Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions

Insights into the Pathogenesis of Enteropathogenic E. coli Using an Improved Intestinal Enterocyte Model

Enteropathogenic E. coli (EPEC) is a human pathogen that targets the small intestine, causing severe and often fatal diarrhoea in infants. A defining feature of EPEC disease is the loss (effacement) of absorptive microvilli (MV) from the surface of small intestinal enterocytes. Much of our understanding of EPEC pathogenesis is derived from studies using cell lines such as Caco-2 - the most extensively used small intestinal model. However, previous work has revealed fundamental differences between Caco-2 cells and in vivo differentiated enterocytes in relation to MV effacement. This, and the high heterogeneity and low transfection efficiency of the Caco-2 cell line prompted the isolation of several sub-clones (NCL-1-12) to identify a more tractable and improved in vivo-like cell model. Along with established Caco-2 clones (TC-7, BBE1), sub-clones were assessed for growth rate, apical surface morphology, epithelial barrier function and transfection efficiency. TC-7 cells provided the best all-round clone and exhibited highest levels of ectopic gene expression following cell polarisation. Novel alterations in EGFP-labelled mitochondria, that were not previously documented in non-polarised cell types, highlighted the potential of the TC-7 model for defining dynamic enterocyte-specific changes during infection. Crucially, the TC-7 cell line also mimicked ex vivo derived enterocytes with regard to MV effacement, enabling a better dissection of the process. Effacement activity caused by the EPEC protein Map in the Caco-2 but not ex vivo model, was linked to a defect in suppressing its Cdc42-dependent functionality. MV effacement activity of the EPEC protein EspF in the TC-7 model was dependent on its N-WASP binding motif, which is also shown to play an essential role in epithelial barrier dysfunction. Together, this study highlights the many advantages of using TC-7 cells as a small intestinal model to study host-pathogen interactions

Secretion systems of pathogenic escherichia coli

Protein secretion plays a central role in modulating the interactions of bacteria with their environments. Bacterial ribosomes synthesize up to 8000 different proteins. Almost half of these become integrated in membranes and are secreted to the periplasm or to the external milieu. Many bacterial processes , such as DNA replication, motility, transport, antibiotic resistance, scavenging of chemicals, and pathogenesis, depend on protein secretion. Thereby, evolutionarily unrelated protein nanomachines have been developed, which allow exported proteins to cross the Gram-negative membranes. Bacterial proteins can be exported directly from the cytoplasm out of the cell by a one-step (cytoplasm to extracellular milieu), including the type I secretion system (T1SS), T3SS, T4SS, and T6SS, or two-step (periplasm translocation step), including the T2SS and T5SS, while the T4SS can use either the one- or two-step mechanism. The T3SS, T5SS, and T6SS are the more common secretion systems in Escherichia coli and most of the secreted substrates are virulence factors related to pathogenic E. coli . In this chapter, we will describe the main characteristic of these last three secretion systems.Inst. de BiotecnologíaFil: Navarro-García, Fernando. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzados.Departamento de Biología Celular; MéxicoFil: Ruiz-Perez, Fernando. University of Virginia School of Medicine. Department of Pediatrics; Estados UnidosFil: Larzabal, Mariano. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin