Characterization of LolA/B-like proteins in Bacteroidetes

Abstract

In most Gram-negative bacteria, the lol system allows the transport of lipoproteins from the IM to the OM. The LolCDE complex extracts mature lipoproteins from the IM and delivers them to LolA, a chaperone that carries them through the periplasm and delivers them to LolB in the OM. Finally, lipoproteins are anchored in the OM where they fulfil many crucial roles such as virulence, adhesion, signalling, envelope biogenesis, etc... In the phylum Bacteroidetes, many OM lipoproteins play important roles, such as in gliding motility, T9 secretion and uptake/metabolism of sugars (SUS-like system). Surprisingly, to date, only homologs of LolA have been reported in this phylum but none of LolB. Interestingly, in our lab, we recently identified two LolA and two LolB homologs in the Bacteroidetes Flavobacterium johnsoniae. Characterization of the function of these LolA/B-like proteins (LolA1/LolB1 and LolA2/LolB2) lead to the observation of a decreased number of some lipoproteins involved in gliding and T9SS in the OM of a lolA1 mutant. In addition, mutation of lolA1 as well as of lolB1 determined lack of motility and T9 secretion. However, while these data suggest an involvement of LolA1 and maybe LolB1 in OM lipoprotein transport, direct evidence of their involvement in OM lipoprotein transport is lacking. Concerning LolA2 and LolB2, no blatant phenotype could be observed for these mutants nor data concerning lipoproteins localization were generated. During my master thesis I tried to better understand the role of LolA/B-like proteins in F. johnsoniae. We formulated two main hypotheses based on the ability of LolA and LolB to accommodate and transport lipid moieties in their hydrophobic cavities. The first hypothesis concerns a putative role in OM lipoprotein transport. To this aim, I performed pull-down assays using LolA/B-like proteins as baits in order to identify putative interacting (lipo)proteins. The data I obtained indicate that while several lipoproteins could be identified when LolA-like proteins were pulled-down, only few lipoproteins seem to interact with LolB-like proteins. Surprisingly, no lipoprotein involved in gliding/T9SS could be identified among LolA1 and LolB1 interactants. In contrast, several (lipo)proteins, part of polysaccharide metabolic processes seem to interact with both LolA/B couples thus suggesting a role of these proteins in their transport. Our second hypothesis concerns the role of LolA/B-like proteins in the transport of some OM lipids unique to Bacteroidetes, sulfonolipids and flexirubin. Interestingly, sulfonolipds are required for gliding motility as well as LolA1 and LolB1. However, phenotypic comparison between these mutants showed that no other phenotype was shared. With the aim to detect sulfonolipids in LolA1 and LolB1 mutant strains OM by MS, we tried to obtain pure OM fractions but unfortunately, we did not succeed. Concerning flexirubin, this hydrophobic orange pigment is synthesized in a unique gene cluster to which lolA2 and lolB2 belong suggesting a putative role of these gene products in flexirubin transport. However, due to the inability to obtain pure OM fractions, we could not test this hypothesis. Further work is required to unravel the role of Bacteroidetes LolA/B-like proteins which might differ from that of most studied bacteria.

Date of Award	20 Jan 2022
Original language	English
Awarding Institution	University of Namur
Supervisor	Francesco Renzi (Supervisor)