In systems biology, material balances, kinetic models, and thermodynamic boundary conditions are increasingly used for metabolic network analysis. It is remarkable that the reversibility of enzyme-catalyzed reactions and the influence of cytosolic conditions are often neglected in kinetic models. In fact, enzyme-catalyzed reactions in numerous metabolic pathways such as in glycolysis are often reversible, i.e., they only proceed until an equilibrium state is reached and not until the substrate is completely consumed. Here, we propose the use of irreversible thermodynamics to describe the kinetic approximation to the equilibrium state in a consistent way with very few adjustable parameters. Using a flux-force approach allowed describing the influence of cytosolic conditions on the kinetics by only one single parameter. The approach was applied to reaction steps 2 and 9 of glycolysis (i.e., the phosphoglucose isomerase reaction from glucose 6-phosphate to fructose 6-phosphate and the enolase-catalyzed reaction from 2-phosphoglycerate to phosphoenolpyruvate and water). The temperature dependence of the kinetic parameter fulfills the Arrhenius relation and the derived activation energies are plausible. All the data obtained in this work were measured efficiently and accurately by means of isothermal titration calorimetry (ITC). The combination of calorimetric monitoring with simple flux-force relations has the potential for adequate consideration of cytosolic conditions in a simple manner.

For systems biology, it is important to describe the kinetic and thermodynamic properties of enzyme-catalyzed reactions and reaction cascades quantitatively under conditions prevailing in the cytoplasm. While in part I kinetic models based on irreversible thermodynamics were tested, here in part II, the influence of the presumably most important cytosolic factors was investigated using two glycolytic reactions (i.e., the phosphoglucose isomerase reaction (PGI) with a uni-uni-mechanism and the enolase reaction with an uni-bi-mechanism) as examples. Crowding by macromolecules was simulated using polyethylene glycol (PEG) and bovine serum albumin (BSA). The reactions were monitored calorimetrically and the equilibrium concentrations were evaluated using the equation of state ePC-SAFT. The pH and the crowding agents had the greatest influence on the reaction enthalpy change. Two kinetic models based on irreversible thermodynamics (i.e., single parameter flux-force and two-parameter Noor model) were applied to investigate the influence of cytosolic conditions. The flux-force model describes the influence of cytosolic conditions on reaction kinetics best. Concentrations of magnesium ions and crowding agents had the greatest influence, while temperature and pH-value had a medium influence on the kinetic parameters. With this contribution, we show that the interplay of thermodynamic modeling and calorimetric process monitoring allows a fast and reliable quantification of the influence of cytosolic conditions on kinetic and thermodynamic parameters.

Macrophages (MΦ) as specialized immune cells are involved in rubella virus (RuV) pathogenesis and enable the study of its interaction with the innate immune system. A similar replication kinetics of RuV in the two human MΦ types, the pro-inflammatory M1-like (or GM-MΦ) and anti-inflammatory M2-like (M-MΦ), was especially in M-MΦ accompanied by a reduction in the expression of the innate immune receptor CD14. Similar to RuV infection, exogenous interferon (IFN) β induced a loss of glycolytic reserve in M-MΦ, but in contrast to RuV no noticeable influence on CD14 expression was detected. We next tested the contribution of CD14 to the generation of cytokines/chemokines during RuV infection of M-MΦ through the application of anti-CD14 blocking antibodies. Blockage of CD14 prior to RuV infection enhanced generation of virus progeny. In agreement with this observation, the expression of IFNs was significantly reduced in comparison to the isotype control. Additionally, the expression of TNF-α was slightly reduced, whereas the chemokine CXCL10 was not altered. In conclusion, the observed downmodulation of CD14 during RuV infection of M-MΦ appears to contribute to virus-host-adaptation through a reduction of the IFN response.

Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C. parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1–11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium, C. parvum infection led to a massive increase in glucose consumption and lactate production. Together, these results point to the important role of both glycolysis and glutaminolysis during C. parvum intracellular replication. Referring to obtained metabolic signatures, we targeted glycolysis as well as glutaminolysis in C. parvum-infected host cells by using the inhibitors lonidamine [inhibitor of hexokinase, mitochondrial carrier protein (MCP) and monocarboxylate transporters (MCT) 1, 2, 4], galloflavin (lactate dehydrogenase inhibitor), syrosingopine (MCT1- and MCT4 inhibitor) and compound 968 (glutaminase inhibitor) under hyperoxic and physioxic conditions. In line with metabolic signatures, all inhibitors significantly reduced parasite replication under both oxygen conditions, thereby proving both energy-related metabolic pathways, glycolysis and glutaminolysis, but also lactate export mechanisms via MCTs as pivotal for C. parvum under in vivo physioxic conditions of mammals.

Staphylococcus aureus causes chronic bacterial pneumonias that are resistant to antimicrobial treatment and carry a high burden of morbidity and mortality. S. aureus persists in the lung by assuming adaptive phenotypes like biofilms, which protect the bacteria from antibiotics and host bacterial clearance. It is well established that staphylococcal adaptation to the host is often driven by immune pressure, but the specific factors that drive S. aureus persistence in the setting of chronic lung infection have not been fully elucidated. One of the critical processes that drives immune cell function is metabolism. In addition to fueling the bioenergetic needs of the cell and competing with pathogens for key resources, immune cell metabolism also generates key regulatory metabolites that can either bolster or dampen inflammation in a process known as immunometabolism. The role of these regulatory immune metabolites in staphylococcal pneumonias has not been explored. This thesis addresses the hypothesis that immune metabolites play an important role in the pathogenesis of S. aureus pneumonias, not only by regulating immune cell function but also by promoting bacterial adaptation to the lung. In Chapter 1, we examine the current understanding of the pathogenesis of staphylococcal lung infections and review the role of immune metabolites in regulating inflammation. In Chapter 2, we describe the methods we used to test our hypothesis. In Chapter 3, we define the immunometabolic response to S. aureus in the lung, identifying the anti-inflammatory metabolite itaconate as one of the most upregulated metabolites in the infected airway. We determine that itaconate production is triggered by bacterial PAMPs, and is driven by host mitochondrial stress in response to bacterial metabolism. We also discover that neutrophils are the main source of itaconate during staphylococcal pneumonia. In Chapter 4, we investigate the impact of itaconate on neutrophils, the major immune cell responsible for controlling S. aureus infection. We establish that itaconate impedes bacterial clearance and limits neutrophil bacterial killing. This occurs through two major mechanisms, including inhibition of neutrophil glycolysis, which impairs neutrophil survival during infection, and inhibition of the oxidative burst. We find that neutrophil itaconate production is still beneficial to the host, as it promotes protective, anti-oxidant and anti-cell death pathways in the epithelial and endothelial cells that are critical for respiration. In Chapter 5, we investigate the impact of itaconate on the metabolic adaptation of S. aureus to the host. We use longitudinal clinical isolates from a patient with chronic staphylococcal pneumonia to define how clonal strains adapt to the inflamed, itaconate-laden lung. The isolates demonstrate that there is selection for strains with reduced bioenergetics but increased biofilm formation. These metabolic changes are recapitulated by exposing a non-adapted S. aureus strain to itaconate, which inhibits staphylococcal bioenergetics via glycolysis, and causes increased utilization of pathways that produce biofilms. Our data demonstrate that the host immune metabolite itaconate promotes bacterial persistence during staphylococcal pneumonia by impeding bacterial clearance and promoting bacterial biofilm formation. In Chapter 6, we discuss the potential impact of these findings, particularly on the current efforts to develop itaconate as an anti-inflammatory therapeutic, and offer directions for future studies that can further explore how metabolic pathways that normally control inflammation can influence pathogen persistence in the host.

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