Biology subjects

Hardt, W.-D.

Publications and source records attributed to Hardt, W.-D..

29 records · Page 2Linked to original sources

bioRxiv2022

Fitness advantage of Bacteroides thetaiotaomicron capsular polysaccharide is dependent on the resident microbiota

Many microbiota-based therapeutics rely on our ability to introduce a microbe of choice into an already-colonized intestine. However, we remain largely blind to the quantitative effects of processes determining colonization success. In this study, we used genetically-barcoded Bacteroides thetaiotaomicron (B.theta) strains in combination with mathematical modeling to quantify population bottlenecks experienced by B.theta during gut colonization. Integrating population bottlenecks sizes with careful quantification of net growth rates in vivo and in vitro allows us to build models describing the events during intestinal colonization in the context of gnotobiotic and complex microbiotas. Using these models, we estimated the decrease in niche size for B.theta colonization with increasing microbiota complexity. In addition, our system can be applied to mechanistically dissect colonization defects of mutant strains. As a proof of concept, we demonstrated that the competitive disadvantage of a B.theta mutant lacking capsular polysaccharide is due to a combination of an increased lag-phase before growth initiation in the gut, combined with an increased clearance rate. Crucially, the requirement for the B.theta capsule depended strongly on microbiota composition, suggesting that the dominant role may be protection from bacterial or phage aggression rather than from host-induced bactericidal mechanisms.

microbiology↗

bioRxiv2021

Circadian patterns of energy metabolism are only partially reconstituted in gnotobiotic mice

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 microbiome to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat-mass than germ-free 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 germ-free 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 per gram lean mass between the different microbiota groups, suggesting that cumulative very 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 germ-free mice the respiratory exchange ratio 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 germ-free mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a germ-free-like drop in liver glycogen storage during the light cycle and both liver and plasma metabolomes of OligoMM12 mice clustered closely with germ-free 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.

microbiology↗

bioRxiv2021

Pathogen invasion-dependent tissue reservoirs and plasmid-encoded antibiotic degradation promote within-host plasmid transfer

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 Salmonella enterica serovar Typhimurium (S.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 S.Tm could serve to diversify tissue-lodged plasmid reservoirs, and thereby promote plasmid spread. Starting with intraperitoneal mouse infections, we demonstrate that S.Tm cells re-seeding the gut lumen initiate clonal expansion. ESBL plasmid-encoded gut luminal antibiotic degradation by donors can foster recipient survival under beta-lactam antibiotic treatment, enhancing transconjugant formation upon re-seeding. S.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.

evolutionary biology↗

bioRxiv2021

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

Salmonella spp. express Salmonella pathogenicity island 1 (SPI-1) genes to mediate the initial phase of interaction with host cells. Prior studies indicate short-chain fatty acids, microbial metabolites at high concentrations in the gastrointestinal tract, limit SPI-1 gene expression. A number of reports show only a subset of Salmonella cells in a population express these genes, suggesting short-chain fatty acids could decrease SPI-1 population-level expression by acting on per-cell expression and/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 SPI-1 expression. Our results show short-chain fatty acids do not repress SPI-1 expression by individual cells. Rather, these compounds act to selectively slow the growth of SPI-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 SPI-1 cell-type proportions, our findings imply gut microbial metabolites act on cooperation between the two cell-types and ultimately influence Salmonellas capacity to establish within a host. Significance StatementEmergence of distinct cell-types in populations of genetically identical bacteria is common. Furthermore, it is becoming increasingly clear that cooperation between cell-types can be beneficial. This is the case during Salmonella infection, in which cooperation between inflammation-inducing virulent and fast-growing avirulent cell-types occurs during infection to aid in colonization of the host gut. Here, we show gut microbiota-derived metabolites slow growth by the virulent cell-type. Our study implies microbial metabolites shape cooperative interactions between the virulent and avirulent cell types, a finding that can help explain the wide array of clinical manifestations of Salmonella infection.

microbiology↗

bioRxiv2021

Cooperative virulence can emerge via horizontal gene transfer but is stabilized by transmission

