Carey Lambert

Bdellovibrio as therapeutic agents: a predatory renaissance?

Bdellovibrio are predatory bacteria that invade the periplasm of other Gram-negative bacteria where they undergo a complex developmental cycle that culminates in killing of the prey cell. Their intracellular niche allows Bdellovibrio to feed without competition and their lytic action can rapidly reduce bacterial populations, including pathogens, making these predatory bacteria interesting potential candidates for therapeutic applications. With the complete genome sequence for one Bdellovibrio strain now available, researchers now have an opportunity to evaluate the therapeutic potential of these predatory bacteria.

Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus

The predatory bacterium Bdellovibrio bacteriovorus swims rapidly by rotation of a single, polar flagellum comprised of a helical filament of flagellin monomers, contained within a membrane sheath and powered by a basal motor complex. Bdellovibrio collides with, enters and replicates within bacterial prey, a process previously suggested to firstly require flagellar motility and then flagellar shedding upon prey entry. Here we show that flagella are not always shed upon prey entry and we study the six fliC flagellin genes of B. bacteriovorus, finding them all conserved and expressed in genome strain HD100 and the widely studied lab strain 109J. Individual inactivation of five of the fliC genes gave mutant Bdellovibrio that still made flagella, and which were motile and predatory. Inactivation of the sixth fliC gene abolished normal flagellar synthesis and motility, but a disordered flagellar sheath was still seen. We find that this non‐motile mutant was still able to predate when directly applied to lawns of YFP‐labelled prey bacteria, showing that flagellar motility is not essential for prey entry but important for efficient encounters with prey in liquid environments.

Effects of orally administered Bdellovibrio bacteriovorus on the well-being and Salmonella colonization of young chicks

ABSTRACT Bdellovibrio bacteriovorus is a bacterium which preys upon and kills Gram-negative bacteria, including the zoonotic pathogens Escherichia coli and Salmonella. Bdellovibrio has potential as a biocontrol agent, but no reports of it being tested in living animals have been published, and no data on whether Bdellovibrio might spread between animals are available. In this study, we tried to fill this knowledge gap, using B. bacteriovorus HD100 doses in poultry with a normal gut microbiota or predosed with a colonizing Salmonella strain. In both cases, Bdellovibrio was dosed orally along with antacids. After dosing non-Salmonella-infected birds with Bdellovibrio, we measured the health and well-being of the birds and any changes in their gut pathology and culturable microbiota, finding that although a Bdellovibrio dose at 2 days of age altered the overall diversity of the natural gut microbiota in 28-day-old birds, there were no adverse effects on their growth and well-being. Drinking water and fecal matter from the pens in which the birds were housed as groups showed no contamination by Bdellovibrio after dosing. Predatory Bdellovibrio orally administered to birds that had been predosed with a gut-colonizing Salmonella enterica serovar Enteritidis phage type 4 strain (an important zoonotic pathogen) significantly reduced Salmonella numbers in bird gut cecal contents and reduced abnormal cecal morphology, indicating reduced cecal inflammation, compared to the ceca of the untreated controls or a nonpredatory ΔpilA strain, suggesting that these effects were due to predatory action. This work is a first step to applying Bdellovibrio therapeutically for other animal, and possibly human, infections.

Predation by Bdellovibrio bacteriovorus HD100 Requires Type IV Pili

Early electron microscopy and more recent studies in our laboratory of Bdellovibrio bacteriovorus cells indicated the presence of narrow fibers at the nonflagellar pole of this unusual predatory bacterium. Analysis of the B. bacteriovorus HD100 genome showed a complete set of genes potentially encoding type IV pili and an incomplete gene set for Flp pili; therefore, the role of type IV pili in the predatory life cycle of B. bacteriovorus HD100 was investigated. Alignment of the predicted PilA protein with known type IV pilins showed the characteristic conserved N terminus common to type IVa pilins. The pilA gene, encoding the type IV pilus fiber protein, was insertionally inactivated in multiple Bdellovibrio replicate cultures, and the effect upon the expression of other pilus genes was monitored by reverse transcriptase PCR. Interruption of pilA in replicate isolates abolished Bdellovibrio predatory capability in liquid prey cultures and on immobilized yellow fluorescent protein-labeled prey, but the mutants could be cultured prey independently. Expression patterns of pil genes involved in the formation of type IV pili were profiled across the predatory life cycle from attack phase predatory Bdellovibrio throughout the intraperiplasmic bdelloplast stages to prey lysis and in prey-independent growth. Taken together, the data show that type IV pili play a critical role in Bdellovibrio predation.

