Gram-negative bacteria are characterized by a unique double-membrane structure consisting of an inner membrane (IM) and an outer membrane (OM), with a periplasmic space enriched with peptidoglycans between them. Both membranes are lipid bilayers in which a wide variety of associated and integral membrane proteins are embedded. Among the OM proteins, porins, structured as β-barrels, facilitate the passive transport of molecules across the membrane (Rollauer et al., 2015). The outer leaflet of the OM is mainly composed of glycolipids and lipopolysaccharides, which serve as a protective barrier and communication interface for the bacteria, whereas the inner leaflet consists of phospholipids. In contrast, both leaflets of the IM consist of phospholipids, and the proteins that span the IM predominantly adopt helical structures, performing critical functions such as translocating proteins from the cytoplasm (Orfanoudaki and Economou, 2014; Green and Mecsas, 2016), mediating metabolite exchange, and driving the electron transport chain to generate energy (Wallin and von Heijne, 1998). Both membranes also contain lipoproteins, which are synthesized as pre-lipoproteins in the cytoplasm, transported over the IM (Green and Mecsas, 2016), and anchored to the lipid bilayer through acyl chains covalently attached to a conserved cysteine residue. This anchoring process involves several enzymatic steps: diacylglycerol transferase (Lgt) catalyzes the attachment of a diacylglyceryl to the sulfhydryl of the cysteine (Sankaran and Wu, 1994) resulting in a diacylated protein, and signal peptidase II (LspA) removes the signal peptide, exposing the acylated cysteine as the first residue (Vogeley et al., 2016). In many bacteria, such as Escherichia coli, the enzyme apolipoprotein N-acyltransferase (Lnt) further acylates the free amino group of the cysteine, resulting in a fully matured lipoprotein with three acyl chains (Hillmann et al., 2011). The fate of the lipoprotein, whether retained in the IM or transported to the OM, depends on a sorting motif after the acylated cysteine. In the case of OM-targeted lipoproteins, the localization of lipoprotein (Lol) system plays a key role (Narita et al., 2004; Okuda and Tokuda, 2011). This system, particularly well-studied in E. coli, consists of the ABC transporter complex, LolCDE. In this complex, LolE extracts the lipoprotein from the IM and transfers it to the chaperone LolA, which is bound to the periplasmic domain of LolC. A dimer of LolD is located on the cytoplasmic side of the LolCE dimer and provides energy to this process via its ATPase activity (Figure 1). LolA then forms a complex with the lipoprotein, transports it across the periplasm and delivers it to LolB, a recipient protein, which itself is a lipoprotein, anchored to the OM. In the next step, LolB transfers the acyl chains of the lipoprotein to the inner leaflet of the OM (Narita et al., 2004; Okuda and Tokuda, 2011). Notably, LolB has only been identified in β- and γ-proteobacteria and appears to be absent in all other bacterial classes. In the α-proteobacteria, Caulobacter vibrioides, LolA (LolA-CV) has been shown to perform both transport and insertion of lipoproteins into the OM (Smith et al., 2023).

Helicobacter pylori is an ε-proteobacterium known for colonizing the human gastric mucosa and for contributing to conditions such as peptic ulcers and gastric inflammation (Suerbaum and Michetti, 2002; Nomura et al., 2002). H. pylori harbors approximately 40 lipoproteins, including CagT, a component of the type IV secretion system that secretes the oncoprotein CagA (Babu et al., 2006; Backert et al., 2015). Despite its reliance on functional lipoprotein transport, the Lol system in H. pylori differs significantly from the canonical system in E. coli. Specifically, H. pylori lacks the LolB receptor protein at the OM and, instead of the LolCE dimer at the IM utilizes a homodimeric LolF (Figure 1), which combines the functions of LolC and LolE (LoVullo et al., 2015). This divergence underscores the critical role of acylation and transport in maintaining bacterial membrane integrity, making these processes attractive targets for antibacterial drug development.

