Abstract Bacterial genomes are dynamic, shaped by gene gain, loss, and horizontal transfer; yet selection preserves the colocalization of functionally coupled genes into recurrent modules known as syntons. Many large-scale approaches do not explicitly account for phylogenetic relatedness between genomes which can inflate support for associations that are simply shared by close relatives. To address this, we introduce a scalable framework that clusters gene-family occurrences by genomic neighborhood and counts co-occurrences across distinct genomic contexts, reducing redundancy from vertical inheritance and uneven database sampling without explicit phylogenetic reconstruction. Applied to 32 791 RefSeq genomes comprising 123 million genes, this approach identifies 15 million syntons across 779 000 synton families, characterizing their diversity and revealing structured associations among antiphage defense systems. Leveraging this context-aware framework, we also introduce Context Residual Entropy (CRE) to quantify gene families neighborhood variability. By prioritizing independent evolutionary events over redundant genome-level counts, our approach provides a scalable, phylogenetic tree-free view of modular organization across bacterial genomes.
Candida albicans is an opportunistic pathogen residing in the gastro-intestinal tract of humans from where it can cause life-threatening systemic infection. Dysbiosis is one predisposing factor for C. albicans overgrowth, indicating that commensal bacteria limit fungal growth and convey colonization resistance. Phocaeicola vulgatus (formerly classified as Bacteroides) is an abundant gut commensal. We show that P. vulgatus can protect enterocytes in vitro from C. albicans damage. The protective effect is most pronounced if the bacteria pre-colonize host cells 6 h prior to addition of C. albicans. Colonization of the enterocytes with P. vulgatus leads to reduced adhesion of C. albicans, shorter hyphae, and increased fungal shedding, while the overall fungal burden is not reduced. The protective effect is contact-dependent but can be elicited to some degree by heat-inactivated bacterial cells. Our findings suggest that multiple mechanisms mediate the protective effect, including activation of self-defenses of the host cells to shed the pathogen, as well as a direct antagonistic interaction between the fungi and the bacteria targeting C. albicans filamentation, and thereby hyphae-associated virulence factors.
Fungi harbor unique primary and secondary metabolic pathways that represent a hidden treasure of biochemical and natural compounds. Their metabolism is central to their ability to interact with, to adapt to and to survive in host environments, and to cause human disease and great harm. Among these is Lichtheimia corymbifera, an emerging causative agent of mucormycosis that has been classified as high priority fungal pathogen by the World Health Organization and is gaining increasing importance as a model organism for research on invasive fungal infections. We reconstructed a genome-scale L. corymbifera metabolic model and show substantial differential metabolic activity to process carbohydrate or amino acid carbon sources. We furthermore adapt the model to proteome changes depending on the presence of human peripheral blood mononuclear cells (PBMCs) and show fungal sphingolipid metabolic activity changes next to changes in cytoskeleton and tight junction associated PBMC activity over three days of cultivation. These insights underline the possibility that L. corymbifera can potentially scavenge host-derived lipids to fortify its own cell membrane. We demonstrate that in silico metabolic predictions can provide testable hypotheses and can lead to the identification of metabolic processes which are essential for the development of targeted antifungal drugs and novel solutions for balancing host challenges.
Neisseria meningitidis is a human-adapted commensal pathogen that must continuously balance nutrient acquisition with stress tolerance. Here, we identify a type II-C CRISPR/Cas-associated small RNA (scaRNA) as a posttranscriptional regulator of the efeUOB operon and oxidative stress responses. Using in vitro RNA binding and structure probing assays, we show that the scaRNA interacts with the 5' untranslated region of efeO mRNA, leading to reduced translation of this component of the ferrous iron transporter EfeUOB. Consistent with this, efeO translational fusions demonstrate repression by the scaRNA, whereas a ΔscaRNA mutant shows increased reporter expression. We further show that meningococcal Cas9 (Nme1Cas9) is able to cleave scaRNA in vitro, but in vivo phenotypes are primarily scaRNA-dependent, indicating that Nme1Cas9 contributes, at most, indirectly to this regulation. In line with this observation, comparative proteomics revealed overlapping but distinct roles of scaRNA and Nme1Cas9 in oxidative stress adaptation, energy metabolism, and ion transport. While steady-state protein abundances did not capture all scaRNA-dependent effects, functional assays confirmed that scaRNA inactivation reduces survival under oxidative stress. Together, our results identify scaRNA-mediated repression of efeO as a novel posttranscriptional mechanism that contributes to stress adaptation in meningococci. These findings expand the functional repertoire of CRISPR-associated elements and suggest a role for small RNA-based regulation in iron-related stress adaptation in a major human pathogen.
