PrsA2 (CD630_35000) of Clostridioides difficile Is an Active Parvulin-Type PPIase and a Virulence Modulator

Clostridioides difficile is the main cause for nosocomial antibiotic associated diarrhea and has become a major burden for the health care systems of industrial countries. Its main virulence factors, the small GTPase glycosylating toxins TcdA and TcdB, are extensively studied. In contrast, the contribution of other factors to development and progression of C. difficile infection (CDI) are only insufficiently understood. Many bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) have been described in the context of virulence. Among them are the parvulin-type PrsA-like PPIases of Gram-positive bacteria. On this basis, we identified CD630_35000 as the PrsA2 homolog in C. difficile and conducted its enzymatic and phenotypic characterization in order to assess its involvement during C. difficile infection. For this purpose, wild type CdPrsA2 and mutant variants carrying amino acid exchanges mainly in the PPIase domain were recombinantly produced. Recombinant CdPrsA2 showed PPIase activity toward the substrate peptide Ala-Xaa-Pro-Phe with a preference for positively charged amino acids preceding the proline residue. Mutation of conserved residues in its active site pocket impaired the enzymatic activity. A PrsA2 deficient mutant was generated in the C. difficile 630Δerm background using the ClosTron technology. Inactivation of prsA2 resulted in a reduced germination rate in response to taurocholic acid, and in a slight increase in resistance to the secondary bile acids LCA and DCA. Interestingly, in the absence of PrsA2 colonization of mice by C. difficile 630 was significantly reduced. We concluded that CdPrsA2 is an active PPIase that acts as a virulence modulator by influencing crucial processes like sporulation, germination and bile acid resistance resulting in attenuated mice colonization

Abbau des Mikrobioms zur Modulation der Wirtsabwehr gegen Clostridioides difficile-Infektionen

The intestinal microbiota consists of numerous microbes forming a dynamic ecosystem in close association with the host. One of the many functions of the microbiota is the colonization resistance against intestinal pathogens. Alterations in the composition of the microbiota, for example caused by antibiotics, may cause the loss of colonization resistance, making the host vulnerable towards infections, e.g. with Clostridioides difficile. C. difficile is a spore-forming anaerobic bacterium which can cause severe diarrhea and life-threatening complications in infected individuals. In many developed countries, C. difficile infection is a major cause of antibiotic-related nosocomial infections. The disease affects mostly elderly individuals. Antibiotics are the standard treatment, however, up to 30% of the patients are suffering from recurrent C. difficile infections. Transfer of fecal matter from a healthy individual to the patient is able to cure these infections. Recent studies have demonstrated that the conversion of primary to secondary bile acids by commensal bacteria is important for colonization resistance, but further evidence suggested that the restoration of the secondary bile acid production may not be sufficient for complete protection. In the present study, the effect of the microbiota on C. difficile infection has been explored. Therefore, a C. difficile mouse model has been established initially by exploring the effect of different ages and microbiota compositions on the severity of C. difficile infections. Second, the effect of specific mutations in the genome of C. difficile on the ability to colonize mice and to produce spores has been examined. Third, the inhibitory effect of specifically isolated commensal bacteria of the microbiota of resistant mouse models on C. difficile infections has been explored in depth in order to create a consortium of bacteria that protects the host against C. difficile. Our findings confirm that the age of the mice, comparable as in humans, is important for the severity of the disease of mice. They also confirm that secondary bile acids are indeed important to protect the host against C. difficile infections. However, in our model, adding a secondary bile acid producer to the microbiota is not sufficient for complete protection of the mice, but together with other commensal bacteria, secondary bile acid producing bacteria are able to provide complete resistance against C. difficile induced pathogenesis.Die intestinale Mikrobiota bezeichnet ein komplexes Ökosystem aus zahlreichen Mikroorganismen, welches in enger Verbindung mit dem Wirt steht. Eine Hauptfunktion der Mikrobiota stellt die Besiedelungsresistenz gegen intestinale Pathogene dar. Veränderungen in der Zusammensetzung kann zum Verlust der Besiedelungsresistenz führen und den Wirt anfällig für Infektionen, mit z.B. Clostridioides difficile machen. C. difficile ist ein sporenbildendes, anaerobes Bakterium, welches besonders in immunsupprimierten und älteren Individuen schwere Durchfälle, bis hin zu lebensbedrohlichen Komplikationen führen kann. In Industrieländern ist C. difficile der häufigste Erreger nosokomialer und Antibiotika- assoziierter Durchfallerkrankungen. Nach Standardtherapie mit Antibiotika leiden bis zu 30% der Patienten an wiederkehrenden C. difficile Infektionen. Eine wirkungsvolle Alternativbehandlung stellen Stuhltransfers dar, bei der Bakterien gesunder Person auf den Patienten übertragen werden. Studien haben belegt, dass die Umwandlung von primären zu sekundären Gallensäuren durch spezifische Bakterien ein wichtiger Faktor für die Besiedelungsresistenz gegen C. difficile darstellt, dieser allein aber nicht ausreichend ist, um einen kompletten Schutz gegen Infektionen mit diesem Pathogen zu erreichen. In dieser Arbeit wurde der Einfluss der Mikrobiota bei C. difficile Infektionen näher untersucht. Im ersten Schritt wurden Mäuse in unterschiedlichem Alter und mit verschiedenen Mikrobiota-Zusammensetzungen hinsichtlich ihrer Anfälligkeit gegenüber C. difficile verglichen. Im zweiten Schritt, wurden spezifische C. difficile Mutanten auf ihre Besiedelungsfähigkeit und Sporenproduktion hin bewertet. Im letzten Schritt, wurden verschiedene kommensale Bakterien aus der Mikrobiota resistenter Mäuse isoliert und ihre potentiell protektiven Eigenschaften gegenüber C. difficile getestet, um ein spezifisches Konsortium schützender Bakterien gegen Infektionen mit C. difficile zu entwickeln. Die Ergebnisse dieser Studie konnten zeigen, dass vergleichbar zum Menschen das Alter der Mäuse und die Fähigkeit sekundäre Gallensäuren zu produzieren wichtige Einflussfaktoren für die Anfälligkeit gegen C. difficile Infektionen sind. In anfälligen Mäusen war die alleinige Gabe von Gallensäure produzierenden Bakterien nicht ausreichend, jedoch konnte durch die zusätzliche Gabe weiterer kommensaler Bakterien ein dauerhafter und kompletter Schutz vor C. difficile induzierter Pathogenese erreicht werden

