a so-called "type IV secretion system" (T4SS), which, by injecting a plethora of "effector proteins" into the host cell, determines pathogen-host interactions and the formation of a distinct intracellular compartment, the "-containing vacuole" (LCV). It is unknown how the T4SS makes contact to the LCV membrane to deliver the effectors. In this study, we identify indentations in the host cell membrane in close proximity to functional T4SSs localizing at the bacterial poles. Our work reveals first insights into the architecture of -LCV contact sites.
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http://dx.doi.org/10.1128/mBio.02180-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8510526PMC
October 2021

Analysis of Salmonella Persister Population Sizes, Dynamics of Gut Luminal Seeding, and Plasmid Transfer in Mouse Models of Salmonellosis.

Methods Mol Biol 2021 ;2357:253-272

Department of Biology, Institute of Microbiology, ETH Zurich, Zürich, Switzerland.

A previously unappreciated link between persisters and the emergence and spread of antibiotic resistance has been recently established. The bulk of this research has been conducted in vitro, but some studies are beginning to elucidate the importance of persister reservoirs in both antibiotic treatment failure and the spread of antibiotic resistance using in vivo models. In order to further this research, careful analyses of the mechanisms of persister reservoir formation as well as the dynamics of persister survival and postantibiotic regrowth are of importance. Here, we present a mouse model to quantitatively study Salmonella persisters in vivo. By using neutral unique sequence barcodes, we describe the quantitative analysis of rare events (aka bottlenecks) associated with persister reservoir formation, survival, and reseeding of the gut lumen. This provides quantitative data for persister-fueled plasmid transfer in vivo. Although this chapter describes analysis of Salmonella persisters in a mouse model, these concepts can be applied to any experimental system provided that tractable experimental systems are present.
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http://dx.doi.org/10.1007/978-1-0716-1621-5_17DOI Listing
January 2022

CXCL12-abundant reticular cells are the major source of IL-6 upon LPS stimulation and thereby regulate hematopoiesis.

Blood Adv 2021 12;5(23):5002-5015

Department of Medical Oncology and Hematology, University of Zurich and University Hospital Zurich, Zurich, Switzerland.

Hematopoiesis is maintained by hematopoietic stem and progenitor cells that are located in the bone marrow (BM) where they are embedded within a complex supportive microenvironment consisting of a multitude of various non-hematopoietic and hematopoietic cell types. The BM microenvironment not only regulates steady-state hematopoiesis by provision of growth factors, cytokines, and cell-cell contact but is also an emerging key player during the adaptation to infectious and inflammatory insults (emergency hematopoiesis). Through a combination of gene expression analyses in prospectively isolated non-hematopoietic BM cell populations and various mouse models, we found that BM CXCL12-abundant reticular (CAR) cells are a major source of systemic and local BM interleukin-6 (IL-6) levels during emergency hematopoiesis after lipopolysaccharide (LPS) stimulation. Importantly, although IL-6 is dispensable during the initial phase of LPS-induced emergency hematopoiesis, it is required to sustain an adequate hematopoietic output during chronic repetitive inflammation. Our data highlight the essential role of the non-hematopoietic BM microenvironment for the sensing and integration of pathogen-derived signals into sustained demand-adapted hematopoietic responses.
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http://dx.doi.org/10.1182/bloodadvances.2021005531DOI Listing
December 2021

Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF.

J Exp Med 2021 11 16;218(11). Epub 2021 Sep 16.

Institute of Microbiology, Department of Biology, Eidgenössische Technische Hochschule Zurich, Zurich, Switzerland.

