Biology subjects

Basler, M.

Publications and source records attributed to Basler, M..

15 recordsLinked to original sources

bioRxiv2026

Correlation of Live Imaging and Fixed-Expansion Microscopy to study intracellular pathogens

Expansion microscopy (ExM) enables high-resolution fluorescence imaging by physically enlarging biological samples, but its application to intracellular bacteria remains challenging. Here, we adapted the Ten-fold Robust Expansion Microscopy protocol to study host-pathogen interactions. Expansion of Burkholderia thailandensis-infected epithelial cells revealed previously unresolved membrane architectures associated with bacterial cell-to-cell dissemination. While host cells expanded approximately 9-fold, bacterial cells displayed incomplete and heterogeneous expansion. We found that fixation conditions strongly influence bacterial expansion, while presence of peptidoglycan partially limits expansion. To connect the membrane ultrastructures revealed by expansion with infection dynamics, we developed Live-to-Fixed Expansion microscopy (LiFEx), a workflow that correlates dynamic events observed by live-cell imaging with high-resolution cellular structures resolved by ExM. By correlating T6SS-5 assembly with subsequent high-resolution membrane architecture, LiFEx revealed membrane alterations developing within minutes following T6SS-5 assembly. Together, our approach enabled the investigation of dynamic host-pathogen interactions across complementary spatial and temporal scales.

microbiology↗

bioRxiv2026

Subpopulations of Pseudomonas aeruginosa inflict distinct tissue damage during infection

P. aeruginosa is a Gram-negative, ubiquitous, opportunistic pathogen that can colonize open wounds and the respiratory airways and is particularly problematic for patients suffering from cystic fibrosis (CF) where it can cause chronic infections. One factor contributing to P. aeruginosa diversity during infection is phenotypic heterogeneity. Phenotypic heterogeneity is the ability of bacteria to express different levels of selected pathways, giving rise to multiple bacterial subpopulations in the same environment. The prevalence and relevance of this phenomenon have been poorly characterized in infection models. In this study, we developed strategies to detect these subpopulations via single cell reporters, flow cytometry and fluorescence microscopy, in simple infection models and a physiologically relevant, transwell-based model of the bronchial epithelium. Next, we optimized cell sorting coupled to low input proteomics approaches to characterize the physiological state of the identified subpopulations, and genetically manipulated P. aeruginosa to lock them in ON or OFF states of expression of phenotypically heterogeneous pathways. We identify that type IVc/Tad pili are expressed in a subset of P. aeruginosa cells and inflict damage to the host tissue, while type Tad pili-negative subpopulations express other virulence factors instead. Lastly, we validated the heterogeneous expression of this machinery across clinical strains. Our findings uncovered the diversity of strategies employed by P. aeruginosa for pathogenesis, where distinct bacterial subpopulations use different virulence factors to damage respiratory epithelia.

microbiology↗

bioRxiv2026

Intranuclear niche: actin-tail mediated nuclear entry by intracellular pathogens

Intracellular pathogens localize to various niches in the host cells to avoid immune detection. However, very little is known about bacteria that enter the host nuclei. Here we report that Burkholderia thailandensis, a facultative intracellular pathogen, can enter eukaryotic nuclei and replicate. Nuclear invasion events were rare, occurring 1 in 500-1,000 infected cells, and inhibition of cell division further reduced the frequency of these events. Moreover, we show that nuclear entry requires actin tail motility, although it is independent of other virulence factors such as the Type III Secretion System, Type VI Secretion System-5, and flagella motility. Inactivation of actin tail motility by deleting bimA or inhibition of actin polymerisation by cytochalasin D abolished nuclear entry. Surprisingly, we observed that accumulation of B. thailandensis in the nucleus activated assembly of the Type VI Secretion Systems-5. We further show that Shigella flexneri also enters nucleus in an actin polymerization dependent mechanism. Together, we show that actin tail forming intracellular pathogens occasionally localize to the nucleus, and while this largely requires host cell division, it may provide pathogens with a protective niche in certain mitotically active cells, such as skin, gut or epithelial cells.

