A network of small RNAs regulates sporulation initiation in Clostridioides difficile.

The obligate anaerobic, enteric pathogen Clostridioides difficile persists in the intestinal tract by forming antibiotic-resistant endospores that contribute to relapsing and recurrent infections. Despite the importance of sporulation for C. difficile pathogenesis, environmental cues and molecular mechanisms that regulate sporulation initiation remain ill-defined. Here, by using RIL-seq to globally capture the Hfq-dependent RNA-RNA interactome, we discovered a network of small RNAs that bind to mRNAs encoding sporulation-related genes. We show that two of these small RNAs, SpoX and SpoY, regulate translation of the master regulator of sporulation, Spo0A, in an opposing manner, which ultimately leads to altered sporulation rates. Infection of antibiotic-treated mice with SpoX and SpoY deletion mutants revealed a global effect on gut colonization and intestinal sporulation. Our work uncovers an elaborate RNA-RNA interactome controlling the physiology and virulence of C. difficile and identifies a complex post-transcriptional layer in the regulation of spore formation in this important human pathogen.

An RNA-centric global view of Clostridioides difficile reveals broad activity of Hfq in a clinically important gram-positive bacterium.

The gram-positive human pathogen Clostridioides difficile has emerged as the leading cause of antibiotic-associated diarrhea. However, little is known about the bacterium's transcriptome architecture and mechanisms of posttranscriptional control. Here, we have applied transcription start site and termination mapping to generate a single-nucleotide-resolution RNA map of C. difficile 5' and 3' untranslated regions, operon structures, and noncoding regulators, including 42 sRNAs. Our results indicate functionality of many conserved riboswitches and predict cis-regulatory RNA elements upstream of multidrug resistance (MDR)-type ATP-binding cassette (ABC) transporters and transcriptional regulators. Despite growing evidence for a role of Hfq in RNA-based gene regulation in C. difficile, the functions of Hfq-based posttranscriptional regulatory networks in gram-positive pathogens remain controversial. Using Hfq immunoprecipitation followed by sequencing of bound RNA species (RIP-seq), we identify a large cohort of transcripts bound by Hfq and show that absence of Hfq affects transcript stabilities and steady-state levels. We demonstrate sRNA expression during intestinal colonization by C. difficile and identify infection-related signals impacting its expression. As a proof of concept, we show that the utilization of the abundant intestinal metabolite ethanolamine is regulated by the Hfq-dependent sRNA CDIF630nc_085. Overall, our study lays the foundation for understanding clostridial riboregulation with implications for the infection process and provides evidence for a global role of Hfq in posttranscriptional regulation in a gram-positive bacterium.

Host-mediated sugar oxidation promotes post-antibiotic pathogen expansion.

Changes in the gut microbiota may underpin many human diseases, but the mechanisms that are responsible for altering microbial communities remain poorly understood. Antibiotic usage elevates the risk of contracting gastroenteritis caused by Salmonella enterica serovars, increases the duration for which patients shed the pathogen in their faeces, and may on occasion produce a bacteriologic and symptomatic relapse. These antibiotic-induced changes in the gut microbiota can be studied in mice, in which the disruption of a balanced microbial community by treatment with the antibiotic streptomycin leads to an expansion of S. enterica serovars in the large bowel. However, the mechanisms by which streptomycin treatment drives an expansion of S. enterica serovars are not fully resolved. Here we show that host-mediated oxidation of galactose and glucose promotes post-antibiotic expansion of S. enterica serovar Typhimurium (S. Typhimurium). By elevating expression of the gene encoding inducible nitric oxide synthase (iNOS) in the caecal mucosa, streptomycin treatment increased post-antibiotic availability of the oxidation products galactarate and glucarate in the murine caecum. S. Typhimurium used galactarate and glucarate within the gut lumen of streptomycin pre-treated mice, and genetic ablation of the respective catabolic pathways reduced S. Typhimurium competitiveness. Our results identify host-mediated oxidation of carbohydrates in the gut as a mechanism for post-antibiotic pathogen expansion.