Intestinal inflammation fuels Salmonella Typhimurium (S.Tm) transmission despite a fitness cost associated with the expression of virulence. Cheater mutants can emerge that profit from inflammation without enduring this cost. Intestinal virulence in S.Tm is therefore a cooperative trait, and its evolution a conundrum. Horizontal gene transfer (HGT) of cooperative alleles may facilitate the emergence of cooperative virulence, despite its instability. To test this hypothesis, we cloned hilD, coding for a master regulator of virulence, into a conjugative plasmid that is highly transferrable during intestinal colonization. We demonstrate that virulence can emerge by hilD transfer between avirulent strains in vivo. However, this was indeed unstable and hilD mutant cheaters arose within a few days. The timing of cheater emergence depended on the cost. We further show that stabilization of cooperative virulence in S.Tm is dependent on transmission dynamics, strengthened by population bottlenecks, leading cheaters to extinction and allowing cooperators to thrive.

evolutionary biology↗

bioRxiv2019

Rationally designed oral vaccines can set an evolutionary trap for Salmonella Typhimurium

Introductory paragraphThe ability of gut bacterial pathogens to escape immunity by antigenic variation, particularly via changes to surface-exposed antigens, is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2, 3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here we demonstrated 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-variation4, 5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen specific IgA and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of oral vaccines, we rationally induced IgA responses blocking all of these trajectories, which selected for Salmonella mutants carrying deletions of the O-antigen polymerase wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents, complement), 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.

immunology↗

bioRxiv2019

Extended-spectrum beta-lactamase antibiotic resistance plasmids have diverse transfer rates and can be spread in the absence of selection

Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global spread of antibiotic resistance. However, the relative contributions of factors that underlie the spread of clinically relevant plasmids are unclear. Here, we quantified conjugative transfer dynamics of Extended Spectrum Beta-Lactamase (ESBL) producing plasmids in the absence of antibiotics. We showed that clinical Escherichia coli strains natively associated with ESBL-plasmids conjugate efficiently with three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain, reaching final transconjugant frequencies of up to 1% within 24 hours in vitro. The variation of final transconjugant frequencies varied among plasmids, donors and recipients and was better explained by variation in conjugative transfer efficiency than by variable clonal expansion. We identified plasmid-specific genetic factors, specifically the presence/absence of transfer genes, that influenced final transconjugant frequencies. Finally, we investigated plasmid spread within the mouse intestine, demonstrating qualitative agreement between plasmid spread in vitro and in vivo. This suggests a potential for the prediction of plasmid spread in the gut of animals and humans, based on in vitro testing. Altogether, this may allow the identification of resistance plasmids with high spreading potential and help to devise appropriate measures to restrict their spread.

microbiology↗

bioRxiv2018

Spatiotemporal proteomics uncovers cathepsin-dependent host cell death during bacterial infection

Immune cells need to swiftly and effectively respond to invading pathogens. This response relies heavily on rapid protein synthesis and accurate cellular targeting to ensure pathogen destruction. In return, pathogens intercept this response to ensure their survival and proliferation. To gain insight into this dynamic interface, we combined click-chemistry with pulsed stable isotope labeling of amino acids (pSILAC-AHA) in cell culture to quantify the newly synthesised host proteome during macrophage infection with the model intracellular bacterial pathogen, Salmonella enterica Typhimurium (STm). We monitored newly synthesised proteins across different host cell compartments and infection stages, and used available proteomics data in response to lipopolysaccharide to deconvolute the STm-specific response. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus, the latter of which correlated with signatures of cell death. Pharmacological cathepsin inhibition suppressed Caspase-11 dependent macrophage cell death, thus demonstrating an active role for cathepsins during STm induced pyroptosis. Our study illustrates that resolving host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.

systems biology↗

bioRxiv2018

Microbiota stability in healthy individuals after single-dose lactulose challenge - a randomized controlled study

