Novel molecular insights about lactobacillar sortase-dependent piliation

One of the more conspicuous structural features that punctuate the outer cell surface of certain bacterial Gram-positive genera and species is the sortase-dependent pilus. As these adhesive and variable-length protrusions jut outward from the cell, they provide a physically expedient and useful means for the initial contact between a bacterium and its ecological milieu. The sortase-dependent pilus displays an elongated macromolecular architecture consisting of two to three types of monomeric protein subunits (pilins), each with their own specific function and location, and that are joined together covalently by the transpeptidyl activity of a pilus-specific C-type sortase enzyme. Sortase-dependent pili were first detected among the Gram-positive pathogens and subsequently categorized as an essential virulence factor for host colonization and tissue invasion by these harmful bacteria. However, the sortase-dependent pilus was rebranded as also a niche-adaptation factor after it was revealed that “friendly” Gram-positive commensals exhibit the same kind of pilus structures, which includes two contrasting gut-adapted species from the Lactobacillus genus, allochthonous Lactobacillus rhamnosus and autochthonous Lactobacillus ruminis. This review will highlight and discuss what has been learned from the latest research carried out and published on these lactobacillar pilus types.Peer reviewe

An in silico pan-genomic probe for the molecular traits behind Lactobacillus ruminis gut autochthony

As an ecological niche, the mammalian intestine provides the ideal habitat for a variety of bacterial microorganisms. Purportedly, some commensal genera and species offer a beneficial mix of metabolic, protective, and structural processes that help sustain the natural digestive health of the host. Among these sort of gut inhabitants is the Gram-positive lactic acid bacterium Lactobacillus ruminis, a strict anaerobe with both pili and flagella on its cell surface, but also known for being autochthonous (indigenous) to the intestinal environment. Given that the molecular basis of gut autochthony for this species is largely unexplored and unknown, we undertook a study at the genome level to pinpoint some of the adaptive traits behind its colonization behavior. In our pan-genomic probe of L. ruminis, the genomes of nine different strains isolated from human, bovine, porcine, and equine host guts were compiled and compared for in silico analysis. For this, we conducted a geno-phenotypic assessment of protein-coding genes, with an emphasis on those products involved with cell-surface morphology and anaerobic fermentation and respiration. We also categorized and examined the core and accessory genes that define the L. ruminis species and its strains. Here, we made an attempt to identify those genes having ecologically relevant phenotypes that might support or bring about intestinal indigenousness.Peer reviewe

