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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.

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.

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.

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.

[This corrects the article DOI: 10.1093/femsml/uqaf041.].

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.