Biology subjects

Fortier, L.-C.

Publications and source records attributed to Fortier, L.-C..

8 recordsLinked to original sources

bioRxiv2026

Genomic and phenotype-based framework for rational design of phage cocktails against Escherichia coli isolates from urinary tract infections

Urinary tract infections (UTIs) are frequently caused by Escherichia coli and are increasingly associated with antimicrobial resistance, highlighting the need for alternative therapies. Although phage therapy is a promising approach, the rational selection of phages and phage cocktails remains challenging, particularly when detailed genomic information on the infecting strain is unavailable. In this study, we isolated 18 phages from environmental samples collected in Sherbrooke, Quebec, Canada. Genome sequencing identified 15 pure lytic phages representing diverse taxonomic groups and revealed prophage-derived contaminants in three isolates, underscoring the importance of genomic validation during phage discovery. Host range was determined using efficiency-of-plating (EOP) assays against UTI-associated E. coli isolates. Based on host-range overlap and cumulative EOP values, we developed a phenotype-based framework for phage cocktail design and classified combinations as redundant or complementary. Complementary cocktails composed of phages with distinct host ranges and high infection effectiveness consistently outperformed redundant combinations in time-kill assays, demonstrating that host-range diversity and lytic effectiveness are independent properties that should be considered separately during cocktail design. To evaluate cocktail performance under physiologically relevant conditions, the most effective combinations were tested in artificial urine. Both cocktails exhibited reduced activity compared with standard laboratory media, indicating that environmental conditions strongly influence phage-host interactions. Together, our results demonstrate that host-range overlap and cumulative infection effectiveness provide a practical framework for identifying effective phage combinations in the absence of detailed bacterial genomic information and may facilitate the rational development of phage therapies for UTIs.

microbiology↗

bioRxiv2025

Deciphering the RNA-based regulation mechanism of the phage-encoded AbiF system in Clostridioides difficile

Clostridioides difficile is the major cause of nosocomial infections associated with antibiotic therapy. The severity of C. difficile infections increased worldwide with the emergence of hypervirulent strains, including 027 ribotype epidemic strains. Many aspects of C. difficiles adaptation strategies during pathogenesis remain poorly understood. This pathogen thrives in gut communities that are rich in microbes and phages. To regulate horizontal transfer of genetic material during its infection cycle, C. difficile relies on diverse mechanisms. More specifically, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas and Toxin-Antitoxin (TA) systems contribute to prophage maintenance, prevention of phage infection, and stress response. Abortive infection (Abi) systems can provide additional lines of anti-phage defense. RNAs have emerged as key components of these systems including CRISPR RNAs and antitoxin RNAs within type I and type III TA. We report here the identification of a new AbiF-like system within a prophage of the hypervirulent C. difficile strain R20291. It is associated with an Abi_2/AbiD/F protein family largely distributed in Bacillota and Pseudomonadota with structural links to ancestral Cas13 proteins at the origin of the RNA-targeting CRISPR-Cas13 systems. We demonstrated toxic activity of the AbiFCd protein in C. difficile and in Escherichia coli and negative regulation of the abiFCd expression by an associated non-coding RNA RCd22. RCd22 contains two conserved abiF motifs and is active both in cis and in trans to neutralize the toxin by direct RNA-protein interaction, similar to RNA antitoxin in type III TA. A mass spectrometry interactomics analysis of protein fractions from MS2-Affinity Purification coupled with RNA sequencing (MAPS) revealed the AbiFCd protein among the most enriched RCd22 partners in C. difficile. Structural modeling of the RNA-protein complex and mutagenesis analysis revealed key positions on both protein and RNA partners for this interaction and toxic activity. In summary, these findings provide valuable insights into the mechanisms of interaction between bacteria and phages, which are pertinent to the advancement of phage therapy, genome editing, epidemiological surveillance, and the formulation of novel therapeutic approaches.

microbiology↗

bioRxiv2024

Protection against Clostridioides difficile disease by a naturally avirulent C. difficile strain