Social behaviour and making attachments: a report from the fifth 'Young Microbiologists Symposium on Microbe Signalling, Organisation and Pathogenesis'.

The fifth Young Microbiologists Symposium was held in Queen's University Belfast, Northern Ireland, in late August 2018. The symposium, focused on 'Microbe signalling, organization and pathogenesis', attracted 121 microbiologists from 15 countries. The meeting allowed junior scientists to present their work to a broad audience, and was supported by the European Molecular Biology Organization, the Federation of European Microbiological Societies, the Society of Applied Microbiology, the Biochemical Society and the Microbiology Society. Sessions covered recent advances in areas of microbiology including gene regulation and signalling, secretion and transport across membranes, infection and immunity, and antibiotics and resistance mechanisms. In this Meeting Report, we highlight some of the most significant advances and exciting developments communicated during talks and poster presentations.

Respiration of Microbiota-Derived 1,2-propanediol Drives Salmonella Expansion during Colitis.

Intestinal inflammation caused by Salmonella enterica serovar Typhimurium increases the availability of electron acceptors that fuel a respiratory growth of the pathogen in the intestinal lumen. Here we show that one of the carbon sources driving this respiratory expansion in the mouse model is 1,2-propanediol, a microbial fermentation product. 1,2-propanediol utilization required intestinal inflammation induced by virulence factors of the pathogen. S. Typhimurium used both aerobic and anaerobic respiration to consume 1,2-propanediol and expand in the murine large intestine. 1,2-propanediol-utilization did not confer a benefit in germ-free mice, but the pdu genes conferred a fitness advantage upon S. Typhimurium in mice mono-associated with Bacteroides fragilis or Bacteroides thetaiotaomicron. Collectively, our data suggest that intestinal inflammation enables S. Typhimurium to sidestep nutritional competition by respiring a microbiota-derived fermentation product.

Colonization resistance: The deconvolution of a complex trait.

Carbapenemase-producing Enterobacteriaceae are an emerging threat to hospitals worldwide, and antibiotic exposure is a risk factor for developing fecal carriage that may lead to nosocomial infection. Here, we review how antibiotics reduce colonization resistance against Enterobacteriaceae to pinpoint possible control points for curbing their spread. Recent work identifies host-derived respiratory electron acceptors as a critical resource driving a post-antibiotic expansion of Enterobacteriaceae within the large bowel. By providing a conceptual framework for colonization resistance against Enterobacteriaceae, these mechanistic insights point to the metabolism of epithelial cells as a possible target for intervention strategies.

Salmonella enterica Serovar Typhi conceals the invasion-associated type three secretion system from the innate immune system by gene regulation.

Delivery of microbial products into the mammalian cell cytosol by bacterial secretion systems is a strong stimulus for triggering pro-inflammatory host responses. Here we show that Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, tightly regulates expression of the invasion-associated type III secretion system (T3SS-1) and thus fails to activate these innate immune signaling pathways. The S. Typhi regulatory protein TviA rapidly repressed T3SS-1 expression, thereby preventing RAC1-dependent, RIP2-dependent activation of NF-κB in epithelial cells. Heterologous expression of TviA in S. enterica serovar Typhimurium (S. Typhimurium) suppressed T3SS-1-dependent inflammatory responses generated early after infection in animal models of gastroenteritis. These results suggest that S. Typhi reduces intestinal inflammation by limiting the induction of pathogen-induced processes through regulation of virulence gene expression.

Formation of the pyruvoyl-dependent proline reductase Prd from Clostridioides difficile requires the maturation enzyme PrdH.

Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacterium Clostridioides difficile. D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostridial toxins TcdA and TcdB. D-proline reduction is catalyzed by the proline reductase Prd, which belongs to the pyruvoyl-dependent enzymes. These enzymes are translated as inactive proenzymes and require subsequent processing to install the covalently bound pyruvate. Whereas pyruvoyl formation by intramolecular serinolysis has been studied in unrelated enzymes, details about pyruvoyl generation by cysteinolysis as in Prd are lacking. Here, we show that Prd maturation requires a small dimeric protein that we have named PrdH. PrdH (CD630_32430) is co-encoded with the PrdA and PrdB subunits of Prd and also found in species producing similar reductases. By producing stable variants of PrdA and PrdB, we demonstrate that PrdH-mediated cleavage and pyruvoyl formation in the PrdA subunit requires PrdB, which can be harnessed to produce active recombinant Prd for subsequent analyses. We further created PrdA- and PrdH-mutants to get insight into the interaction of the components and into the processing reaction itself. Finally, we show that deletion of prdH renders C. difficile insensitive to proline concentrations in culture media, suggesting that this processing factor is essential for proline utilization. Due to the link between Stickland fermentation and pathogenesis, we suggest PrdH may be an attractive target for drug development.

Byproducts of inflammatory radical metabolism provide transient nutrient niches for microbes in the inflamed gut.

Louis Pasteur's experiments on tartaric acid laid the foundation for our understanding of molecular chirality, but major questions remain. By comparing the optical activity of naturally-occurring tartaric acid with chemically-synthesized paratartaric acid, Pasteur realized that naturally-occurring tartaric acid contained only L-tartaric acid while paratartaric acid consisted of a racemic mixture of D- and L-tartaric acid. Curiously, D-tartaric acid has no known natural source, yet several gut bacteria specifically degrade D-tartaric acid. Here, we investigated the oxidation of monosaccharides by inflammatory reactive oxygen and nitrogen species. We found that this reaction yields an array of alpha hydroxy carboxylic acids, including tartaric acid isomers. Utilization of inflammation- derived D- and L-tartaric acid enhanced colonization by Salmonella Typhimurium and E. coli in murine models of gut inflammation. Our findings suggest that byproducts of inflammatory radical metabolism, such as tartrate and other alpha hydroxy carboxylic acids, create transient nutrient niches for enteric pathogens and other potentially harmful bacteria. Furthermore, this work illustrates that inflammatory radicals generate a zoo of molecules, some of which may erroneously presumed to be xenobiotics.

Surface-associated motility, a common trait of clinical isolates of Acinetobacter baumannii, depends on 1,3-diaminopropane.

While flagella-independent motility has long been described in representatives of the genus Acinetobacter, the mechanism of motility remains ambiguous. Acinetobacter baumannii, a nosocomial pathogen appearing increasingly multidrug-resistant, may profit from motility during infection or while persisting in the hospital environment. However, data on the frequency of motility skills among clinical A. baumannii isolates is scarce. We have screened a collection of 83 clinical A. baumannii isolates of different origin and found that, with the exception of one isolate, all were motile on wet surfaces albeit to varying degrees and exhibiting differing morphologies. Screening a collection of transposon mutants of strain ATCC 17978 for motility defects, we identified 2 akinetic mutants carrying transposon insertions in the dat and ddc gene, respectively. These neighbouring genes contribute to synthesis of 1,3-diaminopropane (DAP), a polyamine ubiquitously produced in Acinetobacter. Supplementing semi-solid media with DAP cured the motility defect of both mutants. HPLC analyses confirmed that DAP synthesis was abolished in ddc and dat mutants of different A. baumannii isolates and was re-established after genetic complementation. Both, the dat and ddc mutant of ATCC 17978 were attenuated in the Galleria mellonella caterpillar infection model. Taken together, surface-associated motility is a common trait of clinical A. baumannii isolates that requires DAP and may play a role in its virulence.

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.

The metabolic footprint of Clostridia and Erysipelotrichia reveals their role in depleting sugar alcohols in the cecum.

