Bastian Pasche

Extending the Host Range of Listeria monocytogenes by Rational Protein Design

In causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts. We have emulated this chance occurrence by extending the host range accessible to the human pathogen Listeria monocytogenes by the intestinal route to include the mouse. Analyzing the recognition complex of the listerial invasion protein InlA and its human receptor E-cadherin, we postulated and verified amino acid substitutions in InlA to increase its affinity for E-cadherin. Two single substitutions increase binding affinity by four orders of magnitude and extend binding specificity to include formerly incompatible murine E-cadherin. By rationally adapting a single protein, we thus create a versatile murine model of human listeriosis.

Vitamin D receptor signaling contributes to susceptibility to infection with Leishmania major

We have previously reported that 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3) can selectively suppress key functions of interferon‐gamma (IFN‐γ) activated macrophages. To further explore this mechanism for its relevance in vivo, we investigated an infection model that crucially depends on the function of IFN‐γ activated macrophages, the infection with the intracellular protozoan Leishmania mαjor. 1α,25(OH)2D3 treatment of L. major infected macrophages demonstrated a vitamin D receptor (Vdr) dependent inhibition of macrophage killing activity. Further analysis showed that this was a result of decreased production of nitric oxide by 1α,25(OH)2D3‐treated macrophages due to Vdr‐dependent up‐regulation of arginase 1 ex‐pression, which overrides NO production by Nos2. When analyzing the course of infection in vivo,we found that Vdr‐knockout (Vdr‐KO) mice were more resistant to L. major infection than their wild‐type littermates. This result is in agreement with an inhibitory influence of 1α,25(OH)2D3 on the macrophage mediated host defense. Further investigation showed that Vdr‐KO mice developed an unaltered T helper cell type 1 (Th1) response on infection as indicated by normal production of IFN‐γ by CD4+ and CD8+ T cells. Therefore, we propose that the absence of 1α,25(OH)2D3‐mediated inhibition of macrophage microbicidal activity in Vdr‐KO mice results in increased resistance to Leishmania infection.

Sex-dependent susceptibility to Listeria monocytogenes infection is mediated by differential interleukin-10 production.

ABSTRACT It is well documented that sex-dependent factors affect susceptibility to infection, with most mouse models demonstrating higher resistance in females. We made the unexpected observation that infection with the intracellular bacterium Listeria monocytogenes showed an opposite pattern in several commonly used inbred mouse strains: female C57BL/6J, BALB/c, C3H/HeN, and CBA/J mice were significantly more susceptible to Listeria infection. The pronounced sensitivity of females to Listeria, which was revealed by significantly higher lethality rates, correlated also with increased bacterial numbers in organ tissues (spleen and liver) and several immunological changes in peripheral blood samples. Surprisingly, increased severity of infection in females was associated with elevated interleukin-10 (IL-10) levels in plasma. Experiments using Il10 knockout mice, for which no differences between the susceptibilities of males and females to Listeria infection could be detected, confirmed the important role of this immunosuppressive cytokine for the outcome of disease. Our findings are likely to have clinical relevance, since similar sex differences with regard to infection with Listeria monocytogenes and other intracellular pathogens have been reported for humans.

Schistosoma co-infection protects against brain pathology but does not prevent severe disease and death in a murine model of cerebral malaria

Co-infections of helminths and malaria parasites are common in human populations in most endemic areas. It has been suggested that concomitant helminth infections inhibit the control of malaria parasitemia but down-modulate severe malarial disease. We tested this hypothesis using a murine co-infection model of schistosomiasis and cerebral malaria. C57BL/6 mice were infected with Schistosoma mansoni and 8-9 weeks later, when Schistosoma infection was patent, mice were co-infected with Plasmodium berghei ANKA strain. We found that a concomitant Schistosoma infection increased parasitemia at the beginning of the P. berghei infection. It did not protect against P. berghei-induced weight loss and hypothermia, and P. berghei-mono-infected as well as S. mansoni-P. berghei-co-infected animals showed a high case fatality between days 6 and 8 of malarial infection. However, co-infection significantly reduced P. berghei-induced brain pathology. Over 40% of the S. mansoni-P. berghei-co-infected animals that died during this period were completely protected against haemorrhaging, plugging of blood vessels and infiltration, indicating that mortality in these animals was not related to cerebral disease. Schistosoma mansoni-P. berghei-co-infected mice had elevated plasma concentrations of IL-5 and IL-13 and on day 6 lower levels of IFN-γ, IL-10, monocyte chemoattractant protein-1 (MCP-1) and monokine induced by IFN-γ (MIG) than P. berghei-mono-infected mice. We conclude that in P. berghei infections, disease and early death are caused by distinct pathogenic mechanisms, which develop in parallel and are differentially influenced by the immune response to S. mansoni. This might explain why, in co-infected mice, death could be induced in the absence of brain pathology.