Discrete cyclic di-GMP-dependent control of bacterial predation versus axenic growth in Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways.

The First Bite— Profiling the Predatosome in the Bacterial Pathogen Bdellovibrio

Bdellovibrio bacteriovorus is a Gram-negative bacterium that is a pathogen of other Gram-negative bacteria, including many bacteria which are pathogens of humans, animals and plants. As such Bdellovibrio has potential as a biocontrol agent, or living antibiotic. B. bacteriovorus HD100 has a large genome and it is not yet known which of it encodes the molecular machinery and genetic control of predatory processes. We have tried to fill this knowledge-gap using mixtures of predator and prey mRNAs to monitor changes in Bdellovibrio gene expression at a timepoint of early-stage prey infection and prey killing in comparison to control cultures of predator and prey alone and also in comparison to Bdellovibrio growing axenically (in a prey-or host independent “HI” manner) on artificial media containing peptone and tryptone. From this we have highlighted genes of the early predatosome with predicted roles in prey killing and digestion and have gained insights into possible regulatory mechanisms as Bdellovibrio enter and establish within the prey bdelloplast. Approximately seven percent of all Bdellovibrio genes were significantly up-regulated at 30 minutes of infection- but not in HI growth- implicating the role of these genes in prey digestion. Five percent were down-regulated significantly, implicating their role in free-swimming, attack-phase physiology. This study gives the first post- genomic insight into the predatory process and reveals some of the important genes that Bdellovibrio expresses inside the prey bacterium during the initial attack.

The Structure of an Unconventional HD-GYP Protein from Bdellovibrio Reveals the Roles of Conserved Residues in this Class of Cyclic-di-GMP Phosphodiesterases

ABSTRACT Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins. IMPORTANCE It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by the analysis of several crystal structures, but no information has been available to date for the unrelated HD-GYP class of hydrolases. Here we present the multidomain structure of an unusual member of the HD-GYP family from the predatory bacterium Bdellovibrio bacteriovorus and detail the features that distinguish it from the wider structural family of general HD fold hydrolases. The structure reveals how a binuclear iron center is formed from several conserved residues and provides a basis for understanding HD-GYP family sequence requirements for c-di-GMP hydrolysis. It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by the analysis of several crystal structures, but no information has been available to date for the unrelated HD-GYP class of hydrolases. Here we present the multidomain structure of an unusual member of the HD-GYP family from the predatory bacterium Bdellovibrio bacteriovorus and detail the features that distinguish it from the wider structural family of general HD fold hydrolases. The structure reveals how a binuclear iron center is formed from several conserved residues and provides a basis for understanding HD-GYP family sequence requirements for c-di-GMP hydrolysis.

Injections of Predatory Bacteria Work Alongside Host Immune Cells to Treat Shigella Infection in Zebrafish Larvae

Summary Bdellovibrio bacteriovorus are predatory bacteria that invade and kill a range of Gram-negative bacterial pathogens in natural environments and in vitro [1, 2]. In this study, we investigated Bdellovibrio as an injected, antibacterial treatment in vivo, using zebrafish (Danio rerio) larvae infected with an antibiotic-resistant strain of the human pathogen Shigella flexneri. When injected alone, Bdellovibrio can persist for more than 24 hr in vivo yet exert no pathogenic effects on zebrafish larvae. Bdellovibrio injection of zebrafish containing a lethal dose of Shigella promotes pathogen killing, leading to increased zebrafish survival. Live-cell imaging of infected zebrafish reveals that Shigella undergo rounding induced by the invasive predation from Bdellovibrio in vivo. Furthermore, Shigella-dependent replication of Bdellovibrio was captured inside the zebrafish larvae, indicating active predation in vivo. Bdellovibrio can be engulfed and ultimately eliminated by host neutrophils and macrophages, yet have a sufficient dwell time to prey on pathogens. Experiments in immune-compromised zebrafish reveal that maximal therapeutic benefits of Bdellovibrio result from the synergy of both bacterial predation and host immunity, but that in vivo predation contributes significantly to the survival outcome. Our results demonstrate that successful antibacterial therapy can be achieved via the host immune system working together with bacterial predation by Bdellovibrio. Such cooperation may be important to consider in the fight against antibiotic-resistant infections in vivo.