PrsA2 (CD630_35000) of Clostridioides difficile Is an Active Parvulin-Type PPIase and a Virulence Modulator

Clostridioides difficile is the main cause for nosocomial antibiotic associated diarrhea and has become a major burden for the health care systems of industrial countries. Its main virulence factors, the small GTPase glycosylating toxins TcdA and TcdB, are extensively studied. In contrast, the contribution of other factors to development and progression of C. difficile infection (CDI) are only insufficiently understood. Many bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) have been described in the context of virulence. Among them are the parvulin-type PrsA-like PPIases of Gram-positive bacteria. On this basis, we identified CD630_35000 as the PrsA2 homolog in C. difficile and conducted its enzymatic and phenotypic characterization in order to assess its involvement during C. difficile infection. For this purpose, wild type CdPrsA2 and mutant variants carrying amino acid exchanges mainly in the PPIase domain were recombinantly produced. Recombinant CdPrsA2 showed PPIase activity toward the substrate peptide Ala-Xaa-Pro-Phe with a preference for positively charged amino acids preceding the proline residue. Mutation of conserved residues in its active site pocket impaired the enzymatic activity. A PrsA2 deficient mutant was generated in the C. difficile 630Δerm background using the ClosTron technology. Inactivation of prsA2 resulted in a reduced germination rate in response to taurocholic acid, and in a slight increase in resistance to the secondary bile acids LCA and DCA. Interestingly, in the absence of PrsA2 colonization of mice by C. difficile 630 was significantly reduced. We concluded that CdPrsA2 is an active PPIase that acts as a virulence modulator by influencing crucial processes like sporulation, germination and bile acid resistance resulting in attenuated mice colonization

Sequence and cultivation study of Muribaculaceae reveals novel species, host preference, and functional potential of this yet undescribed family