Clostridioides difficile (C. difficile) strains belonging to the epidemic BI/NAP1/027 (RT027) group have been associated with increased transmissibility and disease severity. In addition to the major toxin A and toxin B virulence factors, RT027 strains also encode the CDT binary toxin. Our lab previously identified a toxigenic RT027 isolate, ST1-75, that is avirulent in mice despite densely colonizing the colon. Here, we show that co-infecting mice with the avirulent ST1-75 and virulent R20291 strains protects mice from colitis due to rapid clearance of the virulent strain and persistence of the avirulent strain. Although avirulence of ST1-75 is due to a mutation in the cdtR gene, which encodes a response regulator that modulates the production of all three C. difficile toxins, the ability of ST1-75 to protect against acute colitis is not directly attributable to the cdtR mutation. Metabolomic analyses indicate that the ST1-75 strain depletes amino acids more rapidly than the R20291 strain and supplementation with amino acids ablates ST1-75s competitive advantage, suggesting that the ST1-75 strain limits the growth of virulent R20291 bacteria by amino acid depletion. Since the germination kinetics and sensitivity to the co-germinant glycine are similar for the ST1-75 and R20291 strains, our results identify the rapidity of in vivo nutrient depletion as a mechanism providing strain-specific, virulence-independent competitive advantages to different BI/NAP1/027 strains. They also suggest that the ST1-75 strain may, as a biotherapeutic agent, enhance resistance to CDI in high-risk patients. ImportanceClostridioides difficile infections (CDI) are prevalent in healthcare settings and are associated with high recurrence rates. Therapies to prevent CDI, including recent FDA-approved live biotherapeutic products, are costly and have not been used to prevent primary infections. While a nontoxigenic C. difficile strain (NTCD-M3) protects against virulent CDI in animals and reduced CDI recurrence in a phase 2 clinical trial, protection against CDI recurrence in humans was variable and required high doses of the nontoxigenic strain. Here we show that an avirulent C. difficile isolate, ST1-75, efficiently outcompetes virulent C. difficile strains in mice when co-infected at a 1:1 ratio. Our data suggest that inter-strain competition results from ST1-75s more rapid depletion of amino acids than the virulent R20291 strain. Our study identifies inter-strain nutrient depletion as a potentially exploitable mechanism to reduce the incidence of CDI.

microbiology↗

bioRxiv2023

Molecular mechanism of bacteriophage tail contraction- structure of an S-layer-penetrating bacteriophage

Viruses that infect bacteria (bacteriophages or phages) attach to the host cell envelope, inject their genetic material into the host cytosol and either persist as prophage or hijack the host machinery to produce progeny virions. Attachment is mediated through phage receptor binding proteins that are specific for different host cell surface molecules. A subset of phage, the myoviruses, possess contractile tails, the outer sheath of which contracts upon receptor binding, driving an inner tail tube through the cell envelope and delivering the phage genome into the host cytosol. The molecular details of phage tail contraction and mode of cell envelope penetration have remained poorly understood and were completely unknown for any phage infecting bacteria enveloped by a proteinaceous S-layer. Here we reveal the extended and contracted atomic structures of an intact contractile-tail phage that binds to and penetrates the protective S-layer of the Gram positive human pathogen Clostridioides difficile. Surprisingly, we find no evidence of the intrinsic enzymatic domains that other phages exploit in cell wall penetration, suggesting that sufficient energy is released upon tail contraction to penetrate the S-layer and the thick cell wall without enzymatic activity. However, it is also notable that the tail sheath subunits move less than those studied in related contractile injection systems such as the model phage T4. Instead, the unusually long tail length and flexibility upon contraction likely contribute towards the required free energy release for envelope penetration. Our results show that the principles of phage contraction and infection as determined in the model system of T4 are not universal. We anticipate that our structures will form a strong foundation to engineer C. difficile phages as therapeutics, and highlight important adaptations made in order to infect S-layer containing pathogens.

microbiology↗

bioRxiv2023

Virulence and genomic diversity among clinical isolates of ST1 (BI/NAP1/027) Clostridioides difficile

Clostridioides difficile (C. difficile), a leading cause of nosocomial infection, produces toxins that damage the colonic epithelium and results in colitis that varies from mild to fulminant. Variation in disease severity is poorly understood and has been attributed to host factors (age, immune competence and intestinal microbiome composition) and/or virulence differences between C. difficile strains, with some, such as the epidemic BI/NAP1/027 (MLST1) strain, being associated with greater virulence. We tested 23 MLST1(ST1) C. difficile clinical isolates for virulence in antibiotic-treated C57BL/6 mice. All isolates encoded a complete Tcd pathogenicity locus and achieved similar colonization densities in mice. Disease severity varied, however, with 5 isolates causing lethal infections, 16 isolates causing a range of moderate infections and 2 isolates resulting in no detectable disease. The avirulent ST1 isolates did not cause disease in highly susceptible Myd88-/- or germ-free mice. Genomic analysis of the avirulent isolates revealed a 69 base-pair deletion in the N-terminus of the cdtR gene, which encodes a response regulator for binary toxin (CDT) expression. Genetic deletion of the 69 base-pair cdtR sequence in the highly virulent ST1 R20291 C. difficile strain rendered it avirulent and reduced toxin gene transcription in cecal contents. Our study demonstrates that a natural deletion within cdtR attenuates virulence in the epidemic ST1 C. difficile strain without reducing colonization and persistence in the gut. Distinguishing strains on the basis of cdtR may enhance the specificity of diagnostic tests for C. difficile colitis.

microbiology↗

bioRxiv2022

The Clostridioides difficile S-Layer Protein A (SlpA) serves as a general phage receptor