microbiology↗

bioRxiv2026

Evolved resistance against the type 6 secretion system is toxin specific

Across ecosystems, microbes face attacks by competitors armed with lethal weapons, including toxin-injecting Type 6 Secretion Systems (T6SSs). This in turn selects for T6SS resistance, reducing the future effectiveness of T6SS weaponry in competition. However, we have only a limited understanding of how resistance to T6SS attacks evolves de novo. A key challenge is that T6SS-armed bacteria are highly diverse in the number and type of toxins they inject, spanning multiple distinct modes of action. Do functionally distinct T6SS toxins select for different mechanisms of resistance? To address this, we combine genomics, resistance assays and fluorescence microscopy, to characterise evolved resistances against common T6SS toxin classes across spatial scales. We discovered that amidase and lipase toxins select for mutations in distinct sets of genes, resulting in toxin-specific resistance phenotypes and fitness costs. We also discovered resistance trade-offs: lipase-evolved E. coli became less vulnerable to lipase membrane damage, at the cost of increased susceptibility to lysis by amidase toxins. Finally, using single-gene knockout mutants from the Keio collection we confirmed that specific genes not previously linked to T6SS resistance, including inner membrane transporters, osmo-sensing systems and stress response pathways, conferred resistance to specific toxins while generating sensitivity to others. The specificity of resistance, and associated trade-offs observed, are likely to constrain de novo evolution of resistance against T6SS attackers armed with multiple functionally distinct toxins, helping to explain why T6SS systems are so widespread in nature.

microbiology↗

bioRxiv2026

Regulation of Type VI secretion systems of Burkholderia thailandensis during transition to intracellular lifestyle

Pathogenic bacteria tightly regulate gene expression in response to environmental cues. In Burkholderia species, the Type VI Secretion Systems (T6SS) mediate interbacterial competition and host cell interactions. Here, we show that B. thailandensis switches from the antibacterial T6SS-1 to the anti-eukaryotic T6SS-5 during phagosomal maturation. While T6SS-1 expression persists in the host cells, its assembly progressively declines with increasing T6SS-5 expression. This switch is controlled by the two-component system VirAG as deletion of virA blocks T6SS-5 expression and restores T6SS-1 assembly in host cells. Notably, T6SS-1 activity in host cells induces mitochondrial fragmentation and apoptosis. Our data suggest that Burkholderia has evolved a mechanism to silence T6SS-1 upon host entry to preserve its replicative niche.

microbiology↗

bioRxiv2026

Aeromonas adhesins facilitate kin and non-kin attachment to enable T6SS-mediated antagonism in liquid

Bacterial ability to deploy the type VI secretion system (T6SS) against rivals requires prolonged cell-cell interactions. Such interactions are facilitated on solid surfaces but are assumed to be absent in liquid, leading to the conventional dismissal of T6SS-mediated competition in liquid environments. Here, we find that Aeromonas jandaei employs its T6SS to eliminate diverse bacterial competitors in liquid media. Using a workflow that monitors interbacterial competition via prey luminescence, we demonstrate that auto-aggregation and co-aggregation, facilitated by distinct adhesins, enable kin and non-kin recognition and intoxication in a T6SS-dependent manner. Furthermore, we show that another marine bacterium, Vibrio coralliilyticus, employs T6SS to intoxicate rivals in liquid media. Collectively, our results indicate that T6SS-mediated competition in liquid is more common in marine bacteria than previously anticipated, and can be facilitated by diverse molecular mechanisms that govern cell aggregation.

microbiology↗

bioRxiv2025

TagF coordinates spike-loading as an intermediate checkpoint in Type VI Secretion System assembly

Diderm bacteria use contact-dependent Type VI secretion systems (T6SS) to gain a competitive advantage within bacterial communities and during infection. Whereas the structural core assembly of T6SS is well characterized, the role of diverse accessory proteins during assembly remains under investigation. One well-conserved accessory protein is TagF, a post-translational inhibitor of T6SS dynamics, which was previously characterized in the context of a kinase-phosphatase signaling relay. Here, we identify a subset of T6SS clusters in which TagF occurs in a distinct regulatory context and without known binding partners. Investigating the role of TagF in the constitutively active Acinetobacter baylyi T6SS, we show that though TagF can suppress assembly dynamics, it primarily acts as an assembly coordinator. Using structured illumination microscopy, we show that TagF coordinates the transition from baseplate assembly to sheath elongation by preventing premature and nonproductive sheath assembly in absence of the VgrG spike. Direct interactions with the conserved T6SS tube protein Hcp suggested that TagF blocks sheath elongation by binding and disrupting Hcp hexamers and thus preventing tube formation. Finally, we show that TagF activity depends on crosstalk with TagZ, a previously uncharacterized membrane-associated accessory protein that recruits TagF to the cell periphery, and demonstrate that this interaction can be specifically disrupted by expression of an artificial TagF-binding protein. Together, our findings establish a novel role of TagF as a checkpoint protein that controls for spike-insertion into the baseplate to ensure effective T6SS assembly.

