Autophagy suppression in DNA damaged cells occurs through a newly identified p53-proteasome-LC3 axis.

Macroautophagy is thought to have a critical role in shaping and refining cellular proteostasis in eukaryotic cells recovering from DNA damage. Here, we report a mechanism by which autophagy is suppressed in cells exposed to bacterial toxin-, chemical-, or radiation-mediated sources of genotoxicity. Autophagy suppression is directly linked to cellular responses to DNA damage, and specifically the stabilization of the tumor suppressor p53, which is both required and sufficient for regulating the ubiquitination and proteasome-dependent reduction in cellular pools of microtubule-associated protein 1 light chain 3 (LC3A/B), a key precursor of autophagosome biogenesis and maturation, in both epithelial cells and an ex vivo organoid model. Our data indicate that suppression of autophagy, through a newly identified p53-proteasome-LC3 axis, is a conserved cellular response to multiple sources of genotoxicity. Such a mechanism could potentially be important for realigning proteostasis in cells undergoing DNA damage repair.

Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses.

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.

Cellular interactions of the cytolethal distending toxins from Escherichia coli and Haemophilus ducreyi.

The cytolethal distending toxins (CDTs) compose a subclass of intracellularly acting genotoxins produced by many Gram-negative pathogenic bacteria that disrupt the normal progression of the eukaryotic cell cycle. Here, the intoxication mechanisms of CDTs from Escherichia coli (Ec-CDT) and Haemophilus ducreyi (Hd-CDT), which share limited amino acid sequence homology, were directly compared. Ec-CDT and Hd-CDT shared comparable in vitro DNase activities of the CdtB subunits, saturable cell surface binding with comparable affinities, and the requirement for an intact Golgi complex to induce cell cycle arrest. In contrast, disruption of endosome acidification blocked Hd-CDT-mediated cell cycle arrest and toxin transport to the endoplasmic reticulum and nucleus, while having no effects on Ec-CDT. Phosphorylation of the histone protein H2AX, as well as nuclear localization, was inhibited for Hd-CdtB, but not Ec-CdtB, in cells expressing dominant negative Rab7 (T22N), suggesting that Hd-CDT, but not Ec-CDT, is trafficked through late endosomal vesicles. In support of this idea, significantly more Hd-CdtB than Ec-CdtB co-localized with Rab9, which is enriched in late endosomal compartments. Competitive binding studies suggested that Ec-CDT and Hd-CDT bind to discrete cell surface determinants. These results suggest that Ec-CDT and Hd-CDT are transported within cells by distinct pathways, possibly mediated by their interaction with different receptors at the cell surface.

Bacillus anthracis spore interactions with mammalian cells: relationship between germination state and the outcome of in vitro.

BACKGROUND: During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium. RESULTS: Using an in vitro model of infection, we evaluated the influence of the germination state of B. anthracis spores, as controlled by defined culture conditions, on the outcome of infection. Spores prepared from B. anthracis Sterne 7702 germinated in a variety of common cell culture media supplemented with fetal bovine serum (FBS) while, in the absence of FBS, germination was strictly dependent on medium composition. RAW264.7 macrophage-like cells internalized spores to the same extent in either germinating or non-germinating media. However, significantly more viable, intracellular B. anthracis were recovered from cells infected under non-germinating conditions compared to germinating conditions. At the same time, RAW264.7 cells demonstrated a significant loss in viability when infected under non-germinating conditions. CONCLUSIONS: These results suggest that the outcome of host cell infection is sensitive to the germination state of spores at the time of uptake. Moreover, this study demonstrates the efficacy of studying B. anthracis spore infection of host cells within a defined, non-germinating, in vitro environment.

Modulation of iron homeostasis in macrophages by bacterial intracellular pathogens.

