A comparative analysis of lipoprotein transport proteins: LolA and LolB from Vibrio cholerae and LolA from Porphyromonas gingivalis.

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.

Bacterial protein MakA causes suppression of tumour cell proliferation via inhibition of PIP5K1α/Akt signalling.

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.

A hyperpromiscuous antitoxin protein domain for the neutralization of diverse toxin domains.

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.

Protein-lipid interaction at low pH induces oligomerization of the MakA cytotoxin from Vibrio cholerae.

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.

A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion.

The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD, and MakE. MakA, MakB, and MakE were readily detected in culture supernatants of wild-type V. cholerae, whereas secretion was very much reduced from a flagellum-deficient mutant. Crystal structures of MakA, MakB, and MakE revealed a structural relationship to a superfamily of bacterial pore-forming toxins. Expression of MakA/B/E in Escherichia coli resulted in toxicity toward Caenorhabditis elegans used as a predatory model organism. None of these Mak proteins alone or in pairwise combinations were cytolytic, but an equimolar mixture of MakA, MakB, and MakE acted as a tripartite cytolytic toxin in vitro, causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining the assembly of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity toward C. elegans Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen Vibrio anguillarum We suggest that the hitherto-unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms.

Structural Insight into a Yeast Maltase-The BaAG2 from Blastobotrys adeninivorans with Transglycosylating Activity.

An early-diverged yeast, Blastobotrys (Arxula) adeninivorans (Ba), has biotechnological potential due to nutritional versatility, temperature tolerance, and production of technologically applicable enzymes. We have biochemically characterized from the Ba type strain (CBS 8244) the GH13-family maltase BaAG2 with efficient transglycosylation activity on maltose. In the current study, transglycosylation of sucrose was studied in detail. The chemical entities of sucrose-derived oligosaccharides were determined using nuclear magnetic resonance. Several potentially prebiotic oligosaccharides with α-1,1, α-1,3, α-1,4, and α-1,6 linkages were disclosed among the products. Trisaccharides isomelezitose, erlose, and theanderose, and disaccharides maltulose and trehalulose were dominant transglycosylation products. To date no structure for yeast maltase has been determined. Structures of the BaAG2 with acarbose and glucose in the active center were solved at 2.12 and 2.13 Å resolution, respectively. BaAG2 exhibited a catalytic domain with a (ß/α)8-barrel fold and Asp216, Glu274, and Asp348 as the catalytic triad. The fairly wide active site cleft contained water channels mediating substrate hydrolysis. Next to the substrate-binding pocket an enlarged space for potential binding of transglycosylation acceptors was identified. The involvement of a Glu (Glu309) at subsite +2 and an Arg (Arg233) at subsite +3 in substrate binding was shown for the first time for α-glucosidases.

High-resolution model of Arabidopsis Photosystem II reveals the structural consequences of digitonin-extraction.

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.

Structural and functional characterization of a plant alpha-actinin.

The Australian tree malletwood (Rhodamnia argentea) is unique. The genome of malletwood is the only known plant genome that contains a gene coding for an α-actinin-like protein. Several organisms predating the animal-plant bifurcation express an α-actinin or α-actinin-like protein. Therefore, it appears that plants in general, but not malletwood, have lost the α-actinin or α-actinin-like gene during evolution. In order to characterize its structure and function, we synthesized the gene and expressed the recombinant R. argentea protein. The results clearly show that this protein has all properties of genuine α-actinin. The N-terminal actin-binding domain (ABD), with two calponin homology motifs, is very similar to the ABD of any α-actinin. The C-terminal calmodulin-like domain, as well as the intervening rod domain, are also similar to the corresponding regions in other α-actinins. The R. argentea α-actinin-like protein dimerises in solution and thereby can cross-link actin filaments. Based on these results, we believe the R. argentea protein represents a genuine α-actinin, making R. argentea unique in the plant world.

Suppression of ß-catenin signaling in colon carcinoma cells by a bacterial protein.

Colorectal cancer is one of the leading causes of cancer-related death worldwide. The adenomatous polyposis coli (APC) gene is mutated in hereditary colorectal tumors and in more than 80% of sporadic colorectal tumors. APC mutations impair ß-catenin degradation, leading to its permanent stabilization and increased transcription of cancer-driving target genes. In colon cancer, impairment of ß-catenin degradation leads to its cytoplasmic accumulation, nuclear translocation, and subsequent activation of tumor cell proliferation. Suppressing ß-catenin signaling in cancer cells therefore appears to be a promising strategy for new anticancer strategies. Recently, we discovered a novel Vibrio cholerae cytotoxin, motility-associated killing factor A (MakA), that affects both invertebrate and vertebrate hosts. It promotes bacterial survival and proliferation in invertebrate predators but has unknown biological role(s) in mammalian hosts. Here, we report that MakA can cause lethality of tumor cells via induction of apoptosis. Interestingly, MakA exhibited potent cytotoxic activity, in particular against several tested cancer cell lines, while appearing less toxic toward nontransformed cells. MakA bound to the tumor cell surface became internalized into the endolysosomal compartment and induced leakage of endolysosomal membranes, causing cytosolic release of cathepsins and activation of proapoptotic proteins. In addition, MakA altered ß-catenin integrity in colon cancer cells, partly through a caspase- and proteasome-dependent mechanism. Importantly, MakA inhibited ß-catenin-mediated tumor cell proliferation. Remarkably, intratumor injection of MakA significantly reduced tumor development in a colon cancer murine solid tumor model. These data identify MakA as a novel candidate to be considered in new strategies for development of therapeutic agents against colon cancer.

