Acetate‐Linked Energy Metabolism as a Determinant of Early Haemophilus influenzae Infection Fitness

Acetate is a major metabolic end‐product of Haemophilus influenzae and is produced via the phosphotransacetylase–acetate kinase (Pta–AckA) pathway, which also generates adenosine triphosphate (ATP). Despite its importance, the contribution of this pathway to H. influenzae physiology and virulence remains poorly understood. Here, we have investigated the individual roles of AckA and Pta for these processes by generating nonpolar single‐gene knockouts in a chronic obstructive pulmonary disease (COPD)‐isolate strain (Hi2019) and characterized their metabolic and infection phenotypes. Both mutants exhibited significant growth impairments under microaerobic and anaerobic conditions, with reductions in growth rate of up to 50% compared with the wild type. Loss of AckA or Pta decreased ATP levels to ~ 50% of wild‐type values and caused marked overoxidation of the NAD + /NADH pool. Metabolomic analyses revealed distinct perturbations at the pyruvate node: the pta mutant produced minimal acetate but accumulated pyruvate and d ‐lactate, while the ackA mutant continued to produce acetate, likely via nonenzymatic breakdown of acetyl‐phosphate. Both mutations also increased sensitivity to oxidative stress and enhanced biofilm formation. In infection models, including bronchial epithelial cells, primary human nasal epithelia, and murine macrophages, intracellular survival of both mutants was significantly reduced during early infection stages, although attenuation diminished over time. These findings demonstrate that the Pta–AckA pathway is critical for metabolic homeostasis, stress resistance, and early intracellular colonization, highlighting its potential as a target for management of early‐stage H. influenzae infections.