Intestinal epithelial cell (IEC) NF-κB signaling regulates the balance between mucosal homeostasis and inflammation. It is not fully understood which signals tune this balance and how bacterial exposure elicits the process. Pure LPS induces epithelial NF-κB activation in vivo. However, we found that in mice, IECs do not respond directly to LPS. Instead, tissue-resident lamina propria intercrypt macrophages sense LPS via TLR4 and rapidly secrete TNF to elicit epithelial NF-κB signaling in their immediate neighborhood. This response pattern is relevant also during oral enteropathogen infection. The macrophage-TNF-IEC axis avoids responses to luminal microbiota LPS but enables crypt- or tissue-scale epithelial NF-κB responses in proportion to the microbial threat. Thereby, intercrypt macrophages fulfill important sentinel functions as first responders to Gram-negative microbes breaching the epithelial barrier. The tunability of this crypt response allows the induction of defense mechanisms at an appropriate scale according to the localization and intensity of microbial triggers.
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http://dx.doi.org/10.1084/jem.20210862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8480669PMC
November 2021

Salmonella effector driven invasion of the gut epithelium: breaking in and setting the house on fire.

Curr Opin Microbiol 2021 12 6;64:9-18. Epub 2021 Sep 6.

Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland. Electronic address:

Salmonella Typhimurium (S.Tm) is a major cause of diarrheal disease. The invasion into intestinal epithelial cells (IECs) is a central step in the infection cycle. It is associated with gut inflammation and thought to benefit S.Tm proliferation also in the intestinal lumen. Importantly, it is still not entirely clear how inflammation is elicited and to which extent it links to IEC invasion efficiency in vivo. In this review, we summarize recent findings explaining IEC invasion by type-three-secretion-system-1 (TTSS-1) effector proteins and discuss their effects on invasion and gut inflammation. In non-polarized tissue culture cells, the TTSS-1 effectors (mainly SopB/E/E2) elicit large membrane ruffles fueling cooperative invasion, and can directly trigger pro-inflammatory signaling. By contrast, in the murine gut, we observe discreet-invasion (mainly via the TTSS-1 effector SipA) and a prominent pro-inflammatory role of the host?"s epithelial inflammasome(s), which sense pathogen associated molecular patterns (PAMPs). We discuss why it has remained a major challenge to tease apart direct and indirect inflammatory effects of TTSS-1 effectors and explain why further research will be needed to fully determine their inflammation-modulating role(s).
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http://dx.doi.org/10.1016/j.mib.2021.08.007DOI Listing
December 2021

Microbiota-derived metabolites inhibit virulent subpopulation development by acting on single-cell behaviors.

Proc Natl Acad Sci U S A 2021 08;118(31)

Department of Environmental Systems Science, ETH Zürich, Zürich 8092, Switzerland.

spp. express pathogenicity island 1 Type III Secretion System 1 (T3SS-1) genes to mediate the initial phase of interaction with their host. Prior studies indicate short-chain fatty acids, microbial metabolites at high concentrations in the gastrointestinal tract, limit population-level T3SS-1 gene expression. However, only a subset of cells in a population express these genes, suggesting short-chain fatty acids could decrease T3SS-1 population-level expression by acting on per-cell expression or the proportion of expressing cells. Here, we combine single-cell, theoretical, and molecular approaches to address the effect of short-chain fatty acids on T3SS-1 expression. Our in vitro results show short-chain fatty acids do not repress T3SS-1 expression by individual cells. Rather, these compounds act to selectively slow the growth of T3SS-1-expressing cells, ultimately decreasing their frequency in the population. Further experiments indicate slowed growth arises from short-chain fatty acid-mediated depletion of the proton motive force. By influencing the T3SS-1 cell-type proportions, our findings imply gut microbial metabolites act on cooperation between the two cell types and ultimately influence 's capacity to establish within a host.
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http://dx.doi.org/10.1073/pnas.2103027118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346864PMC
August 2021

miR-802 regulates Paneth cell function and enterocyte differentiation in the mouse small intestine.

Nat Commun 2021 06 7;12(1):3339. Epub 2021 Jun 7.

Institute of Molecular Health Sciences, ETH Zurich, Zürich, Switzerland.