Background and aimsLactulose is a common food ingredient and widely used as a treatment for constipation or hepatic encephalopathy and a substrate for hydrogen breath tests. Lactulose is fermented by the colon microbiota resulting in the production of hydrogen (H2). H2 is a substrate for enteropathogens including Salmonella Typhimurium (S. Typhimurium) and increased H2 production upon lactulose ingestion might favor the growth of H2-consuming enteropathogens. We aimed to analyze effects of single-dose lactulose ingestion on the growth of intrinsic Escherichia coli (E. coli), which can be efficiently quantified by plating and which share most metabolic requirements with S. Typhimurium.\n\nMethods32 healthy volunteers (18 females, 14 males) were recruited. Participants were randomized for single-dose ingestion of 50 g lactulose or 50 g sucrose (controls). After ingestion, H2 in expiratory air and symptoms were recorded. Stool samples were acquired at days -1, 1 and 14. We analyzed 16S microbiota composition and abundance and characteristics of E. coli isolates.\n\nResultsLactulose ingestion resulted in diarrhea in 14/17 individuals. In 14/17 individuals, H2-levels in expiratory air increased by [≥]20 ppm within 3 hours after lactulose challenge. H2-levels correlated with the number of defecations within 6 hours. E. coli was detectable in feces of all subjects (2 x 102 - 109 CFU/g). However, the number of E. coli colony forming units (CFU) on selective media did not differ between any time point before or after challenge with sucrose or lactulose. The microbiota composition also remained stable upon lactulose exposure.\n\nConclusionIngestion of a single dose of 50 g lactulose does not significantly alter E. coli density in stool samples of healthy volunteers. 50 g lactulose therefore seems unlikely to sufficiently alter growth conditions in the intestine for a significant predisposition to infection with H2-consuming enteropathogens such as S. Typhimurium (www.clinicaltrials.gov NCT02397512).

microbiology↗

bioRxiv2018

Genomic, Proteomic and Phenotypic Heterogeneity in HeLa Cells across Laboratories: Implications for Reproducibility of Research Results

The independent reproduction of research results is a cornerstone of experimental research, yet it is beset by numerous challenges, including the quality and veracity of reagents and materials. Much of life science research depends on life materials, including human tissue culture cells. In this study we aimed at determining the degree of variability in the molecular makeup and the ensuing phenotypic consequences in commonly used human tissue culture cells. We collected 14 stock HeLa aliquots from 13 different laboratories across the globe, cultured them in uniform conditions and profiled the genome-wide copy numbers, mRNAs, proteins and protein turnover rates via genomic techniques and SWATH mass spectrometry, respectively. We also phenotyped each cell line with respect to the ability of transfected Let7 mimics to modulate Salmonella infection.\n\nWe discovered significant heterogeneity between HeLa variants, especially between lines of the CCL2 and Kyoto variety. We also observed progressive divergence within a specific cell line over 50 successive passages. From the aggregate multi-omic datasets we quantified the response of the cells to genomic variability across the transcriptome and proteome. We discovered organelle-specific proteome remodeling and buffering of protein abundance by protein complex stoichiometry, mediated by the adaptation of protein turnover rates. By associating quantitative proteotype and phenotype measurements we identified protein patterns that explained the varying response of the different cell lines to Salmonella infection.\n\nAltogether the results indicate a striking degree of genomic variability, the rapid evolution of genomic variability in culture and its complex translation into distinctive expressed molecular and phenotypic patterns. The results have broad implications for the interpretation and reproducibility of research results obtained from HeLa cells and provide important basis for a general discussion of the value and requirements for communicating research results obtained from human tissue culture cells.

systems biology↗

bioRxiv2018

Enchained growth and cluster dislocation: a possible mechanism for microbiota homeostasis

Immunoglobulin A is a class of antibodies produced by the adaptive immune system and secreted into the gut lumen to fight pathogenic bacteria. We recently demonstrated that the main physical effect of these antibodies is to enchain daughter bacteria, i.e. to cross-link bacteria into clusters as they divide, preventing them from interacting with epithelial cells. These links between bacteria may break over time. Using analytical and numerical calculations on several models to check the results robustness, we study the rate of increase in the number of free bacteria as a function of the replication rate of bacteria, and the resulting distribution of chain sizes. At higher replication rates, the bacteria replicate before the link between daughter bacteria breaks, leading to growing cluster sizes. However at low growth rates two daughter cells have a high probability to break apart. Thus the gut could produce IgA against all the bacteria it has encountered, but the most affected bacteria would be the fast replicating ones, which could destabilize the microbiota.

biophysics↗