Inhibiting components of the Lol system, particularly LolCDE in E. coli, has been explored as a potential strategy for novel antimicrobial agents (Nickerson et al., 2018; Caro et al., 2019). Additionally, LolA has been implicated in the transport of polymyxins—lipopeptide antibiotics used as a last-resort treatment—across the periplasm to the IM (Weerakoon et al., 2021; Pedebos et al., 2021). Previous studies have shown that LolA from bacteria such as Porphyromonas gingivalis and Vibrio cholerae bind polymyxin B, while LolB from V. cholerae does not (Jaiman et al., 2023). To assess the potential of LolA as a broad-spectrum antibacterial target, it is essential to investigate its structure and binding properties across different bacterial classes. If LolA exhibits conserved binding mechanisms, it could serve as a universal target for Gram-negative bacteria. However, structural variability might limit its applicability to specific bacterial species. While crystal structures of LolA have been determined for several γ-proteobacteria, including Pseudomonas aeruginosa, E. coli, Yersinia pestis, and V. cholerae (Jaiman et al., 2023; Remans et al., 2010; Takeda et al., 2003), as well as the Bacteroidota P. gingivalis (Jaiman et al., 2023), structural studies on other bacterial classes remain limited. Additionally, LolA from E. coli (LolA-EC) has been studied in complex with LolC both using crystallography and cryo-electron microscopy (Kaplan et al., 2018; Tang et al., 2021). These studies were recently supplemented with the crystal structure of LolA-EC in complex with a triacylated peptide, offering valuable insights into complex formation and function (Kaplan et al., 2022). The recent determination of a LolA-LolB complex in Xanthomonas campestris (γ-proteobacteria) (Furlanetto and Divne, 2023) further highlights the diversity of the Lol system. In this study, we present the crystal structure of LolA from H. pylori (LolA-HP) and examine its interaction with lipopeptide antibiotics. Our findings provide a foundation for future drug discovery efforts targeting the Lol system across diverse classes of Gram-negative bacteria.

Helicobacter pylori belongs to the class ε-proteobacteria, and its Lol machinery exhibits significant differences compared to the well-characterized system in E. coli, which represents the class γ-proteobacteria. In E. coli, the IM complex LolCDE plays a crucial role: LolE extracts lipoproteins from the membrane, while LolC transfers them to LolA. Conversely, H. pylori utilizes a LolF dimer that integrates both of these functions (LoVullo et al., 2015; McClain et al., 2020). In both organisms, a LolD dimer located on the cytoplasmic side provides energy through its ATPase activity.

Additionally, E. coli and other γ-proteobacteria, feature LolB, a lipoprotein anchored to the inner leaflet of the OM, which receives the lipoprotein cargo from LolA. In this transfer, LolA and LolB are hypothesized to interact through a mouth-to-mouth mechanism, driven by electrostatic attraction between the negatively charged LolA and the positively charged LolB, during which the acyl groups of the lipoprotein bound to LolA slide to the binding cleft of LolB (Okuda and Tokuda, 2009). Subsequently, by an unknown mechanism, the acyl chains are transferred from LolB to the lipid bilayer resulting in the anchoring of the lipoprotein to the membrane. It has been shown that an exposed and conserved Leu in the loop between β3 and β4 in LolB-EC is critical for its ability to anchor lipoproteins (Hayashi et al., 2014). This Leu is not completely conserved in all γ-proteobacteria; instead, X. campestris has a valine in the same position and the crystal structure of its LolA-LolB complex clearly shows that the loop containing the valine is deeply buried in the binding cleft of LolA, in close contact with many hydrophobic residues. Interestingly, a modeled structure of LolA from the α-proteobacteria C. vibrioides, (LolA-CV), a bacterium that does not have a LolB protein, also shows an exposed leucine, but in a long loop connecting strands β8 and β9. It has been demonstrated that LolA-CV can compensate for the functions of both LolA and LolB in E. coli ΔlolA and ΔlolB deletion mutants and that this leucine is particularly important (Smith et al., 2023). Hence, LolA-CV has dual roles which indicates that dual functionality is a possible solution for lipoprotein membrane association in bacteria that lack the LolB protein. H. pylori does not have a LolB and indeed LolA-HP intriguingly possesses a leucine (Leu74), in the same position as LolB-EC, suggesting that LolA-HP also might fulfill a dual function. However, this calls for a more extensive study as leucines are present on several loops in LolA-HP: Leu46 on the β1β2 loop, Leu74 on the β3β4 loop, and Leu92 on loop β5β6.

An alternative hypothesis regarding lipoprotein insertion into the membrane is that certain bacterial species may have evolved other proteins with the capacity to receive lipoproteins at the OM, yet these proteins are too divergent in sequence from the reference protein LolB-EC to be detected via conventional sequence analysis. This could represent an instance of convergent evolution, where proteins with different structures result in the same outcome. For example, Bacteroides fragilis, which lacks the Lnt enzyme, nonetheless exhibits triacylated lipoproteins. In this case, an enzyme named lipoprotein N-acyltransferase in Bacteroides (Lnb) was recently discovered and characterized, displaying similar catalytic activity as Lnt despite a different predicted structure (Armbruster et al., 2024). Further investigation is required to explore these alternatives in greater detail.

