Evidence of Reduced Virulence and Increased Colonization among Pneumococcal Isolates of Serotype 3 Clade II Lineage in Mice.

Recent phylogenetic profiling of pneumococcal serotype 3 (Pn3) isolates revealed a dynamic interplay among major lineages with the emergence and global spread of a variant termed Clade II. The cause of Pn3 clade II dissemination along with epidemiological and clinical ramifications are currently unknown. Here, we sought to explore biological characteristics of dominant Pn3 clades in a mouse model of pneumococcal invasive disease and carriage. Carriage and virulence potential were strain dependent with marked differences among clades. We found that clinical isolates from Pn3 clade II are less virulent and less invasive in mice compared to clade I isolates. We also observed that clade II isolates are carried for longer and at higher bacterial densities in mice compared to clade I isolates. Taken together, our data suggest that the epidemiological success of Pn3 clade II could be related to alterations in the pathogen's ability to cause invasive disease and to establish a robust carriage episode.

Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence.

Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies and that bacteria derived from the isolates display discrete motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein colony morphology regulators RST (CmrRST), which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of flagella and type IV pili, suggesting a novel mechanism of cell migration in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration on surfaces. In a hamster model of acute C. difficile disease, the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.

Inactivation of the riboswitch-controlled GMP synthase GuaA in Clostridioides difficile is associated with severe growth defects and poor infectivity in a mouse model of infection.

Clostridioides difficile is the main cause of nosocomial antibiotic-associated diarrhoea. There is a need for new antimicrobials to tackle this pathogen. Guanine riboswitches have been proposed as promising new antimicrobial targets, but experimental evidence of their importance in C. difficile is missing. The genome of C. difficile encodes four distinct guanine riboswitches, each controlling a single gene involved in purine metabolism and transport. One of them controls the expression of guaA, encoding a guanosine monophosphate (GMP) synthase. Here, using in-line probing and GusA reporter assays, we show that these riboswitches are functional in C. difficile and cause premature transcription termination upon binding of guanine. All riboswitches exhibit a high affinity for guanine characterized by Kd values in the low nanomolar range. Xanthine and guanosine also bind the guanine riboswitches, although with less affinity. Inactivating the GMP synthase (guaA) in C. difficile strain 630 led to cell death in minimal growth conditions, but not in rich medium. Importantly, the capacity of a guaA mutant to colonize the mouse gut was significantly reduced. Together, these results demonstrate the importance of de novo GMP biosynthesis in C. difficile during infection, suggesting that targeting guanine riboswitches with analogues could be a viable therapeutic strategy.

Genome-wide detection of conservative site-specific recombination in bacteria.

The ability of clonal bacterial populations to generate genomic and phenotypic heterogeneity is thought to be of great importance for many commensal and pathogenic bacteria. One common mechanism contributing to diversity formation relies on the inversion of small genomic DNA segments in a process commonly referred to as conservative site-specific recombination. This phenomenon is known to occur in several bacterial lineages, however it remains notoriously difficult to identify due to the lack of conserved features. Here, we report an easy-to-implement method based on high-throughput paired-end sequencing for genome-wide detection of conservative site-specific recombination on a single-nucleotide level. We demonstrate the effectiveness of the method by successfully detecting several novel inversion sites in an epidemic isolate of the enteric pathogen Clostridium difficile. Using an experimental approach, we validate the inversion potential of all detected sites in C. difficile and quantify their prevalence during exponential and stationary growth in vitro. In addition, we demonstrate that the master recombinase RecV is responsible for the inversion of some but not all invertible sites. Using a fluorescent gene-reporter system, we show that at least one gene from a two-component system located next to an invertible site is expressed in an on-off mode reminiscent of phase variation. We further demonstrate the applicability of our method by mining 209 publicly available sequencing datasets and show that conservative site-specific recombination is common in the bacterial realm but appears to be absent in some lineages. Finally, we show that the gene content associated with the inversion sites is diverse and goes beyond traditionally described surface components. Overall, our method provides a robust platform for detection of conservative site-specific recombination in bacteria and opens a new avenue for global exploration of this important phenomenon.

Expanding the repertoire of conservative site-specific recombination in Clostridioides difficile.