The intestinal epithelium is a complex structure that integrates digestive, immunological, neuroendocrine, and regenerative functions. Epithelial homeostasis is maintained by a coordinated cross-talk of different epithelial cell types. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here we show that the intestine-enriched miR-802 is a central regulator of intestinal epithelial cell proliferation, Paneth cell function, and enterocyte differentiation. Genetic ablation of mir-802 in the small intestine of mice leads to decreased glucose uptake, impaired enterocyte differentiation, increased Paneth cell function and intestinal epithelial proliferation. These effects are mediated in part through derepression of the miR-802 target Tmed9, a modulator of Wnt and lysozyme/defensin secretion in Paneth cells, and the downstream Wnt signaling components Fzd5 and Tcf4. Mutant Tmed9 mice harboring mutations in miR-802 binding sites partially recapitulate the augmented Paneth cell function of mice lacking miR-802. Our study demonstrates a broad miR-802 network that is important for the integration of signaling pathways of different cell types controlling epithelial homeostasis in the small intestine.
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http://dx.doi.org/10.1038/s41467-021-23298-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184787PMC
June 2021

A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants.

Nat Microbiol 2021 07 27;6(7):830-841. Epub 2021 May 27.

Institute of Microbiology, Department of Biology, ETH Zürich, Zürich, Switzerland.

The ability of gut bacterial pathogens to escape immunity by antigenic variation-particularly via changes to surface-exposed antigens-is a major barrier to immune clearance. However, not all variants are equally fit in all environments. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.
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http://dx.doi.org/10.1038/s41564-021-00911-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611113PMC
July 2021

Dynamic modelling to identify mitigation strategies for the COVID-19 pandemic.

Swiss Med Wkly 2021 04 4;151:w20487. Epub 2021 May 4.

Department of Mechanical and Process Engineering, Swiss Federal Institute of Technology, IFD, Zurich, Switzerland.

Relevant pandemic-spread scenario simulations can provide guiding principles for containment and mitigation policies. We devised a compartmental model to predict the effectiveness of different mitigation strategies with a main focus on mass testing. The model consists of a set of simple differential equations considering the population size, reported and unreported infections, reported and unreported recoveries, and the number of COVID-19-inflicted deaths. We assumed that COVID-19 survivors are immune (e.g., mutations are not considered) and that the virus is primarily passed on by asymptomatic and pre-symptomatic individuals. Moreover, the current version of the model does not account for age-dependent differences in the death rates, but considers higher mortality rates due to temporary shortage of intensive care units. The model parameters have been chosen in a plausible range based on information found in the literature, but it is easily adaptable, i.e., these values can be replaced by updated information any time. We compared infection rates, the total number of people getting infected and the number of deaths in different scenarios. Social distancing or mass testing can contain or drastically reduce the infections and the predicted number of deaths when compared with a situation without mitigation. We found that mass testing alone and subsequent isolation of detected cases can be an effective mitigation strategy, alone and in combination with social distancing. It is of high practical relevance that a relationship between testing frequency and the effective reproduction number of the virus can be provided. However, unless one assumes that the virus can be globally defeated by reducing the number of infected persons to zero, testing must be upheld, albeit at reduced intensity, to prevent subsequent waves of infection. The model suggests that testing strategies can be equally effective as social distancing, though at much lower economic costs. We discuss how our mathematical model may help to devise an optimal mix of mitigation strategies against the COVID-19 pandemic. Moreover, we quantify the theoretical limit of contact tracing and by how much the effect of testing is enhanced, if applied to sub-populations with increased exposure risk or prevalence.
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http://dx.doi.org/10.4414/smw.2021.20487DOI Listing
April 2021

Bacterial detection by NAIP/NLRC4 elicits prompt contractions of intestinal epithelial cell layers.

