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The focus of this thesis is the engineering and analysis of the enantioselectivity of esterases using 3-phenylbutyric acid (3-PBA) as model substrate. An ultra high throughput assay for identification of enantioselective esterases has been developed, based on the combination of in vivo selection and flow cytometry. The in vivo selection medium consists of a couple of pseudo-enantiomers of 3-PBA; one enantiomer is coupled to glycerol (GE), and hydrolysis of this substrate will enable cell survival. The other enantiomer is coupled to the toxin 2,3-dibromopropanol (BE), the hydrolysis of this substrate will cause cell death. Thus, cell survival is a function of the enantioselectivity of the enzyme expressed. The pseudo-enantiomeric substrates are structurally similar to allow selection for enantioselectivity instead of selection for enzyme substrate affinity. Next, esterase BS2 was chosen as negative control to establish the selection system since it hydrolyses both pseudo-enantiomers with low enantioselectivity (E~3 and 1, respectively). High enantioselective esterases towards 3-PBA: esterases PestE and CL1 (E > 100, both (R)-selective) were identified in a screening and used as positive controls. Further, the hyperthermophilic esterase PestE was crystallized. After elucidation of the enzyme structure, the high enantioselectivity of the enzyme towards 3-PBA could be explained by molecular modelling. The optimal concentration of the pseudo-enantiomeric substrates was set to be 5 mM for GE (higher concentrations were toxic) and 20 mM for BE (lower concentrations did not completely inhibit bacterial growth). The in vivo selection system was established together with the identification of a flow cytometric method to differentiate bacterial physiological status. The combination of Syto9 and PI was chosen as staining technique, because it allowed differentiation of the viable and the dead cell populations, and of these from the background. After viability detection by flow cytometry was established, esterases PestE and BS2 were cultivated in selection ((R)-GE and (S)-BE) and anti-selection medium ((S)-GE and (R)-BE). Clear differences in the culture viability depending on the enantioselectivity of the enzyme expressed appeared: cells expressing the (R)-enantioselective PestE could proliferate in selection medium, but could not proliferate in anti-selection medium. Cells expressing the non-selective BS2 did not grow in any media. Further, cultures containing mixtures of BS2/PestE or BS2/CL1 expressing cells were incubated in selection and anti-selection medium, and the viable clones were detected by flow cytometry analysis, sorted out and plated on agar. When the mixtures were incubated in selection medium, enrichment of the (R)-selective enzyme (PestE or CL1) over the non-selective enzyme (BS2) was observed. When the enzyme mixtures were incubated in anti-selection medium, very few colonies grew on agar, indicating that cell survival was a function of enzyme enantioselectivity. The successfully developed assay was used to identify variants with increased enantioselectivity in a mutant library of esterase PFEI (E ~ 3, (R)-selective) created by saturation mutagenesis. After library expression, 108 clones were in vivo selected and analyzed by flow cytometry. The viable cells were sorted out and plated on agar. The 28 resulting colonies were transferred to one microtiterplate and their activity and enantioselectivity (Eapp) was investigated using p-nitrophenyl derivatives. Four interesting mutants were identified: Table 1. Enantioselectivity of the in vivo selected mutants. Mutant Eapp[a]Etrue[b]Etrue[c]Etrue[d]Etrue[e] Mutations C4 80 4 4 3 1 V121I, F198G, V225A E7 >100 2 n.d. 3 n.d. V121S E8 2 25 16 50 >100 V121S, F198G, V225A F5 5 13 15 18 80 F121I, F198C [a] with separate (R)- or (S)-enantiomers of p-nitrophenyl-3-phenylbutanoate. [b] towards GE with cell lysate or [c] pure enzyme. [d] towards Et-3-PB with cell lysate or [e] pure enzyme. n.d. not determined. The mutants were purified and activity and enantioselectivity were determined in kinetic resolutions towards Et-3-PB and GE (Table 1). Mutants identified as highly enantioselective in the Eapp-assay (C4 and E7) were low selective in kinetic resolutions. On the contrary, mutants E8 and F5, which showed low enantioselectivity towards p-nitrophenyl-3-phenylbutanoate, hydrolyzed the 3-phenylbutyric esters with good to excellent enantioselectivities. This confirms that Eapp values can differ much from Etrue values as “you get what you screen for”, and supports that the here described method is very suitable for identification of enantioselective esterases. In this PhD thesis a novel strategy for identification of enantioselective esterases has been developed. This method allows a very high throughput (≥ 108 mutants/day) and opens the bottleneck of variant analysis, which exists in protein engineering technology.
Viral diseases are a threat to bacteria and enormous animals alike. Vaccines are available against several viruses. However, for some viruses, like ASFV, we still lack vaccines, while for others, like IAV, they are not as effective as we need them to be. To a large extent, this is because we do not fully understand the mechanisms conferring antiviral immunity. To improve our understanding of antiviral immunity, we used a model species that is in many immunological aspects closer to humans than the widely used laboratory mice, pigs. In this thesis, pigs were investigated as a potential biomedical model species for viral respiratory infections in humans and as a natural host for viral infections. Both approaches provide valuable insights into aspects of porcine immunology that can either be used as the foundation for translational research or for the design of targeted therapeutics and vaccines for pigs.