Recent genomic analysis of an epidemic ribotype 027 (RT027) Clostridioides difficile strain revealed the presence of several chromosomal site-specific invertible sites hypothesized to control the expression of adjacent genes in a bimodal on-off mode. This process, named phase variation, is thought to enhance phenotypic variability under homogeneous conditions ultimately increasing population fitness in unpredictable environmental fluctuations. The full extent of phase variation mediated by DNA-inversions in C. difficile is currently unknown. Here, we sought to expand our previous analysis by screening for site-specific inversions in isolates that belong to the rapidly emerging ribotypes RT017 and RT078. We report the finding of one novel inversion site for which we demonstrate the inversion potential and quantify inversion proportions during exponential and stationary growth in both historic and modern isolates of the same ribotype. We then employ a computational approach to assess the prevalence of all sites identified so far in a large collection of sequenced C. difficile isolates. We show that phase-variable loci are widespread with some sites being present in virtually all analyzed strains. Furthermore, in our small subset of RT017 and RT078 strains, we detect no evidence of gain or loss of invertible sites in historic versus modern isolates demonstrating the relative stability of those genomic elements. Overall, our results support the idea that C. difficile has adopted phase variation mediated by DNA inversions as its major generator of diversity which could be beneficial during the pathogenesis process.

High Prevalence and Genetic Diversity of Large phiCD211 (phiCDIF1296T)-Like Prophages in Clostridioides difficile.

Clostridioides difficile (formerly Clostridium difficile) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified in C. difficile so far, with a genome of 131 kbp. It shares morphological and genomic similarity with other large siphophages, like phage 949, infecting Lactococcus lactis, and phage c-st, infecting Clostridium botulinum A PhageTerm analysis indicated the presence of 378-bp direct terminal repeats at the phiCD211 genome termini. Among striking features of phiCD211, the presence of several transposase and integrase genes suggests past recombination events with other mobile genetic elements. Several gene products potentially influence the bacterial lifestyle and fitness, including a putative AcrB/AcrD/AcrF multidrug resistance protein, an EzrA septation ring formation regulator, and a spore protease. We also identified a CRISPR locus and a cas3 gene. We screened 2,584 C. difficile genomes available and detected 149 prophages sharing ≥80% nucleotide identity with phiCD211 (5% prevalence). Overall, phiCD211-like phages were detected in C. difficile strains corresponding to 21 different multilocus sequence type groups, showing their high prevalence. Comparative genomic analyses revealed the existence of several clusters of highly similar phiCD211-like phages. Of note, large chromosome inversions were observed in some members, as well as multiple gene insertions and module exchanges. This highlights the great plasticity and gene coding potential of the phiCD211/phiCDIF1296T genome. Our analyses also suggest active evolution involving recombination with other mobile genetic elements.IMPORTANCEClostridioides difficile is a clinically important pathogen representing a serious threat to human health. Our hypothesis is that genetic differences between strains caused by the presence of integrated prophages could explain the apparent differences observed in the virulence of different C. difficile strains. In this study, we provide a full characterization of phiCD211, also known as phiCDIF1296T, the largest phage known to infect C. difficile so far. Screening 2,584 C. difficile genomes revealed the presence of highly similar phiCD211-like phages in 5% of the strains analyzed, showing their high prevalence. Multiple-genome comparisons suggest that evolution of the phiCD211-like phage community is dynamic, and some members have acquired genes that could influence bacterial biology and fitness. Our study further supports the relevance of studying phages in C. difficile to better understand the epidemiology of this clinically important human pathogen.

The Clostridium difficile cell wall protein CwpV confers phase-variable phage resistance.

