Non-typeable Haemophilus influenzae (NTHi) is a human respiratory pathogen involved in upper and lower respiratory tract diseases, including COPD, cystic fibrosis, and asthma^{[1, 2]}. Despite extensive studies of NTHi virulence, growth substrates used in vivo have not been unambiguously identified. Here, we investigated the role of lactate, a universal metabolite in humans, in NTHi growth. NTHi substrates were investigated using phenotypic microarrays (Omnilog), extracellular metabolomics of NTHi-infected and uninfected submerged tissue cells (TCs), and mutant phenotypic characterisation.

Metabolomic analyses revealed that, in addition to glucose, NTHi consumes TC-derived lactate, which was a highly used substrate in the microarrays. Additionally, the growth rate of Hi2019 and Hi2019 respiration on L-lactate were 1.5-fold and 13-fold greater than on D-lactate, suggesting NTHi primarily uses L-lactate.

We bioinformatically identified two Hi lactate utilisation systems; 84% of strains metabolised L- and D-lactate via membrane-bound L-lactate (LldD) and D-lactate (Dld) dehydrogenases and a cytoplasmic NAD⁺-dependent D-lactate dehydrogenase (LdhA), while 16% contained the L-lactate-specific LutABC system. In Hi2019, LldD activity decreased, while Dld and LdhA activity increased, with reduced oxygen, suggesting LDHs are oxygen-regulated.

Additionally, Hi2019^∆lldD, Hi2019^∆dld, and Hi2019^∆ldhA mutants had impaired L- and D-lactate respiration and, following the removal of one LDH gene, the activities of the remaining LDHs in the mutants was significantly decreased compared to the wild type control. This suggested that LldD, Dld, and LdhA activity is interdependent. Interestingly, only Hi2019^∆lldD had reduced growth, by 2-fold, on lactic acid, suggesting LldD, which was highly expressed in Hi2019 TC co-cultures, is central for NTHi energy generation on lactate and is currently being investigated in co-culture. From this work, we conclude that TC-derived lactate is an important NTHi carbon source and that targeting this lactate metabolism could undermine NTHi growth during infection.