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Pathogenic serotypes of Vibrio cholerae, transmitted through contaminated water and food, are responsible for outbreaks of cholera, an acute diarrheal disease. While the cholera toxin is the primary virulence factor, V. cholerae also expresses other virulence factors, such as the tripartite toxin MakABE that is secreted via the bacterial flagellum. These three proteins are co-expressed with two accessory proteins, MakC and MakD, whose functions remain unknown. Here, we present the crystal structures of MakC and MakD, revealing that they are similar in both sequence and structure but lack other close structural relatives. Our study further investigates the roles of MakC and MakD, focusing on their impact on the expression and secretion of the components of the MakABE tripartite toxin. Through deletion mutant analysis, we found that individual deletions of makC or makD do not significantly affect MakA expression or secretion. However, the deletion of both makC and makD impairs the expression of MakB, which is directly downstream, and decreases the expression of MakE, which is separated from makCD by two genes. Conversely, MakA, encoded by the makA gene located between makB and makE, is expressed normally but its secretion is impaired. Additionally, our findings indicate that MakC, in contrast to MakD, exhibits strong interactions with other proteins. Furthermore, both MakC and MakD were observed to be localized within the cytosol of the bacterial cell. This study provides new insights into the regulatory mechanisms affecting the Mak protein family in V. cholerae and highlights the complex interplay between gene proximity and protein expression.

Lipoproteins are crucial for maintaining the structural integrity of bacterial membranes. In Gram-negative bacteria, the localization of lipoprotein (Lol) system facilitates the transport of these proteins from the inner membrane to the outer membrane. In Helicobacter pylori, an ε-proteobacterium, lipoprotein transport differs significantly from the canonical and well-studied system in Escherichia coli, particularly due to the absence of LolB and the use of a LolF homodimer instead of the LolCE heterodimer. This study presents the crystal structure of the H. pylori lipoprotein chaperone LolA (LolA-HP) and its interaction with lipopeptide antibiotics such as polymyxin B and colistin. Isothermal titration calorimetry revealed that, unlike LolA from Vibrio cholerae and Porphyromonas gingivalis, LolA-HP does not bind to these antibiotics. Structural comparisons showed that LolA-HP has a deeper hydrophobic cleft but lacks the negative electrostatic potential critical for binding polymyxins. These findings offer insights into the structural diversity of LolA across bacterial species and its potential as a target for antibacterial agents.

In Gram-negative bacteria, N-terminal lipidation is a signal for protein trafficking from the inner membrane (IM) to the outer membrane (OM). The IM complex LolCDE extracts lipoproteins from the membrane and moves them to the chaperone LolA. The LolA-lipoprotein complex crosses the periplasm after which the lipoprotein is anchored to the OM. In γ-proteobacteria anchoring is assisted by the receptor LolB, while a corresponding protein has not been identified in other phyla. In light of the low sequence similarity between Lol-systems from different phyla and that they may use different Lol components, it is crucial to compare representative proteins from several species. Here we present a structure-function study of LolA and LolB from two phyla: LolA from Porphyromonas gingivalis (phylum bacteroidota), and LolA and LolB from Vibrio cholerae (phylum proteobacteria). Despite large sequence differences, the LolA structures are very similar, hence structure and function have been conserved throughout evolution. However, an Arg-Pro motif crucial for function in γ-proteobacteria has no counterpart in bacteroidota. We also show that LolA from both phyla bind the antibiotic polymyxin B whereas LolB does not. Collectively, these studies will facilitate the development of antibiotics as they provide awareness of both differences and similarities across phyla.

Toxin–antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids as well as temperate bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analyzed the conservation of genomic neighborhoods using our computational tool FlaGs (for flanking genes), which allows high-throughput detection of TA-like operons. Focusing on the widespread but poorly experimentally characterized antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have named the domain Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralized TA pairs. While the majority of validated PanA-neutralized toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises transcription and translation as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralization mechanisms employed by Type II TA systems. Finally, through directed evolution, we have selected PanA variants that can neutralize noncognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain.

