RNA Interference Analysis of Legionella in Drosophila Cells: Exploitation of Early Secretory Apparatus Dynamics

Legionella pneumophila translocates multiple bacterial effector proteins into host cells to direct formation of a replication vacuole for the bacterium. The emerging consensus is that formation of this compartment involves recruitment of membrane material that traffics between the endoplasmic reticulum (ER) and Golgi. To investigate this model, a targeted approach was used to knock down expression of proteins involved in membrane trafficking, using RNA interference in Drosophila cells. Surprisingly, few single knockdowns of ER–Golgi transport proteins decreased L. pneumophila replication. By analyzing double-stranded RNAs in pairs, combinations were identified that together caused defects in intracellular replication, consistent with the model that membrane traffic funnels into the replication vacuole from multiple sources. In particular, simultaneous depletion of the intermediate compartment and Golgi-tethering factor transport protein particle together with the ER SNARE protein Sec22 reduced replication efficiency, indicating that introduction of lesions at distinct sites in the secretory system reduces replication efficiency. In contrast to knockdowns in secretory traffic, which required multiple simultaneous hits, knockdown of single cytosolic components of ER-associated degradation, including Cdc48/p97 and associated cofactors, was sufficient to inhibit intracellular replication. The requirement for the Cdc48/p97 complex was conserved in mammalian cells, in which replication vacuoles showed intense recruitment of ubiquitinated proteins, the preferred substrates of Cdc48/p97. This complex promoted dislocation of both ubiquitinated proteins and bacterial effectors from the replication vacuole, consistent with the model that maintenance of high-level replication requires surveillance of the vacuole surface. This work demonstrates that L. pneumophila has the ability to gain access to multiple sites in the secretory system and provides the first evidence for a role of the Cdc48/p97 complex in promoting intracellular replication of pathogens and maintenance of replication vacuoles

DNA Damage Triggers Genetic Exchange in Helicobacter pylori

Many organisms respond to DNA damage by inducing expression of DNA repair genes. We find that the human stomach pathogen Helicobacter pylori instead induces transcription and translation of natural competence genes, thus increasing transformation frequency. Transcription of a lysozyme-like protein that promotes DNA donation from intact cells is also induced. Exogenous DNA modulates the DNA damage response, as both recA and the ability to take up DNA are required for full induction of the response. This feedback loop is active during stomach colonization, indicating a role in the pathogenesis of the bacterium. As patients can be infected with multiple genetically distinct clones of H. pylori, DNA damage induced genetic exchange may facilitate spread of antibiotic resistance and selection of fitter variants through re-assortment of preexisting alleles in this important human pathogen

Helicobacter pylori's Unconventional Role in Health and Disease

The discovery of a bacterium, Helicobacter pylori, that is resident in the human stomach and causes chronic disease (peptic ulcer and gastric cancer) was radical on many levels. Whereas the mouth and the colon were both known to host a large number of microorganisms, collectively referred to as the microbiome, the stomach was thought to be a virtual Sahara desert for microbes because of its high acidity. We now know that H. pylori is one of many species of bacteria that live in the stomach, although H. pylori seems to dominate this community. H. pylori does not behave as a classical bacterial pathogen: disease is not solely mediated by production of toxins, although certain H. pylori genes, including those that encode exotoxins, increase the risk of disease development. Instead, disease seems to result from a complex interaction between the bacterium, the host, and the environment. Furthermore, H. pylori was the first bacterium observed to behave as a carcinogen. The innate and adaptive immune defenses of the host, combined with factors in the environment of the stomach, apparently drive a continuously high rate of genomic variation in H. pylori. Studies of this genetic diversity in strains isolated from various locations across the globe show that H. pylori has coevolved with humans throughout our history. This long association has given rise not only to disease, but also to possible protective effects, particularly with respect to diseases of the esophagus. Given this complex relationship with human health, eradication of H. pylori in nonsymptomatic individuals may not be the best course of action. The story of H. pylori teaches us to look more deeply at our resident microbiome and the complexity of its interactions, both in this complex population and within our own tissues, to gain a better understanding of health and disease

Distinct pathologies of <i>H. pylori</i>–induced disease.

<p>(A) Duodenal ulcer disease correlates with high inflammation in the antrum (red bursts), lower levels of inflammation in the corpus, and high acid secretion (+). (B) Gastric ulcer or adenocarcinoma correlates with increased inflammation in the corpus, low acid secretion, and multifocal atrophy (wavy lines).</p

Mechanisms that create genetic diversity in <i>H. pylori</i>.