Microfilariae of the Filarial Nematode Litomosoides sigmodontis Exacerbate the Course of Lipopolysaccharide-Induced Sepsis in Mice

ABSTRACT Helminths facilitate their own survival by actively modulating the immune systems of their hosts. We investigated the impacts that different life cycle stages of the rodent filaria Litomosoides sigmodontis have on the inflammatory responses of mice injected with sublethal doses of lipopolysaccharide (LPS). Mice infected with female adult worms from prepatent infections, worms which have not yet started to release microfilariae, developed lower levels of proinflammatory cytokines in the peripheral blood after LPS challenge than sham-treated controls, demonstrating that female adult worms can mitigate the innate immune response. The presence of microfilariae in mice, however, through either direct injection or implantation of microfilaria-releasing adult female worms, turned the LPS challenge fatal. This lethal outcome was characterized by increased plasma levels of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 12 (IL-12), and IL-6, greater numbers of macrophages and granulocytes in the peripheral blood, and decreased body temperatures in microfilaria-infected mice. Microfilaria-infected mice deficient in IFN-γ receptor and TNF receptor 1 had increased survival rates after LPS challenge compared to immune-competent mice, suggesting that microfilariae worsen LPS-induced sepsis through actions of IFN-γ and TNF-α. In summary, we have demonstrated that infection of mice with L. sigmodontis female adult worms from prepatent infections protects mice injected with LPS whereas microfilariae worsen LPS-induced sepsis through the induction of proinflammatory cytokines and upregulation of granulocytes, NK cells, and monocytes in the peripheral blood.

Influence of Internalin A murinisation on host resistance to orally acquired listeriosis in mice

BackgroundThe bacterial surface protein internalin (InlA) is a major virulence factor of the food-born pathogen Listeria monocytogenes. It plays a critical role in the bacteria crossing the host intestinal barrier by a species-specific interaction with the cell adhesion molecule E-cadherin. In mice, the interaction of InlA with murine E-cadherin is impaired due to sequence-specific binding incompatibilities. We have previously used the approach of ‘murinisation’ to establish an oral listeriosis infection model in mice by exchanging two amino acid residues in InlA. This dramatically increases binding to mouse E-cadherin. In the present study, we have used bioluminescent murinised and non-murinised Listeria strains to examine the spatiotemporal dissemination of Listeria in four diverse mouse genetic backgrounds after oral inoculation.ResultsThe murinised Listeria monocytogenes strain showed enhanced invasiveness and induced more severe infections in all four investigated mouse inbred strains compared to the non-murinised Listeria strain. We identified C57BL/6J mice as being most resistant to orally acquired listeriosis whereas C3HeB/FeJ, A/J and BALB/cJ mice were found to be most susceptible to infection. This was reflected in faster kinetics of Listeria dissemination, higher bacterial loads in internal organs, and elevated serum levels of IL-6, IFN-γ, TNF-α and CCL2 in the susceptible strains as compared to the resistant C57BL/6J strain. Importantly, murinisation of InlA did not cause enhanced invasion of Listeria monocytogenes into the brain.ConclusionMurinised Listeria are able to efficiently cross the intestinal barrier in mice from diverse genetic backgrounds. However, expression of murinized InlA does not enhance listerial brain invasion suggesting that crossing of the blood brain barrier and crossing of the intestinal epithelium are achieved by Listeria monocytogenes through different molecular mechanisms.

TLR7 contributes to the rapid progression but not to the overall fatal outcome of secondary pneumococcal disease following influenza A virus infection.

Increased risk for bacterial superinfections substantially contributes to the mortality caused by influenza A virus (IAV) epidemics. While the mechanistic basis for this lethal synergism is still insufficiently understood, immune modulation through the viral infection has been shown to be involved. Since the pattern-recognition receptor (PRR) toll-like receptor 7 (TLR7) is a major sensor for the viral genome, we studied how IAV recognition by TLR7 influences the development of secondary pneumococcal infection. In a mouse model of IAV, TLR7-deficient hosts induced a potent antiviral response and showed unchanged survival. In secondary pneumococcal infection during acute influenza, TLR7ko mice showed a fatal outcome similar to wild-type (WT) hosts, despite significantly delayed disease progression. Also, when bacterial superinfection occurred after virus clearance, WT and TLR7-deficient hosts showed similar mortality, even though we found the phagocytic activity of alveolar macrophages isolated from IAV-pre-infected hosts to be enhanced in TLR7ko over WT mice. Thus, we show that a virus-sensing PRR modulates the progression of secondary pneumococcal infection following IAV. However, the fatal overall outcome in WT as well as TLR7ko hosts suggests that processes distinct from TLR7-triggering override the contribution of this single PRR.

Influence of Internalin A murinization on host resistance to orally acquired listeriosis in mice

ECI 2012 is co-organized by the European Federation of Immunological Societies (EFIS) and The British Society for Immunology (BSI).2017 IEEE International Symposium on Radio-Frequency Integration Technology 2017 IEEE International Symposium on Radio-Frequency Integration Technology Wednesday, Aug 30 (Workshop Sessions) Session WS_A: RF Integration Technology for 5G Communications (09:30 – 12:30, Gayageum A) Session Chair: Jae-Sung Rieh (Korea University, Korea) WS_A_1 09:30 – 10:10 Recent Research Advances in mmWave 5G Wireless Communications W. Hong (Southeast University, Nanjing, China) WS_A_2 10:10 – 10:50 28 GHz CMOS Beamforming Chipset and Front-end Module for 5G Millimeter wave Beamforming System J. –G. Kim (Kwangwoon University, Korea) Break 10:50 – 11:10 WS_A_3 11:10 – 11:50 Development of mmWave 5G Prototype Systems J. Y. Lee (KAIST, Korea) WS_A_4 11:50 – 12:30 The Present and Future of Multi-modal mmWave 5G Antennas for Mobile Phone Terminals W. Hong (POSTECH, Korea) Lunch 12:30 – 13:30