BACKGROUND: The catabolic activity of the microbiota contributes to health by aiding in nutrition, immune education, and niche protection against pathogens. However, the nutrients consumed by common taxa within the gut microbiota remain incompletely understood. METHODS: Here we combined microbiota profiling with an un-targeted metabolomics approach to determine whether depletion of small metabolites in the cecum of mice correlated with the presence of specific bacterial taxa. Causality was investigated by engrafting germ-free or antibiotic-treated mice with complex or defined microbial communities. RESULTS: We noted that a depletion of Clostridia and Erysipelotrichia from the gut microbiota triggered by antibiotic treatment was associated with an increase in the cecal concentration of sugar acids and sugar alcohols (polyols). Notably, when we inoculated germ-free mice with a defined microbial community of 14 Clostridia and 3 Erysipelotrichia isolates, we observed the inverse, with a marked decrease in the concentrations of sugar acids and polyols in cecal contents. The carbohydrate footprint produced by the defined microbial community was similar to that observed in gnotobiotic mice receiving a cecal microbiota transplant from conventional mice. Supplementation with sorbitol, a polyol used as artificial sweetener, increased cecal sorbitol concentrations in antibiotic-treated mice, which was abrogated after inoculation with a Clostridia isolate able to grow on sorbitol in vitro. CONCLUSIONS: We conclude that consumption of sugar alcohols by Clostridia and Erysipelotrichia species depletes these metabolites from the intestinal lumen during homeostasis. Video abstract.

Grad-seq identifies KhpB as a global RNA-binding protein in Clostridioides difficile that regulates toxin production.

Much of our current knowledge about cellular RNA-protein complexes in bacteria is derived from analyses in gram-negative model organisms, with the discovery of RNA-binding proteins (RBPs) generally lagging behind in Gram-positive species. Here, we have applied Grad-seq analysis of native RNA-protein complexes to a major Gram-positive human pathogen, Clostridioides difficile, whose RNA biology remains largely unexplored. Our analysis resolves in-gradient distributions for ∼88% of all annotated transcripts and ∼50% of all proteins, thereby providing a comprehensive resource for the discovery of RNA-protein and protein-protein complexes in C. difficile and related microbes. The sedimentation profiles together with pulldown approaches identify KhpB, previously identified in Streptococcus pneumoniae, as an uncharacterized, pervasive RBP in C. difficile. Global RIP-seq analysis establishes a large suite of mRNA and small RNA targets of KhpB, similar to the scope of the Hfq targetome in C. difficile. The KhpB-bound transcripts include several functionally related mRNAs encoding virulence-associated metabolic pathways and toxin A whose transcript levels are observed to be increased in a khpB deletion strain. Moreover, the production of toxin protein is also increased upon khpB deletion. In summary, this study expands our knowledge of cellular RNA protein interactions in C. difficile and supports the emerging view that KhpB homologues constitute a new class of globally acting RBPs in Gram-positive bacteria.

Malaria parasite infection compromises colonization resistance to an enteric pathogen by reducing gastric acidity.

Malaria parasite infection weakens colonization resistance against Salmonella enterica serovar (S.) Typhimurium. S. Typhimurium is a member of the Enterobacterales, a taxon that increases in abundance when the colonic microbiota is disrupted or when the colonic mucosa is inflamed. However, here, we show that infection of mice with Plasmodium yoelii enhances S. Typhimurium colonization by weakening host control in the upper GI tract. P. yoelii-infected mice had elevated gastric pH. Stimulation of gastric acid secretion during P. yoelii infection restored stomach acidity and colonization resistance, demonstrating that parasite-induced hypochlorhydria increases gastric survival of S. Typhimurium. Furthermore, blockade of P. yoelii-induced TNF-α signaling was sufficient to prevent elevation of gastric pH and enhance S. Typhimurium colonization during concurrent infection. Collectively, these data suggest that abundance in the fecal microbiota of facultative anaerobes, such as S. Typhimurium, can be increased by suppressing antibacterial defenses in the upper GI tract, such as gastric acid.

