Identification and characterization of three novel LytM proteins of non-typeable Haemophilus influenzae involved in cell division, Outer Membrane Vesicles production and pathogenesis

Metalloproteases are a class of proteins very important for bacterial physiology, they are involved in many different aspects of microbial life and in the recent years they are target of many relevant studies. Through a genomic approach, we identified three hypothetical metalloprotease in Non-typeable Haemophilus influenzae (NTHi): NT013, NT017 and NT022. These proteins belong to LytM family, which is composed by factors mainly involved in cell division and in pathogenesis. The analysis of knockout mutant strains phenotypes confirms the role of protein NT013 and NT022 in cell splitting process; in particular we observed defective phenotypes in terms of cell morphology, formation of chains and bacterial aggregates. Moreover, we demonstrated a direct activity of protein NT013 in peptidoglycan cleavage, meanwhile NT022 seems to have a regulatory function. Furthermore, the alteration in the cell division in the KO strains resulted in an increase in the release of Outer Membrane Vesicles (OMVs), probably due to a decrease in membrane stability. NT017 does not seem to be involved in cell division process, but has a possible role in host colonization, since NT017 deletion reduces the capacity of NTHi to adhere to epithelial cells, to form biofilm and shows a susceptibility to human serum mediated killing. The results obtained so far clearly highlight the importance of LytM factors in NTHi physiology and pathogenesis