microbiology↗

bioRxiv2025

Highly Correlated Activity across Higher-Order Thalamic Nuclei in Awake and Anesthetized States

The thalamus is an egg-shaped structure deep inside the brain and is of central importance for our cognitive functions. The thalamus can be divided into first- and higher-order (HO) nuclei. Unlike first-order thalamic regions which are known to primarily relay sensory signals to the neocortex, the functions of HO thalamic nuclei remain far less clear. Although previous studies indicate HO thalamic nuclei influence cortical processing and consciousness, most studies examined single thalamic nuclei in isolation. To investigate the relation between distinct HO thalamic nuclei, we recorded local field potentials (LFP) and multi-unit activity (MUA) across multiple thalamic nuclei simultaneously in awake and anesthetized mice with sensory stimulation. The effect of voluntary locomotion was also studied in the awake state. Surprisingly, we found that both LFP and MUA were strongly correlated across HO nuclei in both states, which cannot be explained by volume conduction alone but suggests the existence of shared synaptic inputs. Furthermore, in the awake state locomotion and sensory stimuli activated many HO nuclei, including those that have not classically been regarded as sensory or motor. These findings challenge the classical view of the thalamus being the aggregate of isolated, independent nuclei and suggest that the thalamus serves as an ideal bottleneck whose coordination resulted in effective orchestration of the entire brain.

neuroscience↗

bioRxiv2024

Antagonism as a foraging strategy in microbial communities

In natural habitats, nutrient availability limits bacterial growth. We discovered that bacteria can overcome this limitation by acquiring nutrients through lysing neighboring cells via contact-dependent antagonism. Using single-cell live imaging and isotopic markers, we found that during starvation the type VI secretion system, which transfers toxins into neighboring cells, increases growth by enabling the uptake of nutrients from lysing cells. In a spatially structured environment, the gradual lysis of just a few target cells allows for the growth of a new antagonist cell. Genomic adaptations in antagonists, characterized by a reduced metabolic gene repertoire, and the prevalence of the type VI secretion system in natural environments, such as global oceans and soils, suggest that bacterial antagonism has the potential to substantially contribute to nutrient transfer within microbial communities across various ecosystems.

microbiology↗

bioRxiv2024

Mechanisms of P. aeruginosa resistance to Type VI Secretion System attacks

The Type VI Secretion System (T6SS) is a molecular nanomachine that injects toxic effector proteins into the environment or neighbouring cells, playing an important role in interbacterial competition and host antagonism during infection. Pseudomonas aeruginosa encodes three T6SSs. The H1-T6SS delivers toxins in response to attacks mediated by the T6SS of aggressive bacteria, suggesting that P. aeruginosa can resist T6SS assaults. The mechanisms of resistance are poorly characterized. Here, we performed a CRISPRi screen to identify pathways involved in resistance to T6SS effectors of Acinetobacter baylyi and Vibrio cholerae. We show that members of the GacA/GacS regulon, such as the mag operon or aas, and GacA-independent factors, such as the outer membrane protein OprF, confer resistance to different types of T6SS toxins. Interestingly, some of these T6SS resistance mechanisms lead to higher antibiotic susceptibility, suggesting complex evolutionary links between T6SS and antibiotic resistance.

microbiology↗

bioRxiv2024

Multiplicity of Type 6 Secretion System toxins limits the evolution of resistance

The bacterial Type 6 Secretion System (T6SS) is a toxin-injecting nanoweapon that mediates competition in plant- and animal-associated microbial communities. Bacteria can evolve de novo resistance against T6SS attacks, but resistance is far from universal in natural communities, suggesting key features of T6SS weaponry may act to limit its evolution. Here, we combine eco-evolutionary modelling and experimental evolution to examine how toxin type and multiplicity in Acinetobacter baylyi attackers shape resistance evolution in susceptible Escherichia coli competitors. In both our models and experiments, we find that combinations of multiple distinct toxins limit resistance evolution by creating genetic bottlenecks, driving resistant lineages extinct before they can reach high frequency. We also show that, paradoxically, single-toxin attackers often drive the evolution of cross-resistance, protecting bacteria against unfamiliar toxin combinations, even though such evolutionary pathways were inaccessible against multi-toxin attackers. Our findings indicate that, comparable to antimicrobial and anticancer combination therapies, multi-toxin T6SS arsenals function to limit resistance evolution in competing microbes. This helps us to understand why T6SSs remain widespread and effective weapons in microbial communities, and why many bacteria T6SS-armed encode functionally diverse anti-competitor toxins. SignificanceToxin secretion systems, such as T6SSs, are widely used by bacteria to inhibit competing microorganisms. Here, we show that the secretion of multiple toxins in combination can suppress the evolution of resistance to the T6SS, rationalising its continued widespread use across diverse communities. Our work shows that principles of combination therapy--well known in the context of antimicrobial, antiviral and anticancer therapies--also apply in the context of microbial warfare, helping to explain why many bacteria maintain diverse T6SS toxin arsenals. Resistance suppression is also a technologically useful property of T6SS toxin cocktails, which could be harnessed as part of future biocontrol or biotherapeutic applications, using live T6SS-armed bacteria to limit the growth of problem microorganisms.

