Biology subjects

Bakkeren, E.

Publications and source records attributed to Bakkeren, E..

6 recordsLinked to original sources

bioRxiv2025

Reprogrammed SimCells for Antimicrobial Therapy

Antimicrobial resistance (AMR) is a critical global health challenge. In this study, we developed a novel platform based on chromosome-free and non-replicating simple cells (SimCells, size 1-2 {micro}m) and mini-SimCells (size 100-400 nm) for targeted pathogen elimination. Engineered with surface-displayed nanobodies, SimCells and mini-SimCells selectively bind bacteria expressing specific antigens (e.g., OmpA in E. coli). The selective interactions facilitate close SimCell-pathogen proximity, enabling two antimicrobial mechanisms: direct injection of toxic effectors into bacterial cytoplasm via a heterologous expression of type VI secretion system (T6SS), and enzymatic conversion of aspirin into catechol by engineered salicylate hydroxylase, leading to sustained local production of hydrogen peroxide (H2O2). Our results demonstrate that both reprogrammed SimCells and mini-SimCells can eliminate target E. coli with high specificity and efficiency. Multi-dose reprogrammed mini-SimCell treatment led to a 103-fold selective reduction of targeted bacteria in mixed microbial communities, with minimal disruption to non-target bacteria. We demonstrate that reprogrammed mini-SimCells, engineered with nanobody targeting outer membrane protein OmpA of the clinically relevant multidrug resistant pathogen E. coli ST131, achieved elimination efficiencies over 97% at 24 and 48 hrs. This modularised plug-and-play antimicrobial platform provides a highly specific, efficient and adaptable solution for combating diverse AMR pathogens. Significance StatementAntimicrobial-resistant (AMR) bacteria cause millions of deaths worldwide annually, representing a critical global health challenge. In this study, we developed a novel therapeutic platform using two types of chromosome-free, non-replicating engineered bacterial cells: SimCells (1-2 {micro}m) and mini-SimCells (100-400 nm). These SimCells were engineered to selectively bind to targeted E. coli, facilitating precise delivery of toxic proteins via a Type VI secretion system (T6SS) and localised generation of hydrogen peroxide from aspirin. We demonstrate that mini-SimCells eliminated over 97% of a targeted AMR strain within 48 hours. Moreover, multiple-dose administration achieved a selective 103-fold reduction of targeted E. coli in mixed microbial communities. This modular plug-and-play platform offers an adaptable solution against diverse multi-drug-resistant pathogens.

synthetic biology↗

bioRxiv2022

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 this antibiotic resistance crisis. Despite the 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 (S. Tm) strain SL1344. We show that P3 is mobilized by interacting with the conjugation machinery of a second, 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 suggests that S. Tm 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. IMPORTANCES. Tm is a remarkably adaptable and globally abundant bacterial species that rapidly occupies new niches and survives unstable environmental conditions. As an enteric pathogen, it can potentially interact with a broad range of bacterial species residing in the mammalian gut. High abundance of bacteria in the gut lumen facilitate conjugation and spread of plasmid-encoded antibiotic resistance genes. By studying the transfer dynamics of the P3 plasmid in vitro and in vivo, we illustrate the impact of S. Tm-mediated antibiotic resistance spread via conjugation to a variety of relevant environmental and clinical bacterial isolates. Along with temperate phages or naked DNA, plasmids are among the most critical vehicles driving antibiotic resistance spread. Further understanding of the dynamics and drivers of antibiotic resistance transfer, along with identifying the environmental niches where this occurs, is needed to develop effective solutions for slowing down the emerging threat of multidrug-resistant bacterial pathogens.

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

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↗