How to rewire the host cell: A home improvement guide for intracellular bacteria.

Intracellular bacterial pathogens have developed versatile strategies to generate niches inside the eukaryotic cells that allow them to survive and proliferate. Making a home inside the host offers many advantages; however, intracellular bacteria must also overcome many challenges, such as disarming innate immune signaling and accessing host nutrient supplies. Gaining entry into the cell and avoiding degradation is only the beginning of a successful intracellular lifestyle. To establish these replicative niches, intracellular pathogens secrete various virulence proteins, called effectors, to manipulate host cell signaling pathways and subvert host defense mechanisms. Many effectors mimic host enzymes, whereas others perform entirely novel enzymatic functions. A large volume of work has been done to understand how intracellular bacteria manipulate membrane trafficking pathways. In this review, we focus on how intracellular bacterial pathogens target innate immune signaling, the unfolded protein response, autophagy, and cellular metabolism and exploit these pathways to their advantage. We also discuss how bacterial pathogens can alter host gene expression by directly modifying histones or hijacking the ubiquitination machinery to take control of several host signaling pathways

Helicobacter pylori Infection Causes Characteristic DNA Damage Patterns in Human Cells

Infection with the human pathogen Helicobacter pylori (H. pylori) is a major risk factor for gastric cancer. Since the bacterium exerts multiple genotoxic effects, we examined the circumstances of DNA damage accumulation and identified regions within the host genome with high susceptibility to H. pylori-induced damage. Infection impaired several DNA repair factors, the extent of which depends on a functional cagPAI. This leads to accumulation of a unique DNA damage pattern, preferentially in transcribed regions and proximal to telomeres, in both gastric cell lines and primary gastric epithelial cells. The observed pattern correlates with focal amplifications in adenocarcinomas of the stomach and partly overlaps with known cancer genes. We thus demonstrate an impact of a bacterial infection directed toward specific host genomic regions and describe underlying characteristics that make such regions more likely to acquire heritable changes during infection, which could contribute to cellular transformation

A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro

Background and aims: Helicobacter pylori is the causative agent of gastric diseases and the main risk factor in the development of gastric adenocarcinoma. In vitro studies with this bacterial pathogen largely rely on the use of transformed cell lines as infection model. However, this approach is intrinsically artificial and especially inappropriate when it comes to investigating the mechanisms of cancerogenesis. Moreover, common cell lines are often defective in crucial signalling pathways relevant to infection and cancer. A long-lived primary cell system would be preferable in order to better approximate the human in vivo situation. Methods: Gastric glands were isolated from healthy human stomach tissue and grown in Matrigel containing media supplemented with various growth factors, developmental regulators and apoptosis inhibitors to generate long-lasting normal epithelial cell cultures. Results: Culture conditions were developed which support the formation and quasi-indefinite growth of three dimensional (3D) spheroids derived from various sites of the human stomach. Spheroids could be differentiated to gastric organoids after withdrawal of Wnt3A and Rspondin1 from the medium. The 3D cultures exhibit typical morphological features of human stomach tissue. Transfer of sheared spheroids into 2D culture led to the formation of dense planar cultures of polarised epithelial cells serving as a suitable in vitro model of H. pylori infection. Conclusions: A robust and quasi-immortal 3D organoid model has been established, which is considered instrumental for future research aimed to understand the underlying mechanisms of infection, mucosal immunity and cancer of the human stomach

Influence of the isu peptide on cytokine release and gene expression.

<p>(a) PBMCs were incubated for 24 hrs with or without the isu peptide homopolymer. The cytokine array VI (see Material and Methods) was used. The up-regulated cytokines are circled. (b) PBMCs were treated with Con A and incubated with and without the isu peptide homopolymer for three days and the cytokine array I was used. The down-regulated cytokines are circled. Similar results were obtained with PBMCs from ten other donors. (c) Cytokine array measuring simultaneous release of ten different cytokines after incubation of PBMCs from one donor with and without the isu peptide homopolymer at different time points (6 to 24 hrs), confirming and extenting the results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g002" target="_blank">Figure 2a</a>. Control PBMCs were incubated with medium. Compare the increase in IL-10 expression with that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g003" target="_blank">Figure 3a</a>. (d) Genes with the highest up-regulation (upper part) and down-regulation (lower part) of expression in PBMC of one donor in response to the isu peptide treatment. Using specific real-time PCRs for the up- and down-regulated genes, the changes were confirmed in PBMCs of other donors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g003" target="_blank">Figure 3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g004" target="_blank">4</a>, Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s002" target="_blank">Figure S2</a>). Fold changes (Fc) indicates gene expression compared to control cells incubated in medium. The full names of the genes are given in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s008" target="_blank">Table S4</a>.</p

Kinetics of the modulation of cytokine release and gene expression induced by the isu peptide.