Phosphatidic acid-mediated binding and mammalian cell internalization of the Vibrio cholerae cytotoxin MakA.

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.

Porphyromonas gingivalis fimbrial protein Mfa5 contains a von Willebrand factor domain and an intramolecular isopeptide.

The Gram-negative bacterium Porphyromonas gingivalis is a secondary colonizer of the oral biofilm and is involved in the onset and progression of periodontitis. Its fimbriae, of type-V, are important for attachment to other microorganisms in the biofilm and for adhesion to host cells. The fimbriae are assembled from five proteins encoded by the mfa1 operon, of which Mfa5 is one of the ancillary tip proteins. Here we report the X-ray structure of the N-terminal half of Mfa5, which reveals a von Willebrand factor domain and two IgG-like domains. One of the IgG-like domains is stabilized by an intramolecular isopeptide bond, which is the first such bond observed in a Gram-negative bacterium. These features make Mfa5 structurally more related to streptococcal adhesins than to the other P. gingivalis Mfa proteins. The structure reported here indicates that horizontal gene transfer has occurred among the bacteria within the oral biofilm.

Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy.

Autophagy plays an essential role in the defense against many microbial pathogens as a regulator of both innate and adaptive immunity. Some pathogens have evolved sophisticated mechanisms that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy modulation mediated by the recently discovered Vibrio cholerae cytotoxin, motility-associated killing factor A (MakA). pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 (herein referring to MAP1LC3B) lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex, required for LC3 lipidation at the membranous aggregate, resulted in an inhibition of both canonical autophagy and autophagy-related processes, including the unconventional secretion of interleukin-1ß (IL-1ß). These findings identify a novel mechanism of host autophagy modulation and immune modulation employed by V. cholerae during bacterial infection.

Crystallization of Recombinant Fimbrial Proteins of Porphyromonas gingivalis.

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.

Impact of the Liverpool Care Pathway on quality end-of- care in residential care homes and home care-Nurses' perceptions.

AIM: Aim was to describe how Registered Nurses (RNs) and assistant nurses (ANs) working in residential care homes and home care perceived quality end-of-life care after implementation of the Liverpool Care Pathway (LCP) in terms of subjective importance of care aspects and actual care given. DESIGN: Descriptive cross-sectional. METHODS: Registered Nurses (N = 22; 100% response rate) and ANs (N = 120; 59% response rate) working in a Swedish municipality. Data collection with a study-specific questionnaire (50 items) about perceived reality (PR) and subjective importance (SI). Non-parametric statistics. RESULTS: Implementation of the LCP ensured systematic assessment and alleviation of patients' symptoms and needs. The ANs, more than the RNs, perceived that the patients received the best possible nursing and medical care (p = .01). Both groups considered that communication with patients and families as well as the information exchange between the team members was facilitated. Areas for improvement were identified about psychological and existential support and patients and families' participation in care.

The Dps4 from Nostoc punctiforme ATCC 29133 is a member of His-type FOC containing Dps protein class that can be broadly found among cyanobacteria.

Dps proteins (DNA-binding proteins from starved cells) have been found to detoxify H2O2. At their catalytic centers, the ferroxidase center (FOC), Dps proteins utilize Fe2+ to reduce H2O2 and therefore play an essential role in the protection against oxidative stress and maintaining iron homeostasis. Whereas most bacteria accommodate one or two Dps, there are five different Dps proteins in Nostoc punctiforme, a phototrophic and filamentous cyanobacterium. This uncommonly high number of Dps proteins implies a sophisticated machinery for maintaining complex iron homeostasis and for protection against oxidative stress. Functional analyses and structural information on cyanobacterial Dps proteins are rare, but essential for understanding the function of each of the NpDps proteins. In this study, we present the crystal structure of NpDps4 in its metal-free, iron- and zinc-bound forms. The FOC coordinates either two iron atoms or one zinc atom. Spectroscopic analyses revealed that NpDps4 could oxidize Fe2+ utilizing O2, but no evidence for its use of the oxidant H2O2 could be found. We identified Zn2+ to be an effective inhibitor of the O2-mediated Fe2+ oxidation in NpDps4. NpDps4 exhibits a FOC that is very different from canonical Dps, but structurally similar to the atypical one from DpsA of Thermosynechococcus elongatus. Sequence comparisons among Dps protein homologs to NpDps4 within the cyanobacterial phylum led us to classify a novel FOC class: the His-type FOC. The features of this special FOC have not been identified in Dps proteins from other bacterial phyla and it might be unique to cyanobacterial Dps proteins.

