Pulmonary Pathogens Adapt to Immune Signaling Metabolites in the Airway

Abstract

A limited number of pulmonary pathogens are able to evade normal mucosal defenses to establish acute infection and then adapt to cause chronic pneumonias. Pathogens, such as Pseudomonas aeruginosa or Staphylococcus aureus, are typically associated with infection in patients with underlying pulmonary disease or damage, such as cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). To establish infection, bacteria express a well-defined set of so-called virulence factors that facilitate colonization and activate an immune response, gene products that have been identified in murine models. Less well-understood are the adaptive changes that occur over time in vivo, enabling the organisms to evade innate and adaptive immune clearance mechanisms. These colonizers proliferate, generating a population sufficient to provide selection for mutants, such as small colony variants and mucoid variants, that are optimized for long term infection. Such host-adapted strains have evolved in response to selective pressure such as antibiotics and the recruitment of phagocytes at sites of infection and their release of signaling metabolites (e.g., succinate). These metabolites can potentially function as substrates for bacterial growth and but also generate oxidant stress. Whole genome sequencing and quantified expression of selected genes have helped to explain how P. aeruginosa and S. aureus adapt to the presence of these metabolites over the course of in vivo infection. The serial isolation of clonally related strains from patients with cystic fibrosis has provided the opportunity to identify bacterial metabolic pathways that are altered under this immune pressure, such as the anti-oxidant glyoxylate and pentose phosphate pathways, routes contributing to the generation of biofilms. These metabolic pathways and biofilm itself enable the organisms to dissipate oxidant stress, while providing protection from phagocytosis. Stimulation of host immune signaling metabolites by these pathogens drives bacterial adaptation and promotes their persistence in the airways. The inherent metabolic flexibility of P. aeruginosa and S. aureus is a major factor in their success as pulmonary pathogens.

Keywords: COPD; Pseudomonas aeruginosa; Staphylococcus aereus; cystic fibrosis; fumarate; immunometabolism; inflammation; succinate.

Figure 1

The mitochondrial PTEN-succinate axis promotes P. aeruginosa airway infection. (A) Once interacting with surface TLR4 receptor, P. aeruginosa LPS triggers inflammation. Mitochondria become depolarized producing ROS and succinate (yellow circles). This is a tightly regulated process by the CFTR-PTEN complex, which signals through Akt and PI3Kγ/δ to suppress LPS-TLR4-driven inflammation. Succinate leaked into the cytoplasm inhibits PHD, which activates HIF-1α to induce production of pro-IL-1β. In parallel, P. aeruginosa release soluble LPS in the cytoplasm, flagellin or inject T3SS toxins. These activate different inflammasomes, priming caspases and cleavage of pro-IL-1β to produce IL-1β. Gasdermin D form pores in the cell membrane releasing mature IL-1β and causing cell death. Once extracellular, IL-1β induces recruitment of more immune cells to clear infection. (B) Laboratory and P. aeruginosa clinical strains derived from acutely infected patients (e.g., ICU) are more immunostimulatory than host-adapted isolates, such as in CF. (C) The immunosignaling metabolite succinate induces crc-dependent polysaccharide production by repressing glucose catabolism in P. aeruginosa. Extracellular ROS and surface contact also induce c-di-GMP generation, which promotes the synthesis of Pel, Psl and alginate and biofilm production. The pro-gluconeogenic glyoxylate shunt pathway contributes with extracellular polysaccharides production by shunting carbon atoms into glucose synthesis. (D) Insufficient CFTR-PTEN interaction induces excessive succinate release (yellow circles) by immune cells and P. aeruginosa airway adaptation. In healthy individuals (upper panel), the CFTR-PTEN complex regulates the oxidative state of mitochondria. In cystic fibrosis patients (lower panel), lack of membrane CFTR-PTEN induces mitochondrial deregulation, producing ROS and more succinate release. Cytoplasmic succinate is excreted into the extracellular milieu feeding P. aeruginosa. Succinate-stressed P. aeruginosa upregulate anti-oxidant genes, causing metabolic adaptation, extracellular polysaccharide synthesis (color lines) and biofilm production. Biofilm-producing P. aeruginosa attach to the airway parenchyma, which protects them from phagocytes, antibiotics and antibodies.

Figure 2

Metabolic adaptation by S. aureus. (A) S. aureus acquire mutations in genes associated with terminal electron transport chain components resulting in the formation of small colony variants (SCVs) that display reduced metabolism. The decrease in TCA cycle activity from SCVs results in lower electrochemical gradient which is required for the uptake of aminoglycosides, thus rendering SCVs antibiotic resistant. In addition to their metabolic adaptation, SCVs evade the host immune system by intracellular survival and have increased biofilm forming ability that protects from oxidative stress. S. aureus also undergo bacterial metabolic remodeling resulting in population heterogeneity following internalization by bronchial epithelial cells. One bacterial subset replicates and induces host cell death and the other slows down its growth rate, relying on the catabolism of amino acids such as glutamate to supply oxaloacetate by fueling the TCA cycle and gluconeogenesis (red arrows). S. aureus also acquire mutations in the gene csl2 that results in enhanced cardiolipin synthesis and reduced phosphatidyl glycerol, thereby altering bacterial membrane composition and impairing antibiotic penetration. The reduction in phosphatidylglycerol also leads to inhibition of neutrophil chemotaxis. (B) S. aureus SCVs, such as those auxotrophic for heme, adapt to local fumarate accumulation by overexpressing fumC to degrade it. This helps sustain glycolysis given the role of fumarate as a glycolytic inhibitor. Fumarate degradation is also detrimental to the host and abrogates trained immunity, promoting recurrent infections.