In the LolA protein of E. coli, an Arg-Pro motif located between β2 and β3 has been extensively analyzed. The arginine residue within this motif, which faces the base of the binding cleft, is critical for both the binding of the triacylated lipoproteins and their delivery to LolB. Sequence analysis (Supplementary Figure S1) shows that the Arg-Pro motif is relatively conserved in β-and γ-proteobacteria and is suggested to be associated with the presence of a LolB protein for interaction. Again, X. campestris is an exception as it has a Thr-Pro in the equivalent position. Despite these differences, LolA and LolB in X. campestris interact with a similar dissociation constant (Kd) as the E. coli LolA-LolB pair (14.6 μM vs. 30.6 μM) and is the only LolA–LolB pair that has been co-crystallized (Furlanetto and Divne, 2023; Nakada et al., 2009).

In LolA-HP, this motif is replaced by an Ala-Pro motif (Ala62, Pro63), and the space that would be occupied by the arginine side chain in LolA-EC is instead filled by a phenylalanine side chain (Phe107) on the α3-helix. Intriguingly, LolA-CV, which has been reported to have dual LolA–LolB functions has a classical Arg-Pro motif. The variation does however indicate that the sequence conservation is not strictly maintained even within the same phylum and that the Lol system is very variable.

Lol proteins are considered promising drug targets because the transport of lipoproteins is vital for the biogenesis of the membrane and the subsequent survival of Gram-negative bacteria. The main focus has been on targeting the IM protein complex LolCDE. The initial hits pyridinepyrazole (Breidenstein et al., 2024) and pyridineimidazole (Nayar et al., 2015; McLeod et al., 2015) have recently been developed into a promising substance, lolamicin, that selectively spares the gut bacteria (Muñoz et al., 2024). The soluble LolA was predicted to bind and transport acylated peptides such as the antibiotic polymyxin (Weerakoon et al., 2021; Pedebos et al., 2021), and we could indeed show experimentally that both LolA-VC and LolA-PG bind the substance (Jaiman et al., 2023). In the present study, using ITC, we demonstrated that LolA-VC and LolA-PG also bind the lipopeptide colistin with dissociation constants in the micromolar range. They also exhibit weak binding to nonapeptide, which has the same peptide backbone as polymyxin B but lacks the acyl tail. Hence the acyl part of the polymyxins is most important for binding to LolA but also the peptide part contributes to binding (Table 2).

However, binding is not a universal trait for all LolA proteins as LolA-HP does not bind polymyxin B, colistin, or nonapeptide. Comparative analysis of the electrostatic surface potentials of various LolA proteins reveals notable variability. For instance, LolA from E. coli, V. cholerae, and P. gingivalis mainly display negative electrostatic potential around the mouth of the binding cleft. In contrast, LolA from H. pylori and the modeled structure of LolA from C. vibrioides predominantly feature a more hydrophobic, non-polar environment around the binding cleft mouth. This observation aligns with the fact that the tested molecules carry a positive charge, which would preferentially interact with the negatively charged surfaces of LolA-VC and LolA-PG. This difference in electrostatic properties suggests that the binding affinities of LolA proteins are influenced by their surface potentials, which could be exploited in the design of selective antibacterial agents targeting specific Gram-negative pathogens (Figure 4 and Supplementary Figure S7).

Lol proteins are regarded as promising drug targets due to their critical role in lipoprotein transport, which is essential for membrane biogenesis and the survival of Gram-negative bacteria. Despite the low sequence similarity, the high structural conservation of LolA across various bacterial species (Table 1) highlights the potential for designing antibacterial agents that selectively target LolA components in specific pathogens. This approach, similar to the selective binding of lolamicin to pathogenic bacteria, could minimize the impact on commensal organisms (Muñoz et al., 2024). This highlights the importance of studying a wide variety of bacteria to understand what will work.

Our findings significantly enhance and expand the current understanding of lipoprotein transfer diversity in Gram-negative bacteria. Furthermore, they reveal that homologous proteins, which serve as potential drug targets, may exhibit varying affinities for antibacterial agents despite having highly similar biological functions. These findings underscore the vast knowledge that remains to be uncovered regarding the diversity of biological systems in bacteria.