BACKGROUND: Intracellular bacterial pathogens depend on acquisition of iron for their success as pathogens. The host cell requires iron as an essential component for cellular functions that include innate immune defense mechanisms. The transferrin receptor TfR1 plays an important part for delivering iron to the host cell during infection. Its expression can be modulated by infection, but its essentiality for bacterial intracellular survival has not been directly investigated. RESULTS: We identified two distinct iron-handling scenarios for two different bacterial pathogens. Francisella tularensis drives an active iron acquisition program via the TfR1 pathway program with induction of ferrireductase (Steap3), iron membrane transporter Dmt1, and iron regulatory proteins IRP1 and IRP2, which is associated with a sustained increase of the labile iron pool inside the macrophage. Expression of TfR1 is critical for Francisella's intracellular proliferation. This contrasts with infection of macrophages by wild-type Salmonella typhimurium, which does not require expression of TfR1 for successful intracellular survival. Macrophages infected with Salmonella lack significant induction of Dmt1, Steap3, and IRP1, and maintain their labile iron pool at normal levels. CONCLUSION: The distinction between two different phenotypes of iron utilization by intracellular pathogens will allow further characterization and understanding of host-cell iron metabolism and its modulation by intracellular bacteria.

Activation of the abundant nuclear factor poly(ADP-ribose) polymerase-1 by Helicobacter pylori.

Modification of eukaryotic proteins is a powerful strategy used by pathogenic bacteria to modulate host cells during infection. Previously, we demonstrated that Helicobacter pylori modify an unidentified protein within mammalian cell lysates in a manner consistent with the action of a bacterial ADP-ribosylating toxin. Here, we identified the modified eukaryotic factor as the abundant nuclear factor poly(ADP-ribose) polymerase-1 (PARP-1), which is important in the pathologies of several disease states typically associated with chronic H. pylori infection. However, rather than being ADP-ribosylated by an H. pylori toxin, the intrinsic poly(ADP-ribosyl) polymerase activity of PARP-1 is activated by a heat- and protease-sensitive H. pylori factor, resulting in automodification of PARP-1 with polymers of poly(ADP-ribose) (PAR). Moreover, during infection of gastric epithelial cells, H. pylori induce intracellular PAR-production by a PARP-1-dependent mechanism. Activation of PARP-1 by a pathogenic bacterium represents a previously unrecognized strategy for modulating host cell signaling during infection.

Francisella targets cholesterol-rich host cell membrane domains for entry into macrophages.

Francisella tularensis is a pathogen optimally adapted to efficiently invade its respective host cell and to proliferate intracellularly. We investigated the role of host cell membrane microdomains in the entry of F. tularensis subspecies holarctica vaccine strain (F. tularensis live vaccine strain) into murine macrophages. F. tularensis live vaccine strain recruits cholesterol-rich lipid domains ("lipid rafts") with caveolin-1 for successful entry into macrophages. Interference with lipid rafts through the depletion of plasma membrane cholesterol, through induction of raft internalization with choleratoxin, or through removal of raft-associated GPI-anchored proteins by treatment with phosphatidylinositol phospholipase C significantly inhibited entry of Francisella and its intracellular proliferation. Lipid raft-associated components such as cholesterol and caveolin-1 were incorporated into Francisella-containing vesicles during entry and the initial phase of intracellular trafficking inside the host cell. These findings demonstrate that Francisella requires cholesterol-rich membrane domains for entry into and proliferation inside macrophages.

Streptococcus uberis internalizes and persists in bovine mammary epithelial cells.

Streptococcus uberis is one of the most important emerging bovine mastitis pathogens and chronic persistent intramammary infections (IMI) are often described. To define the ability of S. uberis to persist intracellularly, studies on time-dependent internalization and survival of S. uberis strains in bovine mammary epithelial cells were conducted. Two S. uberis strains (UT366 and UT888) and a Staphylococcus aureus strain used as positive control, all isolated from cows with clinical mastitis were cocultured with bovine mammary epithelial cells (MAC-T) and persistent survival in host epithelial cells for extended periods (120 h) studied. Of S. uberis strains tested, UT366 showed highest internalization values at 60 min of incubation whereas at 8 h of incubation the corresponding values for UT888 were the highest. Of both strains of S. uberis tested, UT366 seems to internalize bovine mammary cells more efficiently initially, however, during the first 8 h, UT888 seems to survive intracellularly better than UT366. Results showed that both S. uberis strains could survive intracellularly up to 120 h without apparent loss of host cells viability. S. aureus internalized more efficiently than all strains tested and host cell death was observed after 72 h of incubation. These results indicate that S. uberis can survive within mammary epithelial cells for extended time without apparent loss of host cells viability. Intracellular persistence of S. uberis may be associated with the spread of the infection to deeper tissues and development of persistent IMI.