A Comparison of the ATP Generating Pathways Used by S. Typhimurium to Fuel Replication within Human and Murine Macrophage and Epithelial Cell Lines

Abstract

The metabolism of S. Typhimurium within infected host cells plays a fundamental role in virulence since it enables intracellular proliferation and dissemination and affects the innate immune response. An essential requirement for the intracellular replication of S. Typhimurium is the need to regenerate ATP. The metabolic route used to fulfil this requirement is the subject of the present study. For infection models we used human and murine epithelial and macrophage cell lines. The epithelial cell lines were mICc12, a transimmortalised murine colon enterocyte cell line that shows many of the characteristics of a primary epithelial cell line, and HeLa cells. The model macrophage cell lines were THP-1A human monocyte/macrophages and RAW 264.7 murine macrophages. Using a mutational approach combined with an exometabolomic analysis, we showed that neither fermentative metabolism nor anaerobic respiration play major roles in energy generation in any of the cell lines studied. Rather, we identified overflow metabolism to acetate and lactate as the foremost route by which S. Typhimurium fulfils its energy requirements.

The metabolic pathways utilized by Salmonella Typhimurium during infection of host cells

“…With respect to glucose transport, the S . Typhimurium Δ ptsG Δ manXYZ Δ glk mutant was found to grow as well as the wild type in LoVo intestinal epithelial cells and showed minor, but significant, defects in intracellular growth in the THP‐1A human macrophage cell line which had been deprived of glucose, amino acids, or both 24 h prior to, during and up to 24 h postinfection (Garcia‐Guttierrez et al ., ; Singh et al ., ). Based on the intracellular growth of other sugar uptake mutants, Bowden and colleagues () also noted that at least one other unidentified PTS sugar was available for utilisation by S .…”

“…These conclusions are based on the attenuation of mutants deleted for genes encoding glycolytic enzymes such as pfkAB , fba , pgk , gapA and tpiA (Bowden et al ., ; Paterson et al . ; Eisenreich et al ., ; Götz et al ., ; Götz and Goebel, ; Garcia‐Gutierrez et al ., ). Isotopologue flux analysis has likewise confirmed the importance of glycolysis for replication of S .…”

“…The TCA cycle does not appear to be essential for the successful replication of S . Typhimurium within macrophages (Bowden et al ., ; Garcia‐Gutierrez et al ., ). Deletion of genes encoding several enzymes within the TCA cycle in fact resulted in enhanced survival/replication of S .…”

“…In support of this, the enhanced growth observed in macrophages was not found with infected HeLa or mIC cl2 epithelial cells, which lack the antimicrobial ROS and reactive nitrogen species responses (Bowden et al ., ; Garcia‐Gutierrez et al ., ). Finally, studies with mutants of fermentative pathways indicate that this is not a preferred metabolic route (Garcia‐Gutierrez et al ., ).…”

High-throughput screening of a collection of known pharmacologically active small compounds for inhibitors ofSalmonellainvasion and intracellular replication

“…Up until the 1990s, the most frequently used drugs to treat salmonellosis have been the fluoroquinalones ciprofloxacin and ofloxacin (Cooke 2007). We then performed secondary screens on promising candidates to determine their effect on reducing invasion or intracellular replication of S. Typhimurium in selected host cell lines (Sly et al 2002;Valle and Guiney 2005;Bowden et al 2014;Garcia-Gutierrez et al 2016). Subsequently cephalosporins such as ceftriaxone have been used to treat salmonellosis, however, about 5% of Salmonella strains are now resistant to cephalosporins, largely in reported cases of S. Heidelberg and S. Newport infections; therefore, newer and more effective antibiotics are required.…”