The nucleolus is a multifunctional structure within the nucleus of eukaryotic cells and is the primary site of ribosome biogenesis. Almost all viruses target and disrupt the nucleolus—a feature exclusive to this pathogen group. Here, using a combination of bio-imaging, genetic and biochemical analyses, we demonstrate that the enteropathogenic E. coli (EPEC) effector protein EspF specifically targets the nucleolus and disrupts a subset of nucleolar factors. Driven by a defined N-terminal nucleolar targeting domain, EspF causes the complete loss from the nucleolus of nucleolin, the most abundant nucleolar protein. We also show that other bacterial species disrupt the nucleolus, dependent on their ability to deliver effector proteins into the host cell. Moreover, we uncover a novel regulatory mechanism whereby nucleolar targeting by EspF is strictly controlled by EPEC's manipulation of host mitochondria. Collectively, this work reveals that the nucleolus may be a common feature of bacterial pathogenesis and demonstrates that a bacterial pathogen has evolved a highly sophisticated mechanism to enable spatio-temporal control over its virulence proteins.

SummaryDelivery of effector molecules into LMme v macrophages by enteropathogenic Escherichia coli , via its type three secretion system (T3SS), inhibits bacterial uptake by a phosphatidylinositol-3 (PI-3) kinasedependent pathway. The T3SS system, encoded by the locus of enterocyte effacement (LEE) pathogenicity island, delivers LEE-and non-LEE-encoded effector proteins into host cells.

SummaryIn vivo studies with the mouse-specific member of the attaching and effacing (A/E) family of pathogens raised the possibility that these non-invasive enteric pathogens can specifically inhibit inducible nitric oxide synthase (iNOS) expression to prevent the production of antimicrobial nitric oxide (NO). In this study we use polarized Caco-2 cells, a model of human small intestinal epithelia, to (i) demonstrate conclusively that an A/E member, human specific enteropathogenic Escherichia coli (EPEC), can inhibit cytokine-induced iNOS expression, (ii) show that this activity is dependent on the delivery of effector molecules into host cells and (iii) investigate the mechanism of inhibition. Analysis of the level of iNOSrelated mRNA, protein and NO production demonstrated that EPEC can inhibit iNOS expression at the transcriptional, by direct and indirect mechanisms, and post-transcriptional levels. This transcriptional block was linked to the failure of the iNOS-related transcriptional factor NF-k B, but not STAT1, to undergo phosphorylation-associated activation. A selective pressure to prevent iNOS production was evidenced by the finding that iNOS activity had a potent antimicrobial effect on adherent but not nonadherent bacteria. Moreover, given the central role NF-k B plays in transcribing genes associated with early host immune responses, this inhibitory mechanism presumably represents an important role in pathogenesis. Our study also provides insights into the nature of NO production in response to bacterial infection as well as the role of the locus of enterocyte effacement (LEE)-encoded effector molecules in inhibiting iNOS expression.

Bacterial pathogens deliver multiple effector proteins into eukaryotic cells to subvert host cellular processes and an emerging theme is the cooperation between different effectors. Here, we reveal that a fine balance exists between effectors that are delivered by enteropathogenic E. coli (EPEC) which, if perturbed can have marked consequences on the outcome of the infection. We show that absence of the EPEC effector Tir confers onto the bacterium a potent ability to destroy polarized intestinal epithelia through extensive host cell detachment. This process was dependent on the EPEC effectors EspG and EspG2 through their activation of the host cysteine protease calpain. EspG and EspG2 are shown to activate calpain during EPEC infection, which increases significantly in the absence of Tir – leading to rapid host cell loss and necrosis. These findings reveal a new function for EspG and EspG2 and show that Tir, independent of its bacterial ligand Intimin, is essential for maintaining the integrity of the epithelium during EPEC infection by keeping the destructive activity of EspG and EspG2 in check.

iASPP is a protein mostly known as an inhibitor of p53 pro-apoptotic activity and a predicted regulatory subunit of the PP1 phosphatase, which is often overexpressed in tumors. We report that iASPP associates with the microtubule plus-end binding protein EB1, a central regulator of microtubule dynamics, via an SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the cortex of mitotic cells, leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of the membrane-to-cortex linker Myosin-Ic (Myo1c), which we identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up upon mitosis because of defective cortical stiffness. We propose that by increasing cortical rigidity, iASPP helps cancer cells maintain a spherical geometry suitable for proper mitotic spindle positioning and chromosome partitioning.