Vulvovaginal candidiasis (VVC) is one of the most common fungal infections, affecting most women during their reproductive years. A core characteristic of VVC is the interplay between Candida albicans pathogenicity and dysregulated innate immune responses. While these inflammatory responses generally underlie VVC severity, epithelial type I interferon (IFN) responses have been associated with improved epithelial resistance to infection and dampened downstream neutrophil activation. Yet, the role of interferon-stimulated genes (ISGs) in epithelial resistance to C. albicans infection remains unclear. Here, we investigated the threshold for mounting type I IFN responses in A-431 vulvovaginal epithelial cells (VECs), and the role of interferon-stimulated gene 15 (ISG15) in epithelial resistance and inflammation. We found that increasing C. albicans burdens corresponded to increasing epithelial cytotoxicity and the release of neutrophil chemoattractant IL-8. Further, expression of IFNB1 and ISGs, specifically ISG15 and MX2, was induced at fungal burdens, before the induction of significant cytotoxicity at later time points. We observed intracellular ISG15 accumulation following C. albicans infection, yet detected reduced unconjugated intracellular ISG15, suggesting its conjugation to other proteins. Mechanistically, we show that ISG15 silencing reduced epithelial cytotoxicity and IL-8 responses to C. albicans infection. Concurrently, a C. albicans infection-specific downregulation of the gene encoding the cell death regulator Z-DNA binding protein 1 (ZBP1) was observed. Accordingly, ZBP1 silencing, similar to ISG15, exhibited reduced tissue damage and IL-8 responses. Collectively, our data suggest that ISG15, expressed by VECs upon C. albicans infection, may exert negative feedback on epithelial resistance to infection induced by type I IFNs.
Chemotaxis, the directed movement of an organism towards nutrients or away from noxious agents is a fundamental process for the survival of many micro-organisms. We combined high-resolution imaging, microfluidic gradients, and frame-by-frame tracking to re-evaluate Acanthamoeba chemotaxis to microbial glycans [mannan, mannose, N-acetyl-d-glucosamine (GlcNAc), N-acetyl-muramic acid (MurNAc)] and peptides [N-formyl methionyl-leucyl-phenylalanine (fMLP) and Boc-Phe-Leu-Phe-Leu-Phe (BOC-FLFLF)]. Our quantitative tracking results on Acanthamoeba castellanii confirm the core patterns in the original studies reported by Schuster and Levandowsky; attraction to fMLP and GlcNAc and lack of response to MurNAc or the peptide antagonist BOC-FLFLF, while revealing previously missed attraction to mannan. In contrast, Acanthamoeba polyphaga demonstrated a more restricted response, with significant chemotaxis observed only toward fMLP, and lack of motility in the presence of MurNAc or BOC-FLFLF. Notably, formyl peptide responses were differentially modulated: BOC-FLFLF reduced fMLP-induced directionality in A. castellanii without impairing motility, while in A. polyphaga, it suppressed both velocity and orientation. When considered alongside genomic analyses that do not reveal a canonical metazoan-like formyl peptide receptor, these behavioural differences suggest that formyl peptide sensing in Acanthamoeba relies on a divergent, pattern-recognition-like signalling strategy, rather than a conserved FPR homolog. These distinct chemoattractant "signatures" are consistent with micro-niche adaptation, and we hypothesise that fine scale tuning of receptor thresholds to local prey spectra contributes to the observed differences between the tested strains. By revisiting classical paradigms, this study offers new perspectives on Acanthamoeba chemotaxis and supports emerging models of protist pattern recognition paralleling innate immunity.
Upstream open reading frames (uORFs) in the 5' leader of bacterial mRNAs can modulate gene expression, yet genome-wide identification remains limited. We combined bioinformatic prediction of ribosome-binding sites (RBSs)-a Shine-Dalgarno sequence and a start codon-with experimental validation to uncover new uORFs in Sinorhizobium meliloti 2011. From totally 1106 predicted upstream RBSs (uRBSs), we first examined 15 candidates using eGFP reporters and integrating existing RNA-seq and Ribo-seq data. Translation was detected at 13 sites, with fluorescence intensity broadly correlating with predicted initiation rates. Two uRBSs correspond to gene start sites, thereby refining gene annotations. In nine cases, uRBS mutations affected downstream gene expression in reporter fusions. Among others, the data suggests that a Type I secretion system operon, the RNA chaperone gene hfq, and metabolic genes are regulated by uORFs. Four uORFs acted through translational coupling. We also identified uRBSs that were ribosome-occupied yet (nearly) silent in eGFP assays, and closely spaced to the downstream main RBS (mRBS). These uRBSs probably mediate ribosomal occlusion downregulating lacR and SM2011_RS36230. A re-screen of the prediction set revealed 335 close uRBS/mRBS pairs. Three of them were analyzed, supporting the proposed ribosomal occlusion mechanism for SM2011_RS03630 and SM2011_RS22110, while for glnK translational coupling to an uORF was suggested. These results indicate that uORFs are more widespread in bacteria than previously recognized and suggest that direct ribosomal occlusion of the mRBS is a novel mechanism for down-regulating protein synthesis.
Natural killer (NK) cells contribute to the innate immune system and are pivotal for the defence against opportunistic pathogens, including fungi. Aspergillus fumigatus (AF), a filamentous mold, can cause invasive pulmonary aspergillosis in immunocompromised patients, e.g. in patients after allogeneic stem cell transplantation (alloSCT). In this pilot study, we challenged NK cell samples from alloSCT recipients collected 90, 120, and 180 days after transplantation and from healthy individuals with AF and characterize the proteome response differences. We identified 2259 differentially abundant proteins between the NK cell proteomes of alloSCT recipients and healthy individuals. Among these, 1118 proteins were differentially abundant at all time points and 1931 proteins specifically at day 180 post-alloSCT. Following stimulation of NK cells with AF, we found a profoundly different early proteome (day 90, n=1652 proteins), while at day 180, only 77 proteins remained significantly differentially abundant. We identified, among others, a major differentially abundant protein cluster related to IL27RA (including OAS, STAT1, and MX). Furthermore, for selected markers [granzyme A (GZMA), Neural Cell Adhesion Molecule 1 (NCAM1/CD56), perforin-1 (PRF1)], we confirmed our proteome data by flow cytometry in NK cells from an independent second patient and healthy individual cohort. In conclusion, we demonstrate the advantage of combining comprehensive proteomic profiling with targeted flow cytometry to investigate NK cell responses to AF. Our data analysis connects STAT1 with IL27RA as well as granzyme, IFNg, and NCAM1 activity, which may be exploited towards future therapeutics warranting confirmation in larger study cohorts.
Salmonella enterica is a facultative intracellular pathogen capable of surviving within host cells, where it faces a sophisticated immune arsenal. Within the phagosomal compartment, the bacterium encounters significant stress from copper and reactive chlorine species like N-chlorotaurine (N-ChT) generated during the oxidative burst. We investigated the regulatory mechanisms enabling Salmonella to adapt to this dual copper/oxidative stress, specifically focusing on the regulation and function of CueP. This periplasmic protein was previously proposed to bind copper ions and to transfer them to the superoxide dismutase SodCII. Here, we demonstrated that copper specifically triggered the CueR pathway and that N-ChT activated the Cpx pathway; simultaneous exposure to both stresses resulted in maximum cueP expression levels. Moreover, CueP was shown to be important for copper resistance in the absence of the multicopper oxidase CueO and exhibits high thermostability in the presence of copper. Additionally, in a ΔcueO background, copper is sufficient to activate the Cpx pathway, ensuring robust cueP induction even without external oxidative signals. These findings establish a direct molecular link between a host antimicrobial agent (N-ChT) and the activation of the Cpx-CueP axis, revealing a new layer of bacterial adaptation to innate immunity. Moreover, they highlight an integrated response strategy contributing to bacterial adaptation to the dual copper/oxidative stress.
In heterogeneous environments, the hyphae of filamentous fungi and oomycetes can facilitate the dispersal of other microorganisms. The use of these "fungal highways" (FH) is regulated by both physical and biological factors with their interplay resulting in variable capabilities of different microbes to establish FH. Several devices have been developed to test the movement of bacteria across mycelium. However, these methods are usually time-consuming and cannot be applied at a large scale. In this study, we developed 3D-printed experimental devices that physically separate two environments while allowing hyphal networks to act as bridges for bacterial movement. The final design allows for the simultaneous testing of up to 10 pairs and the inclusion of any culturing media. With these devices, we investigated how fungal-bacterial pairing, nutrient conditions, and inoculation strategies influence FH formation. Bacterial transport was limited in nutrient-rich media but increased under poorer nutrient conditions, consistent with enhanced exploratory growth of the mycelium. Both cis- and trans-inoculation supported FH formation, although bacterial arrival was delayed in the absence of co-inoculation. The devices were used to demonstrate that transport of bacteria by FH was relevant for the colonization of a natural substrate. Finally, we established a novel in planta assay to evaluate FH formation during host colonization. This assay demonstrated that Fusarium graminearum can transport bacteria during wheat spike colonization. Together, these results provide accessible, scalable tools to study hyphal-mediated bacterial dispersal and highlight the combined role of biological specificity and nutrient context in the establishment of FH.
Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and cellular processes in vivo. Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free H. volcanii strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel plasmid system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins-from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1-while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the system's utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal Cas1 colocalizing with DNA-dense cellular regions and significant, dose-dependent changes in Cas1 mobility following UV-light-induced damage, providing evidence for its possible involvement in DNA damage response processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.
Fungi, and particularly fungal pathogens, are having an increasing impact on human health and economy. At the same time, modern high-throughput technologies offer insights deep into the molecular level and thus mechanisms, giving scientists new opportunities to identify fungal biomarkers and essential components for survival and virulence. This wealth of data, however, is most often only analyzed in the context of a specific scientific question, while many more projects may benefit from a multifaceted view on e.g. fungal gene expression under various conditions. The prime challenge is the limited access to readily pre-processed data and circumventing technological biases introduced by different sequencing platforms and software tools across different projects. We here present FungiNetDB, a web platform comprising 139 fungal pathogenicity datasets and statistical analysis of more than 2000 different pairwise gene expression comparisons. FungiNetDB thus resembles a most comprehensive fungal transcriptomics resource, which can be explored without any programming knowledge, allows highly customizable filtering and cross-project comparisons and download of all offered data tables and visualizations.
Understanding host-pathogen interactions at the molecular level requires methods capable of linking spatial context with proteomic information. Here, we present an integrated workflow combining matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and laser microdissection (LMD)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate Aspergillus fumigatus infection in murine lung tissue. Consecutive formalin-fixed, paraffin-embedded (FFPE) tissue sections were used for spatially resolved MALDI-MSI and subsequent LC-MS/MS analysis of laser-microdissected fungal-infected and non-infected regions. MALDI-MSI revealed reproducible m/z features specifically associated with infected areas. Corresponding tissue microregions were microdissected and analyzed by proteomics to identify candidate proteins underlying these spatial signals. Comparative proteomics of fungal-infected with non-infected alveolar lung regions via LC-MS/MS identified host proteins involved in leukocyte recruitment, inflammatory signaling, and reactive oxygen species formation, including a 424-fold increase in formyl peptide receptor 2 (Fpr2) during fungal invasion of the lungs. Fungal regions were also enriched in proteins encoded by the gliotoxin biosynthetic gene cluster. Spatial and proteomic data were linked by matching theoretical peptide-adduct masses to MALDI-MSI features, using a semi-quantitative scoring system to prioritize protein assignments. Fungal regions showed contributions from both host and pathogen proteins. This workflow establishes a conceptual basis for spatial proteomics of host-pathogen-interactions in fungal infections and enables association of characteristic m/z signals with plausible protein candidates.
Many countries with lower research & innovation capacity face persistent constraints in building stable research systems. Chronic underfunding and weak science policy reduce institutional capacity and limit researchers' career prospects. These conditions encourage brain drain, particularly among early-career scientists who seek predictable funding, transparent evaluation, and merit-based advancement. As a result, research institutions lose skilled personnel, which weakens scientific training, governance, and research output. Additionally, within this environment, predatory publishing practices create further damage. These scientific outlets reward volume over quality, thus distorting evaluation criteria. They promote negative selection by favouring speed of publication at the expense of rigorous peer review. Over time, this weakens academic standards and undermines trust in the research output. The result is a decline in scientific credibility and an overall reduction in international competitiveness. Although predatory publishing is motivated by financial gain, it results in serious institutional consequences. It directly reshapes hiring, promotion, and funding decisions in ways that disadvantage high-quality research. This contributes to the erosion of both research integrity and academic communities.