<p>a, Kinetics of the IL-10 and IL-6 release and expression of IL-10 and IL-6 mRNA. PBMCs were incubated with (gray) or without (light gray) isu peptide homopolymers and supernatants as well as mRNA were collected at different time points between 0–24 hours. The p values were calculated using the Student’s t-test, n = 3. When comparing the IL-10 release induced by the isu peptide homopolymer and that by medium alone, the p value at the peak release (15 hrs) is p = 2.16E-05, the p value for IL-10 RNA at 24 hrs is p = 0.09. All other values were accordingly. b, c, Kinetics of the expression of MMP-1, TREM-1, FCN1, CXCL9 and SEPP-1 in PBMCs incubated with (dotted line) or without (straight line) isu-peptide homopolymers. The figures show a representative result obtained with PBMCs from more than five donors. The p values were calculated using the Student’s t-test, n = 3, the p-value of sTREM-1 release at 24 hrs is p = 0.02.</p

Summary of the changes in cytokine expression.

<p>Cytokine release was measured 24 hrs or 3 days after incubation of normal PBMCs with isu-peptide homopolymers alone or in the presence of a mitogen, respectively. The full name of the abbreviated molecules is given in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s008" target="_blank">Table S4</a>.</p

Helicobacter pylori vacA genotype is a predominant determinant of immune response to Helicobacter pylori CagA.

To evaluate the frequency of Helicobacter pylori (H. pylori) CagA antibodies in H. pylori infected subjects and to identify potential histopathological and bacterial factors related to H. pylori CagA-immune response

Donor-dependence of IL-10 and MMP-1 release.

<p>a, PBMCs from three donors were incubated with the same batch of the isu peptide polymer or medium and IL-10 release and MMP-1 mRNA expression were measured simultaneously. The donor-dependence of IL-10 release was shown using PBMCs from more than 50 donors some are shown in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s002" target="_blank">Figure S2</a>. b, Kinetic date of IL-10 release from PBMCs from a high responder donor A (column 1, dark grey) and low responder donor B (column 3, dark grey), both treated with the isu peptide homopopymer. Untreated medium control cells from both donors (Colum 2 and 4, light grey) did not release IL-10. The p values were calculated using the Student’s t-test, n = 3, p = 0.00015 in the case of a high responder and p = 0.03 in the case of a low responder.</p

Localisation and activity of the immunosuppressive (isu) domain of gp41 of HIV-1.

<p>(a) Functional domains in gp41 of HIV-1 (accession-nr. NCBI K03455): FP, fusion peptide; FPPR, fusion peptide proximal region; NHR, N-terminal helical region; ISU, isu domain; S-S, cystein-cystein loop; CHR, C-terminal helical region; MPER, membrane proximal external region; MSD, membrane spanning domain, 3S, domain binding to the gC1qR and inducing NKp44L expression. In the amino acid sequence of the isu domain stars (*) indicate NH₂-groups, points (.) mark COOH groups relevant for polymerisation. (b) Localisation of the isu domain after interaction of the NHR with the CHR generating a six helix bundle (only one molecule of the trimer is shown). (c) Influence of the isu-peptide on the proliferation of PHA stimulated PBMCs from a healthy donor. Cell proliferation was measured by ³H-thymidine incorporation. ³H-thymidine was added on day zero, one, two or three and cells were then harvested one the next day and the counts per minute were determined, gray - medium control, dark gray – isu peptide-BSA conjugates, added at day 0. (d) Dose dependent induction of IL-6 and Il-10 release by the isu peptide homopolymer (triangle) as measured in ELISAs. In contrast, the amidated control peptide (square) is inactive. (e) Comparative ELISA analysis of IL-10 release from PBMCs of seven donors all treated with the same amount and batch of the isu-peptide homopolymer, the IL-10 release of their PBMCs incubated with medium alone was zero. The p values were calculated using the Student's t-test, n = 3. The p value for the high responder donor 1 was p = 0.001, that of the low responder donor 7 p = 0.03.</p