Proc Natl Acad Sci U S A 2021 04;118(16)

Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden;

The gut epithelium serves to maximize the surface for nutrient and fluid uptake, but at the same time must provide a tight barrier to pathogens and remove damaged intestinal epithelial cells (IECs) without jeopardizing barrier integrity. How the epithelium coordinates these tasks remains a question of significant interest. We used imaging and an optical flow analysis pipeline to study the dynamicity of untransformed murine and human intestinal epithelia, cultured atop flexible hydrogel supports. Infection with the pathogen Typhimurium (Tm) within minutes elicited focal contractions with inward movements of up to ∼1,000 IECs. Genetics approaches and chimeric epithelial monolayers revealed contractions to be triggered by the NAIP/NLRC4 inflammasome, which sensed type-III secretion system and flagellar ligands upon bacterial invasion, converting the local tissue into a contraction epicenter. Execution of the response required swift sublytic Gasdermin D pore formation, ion fluxes, and the propagation of a myosin contraction pulse across the tissue. Importantly, focal contractions preceded, and could be uncoupled from, the death and expulsion of infected IECs. In both two-dimensional monolayers and three-dimensional enteroids, multiple infection-elicited contractions coalesced to produce shrinkage of the epithelium as a whole. Monolayers deficient for Caspase-1(-11) or Gasdermin D failed to elicit focal contractions but were still capable of infected IEC death and expulsion. Strikingly, these monolayers lost their integrity to a markedly higher extent than wild-type counterparts. We propose that prompt NAIP/NLRC4/Caspase-1/Gasdermin D/myosin-dependent contractions allow the epithelium to densify its cell packing in infected regions, thereby preventing tissue disintegration due to the subsequent IEC death and expulsion process.
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http://dx.doi.org/10.1073/pnas.2013963118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072224PMC
April 2021

High throughput sequencing provides exact genomic locations of inducible prophages and accurate phage-to-host ratios in gut microbial strains.

Microbiome 2021 03 29;9(1):77. Epub 2021 Mar 29.

Department of Biology, Institute of Microbiology and Swiss Institute of Bioinformatics, ETH Zürich, Zürich, Switzerland.

Background: Temperate phages influence the density, diversity and function of bacterial populations. Historically, they have been described as carriers of toxins. More recently, they have also been recognised as direct modulators of the gut microbiome, and indirectly of host health and disease. Despite recent advances in studying prophages using non-targeted sequencing approaches, methodological challenges in identifying inducible prophages in bacterial genomes and quantifying their activity have limited our understanding of prophage-host interactions.

Results: We present methods for using high-throughput sequencing data to locate inducible prophages, including those previously undiscovered, to quantify prophage activity and to investigate their replication. We first used the well-established Salmonella enterica serovar Typhimurium/p22 system to validate our methods for (i) quantifying phage-to-host ratios and (ii) accurately locating inducible prophages in the reference genome based on phage-to-host ratio differences and read alignment alterations between induced and non-induced prophages. Investigating prophages in bacterial strains from a murine gut model microbiota known as Oligo-MM or sDMDMm2, we located five novel inducible prophages in three strains, quantified their activity and showed signatures of lateral transduction potential for two of them. Furthermore, we show that the methods were also applicable to metagenomes of induced faecal samples from Oligo-MM mice, including for strains with a relative abundance below 1%, illustrating its potential for the discovery of inducible prophages also in more complex metagenomes. Finally, we show that predictions of prophage locations in reference genomes of the strains we studied were variable and inconsistent for four bioinformatic tools we tested, which highlights the importance of their experimental validation.

Conclusions: This study demonstrates that the integration of experimental induction and bioinformatic analysis presented here is a powerful approach to accurately locate inducible prophages using high-throughput sequencing data and to quantify their activity. The ability to generate such quantitative information will be critical in helping us to gain better insights into the factors that determine phage activity and how prophage-bacteria interactions influence our microbiome and impact human health. Video abstract.
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http://dx.doi.org/10.1186/s40168-021-01033-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008629PMC
March 2021

Epithelium-autonomous NAIP/NLRC4 prevents TNF-driven inflammatory destruction of the gut epithelial barrier in Salmonella-infected mice.

Mucosal Immunol 2021 05 17;14(3):615-629. Epub 2021 Mar 17.

Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland.

The gut epithelium is a critical protective barrier. Its NAIP/NLRC4 inflammasome senses infection by Gram-negative bacteria, including Salmonella Typhimurium (S.Tm) and promotes expulsion of infected enterocytes. During the first ~12-24 h, this reduces mucosal S.Tm loads at the price of moderate enteropathy. It remained unknown how this NAIP/NLRC4-dependent tradeoff would develop during subsequent infection stages. In NAIP/NLRC4-deficient mice, S.Tm elicited severe enteropathy within 72 h, characterized by elevated mucosal TNF (>20 pg/mg) production from bone marrow-derived cells, reduced regeneration, excessive enterocyte loss, and a collapse of the epithelial barrier. TNF-depleting antibodies prevented this destructive pathology. In hosts proficient for epithelial NAIP/NLRC4, a heterogeneous enterocyte death response with both apoptotic and pyroptotic features kept S.Tm loads persistently in check, thereby preventing this dire outcome altogether. Our results demonstrate that immediate and selective removal of infected enterocytes, by locally acting epithelium-autonomous NAIP/NLRC4, is required to avoid a TNF-driven inflammatory hyper-reaction that otherwise destroys the epithelial barrier.
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http://dx.doi.org/10.1038/s41385-021-00381-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8075861PMC
May 2021

Long-term evolution and short-term adaptation of microbiota strains and sub-strains in mice.

Cell Host Microbe 2021 04 3;29(4):650-663.e9. Epub 2021 Mar 3.

Maurice Müller Laboratories, Department for Biomedical Research, University of Bern, 3008 Bern, Switzerland; Department of Visceral Surgery and Medicine, Bern University Hospital, University of Bern, 3008 Bern, Switzerland. Electronic address:

Isobiotic mice, with an identical stable microbiota composition, potentially allow models of host-microbial mutualism to be studied over time and between different laboratories. To understand microbiota evolution in these models, we carried out a 6-year experiment in mice colonized with 12 representative taxa. Increased non-synonymous to synonymous mutation rates indicate positive selection in multiple taxa, particularly for genes annotated for nutrient acquisition or replication. Microbial sub-strains that evolved within a single taxon can stably coexist, consistent with niche partitioning of ecotypes in the complex intestinal environment. Dietary shifts trigger rapid transcriptional adaptation to macronutrient and micronutrient changes in individual taxa and alterations in taxa biomass. The proportions of different sub-strains are also rapidly altered after dietary shift. This indicates that microbial taxa within a mouse colony adapt to changes in the intestinal environment by long-term genomic positive selection and short-term effects of transcriptional reprogramming and adjustments in sub-strain proportions.
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http://dx.doi.org/10.1016/j.chom.2021.02.001DOI Listing
April 2021

Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo.

ISME J 2021 03 4;15(3):862-878. Epub 2020 Nov 4.

Institute of Integrative Biology, Department of Environmental Systems Science, ETH Zurich, Zurich, Switzerland.

Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.
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http://dx.doi.org/10.1038/s41396-020-00819-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026971PMC
March 2021

Epithelial inflammasomes in the defense against Salmonella gut infection.

Curr Opin Microbiol 2021 02 28;59:86-94. Epub 2020 Oct 28.

Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland. Electronic address:

The gut epithelium prevents bacterial access to the host's tissues and coordinates a number of mucosal defenses. Here, we review the function of epithelial inflammasomes in the infected host and focus on their role in defense against Salmonella Typhimurium. This pathogen employs flagella to swim towards the epithelium and a type III secretion system (TTSS) to dock and invade intestinal epithelial cells. Flagella and TTSS components are recognized by the canonical NAIP/NLRC4 inflammasome, while LPS activates the non-canonical Caspase-4/11 inflammasome. The relative contributions of these inflammasomes, the activated cell death pathways and the elicited mucosal defenses are subject to environmental control and appear to change along the infection trajectory. It will be an important future task to explain how this may enable defense against the challenges imposed by diverse bacterial enteropathogens.
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http://dx.doi.org/10.1016/j.mib.2020.09.014DOI Listing
February 2021

Elucidating host-microbe interactions in vivo by studying population dynamics using neutral genetic tags.