<p>Colored arrows represent different genes, and the correspondingly colored triangles, rectangles, and circles represent the proteins encoded by these genes. Diversification mechanisms (right side of figure) include spontaneous point mutations, slipped-strand mispairing, and intragenomic recombination. Allelic changes involving nonsynonymous point mutations and mosaic genes resulting from intragenomic recombination can alter the function and/or the antigenic epitopes of the encoded protein. Gene expression can also be regulated by gene conversion resulting from intragenomic recombination, and phase variation mediated by slipped-strand mispairing. Reassortment of genes (left side of figure) by natural transformation with exogenous DNA also contributes to genetic diversity. Natural transformation with DNA from a superinfecting strain, for example, can introduce new genes and new alleles of already present genes (horizontal gene transfer). Similarly, natural transformation with DNA from a variant clone of the same strain can further propagate an advantageous allele acquired by within-genome diversification.</p

The Proteasome Promotes L. pneumophila Replication and Translocation of Bacterial Effectors causes Ubiquitination of the LCV

<div><p>(A) Mouse BMDMs were pretreated for 1 h with proteasome inhibitor MG-132 or DMSO, incubated with L. pneumophila for 1 h at MOI = 1, washed, and incubated for 14 h. Infectious centers assay was performed as described.</p><p>(B) Mouse BMDMs were incubated with L. pneumophila for 1 h at MOI = 1, fixed at the indicated time, and immunostained with antipolyubiquitin and anti–<i>L. pneumophila.</i></p></div

Cdc48/p97 Localizes to the L. pneumophila Vacuole

<p>(A) Mouse BMDMs were incubated with L. pneumophila for 1 h at MOI = 1, fixed, and immunostained for human Cdc48/p97 and L. pneumophila. Inset is an enlargement of bacterium in panel. Cdc48/p97 associated with 63% ± 11% vacuoles harboring wild-type bacteria and 1.3% ± 1% <i>dotA</i> bacteria. (B) Kc167 cells were incubated with L. pneumophila for 1 h at MOI = 1; replication vacuoles were isolated, fixed, and immunostained with antibodies against <i>Drosophila</i> Cdc48/p97 (Ter94) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020034#ppat-0020034-b035" target="_blank">35</a>] and L. pneumophila. Cdc48/p97 associated with 77% ± 6.9% vacuoles harboring wild-type bacteria and 1.3% ± 0.3% <i>dotA</i> bacteria.</p

RNAi Targeting Cdc48/p97 and Cofactors Decreases L. pneumophila Replication in <i>Drosophila</i> and Human Tissue-Culture Cells

<div><p>(A) dsRNA treated Kc167 cells were incubated for 1 h with L. pneumophila at MOI = 1, washed, and incubated for 30 h prior to microscopic examination of replication vacuoles; mean ± standard error.</p><p>(B) <i>Drosophila</i> Kc167 treated with dsRNA directed against Cdc48/p97, p47 or no dsRNA [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020034#ppat-0020034-b030" target="_blank">30</a>]. Cdc48/p97-treated cells (1 × 10⁶) were lysed in SDS-PAGE buffer, separated by SDS-PAGE electorphoresis, and subjected to Western blotting with anti-Cdc48/p97 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020034#ppat-0020034-b035" target="_blank">35</a>] and antitubulin (Serotec) as a loading control. RNA from p47-treated cells was collected using trizol reagent (Invitrogen), cDNA was prepared using Superscript reverse transcriptase (Invitrogen) and 25 cycles of PCR were completed against either actin or p47.</p><p>(C) HEK 293 cells were silenced for p47 or Cdc48/p97 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020034#ppat-0020034-b039" target="_blank">39</a>], incubated with L. pneumophila at MOI = 2, and washed and incubated for 11 h prior to microscopic examination of replication vacuoles.</p><p>(D) HEK 293 cells were silenced for Cdc48/p97 or p47 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020034#ppat-0020034-b039" target="_blank">39</a>] and subjected to Western blotting with anti-p97 or anti-p47. Antitubulin (Serotec) served as a loading control.</p><p>(E) Network of genes that interact with Cdc48/p97. Lines are color coded and weighted according to predicted confidence score for each interaction [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020034#ppat-0020034-b036" target="_blank">36</a>].</p><p>(F) RNAi of Cdc48/p97 cofactors as in (A), except only the mature (11+ bacteria) vacuoles were plotted.</p></div

Cdc48/p97 Removes Polyubiquitinated Proteins from the Replication Vacuole

<div><p>(A) HEK 293 cells were silenced for Cdc48/p97 or p47 and incubated with wild-type L. pneumophila at MOI = 5 for 2 hours in CM, washed, and fixed, or further incubated in CM for 16 h. Intact cells were immunostained for polyubiquitinated proteins and <i>L. pneumophila.</i></p><p>(B) Quantification of polyubiquitin positive vacuoles from (A). Untreated (black), si p47 (white), si Cdc48/p97 (gray). Mean of three replicates ± standard error; at least 100 cells counted per replicate.</p></div