microbiology↗

bioRxiv2023

Lipopolysaccharide integrity primes bacterial sensitivity to a cell wall-degrading intermicrobial toxin

Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the molecular basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa, Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, surface lipopolysaccharide, that modulate Tae1 toxicity in vivo. Disruption of lipopolysaccharide synthesis provided Escherichia coli (Eco) with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study highlights the consequences of co-regulating essential pathways on recipient fitness during interbacterial competition, and how antibacterial toxins leverage cellular vulnerabilities that are both direct and indirect to their specific targets in vivo.

microbiology↗

bioRxiv2021

reComBat: Batch effect removal in large-scale, multi-source omics data integration

MotivationWith the steadily increasing abundance of omics data produced all over the world under vastly different experimental conditions residing in public databases, a crucial step in many data-driven bioinformatics applications is that of data integration. The challenge of batch-effect removal for entire databases lies in the large number of batches and biological variation which can result in design matrix singularity. This problem can currently not be solved satisfactorily by any common batch-correction algorithm. ResultsWe present reComBat, a regularized version of the empirical Bayes method to overcome this limitation and benchmark it against popular approaches for the harmonization of public gene expression data (both microarray and bulkRNAsq) of the human opportunistic pathogen Pseudomonas aeruginosa. Batch-effects are successfully mitigated while biologically meaningful gene expression variation is retained. reComBat fills the gap in batch-correction approaches applicable to large-scale, public omics databases and opens up new avenues for data-driven analysis of complex biological processes beyond the scope of a single study. Contactmichael.adamer@bsse.ethz.ch AvailabilityThe code is available at https://github.com/BorgwardtLab/reComBat, all data and evaluation code can be found at https://github.com/BorgwardtLab/batchCorrectionPublicData

bioinformatics↗

bioRxiv2020

Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut "anaerobes" are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen (1), with oxygen available to bacteria growing at the mucus layer. Using a combination of analyses, we show that Bacteroides species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 - 0.14% oxygen, defined as nanaerobic conditions (2). Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in Bacteroides species. Type strains of four different Bacteroides species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields high fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic Type VI secretion system processes in metabolically active B. fragilis. The ability to visualize fluorescently-labeled Bacteroides and fluorescently-linked proteins in actively growing nanaerobic gut symbionts ushers in a new age of imaging analyses in these bacteria. SignificanceDespite many recent technological advances to study the human gut microbiota, we still lack a facile system to image dynamic cellular processes in most abundant gut species due to the requirement of oxygen for chromophore maturation of commonly used fluorescent proteins. Here, we took advantage of the ability of anaerobes of the gut microbiota to respire aerobically and grow robustly at 0.10- 0.14% oxygen. This physiologic concentration of oxygen is sufficient for fluorescent proteins to mature, allowing for visualization of biological processes never before imaged in these bacteria. This advance will allow for numerous types of analyses in actively-growing "nanaerobic" gut bacteria including subcellular protein localizations, single-cell analyses, biofilm imaging, and protein interactions with other microbes and the host.

microbiology↗

bioRxiv2017

VipA N-terminal linker and VipB-VipB interaction modulate the contraction of Type VI secretion system sheath

Secretion systems are essential for bacteria to survive and manipulate their environment. The bacterial Type VI Secretion System (T6SS) generates the force needed for protein translocation by the contraction of a long polymer called sheath, which is composed of interconnected VipA/VipB subunits forming a six-start helix. The mechanism of T6SS sheath contraction and the structure of its extended state are unknown. Here we show that elongating the N-terminal VipA linker or eliminating charge of a specific VipB residue abolished sheath contraction and delivery of effectors into target cells. The assembly of the non-contractile sheaths was dependent on the baseplate component TssE and mass-spectrometry analysis identified Hcp, VgrG and other components of the T6SS baseplate specifically associated with stable non-contractile sheaths. The ability to lock T6SS in the pre-firing state opens new possibilities for understanding its mode of action.

microbiology↗