Immunology 2021 04 19;162(4):341-356. Epub 2020 Oct 19.

Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland.

Host-microbe interactions are highly dynamic in space and time, in particular in the case of infections. Pathogen population sizes, microbial phenotypes and the nature of the host responses often change dramatically over time. These features pose particular challenges when deciphering the underlying mechanisms of these interactions experimentally, as traditional microbiological and immunological methods mostly provide snapshots of population sizes or sparse time series. Recent approaches - combining experiments using neutral genetic tags with stochastic population dynamic models - allow more precise quantification of biologically relevant parameters that govern the interaction between microbe and host cell populations. This is accomplished by exploiting the patterns of change of tag composition in the microbe or host cell population under study. These models can be used to predict the effects of immunodeficiencies or therapies (e.g. antibiotic treatment) on populations and thereby generate hypotheses and refine experimental designs. In this review, we present tools to study population dynamics in vivo using genetic tags, explain examples for their implementation and briefly discuss future applications.
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http://dx.doi.org/10.1111/imm.13266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968395PMC
April 2021

How Food Affects Colonization Resistance Against Enteropathogenic Bacteria.

Annu Rev Microbiol 2020 09 21;74:787-813. Epub 2020 Jul 21.

Institute of Microbiology, Department of Biology, ETH Zurich, CH-8093 Zurich, Switzerland; email:

Food has a major impact on all aspects of health. Recent data suggest that food composition can also affect susceptibility to infections by enteropathogenic bacteria. Here, we discuss how food may alter the microbiota as well as mucosal defenses and how this can affect infection. Typhimurium diarrhea serves as a paradigm, and complementary evidence comes from other pathogens. We discuss the effects of food composition on colonization resistance, host defenses, and the infection process as well as the merits and limitations of mouse models and experimental foods, which are available to decipher the underlying mechanisms.
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http://dx.doi.org/10.1146/annurev-micro-020420-013457DOI Listing
September 2020

Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection.

Nat Microbiol 2020 09 8;5(9):1119-1133. Epub 2020 Jun 8.

European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.
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http://dx.doi.org/10.1038/s41564-020-0736-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610801PMC
September 2020

Evolutionary causes and consequences of bacterial antibiotic persistence.

Nat Rev Microbiol 2020 09 27;18(9):479-490. Epub 2020 May 27.

Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland.

Antibiotic treatment failure is of growing concern. Genetically encoded resistance is key in driving this process. However, there is increasing evidence that bacterial antibiotic persistence, a non-genetically encoded and reversible loss of antibiotic susceptibility, contributes to treatment failure and emergence of resistant strains as well. In this Review, we discuss the evolutionary forces that may drive the selection for antibiotic persistence. We review how some aspects of antibiotic persistence have been directly selected for whereas others result from indirect selection in disparate ecological contexts. We then discuss the consequences of antibiotic persistence on pathogen evolution. Persisters can facilitate the evolution of antibiotic resistance and virulence. Finally, we propose practical means to prevent persister formation and how this may help to slow down the evolution of virulence and resistance in pathogens.
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http://dx.doi.org/10.1038/s41579-020-0378-zDOI Listing
September 2020

Import of Aspartate and Malate by DcuABC Drives H/Fumarate Respiration to Promote Initial Salmonella Gut-Lumen Colonization in Mice.

Cell Host Microbe 2020 06 15;27(6):922-936.e6. Epub 2020 May 15.

Institute of Microbiology, D-BIOL, ETH Zürich, 8093 Zürich, Switzerland. Electronic address:

Initial enteropathogen growth in the microbiota-colonized gut is poorly understood. Salmonella Typhimurium is metabolically adaptable and can harvest energy by anaerobic respiration using microbiota-derived hydrogen (H) as an electron donor and fumarate as an electron acceptor. As fumarate is scarce in the gut, the source of this electron acceptor is unclear. Here, transposon sequencing analysis along the colonization trajectory of S. Typhimurium implicates the C4-dicarboxylate antiporter DcuABC in early murine gut colonization. In competitive colonization assays, DcuABC and enzymes that convert the C4-dicarboxylates aspartate and malate into fumarate (AspA, FumABC), are required for fumarate/H-dependent initial growth. Thus, S. Typhimurium obtains fumarate by DcuABC-mediated import and conversion of L-malate and L-aspartate. Fumarate reduction yields succinate, which is exported by DcuABC in exchange for L-aspartate and L-malate. This cycle allows S. Typhimurium to harvest energy by H/fumarate respiration in the microbiota-colonized gut. This strategy may also be relevant for commensal E. coli diminishing the S. Typhimurium infection.
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http://dx.doi.org/10.1016/j.chom.2020.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292772PMC
June 2020

Salmonella Typhimurium discreet-invasion of the murine gut absorptive epithelium.

PLoS Pathog 2020 05 4;16(5):e1008503. Epub 2020 May 4.

Institute of Microbiology, Department of Biology, ETH Zürich, Zürich, Switzerland.

Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host's gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through "discreet-invasion". This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context.
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http://dx.doi.org/10.1371/journal.ppat.1008503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7224572PMC
May 2020

Germ-free and microbiota-associated mice yield small intestinal epithelial organoids with equivalent and robust transcriptome/proteome expression phenotypes.

Cell Microbiol 2020 06 4;22(6):e13191. Epub 2020 Mar 4.

Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland.

Intestinal epithelial organoids established from gut tissue have become a widely used research tool. However, it remains unclear how environmental cues, divergent microbiota composition and other sources of variation before, during and after establishment confound organoid properties, and how these properties relate to the original tissue. While environmental influences cannot be easily addressed in human organoids, mice offer a controlled assay-system. Here, we probed the effect of donor microbiota differences, previously identified as a confounding factor in murine in vivo studies, on organoids. We analysed the proteomes and transcriptomes of primary organoid cultures established from two colonised and one germ-free mouse colony of C57BL/6J genetic background, and compared them to their tissue of origin and commonly used cell lines. While an imprint of microbiota-exposure was observed on the proteome of epithelial samples, the long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead, stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts and closely mimicked expression patterns in the gut epithelium. This included the inflammasome components ASC, Naip1-6, Nlrc4 and Caspase-1, which were highly expressed in all organoids compared to the reference cell line m-IC or mouse embryonic fibroblasts. Taken together, these results reveal that the donor microbiota has little effect on the organoid phenotype and suggest that organoids represent a more suitable culture model than immortalised cell lines, in particular for studies of intestinal epithelial inflammasomes.
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http://dx.doi.org/10.1111/cmi.13191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317401PMC
June 2020

Intestinal epithelial NAIP/NLRC4 restricts systemic dissemination of the adapted pathogen Salmonella Typhimurium due to site-specific bacterial PAMP expression.

Mucosal Immunol 2020 05 17;13(3):530-544. Epub 2020 Jan 17.

Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland.

Inflammasomes can prevent systemic dissemination of enteropathogenic bacteria. As adapted pathogens including Salmonella Typhimurium (S. Tm) have evolved evasion strategies, it has remained unclear when and where inflammasomes restrict their dissemination. Bacterial population dynamics establish that the NAIP/NLRC4 inflammasome specifically restricts S. Tm migration from the gut to draining lymph nodes. This is solely attributable to NAIP/NLRC4 within intestinal epithelial cells (IECs), while S. Tm evades restriction by phagocyte NAIP/NLRC4. NLRP3 and Caspase-11 also fail to restrict S. Tm mucosa traversal, migration to lymph nodes, and systemic pathogen growth. The ability of IECs (not phagocytes) to mount a NAIP/NLRC4 defense in vivo is explained by particularly high NAIP/NLRC4 expression in IECs and the necessity for epithelium-invading S. Tm to express the NAIP1-6 ligands-flagella and type-III-secretion-system-1. Imaging reveals both ligands to be promptly downregulated following IEC-traversal. These results highlight the importance of intestinal epithelial NAIP/NLRC4 in blocking bacterial dissemination in vivo, and explain why this constitutes a uniquely evasion-proof defense against the adapted enteropathogen S. Tm.
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http://dx.doi.org/10.1038/s41385-019-0247-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181392PMC
May 2020