Insights into fundamental characteristics of the porcine immune system form the basis for translational studies. Paper I pioneered a detailed characterization of porcine iNKT cells. To make pigs and porcine iNKT cells more available for scientific investigations, we established multicolor flow cytometry analysis platforms that allow for a more detailed investigation of these cells than previously possible. We found porcine iNKT cells circulating in peripheral blood to be a rare population among CD3+ lymphocytes that displays a pre-activated effector state and can be divided into at least three functional subsets. Upon antigenic activation, they proliferated rapidly, secreted pro-inflammatory cytokines, and exerted cytotoxicity. Moreover, we provided first evidence for a role of iNKT cells in porcine IAV and ASFV infections, which we investigated in more detail in paper IV. Central characteristics, i.e., phenotype and functional properties, exhibit a high degree of similarity between humans and pigs. Moreover, differences between human and murine iNKT cells are more pronounced than between humans and pigs.
Based on the results obtained in paper II, the established biomedical model could be used for further studies of infectious respiratory diseases. IAV infections pave the way for secondary co-infections with increased morbidity and lethality. These bactoviral co-infections are a threat to both pigs and humans. The shared susceptibility as well as homologies on the physiological and immunological level make pigs exceptionally suitable animal models for studies of these infections. Paper I and II can also be interpreted under translational aspects. Activation of iNKT cells in porcine vaccination studies showed promising results. Based on these and our findings, this might be a suitable approach for humans as well. Along with other studies, our results suggest that pigs might be a well-suited large animal model for research in infectious diseases. This is true especially for respiratory infections, such as seasonal IAV infections, for which pigs are natural hosts and contribute to viral spread and emergence as “mixing vessels”, which can result in pandemic strains like H1N1pdm09. We could show that porcine iNKT cells as well as the antiviral responses of cTC against H1N1pdm09 in pigs are comparable to human cells and processes. The increased implementation of pigs in basic and applied research might enable an improved translation of scientific knowledge to human and veterinary medicine.
In two further studies, papers III and IV, we investigated T-cell responses during a viral infection, ASF, for which pigs are the only natural hosts. Immune responses were similar after highly and moderately virulent ASFV infection in domestic pigs and wild boar, respectively. However, they differed between both species. Antiviral immunity in domestic pigs was predominantly exerted by αβ T cells, CD8α+ and DP αβ T cells, while the response in wild boar was dominated by γδ T cells, mainly CD8α+ effector cells. Since wild boar show a higher disease severity and lethality, even during infection with moderately virulent ASFV “Estonia2014”, a shift to γδ T cells seems to be detrimental. In contrast, domestic pigs survive infections with moderately virulent ASFV “Estonia2014”, which indicates that CD8α+ or DP αβ T cells confer protection at least in infections with non-highly virulent ASFV strains. Interestingly, in paper V we found higher and prolonged inflammation in domestic pigs, correlating with increased T-cell influx. However, histopathological analyses revealed no direct explanation for the differences in disease progression and lethality in domestic pigs and wild boar. These findings require further studies to elucidate the underlying mechanisms.
The lack of basic data about immunological differences between domestic pigs and wild boar hampers attempts to understand immunity against ASFV. We found differences between both suid subspecies already at steady state and even more prominent during ASFV infections in papers III-V. Most apparently, T-cell responses in wild boar were heavily biased towards γδ T cells, while immune responses in domestic pigs were based on αβ T cells. However, information about even basic characteristics, like the composition, phenotypes, and functional qualities of wild boar’s immune system, is missing. Therefore, essential baseline data must be obtained in order to adequately assess changes in future studies.
Analyses like these reveal major advantages of pigs as a biomedical model. On the one hand, similar to conventional model species, researchers can investigate every tissue at any desired time. Tissue from human patients is often scarce or not at all available, so models that can be investigated at specific times after infection are needed. On the other hand, results obtained in pigs are more comparable to humans than data from murine studies. Moreover, pigs are susceptible to similar pathogens as humans and experimental infections can be investigated without the need for major genetic manipulations. However, there are also limitations of the porcine model system. Analysis tools are not as advanced as they are for mice, especially in terms of availability of mAbs or genetically modified organisms. Still, given the major advantages that become more and more obvious, efforts should be made to make pigs more applicable for basic and translational research. In addition, findings derived from pigs can be used for the species itself. Pigs are a major livestock species and new treatments, or vaccines could also be used for them. Therefore, this research could eventually also improve animal welfare.
In summary, the presented thesis significantly enhanced our knowledge of porcine immune processes for cTC in general and iNKT cells in particular. Results were obtained both at steady state and in the context of IAV and ASFV infections, and thus, made pigs more available as a model for future research. The use of multicolor flow cytometry provided a broad overview of the ongoing immune reactions and enables further, more wide-ranging studies that can also address open questions in even more complex infection scenarios.