FUNCTIONAL CHARACTERIZATION OF ENVIRONMENTAL LACTATE UTILIZATION DURING NTHi in vitro INFECTION OF HUMAN EPITHELIA

Non-typeable Haemophilus influenzae (NTHi) is a small acapsulate gram-negative coccobacillus and a human restricted nasopharynx commensal. Nevertheless, it is the main cause of chronic obstructive pulmonary disease (COPD) exacerbation. A previous dual RNA-sequencing study highlighted the upregulation of an L-lactate permease gene, suggesting a role for environmental lactate during the infection. Furthermore, a strong downregulation of the sialic acid-specific tripartite ATP-independent periplasmic transporter (TRAP system) was reported, while a gene (pst) with 60% similarity with nanT, an N-acetyl neuraminic acid (NANA) transporter in E. coli, was found to be upregulated during the infection. In order to explore the impact of environmental lactate and NANA utilization during NTHi mucosal colonization, we constructed specific knock out strains for two uptake systems and analysed bacterial fitness and their resistance to complement-mediated killing during infection of human bronchial epithelia in vitro. In vitro assays were performed to analyse NTHi Δpst resistance to serum killing. The pst gene mutant strain did not show any alteration in the complement mediated killing resistance, even when the test was performed after an adaptation of the bacterium by infection of human bronchial epithelial cells. These data suggested a lack of mutuality between the TRAP system and the pst gene as sialic acid transporter during the infection stages. For what concerns the lactate permease knock out (ΔlctP) we observed a faster grow rate compared to the WT strain when lactate is the sole available carbon source. We also reported an impaired membrane composition which may cause this cell instability, more precisely we showed a lipid A decrement in a “lactate environment”. The altered membrane composition results in a strong aggregative phenotype both in bacteria-bacteria and bacteria-epithelia interactions. Despite the low endotoxin levels NTHi ΔlctP was still able to resist to the complement mediated killing when NANA was available. These data open to unknown complement killing evasion systems. In conclusion, our work is aimed to better characterize NTHi infection factors involved in the colonization of the human epithelium.