Abstract Background Bacteria within family S24-7 (phylum Bacteroidetes) are dominant in the mouse gut microbiota and detected in the intestine of other animals. Because they had not been cultured until recently and the family classification is still ambiguous, interaction with their host was difficult to study and confusion still exists regarding sequence data annotation. Methods We investigated family S24-7 by combining data from large-scale 16S rRNA gene analysis and from functional and taxonomic studies of metagenomic and cultured species. Results A total of 685 species was inferred by full-length 16S rRNA gene sequence clustering. While many species could not be assigned ecological habitats (93,045 samples analyzed), the mouse was the most commonly identified host (average of 20% relative abundance and nine co-occurring species). Shotgun metagenomics allowed reconstruction of 59 molecular species, of which 34 were representative of the 16S rRNA gene-derived species clusters. In addition, cultivation efforts allowed isolating five strains representing three species, including two novel taxa. Genome analysis revealed that S24-7 spp. are functionally distinct from neighboring families and versatile with respect to complex carbohydrate degradation. Conclusions We provide novel data on the diversity, ecology, and description of bacterial family S24-7, for which the name Muribaculaceae is proposed

Variations in microbiota composition of laboratory mice influence Citrobacter rodentium infection via variable short-chain fatty acid production.

The composition of the intestinal microbiota influences the outcome of enteric infections in human and mice. However, the role of specific members and their metabolites contributing to disease severity is largely unknown. Using isogenic mouse lines harboring distinct microbiota communities, we observed highly variable disease kinetics of enteric Citrobacter rodentium colonization after infection. Transfer of communities from susceptible and resistant mice into germ-free mice verified that the varying susceptibilities are determined by microbiota composition. The strongest differences in colonization were observed in the cecum and could be maintained in vitro by coculturing cecal bacteria with C. rodentium. Cohousing of animals as well as the transfer of cultivable bacteria from resistant to susceptible mice led to variable outcomes in the recipient mice. Microbiome analysis revealed that a higher abundance of butyrate-producing bacteria was associated with the resistant phenotype. Quantification of short-chain fatty acid (SCFA) levels before and after infection revealed increased concentrations of acetate, butyrate and propionate in mice with delayed colonization. Addition of physiological concentrations of butyrate, but not of acetate and/or propionate strongly impaired growth of C. rodentium in vitro. In vivo supplementation of susceptible, antibiotic-treated and germ-free mice with butyrate led to the same level of protection, notably only when cecal butyrate concentration reached a concentration higher than 50 nmol/mg indicating a critical threshold for protection. In the recent years, commensal-derived primary and secondary bacterial metabolites emerged as potent modulators of hosts susceptibility to infection. Our results provide evidence that variations in SCFA production in mice fed fibre-rich chow-based diets modulate susceptibility to colonization with Enterobacteriaceae not only in antibiotic-disturbed ecosystems but even in undisturbed microbial communities. These findings emphasise the need for microbiota normalization across laboratory mouse lines for infection experiments with the model-pathogen C. rodentium independent of investigations of diet and antibiotic usage

Critical Assessment of Metagenome Interpretation: the second round of challenges.

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the Initiative for the Critical Assessment of Metagenome Interpretation (CAMI). The CAMI II challenge engaged the community to assess methods on realistic and complex datasets with long- and short-read sequences, created computationally from around 1,700 new and known genomes, as well as 600 new plasmids and viruses. Here we analyze 5,002 results by 76 program versions. Substantial improvements were seen in assembly, some due to long-read data. Related strains still were challenging for assembly and genome recovery through binning, as was assembly quality for the latter. Profilers markedly matured, with taxon profilers and binners excelling at higher bacterial ranks, but underperforming for viruses and Archaea. Clinical pathogen detection results revealed a need to improve reproducibility. Runtime and memory usage analyses identified efficient programs, including top performers with other metrics. The results identify challenges and guide researchers in selecting methods for analyses

Critical Assessment of Metagenome Interpretation: the second round of challenges

Meyer F, Fritz A, Deng Z-L, et al. Critical Assessment of Metagenome Interpretation: the second round of challenges. Nature Methods. 2022;19:429-440.Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the Initiative for the Critical Assessment of Metagenome Interpretation (CAMI). The CAMI II challenge engaged the community to assess methods on realistic and complex datasets with long- and short-read sequences, created computationally from around 1,700 new and known genomes, as well as 600 new plasmids and viruses. Here we analyze 5,002 results by 76 program versions. Substantial improvements were seen in assembly, some due to long-read data. Related strains still were challenging for assembly and genome recovery through binning, as was assembly quality for the latter. Profilers markedly matured, with taxon profilers and binners excelling at higher bacterial ranks, but underperforming for viruses and Archaea. Clinical pathogen detection results revealed a need to improve reproducibility. Runtime and memory usage analyses identified efficient programs, including top performers with other metrics. The results identify challenges and guide researchers in selecting methods for analyses