Bacteriophages are present in virtually all ecosystems, and bacteria have developed multiple antiphage strategies to counter their attacks. Clostridium difficile is an important pathogen causing severe intestinal infections in humans and animals. Here we show that the conserved cell-surface protein CwpV provides antiphage protection in C. difficile. This protein, for which the expression is phase-variable, is classified into five types, each differing in their repeat-containing C-terminal domain. When expressed constitutively from a plasmid or the chromosome of locked 'ON' cells of C. difficile R20291, CwpV conferred antiphage protection. Differences in the level of phage protection were observed depending on the phage morphological group, siphophages being the most sensitive with efficiency of plaquing (EOP) values of < 5 × 10(-7) for phages ϕCD38-2, ϕCD111 and ϕCD146. Protection against the myophages ϕMMP01 and ϕCD52 was weaker, with EOP values between 9.0 × 10(-3) and 1.1 × 10(-1). The C-terminal domain of CwpV carries the antiphage activity and its deletion, or part of it, significantly reduced the antiphage protection. CwpV does not affect phage adsorption, but phage DNA replication is prevented, suggesting a mechanism reminiscent of superinfection exclusion systems normally encoded on prophages. CwpV thus represents a novel ubiquitous host-encoded and phase-variable antiphage system in C. difficile.

Global transcriptional response of Clostridium difficile carrying the CD38 prophage.

Clostridium difficile is one of the most dangerous pathogens in hospital settings. Most strains of C. difficile carry one or more prophages, and some of them, like CD38-2 and CD119, can influence the expression of toxin genes. However, little is known about the global host response in the presence of a given prophage. In order to fill this knowledge gap, we used high-throughput RNA sequencing (RNA-seq) to conduct a genome-wide transcriptomic analysis of the epidemic C. difficile strain R20291 carrying the CD38-2 prophage. A total of 39 bacterial genes were differentially expressed in the R20291 lysogen, 26 of them being downregulated. Several of the regulated genes encode transcriptional regulators and phosphotransferase system (PTS) subunits involved in glucose, fructose, and glucitol/sorbitol uptake and metabolism. CD38-2 also upregulated the expression of a group of regulatory genes located in phi-027, a resident prophage common to most ribotype 027 isolates. The most differentially expressed gene was that encoding the conserved phase-variable cell wall protein CwpV, which was upregulated 20-fold in the lysogen. Quantitative PCR and immunofluorescence showed that the increased cwpV expression results from a greater proportion of cells actively transcribing the gene. Indeed, 95% of f lysogenic cells express cwpV, as opposed to only 5% of wild-type cells. Furthermore, the higher proportion of cells expressing cwpV results from a higher frequency of recombination of the genetic switch controlling phase variation, which we confirmed to be dependent on the host-encoded recombinase RecV. In summary, CD38-2 interferes with phase variation of the surface protein CwpV and the expression of metabolic genes.

Characterization of temperate phages infecting Clostridium difficile isolates of human and animal origins.

Clostridium difficile is a Gram-positive pathogen infecting humans and animals. Recent studies suggest that animals could represent potential reservoirs of C. difficile that could then transfer to humans. Temperate phages contribute to the evolution of most bacteria, for example, by promoting the transduction of virulence, fitness, and antibiotic resistance genes. In C. difficile, little is known about their role, mainly because suitable propagating hosts and conditions are lacking. Here we report the isolation, propagation, and preliminary characterization of nine temperate phages from animal and human C. difficile isolates. Prophages were induced by UV light from 58 C. difficile isolates of animal and human origins. Using soft agar overlays with 27 different C. difficile test strains, we isolated and further propagated nine temperate phages: two from horse isolates (ΦCD481-1 and ΦCD481-2), three from dog isolates (ΦCD505, ΦCD506, and ΦCD508), and four from human isolates (ΦCD24-2, ΦCD111, ΦCD146, and ΦCD526). Two phages are members of the Siphoviridae family (ΦCD111 and ΦCD146), while the others are Myoviridae phages. Pulsed-field gel electrophoresis and restriction enzyme analyses showed that all of the phages had unique double-stranded DNA genomes of 30 to 60 kb. Phages induced from human C. difficile isolates, especially the members of the Siphoviridae family, had a broader host range than phages from animal C. difficile isolates. Nevertheless, most of the phages could infect both human and animal strains. Phage transduction of antibiotic resistance was recently reported in C. difficile. Our findings therefore call for further investigation of the potential risk of transduction between animal and human C. difficile isolates.

Evidence of in vivo prophage induction during Clostridium difficile infection.