Flagella-mediated secretion of a novel Vibrio cholerae cytotoxin affecting both vertebrate and invertebrate hosts.

Using Caenorhabditis elegans as an infection host model for Vibrio cholerae predator interactions, we discovered a bacterial cytotoxin, MakA, whose function as a virulence factor relies on secretion via the flagellum channel in a proton motive force-dependent manner. The MakA protein is expressed from the polycistronic makDCBA (motility-associated killing factor) operon. Bacteria expressing makDCBA induced dramatic changes in intestinal morphology leading to a defecation defect, starvation and death in C. elegans. The Mak proteins also promoted V. cholerae colonization of the zebrafish gut causing lethal infection. A structural model of purified MakA at 1.9 Å resolution indicated similarities to members of a superfamily of bacterial toxins with unknown biological roles. Our findings reveal an unrecognized role for V. cholerae flagella in cytotoxin export that may contribute both to environmental spread of the bacteria by promoting survival and proliferation in encounters with predators, and to pathophysiological effects during infections.

The No-Nonsens SDS-PAGE.

The discontinuous polyacrylamide gel electrophoresis system devised by Laemmli (Nature 227:680-685, 1970) has not only been used in numerous laboratories but has also been modified in several ways since its birth. In our laboratories, we use a modified Laemmli SDS-PAGE system for following protein purification as well as for analysis of certain protein-protein interactions, mainly involving filametous actin.

Crystallization of Recombinant α-Actinin and Related Proteins.

When it comes to crystallization each protein is unique. It can never be predicted beforehand in which condition the particular protein will crystallize or even if it is possible to crystallize. Still, by following some simple checkpoints the chances of obtaining crystals are increased. The primary checkpoints are purity, stability, concentration, and homogeneity. High-quality protein crystals are needed. This protocol will allow an investigator to: clone, express, and crystallize a protein of interest.

Structural and functional characterization of shaft, anchor, and tip proteins of the Mfa1 fimbria from the periodontal pathogen Porphyromonas gingivalis.

Very little is known about how fimbriae of Bacteroidetes bacteria are assembled. To shed more light on this process, we solved the crystal structures of the shaft protein Mfa1, the regulatory protein Mfa2, and the tip protein Mfa3 from the periodontal pathogen Porphyromonas gingivalis. Together these build up part of the Mfa1 fimbria and represent three of the five proteins, Mfa1-5, encoded by the mfa1 gene cluster. Mfa1, Mfa2 and Mfa3 have the same overall fold i.e., two ß-sandwich domains. Upon polymerization, the first ß-strand of the shaft or tip protein is removed by indigenous proteases. Although the resulting void is expected to be filled by a donor-strand from another fimbrial protein, the mechanism by which it does so is still not established. In contrast, the first ß-strand in Mfa2, the anchoring protein, is firmly attached by a disulphide bond and is not cleaved. Based on the structural information, we created multiple mutations in P. gingivalis and analysed their effect on fimbrial polymerization and assembly in vivo. Collectively, these data suggest an important role for the C-terminal tail of Mfa1, but not of Mfa3, affecting both polymerization and maturation of downstream fimbrial proteins.

Streptococcus Mutans Adhesin Biotypes that Match and Predict Individual Caries Development.

Dental caries, which affects billions of people, is a chronic infectious disease that involves Streptococcus mutans, which is nevertheless a poor predictor of individual caries development. We therefore investigated if adhesin types of S.mutans with sucrose-independent adhesion to host DMBT1 (i.e. SpaP A, B or C) and collagen (i.e. Cnm, Cbm) match and predict individual differences in caries development. The adhesin types were measured in whole saliva by qPCR in 452 12-year-old Swedish children and related to caries at baseline and prospectively at a 5-year follow-up. Strains isolated from the children were explored for genetic and phenotypic properties. The presence of SpaP B and Cnm subtypes coincided with increased 5-year caries increment, and their binding to DMBT1 and saliva correlated with individual caries scores. The SpaP B subtypes are enriched in amino acid substitutions that coincided with caries and binding and specify biotypes of S. mutans with increased acid tolerance. The findings reveal adhesin subtypes of S. mutans that match and predict individual differences in caries development and provide a rationale for individualized oral care.