Infection leaves a genetic and functional mark on the gut population of a commensal bacterium.

Gastrointestinal infection changes microbiome composition and gene expression. In this study, we demonstrate that enteric infection also promotes rapid genetic adaptation in a gut commensal. Measurements of Bacteroides thetaiotaomicron population dynamics within gnotobiotic mice reveal that these populations are relatively stable in the absence of infection, and the introduction of the enteropathogen Citrobacter rodentium reproducibly promotes rapid selection for a single-nucleotide variant with increased fitness. This mutation promotes resistance to oxidative stress by altering the sequence of a protein, IctA, that is essential for fitness during infection. We identified commensals from multiple phyla that attenuate the selection of this variant during infection. These species increase the levels of vitamin B6 in the gut lumen. Direct administration of this vitamin is sufficient to significantly reduce variant expansion in infected mice. Our work demonstrates that a self-limited enteric infection can leave a stable mark on resident commensal populations that increase fitness during infection.

RNA landscape of the emerging cancer-associated microbe Fusobacterium nucleatum.

Fusobacterium nucleatum, long known as a constituent of the oral microflora, has recently garnered renewed attention for its association with several different human cancers. The growing interest in this emerging cancer-associated bacterium contrasts with a paucity of knowledge about its basic gene expression features and physiological responses. As fusobacteria lack all established small RNA-associated proteins, post-transcriptional networks in these bacteria are also unknown. In the present study, using differential RNA-sequencing, we generate high-resolution global RNA maps for five clinically relevant fusobacterial strains-F. nucleatum subspecies nucleatum, animalis, polymorphum and vincentii, as well as F. periodonticum-for early, mid-exponential growth and early stationary phase. These data are made available in an online browser, and we use these to uncover fundamental aspects of fusobacterial gene expression architecture and a suite of non-coding RNAs. Developing a vector for functional analysis of fusobacterial genes, we discover a conserved fusobacterial oxygen-induced small RNA, FoxI, which serves as a post-transcriptional repressor of the major outer membrane porin FomA. Our findings provide a crucial step towards delineating the regulatory networks enabling F. nucleatum adaptation to different environments, which may elucidate how these bacteria colonize different compartments of the human body.

Stress-induced host membrane remodeling protects from infection by non-motile bacterial pathogens.

While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of Shigella flexneri to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress-dependent inhibition of Shigella binding to host cells. Interestingly, stress caused by intracellular Shigella replication also results in remodeling of the host cell membrane, in vitro and in vivo, which precludes re-infection by this and other non-motile pathogens. In contrast, Salmonella Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress-responsive cell-autonomous defense mechanism that protects epithelial cells from infection by non-motile bacterial pathogens.

A systematic analysis of the RNA-targeting potential of secreted bacterial effector proteins.

Many pathogenic bacteria utilize specialized secretion systems to deliver proteins called effectors into eukaryotic cells for manipulation of host pathways. The vast majority of known effector targets are host proteins, whereas a potential targeting of host nucleic acids remains little explored. There is only one family of effectors known to target DNA directly, and effectors binding host RNA are unknown. Here, we take a two-pronged approach to search for RNA-binding effectors, combining biocomputational prediction of RNA-binding domains (RBDs) in a newly assembled comprehensive dataset of bacterial secreted proteins, and experimental screening for RNA binding in mammalian cells. Only a small subset of effectors were predicted to carry an RBD, indicating that if RNA targeting was common, it would likely involve new types of RBDs. Our experimental evaluation of effectors with predicted RBDs further argues for a general paucity of RNA binding activities amongst bacterial effectors. We obtained evidence that PipB2 and Lpg2844, effector proteins of Salmonella and Legionella species, respectively, may harbor novel biochemical activities. Our study presenting the first systematic evaluation of the RNA-targeting potential of bacterial effectors offers a basis for discussion of whether or not host RNA is a prominent target of secreted bacterial proteins.

Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia.

MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.

HK022 Nun Requires Arginine-Rich Motif Residues Distinct from λ N.

UNLABELLED: Bacteriophage λ N protein binds boxB RNA hairpins in the nut (N utilization) sites of immediate early λ transcripts and interacts with host factors to suppress transcriptional termination at downstream terminators. In opposition to λ N, the Nun protein of HK022 binds the boxBs of coinfecting λ transcripts, interacts with a similar or identical set of host factors, and terminates transcription to suppress λ replication. Comparison of N-boxB and Nun-boxB nuclear magnetic resonance (NMR) structural models suggests similar interactions, though limited mutagenesis of Nun is available. Here, libraries of Nun's arginine-rich motif (ARM) were screened for the ability to exclude λ coinfection, and mutants were assayed for Nun termination with a boxB plasmid reporter system. Several Nun ARM residues appear to be immutable: Asp26, Arg28, Arg29, Arg32, Trp33, and Arg36. Asp26 and Trp33 appear to be unable to contact boxB and are not found at equivalent positions in λ N ARM. To understand if the requirement of Asp26, Trp33, and Arg36 indicated differences between HK022 Nun termination and λ N antitermination complexes, the same Nun libraries were fused to the activation domain of λ N and screened for clones able to complement N-deficient λ. Mutants were assayed for N antitermination. Surprisingly, Asp26 and Trp33 were still essential when Nun ARM was fused to N. Docking suggests that Nun ARM contacts a hydrophobic surface of the NusG carboxy-terminal domain containing residues necessary for Nun function. These findings indicate that Nun ARM relies on distinct contacts in its ternary complex and illustrate how protein-RNA recognition can evolve new regulatory functions. IMPORTANCE: λ N protein interacts with host factors to allow λ nut-containing transcripts to elongate past termination signals. A competing bacteriophage, HK022, expresses Nun protein, which causes termination of λ nut transcripts. λ N and HK022 Nun use similar arginine-rich motifs (ARMs) to bind the same boxB RNAs in nut transcripts. Screening libraries of Nun ARM mutants, both in HK022 Nun and in a λ N fusion, revealed amino acids essential to Nun that could bind one or more host factors. Docking suggests that NusG, which is present in both Nun termination and N antitermination, is a plausible partner. These findings could help understand how transcription elongation is regulated and illustrate how subtle differences allow ARMs to evolve new regulatory functions.