Escherichia coli limits Salmonella Typhimurium infections after diet shifts and fat-mediated microbiota perturbation in mice.

Nat Microbiol 2019 12 7;4(12):2164-2174. Epub 2019 Oct 7.

Institute of Microbiology, D-BIOL, ETH Zürich, Zürich, Switzerland.

The microbiota confers colonization resistance, which blocks Salmonella gut colonization. As diet affects microbiota composition, we studied whether food composition shifts enhance susceptibility to infection. Shifting mice to diets with reduced fibre or elevated fat content for 24 h boosted Salmonella Typhimurium or Escherichia coli gut colonization and plasmid transfer. Here, we studied the effect of dietary fat. Colonization resistance was restored within 48 h of return to maintenance diet. Salmonella gut colonization was also boosted by two oral doses of oleic acid or bile salts. These pathogen blooms required Salmonella's AcrAB/TolC-dependent bile resistance. Our data indicate that fat-elicited bile promoted Salmonella gut colonization. Both E. coli and Salmonella show much higher bile resistance than the microbiota. Correspondingly, competitive E. coli can be protective in the fat-challenged gut. Diet shifts and fat-elicited bile promote S. Typhimurium gut infections in mice lacking E. coli in their microbiota. This mouse model may be useful for studying pathogen-microbiota-host interactions, the protective effect of E. coli, to analyse the spread of resistance plasmids and assess the impact of food components on the infection process.
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http://dx.doi.org/10.1038/s41564-019-0568-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881180PMC
December 2019

Salmonella persisters promote the spread of antibiotic resistance plasmids in the gut.

Nature 2019 09 4;573(7773):276-280. Epub 2019 Sep 4.

Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland.

The emergence of antibiotic-resistant bacteria through mutations or the acquisition of genetic material such as resistance plasmids represents a major public health issue. Persisters are subpopulations of bacteria that survive antibiotics by reversibly adapting their physiology, and can promote the emergence of antibiotic-resistant mutants. We investigated whether persisters can also promote the spread of resistance plasmids. In contrast to mutations, the transfer of resistance plasmids requires the co-occurrence of both a donor and a recipient bacterial strain. For our experiments, we chose the facultative intracellular entero-pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) and Escherichia coli, a common member of the microbiota. S. Typhimurium forms persisters that survive antibiotic therapy in several host tissues. Here we show that tissue-associated S. Typhimurium persisters represent long-lived reservoirs of plasmid donors or recipients. The formation of reservoirs of S. Typhimurium persisters requires Salmonella pathogenicity island (SPI)-1 and/or SPI-2 in gut-associated tissues, or SPI-2 at systemic sites. The re-seeding of these persister bacteria into the gut lumen enables the co-occurrence of donors with gut-resident recipients, and thereby favours plasmid transfer between various strains of Enterobacteriaceae. We observe up to 99% transconjugants within two to three days of re-seeding. Mathematical modelling shows that rare re-seeding events may suffice for a high frequency of conjugation. Vaccination reduces the formation of reservoirs of persisters after oral infection with S. Typhimurium, as well as subsequent plasmid transfer. We conclude that-even without selection for plasmid-encoded resistance genes-small reservoirs of pathogen persisters can foster the spread of promiscuous resistance plasmids in the gut.
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http://dx.doi.org/10.1038/s41586-019-1521-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744281PMC
September 2019
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