Peptidoglycan cross-linking by L,D-transpeptidases in Clostridioides difficile: role in antibiotic resistance and toxin release – DIFFICROSS

We identified three LDTS encoded in C. difficile genome. In order to study the function of these different enzymes, we will create a combination of multiple deletion mutants of the corresponding genes. Although generation of mutants remains a challenging task in C. difficile, we recently engineered a novel system for highly efficient allelic exchange in this bacterium that supports creation of serial deletions and insertions in C. difficile. The nature of the cross-links in the different mutant strains will be determined by analysis of the PG structure of the vegetative cells of C. difficile as well as of the cortex of the spores using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Cell morphology and cell envelope integrity of the mutants will be examined by phase-contrast and electron microscopy. Susceptibility of the mutants to ß-lactams will also be investigated by determination of the minimal inhibitory concentration (MIC) for diverse ß-lactam antibiotics. We will develop the use of fluorogenic D-amino-acids (FDAA) in C. difficile to monitor PG synthesis in live bacterial cells with high-resolution microscopy. In addition, we will construct and visualize fluorescent fusions of LDTs using SNAP and HALO reporters. We will employ transposon-directed insertion site sequencing (TraDIS) on a comprehensive transposon library to identify all genes required for ß-lactams resistance. A mariner delivery vector will be used to create a random and high-density mutant library. The mutant library will then be cultivated in the presence of subinhibitory concentrations of ß-lactam antibiotics to establish a comprehensive list of genes involved in ß-lactams resistance. Relationship between the genes involved in ß-lactams resistance and the L,D-transpeptidation pathway will then be investigated. Mutants of the selected genes will be constructed and we will determine the impact of the mutations on the abundance cross-links generated by LDT by LC-MS/MS. For regulatory proteins, we will perform comparative transcriptomic analysis using RNA-seq to globally identify gene expression changes in the mutant strain of each regulator To test the involvement of LDT in toxin release, we will will monitor toxin release by ELISA in the ldt mutant strains. A hamster model of infection will also be used to determine the contribution of LDTs to C. difficile virulence. We will study the localization of toxins in the ldt and tcdE mutants. Subcellular localization of toxins will be assayed by immunoblotting of cellular fractions. Spatial distribution of toxins will also be examined by using fluorescent-fusions of toxins with the SNAP reporter. Finally, to identify the endolysin transported by the TcdE holin, proteins in the cell wall and cytosol fractions of the ldt and tcdE mutants will be identified by LC-MS/MS and compared.

The construction of the ldt mutants is the first essential step for the success of this project. For this purpose, we used a technique that we have recently developed and that is proving to be very efficient for the laboratory strain 630?erm. We first attempted to generate mutants in the epidemic strain C. difficile UK1. However, for technical reasons, generating mutants in this strain proved much more difficult than expected and we recently decided to pursue the study in the 630?erm strain. Two single mutants have already been obtained and mutant combinations are in progress. These difficulties have delayed the progress of the project but we have used this time to optimize the methods we will use next. This will allow a faster progress in the second period. Analysis of the peptidoglycan composition of strain UK1 by LC-MS/MS revealed a structure very similar to that of the reference strain. We also developed an extraction technique for peptidoglycan from C. difficile spores that allowed us to establish its fine structure. Analysis of the abundance of the peptidoglycan cross-links generated by the LDTs in the spore peptidoglycan is in progress. We have also developed the use of fluorescent amino acids for monitoring peptidoglycan synthesis and optimized the use of Halo and SNAP fluorescent tag codons to obtain better sensitivity when used in C. difficile. We expressed the 3 LDTs fused to these tags and undertook their cellular localization. The image capture was performed at the photonic imaging platform of our research institute, the I2BC, using a spinning-disk fluorescence microscope. The analysis of the obtained images is in progress We have undertaken the study of the non-lytic mechanism of toxin release. We were able to obtain a mutant of the gene encoding the TcdE holin in the C difficile strain UK1. ELISA toxin detection assays showed that TcdE plays an essential role in toxin release and cell fractionation revealed that toxins accumulate between the membrane and the peptidoglycan in the tcdE mutant. These data are consistent with our hypothesis that TcdE carries an endolysin involved in toxin transport through the thick peptidoglycan layer. To identify the endolysin of interest, we conducted a bioinformatic analysis to establish a list of candidates. In parallel, we performed a proteomic analysis by LC-MS/MS on the different cell fractions of the wild type and the tcdE mutant to identify the endolysin involved in the process. This experiment, which allowed us to identify candidates, will be repeated under different growth conditions in order to reinforce our results.