Therapeutic bacteriophages (phages) are being considered as alternatives in the fight against Clostridioides difficile infections. To be efficient, phages should have a wide host range, but the lack of knowledge about the cell receptor used by C. difficile phages hampers the rational design of phage cocktails. Recent reports suggested that the C. difficile surface layer protein A (SlpA) is an important phage receptor, but clear and unambiguous experimental evidence is lacking. Here, using the epidemic R20291 strain and its FM2.5 mutant derivative lacking a functional S-layer, we show that the absence of SlpA renders cells completely resistant to infection by phiCD38-2, phiCDIII and phiCD146, which normally infect the parental strain. Complementation assays with 12 different Slayer Cassette Types (SLCTs) expressed from a plasmid revealed that SLCT-6 also allowed infection by phiCD111, and SLCT-11 enabled infection by phiCD38-2 and phiCD146. Of note, expression of SLCTs 1, 6, 8, 9, 10 or 12 conferred susceptibility to infection by 5 myophages that normally do not infect the R20291 strain, namely phiMMP02, phiMMP03, phiMMP04, phiCD506 and phiCD508. Adsorption assays showed that >50% adsorption was required for productive phage infection. Altogether, our data suggest that many phages use SlpA as their receptor and most importantly, morphologically distinct phages of the Siphoviridae and Myoviridae families target SlpA despite major differences in their tail structures. Our study therefore represents an important breakthrough in our understanding of the molecular interaction between C. difficile and its phages. IMPORTANCEPhage therapy represents an interesting alternative to treat Clostridioides difficile infections because contrary to antibiotics, most phages are highly species-specific, thereby sparing the beneficial gut microbes that protect from infection. However, currently available phages against C. difficile have a narrow host range and target members from only one or a few PCR ribotypes. Without a clear comprehension of the factors that define host specificity, and in particular the host receptor recognized by phages, it is hard to develop therapeutic cocktails in a rational manner. In our study, we provide clear and unambiguous experimental evidence that SlpA is a common receptor used by many siphophages and myophages. Although work is still needed to define how a particular phage RBP binds to a specific SLCT, identification of SlpA as a common receptor is a major keystone that will facilitate the rational design of therapeutic phage cocktails against clinically important strains.

microbiology↗

bioRxiv2020

Type I toxin-antitoxin systems contribute to mobile genetic elements maintenance in Clostridioides difficile and can be used as a counter-selectable marker for chromosomal manipulation

Toxin-antitoxin (TA) systems are widespread on mobile genetic elements as well as in bacterial chromosomes. According to the nature of the antitoxin and its mode of action for toxin inhibition, TA systems are subdivided into different types. The first type I TA modules were recently identified in the human enteropathogen Clostridioides (formerly Clostridium) difficile. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA during normal growth. Here, we report the characterization of five additional type I TA systems present within phiCD630-1 and phiCD630-2 prophage regions of C. difficile 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest. Growth is restored when the antitoxin RNAs, transcribed from the opposite strand, are co-expressed together with the toxin genes. In addition, we show that type I TA modules located within the phiCD630-1 prophage contribute to its stability and mediate phiCD630-1 heritability. Type I TA systems were found to be widespread in genomes of C. difficile phages, further suggesting their functional importance. We have made use of a toxin gene from one of type I TA modules of C. difficile as a counter-selectable marker to generate an efficient mutagenesis tool for this bacterium. This tool enabled us to delete all identified toxin genes within the phiCD630-1 prophage, thus allowing investigation of the role of TA in prophage maintenance. Furthermore, we were able to delete the large 49 kb phiCD630-2 prophage region using this improved procedure.

microbiology↗

bioRxiv2017

PhageTerm: a Fast and User-friendly Software to Determine Bacteriophage Termini and Packaging Mode using randomly fragmented NGS data

Bacteriophages are the most abundant viruses on earth and display an impressive genetic as well as morphologic diversity. Among those, the most common order of phages is the Caudovirales, whose viral particles packages linear double stranded DNA (dsDNA). In this study we investigated how the information gathered by high throughput sequencing technologies can be used to determine the DNA termini and packaging mechanisms of dsDNA phages. The wet-lab procedures traditionally used for this purpose rely on the identification and cloning of restriction fragment which can be delicate and cumbersome. Here, we developed a theoretical and statistical framework to analyze DNA termini and phage packaging mechanisms using next-generation sequencing data. Our methods, implemented in the PhageTerm software, work with sequencing reads in fastq format and the corresponding assembled phage genome.\n\nPhageTerm was validated on a set of phages with well-established packaging mechanisms representative of the termini diversity: 5cos (lambda), 3cos (HK97), pac (P1), headful without a pac site (T4), DTR (T7) and host fragment (Mu). In addition, we determined the termini of 9 Clostridium difficile phages and 6 phages whose sequences where retrieved from the sequence read archive (SRA).\n\nA direct graphical interface is available as a Galaxy wrapper version at https://galaxy.pasteur.fr and a standalone version is accessible at https://sourceforge.net/projects/phageterm/.

microbiology↗