Interaction of GluA1 AMPA receptor with Synapse-Associated Protein 97

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors are glutamate-gated cation channels and mediators of fast excitatory neurotransmission in the mammalian central nervous system. Trafficking and functional regulation of AMPA receptors GluA1-4 is carried out through numerous intracellular protein interactions and post-translational modifications. The aim of this thesis work was to study the selective interaction between AMPA receptor subunit GluA1 and synapse-associated protein 97 (SAP97), a protein scaffold belonging to the protein family of membrane associated guanylate kinase homologs. The interaction between SAP97 and GluA1 has been implicated in AMPA receptor trafficking, neuronal development and synaptic plasticity, while disturbances in normal levels of both GluA1 and SAP97 have been linked to neuropathologies such as Alzheimer s disease and schizophrenia. In the present study, a combination of biochemical and structural work was employed to gain detailed information on the selective interaction of GluA1 with SAP97 identifying molecular determinants involved in and regulating the interaction. X-ray crystallization screens of the second PDZ domain of SAP97 (SAP97PDZ2) yielded well-diffracting crystals both for the apo and ligand bound form. The solved crystal structure of the SAP97PDZ2-GluA1 peptide complex conformed to a conventional class I PDZ interaction with hydrogen bonds forming between the carboxylate group of the ultimate C-terminal residue of the peptide and main chain nitrogens in the carboxylate binding loop of the PDZ domain, and a hydrogen bond between the antepenultimate residue of the peptide and a conserved histidine in the αB helix lining the peptide binding groove. Beside these typical PDZ interactions, as a novel finding we observed contacts within the PDZ domain reorganizing upon peptide binding leading to a slight opening of the peptide binding groove facilitating better accommodation of the ligand. In vitro binding analysis of isolated PDZ domains and short GluA1 peptides showed that, in addition to the prototypic PDZ interaction, a C-terminal cysteine, C893 located upstream from the short PDZ binding motif on GluA1 participated in the interaction through a disulfide bond formed with cysteine C378 of SAP97 under in vitro conditions. Streptavidin pull-down experiments with full-length molecules expressed in cultured cells showed that the C893S mutation leads to a substantial reduction in binding of GluA1 to SAP97, confirming the involvement of C893 in the regulation of the interaction in live cells. Reactive cysteines, like C893, can in addition to disulfide bonds participate in other thiol modifications. In our work we constructed several deletion and cysteine replacement mutants of GluA1 and tested their sensitivity to S-nitrosylating agents nitrosoglutathione and nitrosocysteine. Out of the three C-terminal cysteine residues in GluA1, we identified C893 as the sole cysteine residue sensitive to a post-translational modification by nitric oxide (NO). Furthermore, we found evidence of a physical link between GluA1 and the NO generating neuronal enzyme nitric oxide synthase nNOS via SAP97. The results of the present study provide, for the first time, detailed structural information on the interaction between GluA1 AMPA receptor and SAP97. In addition to a canonical PDZ interaction, the association with SAP97 involves a reactive cysteine residue, C893, in GluA1 C-terminal tail, a potential regulatory target for nitric oxide and oxidative conditions.AMPA receptorer, bestående av underenheterna GluA1-4, är katjonkanaler vars genomsläpplighet styrs av till dem bundet glutamat och de ansvarar för snabb stimulerande signalöverföring i centrala nervsystemets synapser hos däggdjur. Reglering av AMPA receptorers funktion samt deras intracellulära trafik dirigeras av växelverkande proteiner och posttranslationella proteinmodifieringar. Syftet för denna doktorsavhandling var att studera den selektiva interaktionen mellan AMPA receptor GluA1 och SAP97, ett protein tillhörande proteinfamiljen av membran-associerade guanylatkinas homologer. Växelverkan mellan SAP97 och GluA1 påverkar AMPA receptorers intracellulära trafik, den neuronala utvecklingen och synaptisk plasticitet, medan störningar i normala nivåer av GluA1 och SAP97 har länkats till olika sjukdomstillstånd så som Alzheimers sjukdom och schizofreni. I denna studie tillämpades biokemiska metoder i kombination med röntgenkristallografi och NMR för att få detaljerad information om den selektiva interaktionen mellan GluA1 och SAP97 samt identifiera specifikt de molekyler som reglerar växelverkan mellan proteinerna. Med hjälp av röntgenkristallografi löstes kristallstrukturen för den andra PDZ-domänen av SAP97 (SAP97PDZ2) både i sin fria och ligandbundna form. Kristallstrukturen av SAP97PDZ2 tillsammans med GluA1 påvisade en konventionell klass I PDZ-interaktion där GluA1-peptidens yttersta C-terminala aminosyras karboxylatgrupp genom vätebindningar binds till den karboxylatbindande slingan i PDZ-domänen och GluA1-peptidens tredje sista aminosyra binds med ytterligare en vätebindning till en konserverad histidin-aminosyra i αB-helixen som kantar öppningen till den peptidbindande fåran. Förutom de typiska PDZ-interaktionerna upptäcktes det att intramolekylära kontakter inom PDZ-domänen omorganiseras då peptiden binds vilket leder till att den peptid-bindande fåran öppnas en aning för att ge bättre utrymme för peptiden. In vitro- analys av isolerade PDZ-domäner och GluA1-peptider avslöjade förutom den prototypiska PDZ-interaktionen en disulfidbindning som uppstår mellan ett C-terminalt cystein C893 uppströms från det korta PDZ-bindande motivet i GluA1 och cystein C378 i SAP97 under in vitro förhållanden. Från att ha studerat enskilda SAP97 PDZ-domäner och korta GluA1-peptider övergick vi till att uttrycka hela rekombinanta proteiner i odlade celler. Mutationen C893S i GluA1 ledde till en betydlig reducering av bindningsstyrkan till SAP97 vilket bekräftar att C893 deltar i regleringen av interaktionen i levande celler. Reaktiva cysteiner, likt C893, kan förutom disulfidbindningar också delta i andra tiolmodifieringar. I studien konstruerades flera muterade versioner av GluA1, både N- och C-terminala deletioner samt mutantproteiner vars enskilda cysteiner ersatts med aminosyran serin. Genom att undersöka mutantproteinernas känslighet för nitrosoglutation och nitrosocystein kunde vi fastställa att C893, som det enda cysteinet utav tre i den C-terminala cytosoliska svansen av GluA1, är känslig för en posttranslationell modifiering genom kväveoxid (NO). Därutöver, fann vi bevis för en fysisk länk mellan GluA1 och det NO-genererande enzymet neuronalt kväveoxidsyntas (nNOS) via SAP97. Resultaten av denna studie förser oss med, för första gången, detaljerad strukturell information om interaktionen mellan AMPA receptor GluA1 och SAP97. Förutom den kanoniska PDZ-interaktionen innefattar bindningen med SAP97 ett reaktivt cystein, C893, i den C-terminala ändan av GluA1, ett potentiellt föremål för reglering genom kväveoxid och oxidativa förhållanden

Purification, crystallization and preliminary crystallographic analysis of the SpaA backbone-pilin subunit from probiotic Lactobacillus rhamnosus GG