At this stage of the project, our delay in realizing the key mutants of the study does not allow us to answer the main questions raised, but we expect rapid progress in the coming months through our anticipated optimization of the methods we will use next. Our results revealed that the peptidoglycan structure of vegetative cells and spores are very similar in the historical laboratory strain 630?erm and the epidemic strain UK1. This suggests that the findings of this project will be applicable to many strains of C. difficile and reinforces that the use of the laboratory strain as a model is justified. Concerning the mechanism of toxin release, we were able to confirm the importance of the TcdE holine in the process. The accumulation of toxins between the membrane and the peptidoglycan in a tcdE mutant argues for the involvement of an endolysin to locally digest the peptidoglycan and allow the release of toxins. We were able to identify candidate endolysins and the upcoming mutation of the corresponding genes will allow us to study their involvement. On a global scale, a better understanding of mechanisms involved in antimicrobial resistance and virulence is a prerequisite for future development of adapted preventive/therapeutic strategies. New drugs specifically targeting toxin release or lowering the resistance of C. difficile to antimicrobial compounds need to be developed as they may be administrated in prevention to hospitalized patients where patients are the most vulnerable. Such strategies would be of significant economic and societal benefit, as they would improve the wellness of patients and quality of life.

Clostridioides difficile incidence has tripled over the past 15 years and this bacterium has become the main causative agent of antibiotic-associated and health care-associated diarrhea. The impact of C. difficile infection (CDI) in hospitals is considerable in terms of mortality, morbidity and disease management as well as for its financial burden. Virulent strains of C. difficile generally produce two toxins that have been identified as the major virulence factors. However, the mechanism for toxin release is still poorly understood. CDI is difficult to prevent and treat, as the pathogen is resistant to many antimicrobial agents. Although resistance of C. difficile to ß-lactams, the most commonly prescribed class of antibiotics, is a leading contributor to the development of CDI, the underlying mechanisms of resistance are currently unknown. Due to the unsatisfactory nature of standard therapy for CDI, a current imperative is to better understand the molecular mechanisms involved in antibiotic resistance and controlling the key steps of CDI in the perspective of developing novel preventive and curative therapies. In C. difficile, the majority of the peptidoglycan (PG) cross-links is synthesized by non-classical transpeptidases, namely the L,D-transpeptidases (LDTs), insensitive to inhibition by most ß-lactam antibiotics. Our central hypothesis is that LDTs are key enzymes for PG biosynthesis that confer ß-lactam resistance and play a crucial role in toxin release in C. difficile. The overall aim of this project is therefore to characterize the three LDTs identified in C. difficile and the role of each of them. Our first objective will be to elucidate the functions of LDTs in the cell wall biology of C. difficile. We will study the relative roles of the unusual L,D-transpeptidation and the classical D,D-transpeptidation pathways in the formation of cross-links during the expansion and maturation of the C. difficile PG. We will also determine to which extent LDTs are important for cell morphology and ß-lactams resistance. Through the use of a genome-wide approach, our second objective will be to identify novel genes regulating the L,D-transpeptidation pathway. A subset of these genes will be selected for further characterization. Toxin release in C. difficile relies on PG-degrading enzymes, whose activity is controlled by structural alterations of the PG substrate. Our third objective will therefore be to evaluate the impact of LDTs on toxin release in-vitro and on virulence in-vivo. We will also identify molecular actors involved in toxin release in C. difficile. Overall, this work will provide fundamental knowledge on C. difficile PG biosynthesis, a field of research still neglected in this bacterium. Since cell wall synthesis is one of the most important antibiotic targets, a better understanding of the cell wall biology of C. difficile may ultimately help in developing procedures to overcome antibiotic resistance in this bacterium and identifying novel drug targets for the specific eradication of C. difficile. Ultimately, principles from this work will have wider implications beyond C. difficile research and will greatly enhance our current understanding of PG biosynthetic control and dynamics.