Recently, we demonstrated that a novel bacterial cytotoxin, the protein MakA which is released by Vibrio cholerae, is a virulence factor, causing killing of Caenorhabditis elegans when the worms are grazing on the bacteria. Studies with mammalian cell cultures in vitro indicated that MakA could affect eukaryotic cell signalling pathways involved in lipid biosynthesis. MakA treatment of colon cancer cells in vitro caused inhibition of growth and loss of cell viability. These findings prompted us to investigate possible signalling pathways that could be targets of the MakA-mediated inhibition of tumour cell proliferation. Initial in vivo studies with MakA producing V. cholerae and C. elegans suggested that the MakA protein might target the PIP5K1α phospholipid-signalling pathway in the worms. Intriguingly, MakA was then found to inhibit the PIP5K1α lipid-signalling pathway in cancer cells, resulting in a decrease in PIP5K1α and pAkt expression. Further analyses revealed that MakA inhibited cyclin-dependent kinase 1 (CDK1) and induced p27 expression, resulting in G2/M cell cycle arrest. Moreover, MakA induced downregulation of Ki67 and cyclin D1, which led to inhibition of cell proliferation. This is the first report about a bacterial protein that may target signalling involving the cancer cell lipid modulator PIP5K1α in colon cancer cells, implying an anti-cancer effect.

The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.

Vibrio cholerae, responsible for outbreaks of cholera disease, is a highly motile organism by virtue of a single flagellum. We describe how the flagellum facilitates the secretion of three V. cholerae proteins encoded by a hitherto-unrecognized genomic island. The proteins MakA/B/E can form a tripartite toxin that lyses erythrocytes and is cytotoxic to cultured human cells. A structural basis for the cytolytic activity of the Mak proteins was obtained by X-ray crystallography. Flagellum-facilitated secretion ensuring spatially coordinated delivery of Mak proteins revealed a role for the V. cholerae flagellum considered of particular significance for the bacterial environmental persistence. Our findings will pave the way for the development of diagnostics and therapeutic strategies against pathogenic Vibrionaceae.

Porphyromonas gingivalis fimbriae play a critical role in colonization. Elucidation of the fimbrial structure in atomic detail is important for understanding the colonization mechanism and to provide means to combat periodontitis. X-ray crystallography is a technique that is used to obtain detailed information of proteins along with bound ligands and ions. Crystallization of the protein of interest is the first step toward structure determination. Unfortunately it is not possible to predict the crystallization condition of a certain protein or even if the protein can be crystallized. Protein crystallization is, on the contrary, a matter of trial and error. However, the best strategy for success is to focus on the protein purification step to obtain a sample that is pure, stable, homogeneous and of high concentration. This chapter addresses general methods for crystallization of fimbrial proteins.

In higher plants, the photosynthetic process is performed and regulated by Photosystem II (PSII). Arabidopsis thaliana was the first higher plant with a fully sequenced genome, conferring it the status of a model organism; nonetheless, a high-resolution structure of its Photosystem II is missing. We present the first Cryo-EM high-resolution structure of Arabidopsis PSII supercomplex with average resolution of 2.79 Å, an important model for future PSII studies. The digitonin extracted PSII complexes demonstrate the importance of: the LHG2630-lipid-headgroup in the trimerization of the light-harvesting complex II; the stabilization of the PsbJ subunit and the CP43-loop E by DGD520-lipid; the choice of detergent for the integrity of membrane protein complexes. Furthermore, our data shows at the anticipated Mn4CaO5-site a single metal ion density as a reminiscent early stage of Photosystem II photoactivation.

Vibrio cholerae is a noninvasive intestinal pathogen extensively studied as the causative agent of the human disease cholera. Our recent work identified MakA as a potent virulence factor of V. cholerae in both Caenorhabditis elegans and zebrafish, prompting us to investigate the potential contribution of MakA to pathogenesis also in mammalian hosts. In this study, we demonstrate that the MakA protein could induce autophagy and cytotoxicity of target cells. In addition, we observed that phosphatidic acid (PA)-mediated MakA-binding to the host cell plasma membranes promoted macropinocytosis resulting in the formation of an endomembrane-rich aggregate and vacuolation in intoxicated cells that lead to induction of autophagy and dysfunction of intracellular organelles. Moreover, we functionally characterized the molecular basis of the MakA interaction with PA and identified that the N-terminal domain of MakA is required for its binding to PA and thereby for cell toxicity. Furthermore, we observed that the ΔmakA mutant outcompeted the wild-type V. cholerae strain A1552 in the adult mouse infection model. Based on the findings revealing mechanistic insights into the dynamic process of MakA-induced autophagy and cytotoxicity we discuss the potential role played by the MakA protein during late stages of cholera infection as an anti-colonization factor.