The Crohn's disease-associated Escherichia coli strain LF82 relies on SOS and stringent responses to survive, multiply and tolerate antibiotics within macrophages.

Adherent Invasive Escherichia coli (AIEC) strains recovered from Crohn's disease lesions survive and multiply within macrophages. A reference strain for this pathovar, AIEC LF82, forms microcolonies within phagolysosomes, an environment that prevents commensal E. coli multiplication. Little is known about the LF82 intracellular growth status, and signals leading to macrophage intra-vacuolar multiplication. We used single-cell analysis, genetic dissection and mathematical models to monitor the growth status and cell cycle regulation of intracellular LF82. We found that within macrophages, bacteria may replicate or undergo non-growing phenotypic switches. This switch results from stringent response firing immediately after uptake by macrophages or at later stages, following genotoxic damage and SOS induction during intracellular replication. Importantly, non-growers resist treatment with various antibiotics. Thus, intracellular challenges induce AIEC LF82 phenotypic heterogeneity and non-growing bacteria that could provide a reservoir for antibiotic-tolerant bacteria responsible for relapsing infections.

Ent2 Governs Morphogenesis and Virulence in Part through Regulation of the Cdc42 Signaling Cascade in the Fungal Pathogen Candida albicans.

The ability to transition between yeast and filamentous growth states is critical for virulence of the leading human fungal pathogen Candida albicans. Large-scale genetic screens have identified hundreds of genes required for this morphological switch, but the mechanisms by which many of these genes orchestrate this developmental transition remain largely elusive. In this study, we characterized the role of Ent2 in governing morphogenesis in C. albicans. We showed that Ent2 is required for filamentous growth under a wide range of inducing conditions and is also required for virulence in a mouse model of systemic candidiasis. We found that the epsin N-terminal homology (ENTH) domain of Ent2 enables morphogenesis and virulence and does so via a physical interaction with the Cdc42 GTPase-activating protein (GAP) Rga2 and regulation of its localization. Further analyses revealed that overexpression of the Cdc42 effector protein Cla4 can overcome the requirement for the ENTH-Rga2 physical interaction, indicating that Ent2 functions, at least in part, to enable proper activation of the Cdc42-Cla4 signaling pathway in the presence of a filament-inducing cue. Overall, this work characterizes the mechanism by which Ent2 regulates hyphal morphogenesis in C. albicans, unveils the importance of this factor in enabling virulence in an in vivo model of systemic candidiasis and adds to the growing understanding of the genetic control of a key virulence trait. IMPORTANCE Candida albicans is a leading human fungal pathogen that can cause life-threatening infections in immunocompromised individuals, with mortality rates of ~40%. The ability of this organism to grow in both yeast and filamentous forms is critical for the establishment of systemic infection. Genomic screens have identified many genes required for this morphological transition, yet our understanding of the mechanisms that regulate this key virulence trait remains incomplete. In this study, we characterized Ent2 as a core regulator of C. albicans morphogenesis. We show that Ent2 regulates hyphal morphogenesis through an interaction between its ENTH domain and the Cdc42 GAP, Rga2, which signals through the Cdc42-Cla4 signaling pathway. Finally, we show that the Ent2 protein, and specifically its ENTH domain, is required for virulence in a mouse model of systemic candidiasis. Overall, this work identifies Ent2 as a key regulator of filamentation and virulence in C. albicans.

The Crohn's disease-related bacterial strain LF82 assembles biofilm-like communities to protect itself from phagolysosomal attack.

Patients with Crohn's disease exhibit abnormal colonization of the intestine by adherent invasive E. coli (AIEC). They adhere to epithelial cells, colonize them and survive inside macrophages. It appeared recently that AIEC LF82 adaptation to phagolysosomal stress involves a long lag phase in which many LF82 cells become antibiotic tolerant. Later during infection, they proliferate in vacuoles and form colonies harboring dozens of LF82 bacteria. In the present work, we investigated the mechanism sustaining this phase of growth. We found that intracellular LF82 produced an extrabacterial matrix that acts as a biofilm and controls the formation of LF82 intracellular bacterial communities (IBCs) for several days post infection. We revealed the crucial role played by the pathogenicity island encoding the yersiniabactin iron capture system to form IBCs and for optimal LF82 survival. These results illustrate that AIECs use original strategies to establish their replicative niche within macrophages.

Imaging the Cell Cycle of Pathogen E. coli During Growth in Macrophage.

The study of the bacterial cell cycle at the single cell level can not only give insights on the fitness of the bacterial population but also reveal heterogeneous behavior. Typically, the DNA replication, the cell division, and the nucleoid conformation are appropriate representatives of the bacterial cell cycle. Because bacteria rapidly adapt their growth rate to environmental changes, the measure of cell cycle parameters gives valuable insights for the study of bacterial stress response or host-pathogen interactions. Here we describe methods to first introduce fluorescent fusion proteins and fluorescent tag within the chromosome of pathogenic bacteria to study these cell cycle steps; then to follow them within macrophages using a confocal spinning disk microscope.