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Pseudomonas aeruginosa is one of the main causative agents of nosocomial infections and the spread of multidrug-resistant strains is rising. Therefore, novel strategies for therapy are urgently required. The outer membrane composition of Gram-negative pathogens and especially of Pa restricts the efficacy of antibiotic entry into the cell and determines virulence. For efficient outer membrane protein biogenesis, the β-barrel assembly machinery (BAM) complex in the outer membrane and periplasmic chaperones like Skp and SurA are crucial. Previous studies indicated that the importance of individual proteins involved in outer membrane protein biogenesis may vary between different Gram-negative species. In addition, since multidrug-resistant Pa strains pose a serious global threat, the interference with both virulence and antibiotic resistance by disturbing outer membrane protein biogenesis might be a new strategy to cope with this challenge. Therefore, deletion mutants of the non-essential BAM complex components bamB and bamC, of the skp homolog hlpA as well as a conditional mutant of surA were investigated. The most profound effects for both traits were associated with reduced levels of SurA, characterized by increased membrane permeability, enhanced sensitivity to antibiotic treatment and attenuation of virulence in a Galleria mellonella infection model. Strikingly, the depletion of SurA in a multidrug-resistant clinical bloodstream isolate re-sensitized the strain to antibiotic treatment. From our data we conclude that SurA of Pa serves as a promising target for developing a drug that shows antiinfective activity and re-sensitizes multidrug-resistant strains to antibiotics.

Complement resistance is an important virulence trait of Yersinia enterocolitica (Ye). The predominant virulence factor expressed by Ye is Yersinia adhesin A (YadA), which enables bacterial attachment to host cells and extracellular matrix and additionally allows the acquisition of soluble serum factors. The serum glycoprotein vitronectin (Vn) acts as an inhibitory regulator of the terminal complement complex by inhibiting the lytic pore formation. Here, we show YadA-mediated direct interaction of Ye with Vn and investigated the role of this Vn binding during mouse infection in vivo. Using different Yersinia strains, we identified a short stretch in the YadA head domain of Ye O:9 E40, similar to the ‘uptake region' of Y. pseudotuberculosis YPIII YadA, as crucial for efficient Vn binding. Using recombinant fragments of Vn, we found the C-terminal part of Vn, including heparin-binding domain 3, to be responsible for binding to YadA. Moreover, we found that Vn bound to the bacterial surface is still functionally active and thus inhibits C5b-9 formation. In a mouse infection model, we demonstrate that Vn reduces complement-mediated killing of Ye O:9 E40 and, thus, improved bacterial survival. Taken together, these findings show that YadA-mediated Vn binding influences Ye pathogenesis. © 2017 Karger Publishers

Type I IFN signaling amplifies the secretion of LPS-induced proinflammatory cytokines such as TNF-α or IL-6 and might thus contribute to the high mortality associated with Gram-negative septic shock in humans. The underlying molecular mechanism, however, is ill defined. In this study, we report the generation of mice deficient in IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and demonstrate that Ifit2 is a critical signaling intermediate for LPS-induced septic shock. Ifit2 expression was significantly upregulated in response to LPS challenge in an IFN-α receptor- and IFN regulatory factor (Irf)9-dependent manner. Also, LPS induced secretion of IL-6 and TNF-α by bone marrow-derived macrophages (BMDMs) was significantly enhanced in the presence of Ifit2. In accordance, Ifit2-deficient mice exhibited significantly reduced serum levels of IL-6 and TNF-α and reduced mortality in an endotoxin shock model. Investigation of the underlying signal transduction events revealed that Ifit2 upregulates Irf3 phosphorylation. In the absence of Irf3, reduced Ifn-β mRNA expression and Ifit2 protein expression after LPS stimulation was found. Also, Tnf-α and Il-6 secretion but not Tnf-α and Il-6 mRNA expression levels were reduced. Thus, IFN-stimulated Ifit2 via enhanced Irf3 phosphorylation upregulates the secretion of proinflammatory cytokines. It thereby amplifies LPS-induced cytokine production and critically influences the outcome of endotoxin shock.

Introduction: Trefoil factor family 2 (TFF2) protein is expressed predominantly in the mucous cells of gastric mucosa contributing to its protection as an effector molecule. It’s not so prominent presence in immune cells seems to be more relevant for protection of gastric mucosa due to its role as a regulator of immune response. To estimate contribution of TFF2 in defence of intestinal mucosa to bacterial infection we exposed Tff2 deficient mice to Y.enterocolitica infection and monitored several relevant parameters. Materials and Methods: Specific pathogen free, six to eight weeks Tff2-/- and Tff2+/+ mice were orally infected with Y.enterocolitica-WA314. Animal survival was monitored twice a day over a period of 15 days. The group of animals was sacrificed on 3rd day post infection. To determine the concentration of the bacteria per organ (CFU/organ), serial dilutions of homogenized Ileal peyer’s patches were plated out on Yersinia selective media (LB agar) and incubated at 27°C for 48 hours. Different regions of GI tract were collected for RNA isolation to estimate expression of various genes involved in mucosal immune response : Crbp1, Gus, Timp, Psmb5, Bag2, Tap1, Lysosyme P, Tff3, Mmp7, Cathepsin C, Crip. Various organs (stomach, duodenum, jejunum, ileum, caecum, colon, peyer’s patches, spleen, liver, kidney and lung) were collected to monitor histopathological changes. Presence of macrophages in intestinal mucosa was investigated by immunohistochemistry using Mac3 antibody. Results and conclusions: 1. Survival curve showed significantly lower survival ratio for Tff2 deficient mice. All Tff2-/- mice died on day 7 upon infection while 80 % of Tff2+/+ mice survived at that stage. 2. Bacterial titer indicated a higher bacterial load in the illeal Peyer's patches of Tff2-/- mice as compared to Tff2+/+ mice. 3. Payer’s patches Tff2-/- mice had more macrophages (stained with Mac 3 antibody) compared to wild type mice. 4. Histopathological analysis showed a sever structural impairment of the spleen in Tff2-/- mice as compared to Tff2+/+ mice. 5. Ileal genes involved in immune response of mucosa that previously showed modulated expression in comparison of healthy Tff2-/- and Tff2-/- animals, had the same level of expression in Y.enterocolitica challenged animals. These new data show that Tff2 has an important role in immune system by protecting the organism from consequences of infection with Y.enterocolitica-WA314.