Type IV secretion systems (T4SS) are found in both monoderm and diderm bacteria. The broad-host-range conjugative plasmid pIP501 from Enterococcus faecalis harbors a T4SS encoding 15 tra genes responsible for the spread of antimicrobial resistance genes among diverse G+ pathogens. Eight Tra proteins (TraB, TraCB3, TraF, TraHB8, TraI, TraK, TraLB6, and TraMB8) are postulated to form the mating pair formation (MPF) complex representing the central DNA translocation pore. One of these proteins is TraF, a 52.8 kDa transmembrane protein, which lacks any homologs in other well described T4SSs. In this study, TraF was proven to be an essential conjugative transfer protein. The TraF pulldown co-eluted all Tra proteins except TraGB1 and TraN. Bacterial-two-hybrid assay showed a strong interaction between TraF and TraMB8. We present a 1.25 Å resolution crystal structure of the N-terminal domain of TraF, which adopts a pseudokinase fold. AlphaFold predictions of full-length TraF with membrane mimetics show a transmembrane protein with two distinct soluble domains. FoldSeek revealed a strong similarity to YukC (EssB), a transmembrane pseudokinase from type VII secretion system (T7SS). YukC was shown to function as an interaction hub by mediating contacts between its pseudokinase domain and other T7SS proteins as part of the central membrane core complex. We postulate that TraF might play an important role in T4SS complex formation.
Throughout the history of molecular biology, surprising advances have come from the study of all sorts of microbes. The first description of DNA polymerase came from the bacterial workhorse Escherichia coli, reverse transcriptase was revealed by studies of the Rous Sarcoma Virus, and even the initial discovery of DNA as the hereditary molecule was determined using strains of pneumococci in the classic Griffith and Avery-MacLeod-McCarty experiments. Here, we build from these foundational discoveries to discuss the rapid development of molecular tools to study microbes themselves, with a focus on nucleic acid biology. We use fungal pathogens as a case study, as their diversity, complexity, and emerging appreciation as a global threat to society makes them particularly compelling and informative. In this review, we will address how advancements in methods to probe nucleic acids are now informing our understanding of fungal pathogens and the way we fight them in both the clinic and agriculture. We begin with DNA, taking a close look at the exciting progress in the fields of genetic engineering and chromatin biology, and their impact on the elucidation of virulence-associated cellular processes. Emerging RNA-based technologies follow, highlighting the value provided by biochemical advances and large-scale -omics approaches. We end by speculating on the future of molecular mycology and how these new approaches may facilitate generation of novel antifungals and diagnostic strategies.
Intracellular Gram-negative pathogens employ either type IVA or type IVB secretion systems (T4SSs) to translocate effector proteins into host cells, where they modulate cellular processes to facilitate infection and promote intracellular survival. Roughly one-third of these effectors harbor hydrophobic transmembrane domains and are thus destined for integration into host cell membranes during infection. Many of these transmembrane domain-containing effectors (TMEs) localize to the membrane of the pathogen-containing vacuole, thereby contributing to its formation and remodeling. Despite the biological relevance of TMEs, the detailed molecular mechanisms governing their translocation via T4SSs and subsequent membrane integration in the host cell remain insufficiently understood. In this review, the biophysical characteristics of T4SS-secreted TMEs are systematically examined, including predictions of membrane topology and hydrophobicity. These analyses are then contextualized through comparison with recent structural analysis of both T4ASS and T4BSS machineries, as well as with mechanistic principles of eukaryotic membrane protein biogenesis. This integrative approach enables the conceptual reconstruction of the potential pathways by which TMEs are translocated through the T4SS and subsequently targeted and inserted into host membranes, offering new mechanistic insights into the poorly understood handling of bacterial TMEs from both the pathogen and host perspectives.
Microproteins (≤70 amino acids) have important and often essential roles in all kingdoms of life, influencing cell motility, regulation of membrane transport and as transcription factors. In the halophilic archaeon and model system Haloferax volcanii a significant number of µ-proteins were predicted to be zinc finger proteins. Here we used mass spectrometry-based proteomics to systematically investigate the impact of single gene deletions of 19 zinc finger µ-proteins on the proteome of H. volcanii grown in synthetic medium with glucose as sole carbon and energy source. We employed a state-of-the-art dia-PASEF acquisition strategy, detecting over 3400 proteins across the 19 deletion strains and the wild type. The comprehensive proteome coverage enabled a systematic analysis of proteome remodeling. We found that in 11 out of the 19 mutants the proteome remodeling involved proteins annotated to play a role in cell motility, matching swarming and growth rate phenotypes we observed for these strains. Taken together, our data provide the most comprehensive proteome coverage of H. volcanii to date, and the effect of 19 different zinc-finger µ-proteins deletion strains on the proteome of this organism. The combined data (available via ProteomeXchange with identifier PXD066008) provide a valuable resource for future research in the field.