Orbus hercynius gen. nov., sp. nov., isolated from faeces of wild boar, is most closely related to members of the orders 'Enterobacteriales' and Pasteurellales.

A novel gammaproteobacterium, strain CN3(T), was isolated from the faeces of wild boar. Strain CN3(T) was facultatively anaerobic and appeared coccoid or rod-shaped. The partial 16S rRNA gene sequence determined for strain CN3(T) suggested a distant relationship with members of the orders 'Enterobacteriales' and Pasteurellales. The gene sequence showed highest similarity (90.3 %) with Obesumbacterium proteus DSM 2777(T), a member of the family Enterobacteriaceae. The closest relatives outside the order 'Enterobacteriales' according to 16S rRNA gene sequence analysis were members of the order Pasteurellales with 88.7 % similarity (Mannheimia haemolytica NCTC 9380(T) and Actinobacillus lignieresii NCTC 4189(T)). In contrast to most members of the order 'Enterobacteriales', strain CN3(T) was oxidase-positive. The pattern of fatty acids, in particular the high relative abundance of C(18 : 1)ω7c (38.5 %), was clearly distinct from the conserved pattern found for members of the order Pasteurellales. EcoRI ribotyping of strain CN3(T) yielded no significant similarity to existing database entries. The major ubiquinone of strain CN3(T) was Q-8. The DNA G+C content was 36.4 mol%. Strain CN3(T) hosted a phage and secreted considerable amounts of three proteins into the culture supernatant. A spontaneous mutant of strain CN3(T) was isolated which formed long filaments. Microscopic studies revealed the presence of a capsule that the mutant strain was unable to partition after cell division. Strain CN3(T) thus represents a novel species within a new genus, for which the name Orbus hercynius gen. nov., sp. nov. is proposed. The type strain of the type species is CN3(T) (=DSM 22228(T)=CCUG 57622(T)). Classification of the novel species to the family and order level will require further investigations.

Antibacterial Anacardic Acid Derivatives.

We report on the antibacterial activity of five phenolic lipids derived from anacardic acid characterized by increasing alkyl chain lengths with 6, 8, 10, 12, or 14 carbon atoms. The compounds were profiled for their physicochemical properties, transport across epithelial monolayers, cytotoxicity, and antibacterial activity as compared to common antibiotics. No cytotoxicity was reported in cell lines of fibroblast, hepatic, colorectal, or renal origin. C10 and C12 significantly increased the survival in a Galleria mellonella model infected with multi-drug-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococci (VRE) as compared to the untreated control group. Future studies are required to corroborate these findings in relevant animal model systems of infection.

CsrA and CsrB are required for the post-transcriptional control of the virulence-associated effector protein AvrA of Salmonella enterica.

The virulence-associated effector protein AvrA of Salmonella enterica is an ubiquitin-like acetyltransferase/cysteine protease, which interferes with the first line of immune response of the target organism. In contrast to translation of the AvrA protein in S. enterica strains, which takes place either constitutively (class 1 strains), or after acid induction (class 2 strains), or not at all (class 3 strains); the constitutive transcription of the respective avrA genes occurs regardless of these defined expression classes. When the number of avrA genes and mRNA molecules is raised experimentally using plasmids carrying the respective cloned avrA genes together with their promoter regions, the translation of avrA mRNA takes place very strongly in all respective AvrA expression classes. This kind of copy-dependent, post-transcriptional control of AvrA was shown to be dependent on the regulatory action of the CsrA/CsrB system since the deletion of both genes completely abolished the translation in the tested S. enterica strains, whereas the transcription remained unaffected. Moreover, AvrA production in strains carrying the cloned avrA genes on plasmids remained dependent on the presence of CsrA but unaffected in csrB mutant strains. On the other hand, overproduction of the regulatory molecules CsrA and CsrB in S. enterica strains carrying cloned csrA and csrB genes on plasmids ceased the expression of AvrA again. Therefore, the expression of avrA is suggested to be regulated in a post-transcriptional manner by critical and effective concentrations of CsrA (see-saw regulation), which is achieved through the sequestering activity of CsrB.