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Purification, crystallization, and preliminary X-ray diffraction analysis of SpaD, a backbone-pilin subunit encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG

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Bent conformation of a backbone pilin N-terminal domain supports a three-stage pilus assembly mechanism

Journal editors’ pick of their favorite papers from the first year of publishing.Peer reviewe

Cysteine 893 is a target of regulatory thiol modifications of GluA1 AMPA receptors

Recent studies indicate that glutamatergic signaling involves, and is regulated by, thiol modifying and redox-active compounds. In this study, we examined the role of a reactive cysteine residue, Cys-893, in the cytosolic C-terminal tail of GluA1 AMPA receptor as a potential regulatory target. Elimination of the thiol function by substitution of serine for Cys-893 led to increased steady-state expression level and strongly reduced interaction with SAP97, a major cytosolic interaction partner of GluA1 C-terminus. Moreover, we found that of the three cysteine residues in GluA1 C-terminal tail, Cys-893 is the predominant target for Snitrosylation induced by exogenous nitric oxide donors in cultured cells and lysates. Co-precipitation experiments provided evidence for native association of SAP97 with neuronal nitric oxide synthase (nNOS) and for the potential coupling of Ca2+- permeable GluA1 receptors with nNOS via SAP97. Our results show that Cys-893 can serve as a molecular target for regulatory thiol modifications of GluA1 receptors, including the effects of nitric oxide.Peer reviewe

Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adapted Lactobacillus rhamnosus GG

SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensal Lactobacillus rhamnosus GG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria, L. rhamnosus GG SpaE for crystallization was produced by recombinant expression in Escherichia coli. Although several attempts to crystallize the SpaE protein were unsuccessful, trigonal crystals that diffracted to a resolution of 3.1 angstrom were eventually produced using PEG 3350 as a precipitant and high protein concentrations. Further optimization with a combination of additives led to the generation of SpaE crystals in an orthorhombic form that diffracted to a higher resolution of 1.5 angstrom. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 angstrom resolution.Peer reviewe

Crystal structure of the atypically adhesive SpaB basal pilus subunit : Mechanistic insights about its incorporation in lactobacillar SpaCBA pili

To successfully colonize a host or environment, certain genera and species of Gram-positive bacteria have evolved to utilize the so-called sortase-dependent pilus, a long multi-subunit and non-flagellar surface adhesin. One example of this is Lactobacillus rhamnosus GG, a gut-adapted probiotic strain that produces SpaCBA pili. These structures are covalent hetero-oligomers built from three types of pilin subunit, each with a specific location and function (i.e., backbone SpaA for length, tip SpaC for adhesion, and basal SpaB for anchoring). Functionally, the SpaCBA pilus exhibits a promiscuous affinity for components on intestinal surfaces (e.g., mucus, collagen, and epithelial cells), which is largely attributed to the SpaC subunit. Then again, the basal SpaB pilin, in addition to acting as the terminal subunit during pilus assembly, displays an out of character mucoadhesive function. To address the structural basis of this unusual dual functionality, we reveal the 2.39 A resolution crystal structure of SpaB. SpaB consists of one immunoglobulin-like CnaB domain and contains a putative intermolecular isopeptide bond-linking lysine and internal isopeptide bond-asparagine in an FPKN pilin motif within the C-terminal end. Remarkably, we found that a C-terminal stretch of positively charged lysine and arginine residues likely accounts for the atypical mucoadhesiveness of SpaB. Although harboring an autocatalytic triad of residues for a potential internal isopeptide interaction, the SpaB crystal structure lacked the visible electron density for intact bond formation, yet its presence was subsequently confirmed by mass spectral analysis. Finally, we propose a structural model that captures the exclusive basal positioning of SpaB in the SpaCBA pilus.Peer reviewe

Bacteriophages fEV-1 and fD1 Infect Yersinia pestis

Bacteriophages vB_YpeM_fEV-1 (fEV-1) and vB_YpeM_fD1 (fD1) were isolated from incoming sewage water samples in Turku, Finland, using Yersinia pestis strains EV76 and KIM D27 as enrichment hosts, respectively. Genomic analysis and transmission electron microscopy established that fEV-1 is a novel type of dwarf myovirus, while fD1 is a T4-like myovirus. The genome sizes are 38 and 167 kb, respectively. To date, the morphology and genome sequences of some dwarf myoviruses have been described; however, a proteome characterization such as the one presented here, has currently been lacking for this group of viruses. Notably, fEV-1 is the first dwarf myovirus described for Y. pestis. The host range of fEV-1 was restricted strictly to Y. pestis strains, while that of fD1 also included other members of Enterobacterales such as Escherichia coli and Yersinia pseudotuberculosis. In this study, we present the life cycles, genomes, and proteomes of two Yersinia myoviruses, fEV-1 and fD1