Genome analysis of a simultaneously predatory and prey-independent, novel Bdellovibrio bacteriovorus from the River Tiber, supports in silico predictions of both ancient and recent lateral gene transfer from diverse bacteria

BackgroundEvolution equipped Bdellovibrio bacteriovorus predatory bacteria to invade other bacteria, digesting and replicating, sealed within them thus preventing nutrient-sharing with organisms in the surrounding environment. Bdellovibrio were previously described as “obligate predators” because only by mutations, often in gene bd0108, are 1 in ~1x107 of predatory lab strains of Bdellovibrio converted to prey-independent growth. A previous genomic analysis of B. bacteriovorus strain HD100 suggested that predatory consumption of prey DNA by lytic enzymes made Bdellovibrio less likely than other bacteria to acquire DNA by lateral gene transfer (LGT). However the Doolittle and Pan groups predicted, in silico, both ancient and recent lateral gene transfer into the B. bacteriovorus HD100 genome.ResultsTo test these predictions, we isolated a predatory bacterium from the River Tiber- a good potential source of LGT as it is rich in diverse bacteria and organic pollutants- by enrichment culturing with E. coli prey cells. The isolate was identified as B. bacteriovorus and named as strain Tiberius. Unusually, this Tiberius strain showed simultaneous prey-independent growth on organic nutrients and predatory growth on live prey. Despite the prey-independent growth, the homolog of bd0108 did not have typical prey-independent-type mutations. The dual growth mode may reflect the high carbon content of the river, and gives B. bacteriovorus Tiberius extended non-predatory contact with the other bacteria present. The HD100 and Tiberius genomes were extensively syntenic despite their different cultured-terrestrial/freshly-isolated aquatic histories; but there were significant differences in gene content indicative of genomic flux and LGT. Gene content comparisons support previously published in silico predictions for LGT in strain HD100 with substantial conservation of genes predicted to have ancient LGT origins but little conservation of AT-rich genes predicted to be recently acquired.ConclusionsThe natural niche and dual predatory, and prey-independent growth of the B. bacteriovorus Tiberius strain afforded it extensive non-predatory contact with other marine and freshwater bacteria from which LGT is evident in its genome. Thus despite their arsenal of DNA-lytic enzymes; Bdellovibrio are not always predatory in natural niches and their genomes are shaped by acquiring whole genes from other bacteria.

Roles of multiple flagellins in flagellar formation and flagellar growth post bdelloplast lysis in Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus cells have a single polar flagellum whose helical pitch and diameter characteristically change near the midpoint, resulting in a tapered wave. There are six flagellin genes in the genome: fliC1 to fliC6. Accordingly, the flagellar filament is composed of several similar flagellin species. We have used knockout mutants of each gene and analyzed the mutational effects on the filament length and on the composition and localization of each flagellin species in the filament by electron microscopy and one- and two-dimensional polyacrylamide gel electrophoresis. The location and amounts of flagellins in a filament were determined to be as follows: a small amount of FliC3 at the proximal end, followed by a large amount of FliC5, a large amount of FliC1, a small amount of FliC2 in this order, and a large amount of FliC6 at the distal end. FliC4 was present at a low level, but the location was not determined. Filament lengths of newly born progeny cells increased during prolonged incubation in nutrient-deficient buffer. The newly formed part of the elongated filament was composed of mainly FliC6. Reverse transcription PCR analysis of flagellar gene expression over 5 days in buffer showed that fliC gene expression tailed off over 5 days in the wild-type cells, but in the fliC5 mutant, expression of the fliC2, fliC4, and fliC6 genes was elevated on day 5, suggesting that they may be expressed to compensate for the absence of a major component, FliC5.