Prophages contribute to the evolution and virulence of most bacterial pathogens, but their role in Clostridium difficile is unclear. Here we describe the isolation of four Myoviridae phages, ΦMMP01, ΦMMP02, ΦMMP03, and ΦMMP04, that were recovered as free viral particles in the filter-sterilized stool supernatants of patients suffering from C. difficile infection (CDI). Furthermore, identical prophages were found in the chromosomes of C. difficile isolated from the corresponding fecal samples. We therefore provide, for the first time, evidence of in vivo prophage induction during CDI. We completely sequenced the genomes of ΦMMP02 and ΦMMP04, and bioinformatics analyses did not reveal the presence of virulence factors but underlined the unique character of ΦMMP04. We also studied the mobility of ΦMMP02 and ΦMMP04 prophages in vitro. Both prophages were spontaneously induced, with 4 to 5 log PFU/ml detected in the culture supernatants of the corresponding lysogens. When lysogens were grown in the presence of subinhibitory concentrations of ciprofloxacin, moxifloxacin, levofloxacin, or mitomycin C, the phage titers further increased, reaching 8 to 9 log PFU/ml in the case of ΦMMP04. In summary, our study highlights the extensive genetic diversity and mobility of C. difficile prophages. Moreover, antibiotics known to represent risk factors for CDI, such as quinolones, can stimulate prophage mobility in vitro and probably in vivo as well, which underscores their potential impact on phage-mediated horizontal gene transfer events and the evolution of C. difficile.

Prophage-stimulated toxin production in Clostridium difficile NAP1/027 lysogens.

TcdA and TcdB exotoxins are the main virulence factors of Clostridium difficile, one of the most deadly nosocomial pathogens. Recent data suggest that prophages can influence the regulation of toxin expression. Here we present the characterization of ϕCD38-2, a pac-type temperate Siphoviridae phage that stimulates toxin expression when introduced as a prophage into C. difficile. Host range analysis showed that ϕCD38-2 was able to infect 99/207 isolates of C. difficile representing 11 different PCR ribotypes. Of 89 isolates corresponding to the NAP1/027 hypervirulent strain, which recently caused several outbreaks in North America and Europe, 79 (89%) were sensitive to ϕCD38-2. The complete double-stranded DNA (dsDNA) genome was determined, and a putative function could be assigned to 24 of the 55 open reading frames. No toxins or virulence factors could be identified based on bioinformatics analyses. Our data also suggest that ϕCD38-2 replicates as a circular plasmid in C. difficile lysogens. Upon introduction of ϕCD38-2 into a NAP1/027 representative isolate, up to 1.6- and 2.1-fold more TcdA and TcdB, respectively, were detected by immunodot blotting in culture supernatants of the lysogen than in the wild-type strain. In addition, real-time quantitative reverse transcriptase PCR (qRT-PCR) analyses showed that the mRNA levels of all five pathogenicity locus (PaLoc) genes were higher in the CD274 lysogen. Our study provides the first genomic sequence of a new pac-type Siphoviridae phage family member infecting C. difficile and brings further evidence supporting the role of prophages in toxin production in this important nosocomial pathogen.

Multiple Regulatory Mechanisms Control the Production of CmrRST, an Atypical Signal Transduction System in Clostridioides difficile.

Clostridioides difficile, an intestinal pathogen and leading cause of nosocomial infection, exhibits extensive phenotypic heterogeneity through phase variation. The signal transduction system CmrRST, which encodes two response regulators (CmrR and CmrT) and a sensor kinase (CmrS), impacts C. difficile cell and colony morphology, surface and swimming motility, biofilm formation, and virulence in an animal model. CmrRST is subject to phase variation through site-specific recombination and reversible inversion of the "cmr switch," and expression of cmrRST is also regulated by cyclic diguanylate (c-di-GMP) through a riboswitch. The goal of this study was to determine how the cmr switch and c-di-GMP work together to regulate cmrRST expression. We generated "phase-locked" strains by mutating key residues in the right inverted repeat flanking the cmr switch. Phenotypic characterization of these phase-locked cmr-ON and -OFF strains demonstrates that they cannot switch between rough and smooth colony morphologies, respectively, or other CmrRST-associated phenotypes. Manipulation of c-di-GMP levels in these mutants showed that c-di-GMP promotes cmrRST expression and associated phenotypes independently of cmr switch orientation. We identified multiple promoters controlling cmrRST transcription, including one within the ON orientation of the cmr switch and another that is positively autoregulated by CmrR. Overall, this work reveals a complex regulatory network that governs cmrRST expression and a unique intersection of phase variation and c-di-GMP signaling. These findings suggest that multiple environmental signals impact the production of this signaling transduction system. IMPORTANCE Clostridioides difficile is a leading cause of hospital-acquired intestinal infections in the United States. The CmrRST signal transduction system controls numerous physiological traits and processes in C. difficile, including cell and colony morphology, motility, biofilm formation, and virulence. Here, we define the complex, multilevel regulation of cmrRST expression, including stochastic control through phase variation, modulation by the second messenger c-di-GMP, and positive autoregulation by CmrR. The results of this study suggest that multiple, distinct environmental stimuli and selective pressures must be integrated to appropriately control cmrRST expression.

Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis.

Clostridioides (formerly Clostridium) difficile is a leading cause of healthcare-associated infections. Although considerable progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we perform a comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observe a high level of epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity, the corresponding gene of which is highly conserved across our dataset and in all of the approximately 300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacts sporulation, a key step in C. difficile disease transmission, and these results are consistently supported by multiomics data, genetic experiments and a mouse colonization model. Further experimental and transcriptomic analyses suggest that epigenetic regulation is associated with cell length, biofilm formation and host colonization. These findings provide a unique epigenetic dimension to characterize medically relevant biological processes in this important pathogen. This study also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomic studies.

Characterization of Functional Prophages in Clostridium difficile.

Bacteriophages (phages) are present in almost, if not all ecosystems. Some of these bacterial viruses are present as latent "prophages," either integrated within the chromosome of their host, or as episomal DNAs. Since prophages are ubiquitous throughout the bacterial world, there has been a sustained interest in trying to understand their contribution to the biology of their host. Clostridium difficile is no exception to that rule and with the recent release of hundreds of bacterial genome sequences, there has been a growing interest in trying to identify and classify these prophages. Besides their identification in bacterial genomes, there is also growing interest in determining the functionality of C. difficile prophages, i.e., their capacity to escape their host and reinfect a different strain, thereby promoting genomic evolution and horizontal transfer of genes through transduction, for example of antibiotic resistance genes. There is also some interest in using therapeutic phages to fight C. difficile infections.The objective of this chapter is to share with the broader C. difficile research community the expertise we developed in the study of C. difficile temperate phages. In this chapter, we describe a general "pipeline" comprising a series of experiments that we use in our lab to identify, induce, isolate, propagate, and characterize prophages. Our aim is to provide readers with the necessary basic tools to start studying C. difficile phages.

Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile.

UNLABELLED: Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. IMPORTANCE: Clostridium difficile is the major cause of nosocomial infections associated with antibiotic therapy worldwide. To survive in bacteriophage-rich gut communities, enteropathogens must develop efficient systems for defense against foreign DNA elements. CRISPR-Cas systems have recently taken center stage among various anti-invader bacterial defense systems. We provide experimental evidence for the function of the C. difficile CRISPR system against plasmid DNA and bacteriophages. These data demonstrate the original features of active C. difficile CRISPR system and bring important insights into the interactions of this major enteropathogen with foreign DNA invaders during its infection cycle.

Importance of prophages to evolution and virulence of bacterial pathogens.

Bacteriophages, or simply phages, are viruses infecting bacteria. With an estimated 10 ( 31) particles in the biosphere, phages outnumber bacteria by a factor of at least 10 and not surprisingly, they influence the evolution of most bacterial species, sometimes in unexpected ways. "Temperate" phages have the ability to integrate into the chromosome of their host upon infection, where they can reside as "quiescent" prophages until conditions favor their reactivation. Lysogenic conversion resulting from the integration of prophages encoding powerful toxins is probably the most determinant contribution of prophages to the evolution of pathogenic bacteria. We currently grasp only a small fraction of the total phage diversity. Phage biologists keep unraveling novel mechanisms developed by phages to parasitize their host. The purpose of this review is to give an overview of some of the various ways by which prophages change the lifestyle and boost virulence of some of the most dangerous bacterial pathogens.