Endogenous Enterobacteriaceae underlie variation in susceptibility to Salmonella infection.

Lack of reproducibility is a prominent problem in biomedical research. An important source of variation in animal experiments is the microbiome, but little is known about specific changes in the microbiota composition that cause phenotypic differences. Here, we show that genetically similar laboratory mice obtained from four different commercial vendors exhibited marked phenotypic variation in their susceptibility to Salmonella infection. Faecal microbiota transplant into germ-free mice replicated donor susceptibility, revealing that variability was due to changes in the gut microbiota composition. Co-housing of mice only partially transferred protection against Salmonella infection, suggesting that minority species within the gut microbiota might confer this trait. Consistent with this idea, we identified endogenous Enterobacteriaceae, a low-abundance taxon, as a keystone species responsible for variation in the susceptibility to Salmonella infection. Protection conferred by endogenous Enterobacteriaceae could be modelled by inoculating mice with probiotic Escherichia coli, which conferred resistance by using its aerobic metabolism to compete with Salmonella for resources. We conclude that a mechanistic understanding of phenotypic variation can accelerate development of strategies for enhancing the reproducibility of animal experiments.

Commensal Enterobacteriaceae Protect against Salmonella Colonization through Oxygen Competition.

Neonates are highly susceptible to infection with enteric pathogens, but the underlying mechanisms are not resolved. We show that neonatal chick colonization with Salmonella enterica serovar Enteritidis requires a virulence-factor-dependent increase in epithelial oxygenation, which drives pathogen expansion by aerobic respiration. Co-infection experiments with an Escherichia coli strain carrying an oxygen-sensitive reporter suggest that S. Enteritidis competes with commensal Enterobacteriaceae for oxygen. A combination of Enterobacteriaceae and spore-forming bacteria, but not colonization with either community alone, confers colonization resistance against S. Enteritidis in neonatal chicks, phenocopying germ-free mice associated with adult chicken microbiota. Combining spore-forming bacteria with a probiotic E. coli isolate protects germ-free mice from pathogen colonization, but the protection is lost when the ability to respire oxygen under micro-aerophilic conditions is genetically ablated in E. coli. These results suggest that commensal Enterobacteriaceae contribute to colonization resistance by competing with S. Enteritidis for oxygen, a resource critical for pathogen expansion.

Genetic Ablation of Butyrate Utilization Attenuates Gastrointestinal Salmonella Disease.

Salmonella enterica serovar (S.) Typhi is an extraintestinal pathogen that evolved from Salmonella serovars causing gastrointestinal disease. Compared with non-typhoidal Salmonella serovars, the genomes of typhoidal serovars contain various loss-of-function mutations. However, the contribution of these genetic differences to this shift in pathogen ecology remains unknown. We show that the ydiQRSTD operon, which is deleted in S. Typhi, enables S. Typhimurium to utilize microbiota-derived butyrate during gastrointestinal disease. Unexpectedly, genetic ablation of butyrate utilization reduces S. Typhimurium epithelial invasion and attenuates intestinal inflammation. Deletion of ydiD renders S. Typhimurium sensitive to butyrate-mediated repression of invasion gene expression. Combined with the gain of virulence-associated (Vi) capsular polysaccharide and loss of very-long O-antigen chains, two features characteristic of S. Typhi, genetic ablation of butyrate utilization abrogates S. Typhimurium-induced intestinal inflammation. Thus, the transition from a gastrointestinal to an extraintestinal pathogen involved discrete genetic changes, providing insights into pathogen evolution and emergence.