Engulfment, persistence and fate of Bdellovibrio bacteriovorus predators inside human phagocytic cells informs their future therapeutic potential.

In assessing the potential of predatory bacteria, such as Bdellovibrio bacteriovorus, to become live therapeutic agents against bacterial infections, it is crucial to understand and quantify Bdellovibrio host cell interactions at a molecular level. Here, we quantify the interactions of live B. bacteriovorus with human phagocytic cells, determining the uptake mechanisms, persistence, associated cytokine responses and intracellular trafficking of the non-growing B. bacteriovorus in PMA-differentiated U937 cells. B. bacteriovorus are engulfed by U937 cells and persist for 24 h without affecting host cell viability and can be observed microscopically and recovered and cultured post-uptake. The uptake of predators is passive and depends on the dynamics of the host cell cytoskeleton; the engulfed predators are eventually trafficked through the phagolysosomal pathway of degradation. We have also studied the prevalence of B. bacteriovorus specific antibodies in the general human population. Together, these results quantify a period of viable persistence and the ultimate fate of B. bacteriovorus inside phagocytic cells. They provide new knowledge on predator availability inside hosts, plus potential longevity and therefore potential efficacy as a treatment in humans and open up future fields of work testing if predators can prey on host-engulfed pathogenic bacteria.

[Bdellovibrio and like organisms: outstanding predators!] / Bdellovibrio et organismes apparentés - Des prédateurs bactériens hors du commun !

Obligate predatory bacteria, i.e. bacteria requiring a Gram negative prey cell in order to complete their cell cycle, belong to the polyphyletic group referred to as the Bdellovibrio And Like Organisms (BALO). Predatory interactions between bacteria are complex, yet their dynamics and impact on bacterial communities in the environment are becoming better understood. BALO have unique life cycles: they grow epibiotically with the predator remaining attached to the prey's envelope, dividing in a binary manner or periplasmically, i.e. by penetrating the prey's periplasm to generate a number of progeny cells. The periplasmic life cycle includes unique gene and protein patterns and unique signaling features. These ecological and cellular features, along with applications of the BALO in the medical, agricultural and environmental fields are surveyed.

Analysis of gene gain and loss in the evolution of predatory bacteria.

Predatory bacteria are ubiquitously distributed in nature in including in aquatic environments, sewage, intestinal tracts of animals and humans, rhizophere and, soils. However, our understanding of their evolutionary history is limited. Results of recent studies have shown that acquiring novel genes is a major force driving bacterial evolution. Therefore, to gain a better understanding of the impact of gene gain and loss in the evolution of bacterial predators, this study employed comparative genomic approaches to identify core-set gene families and species-specific gene families, and model gene gain and loss events among 11 genomes that represented diverse lineages. In total, 1977 gene families were classified. Of these 509 (pattern 11111111111) were present all of the 11 species. Among the non-core set gene families, 52 were present only in saltwater bacteria predators and had no ortholog in the other genomes. Similarly 109 and 44 were present only in the genomes of Micavibrio spp. and Bdellovibrio spp., respectively. In this study, the gain loss mapping engine GLOOME was selected to analyze and estimate the expectations and probabilities of both gain and loss events in the predatory bacteria. In total, 354 gene families were involved in significant gene gain events, and 407 gene families were classified into gene loss events with high supported value. Moreover, 18 families from the core set gene family were identified as putative genes under positive selection. The results of this study suggest that acquisition of particular genes that encode functional proteins in metabolism and cellular processes and signaling, especially ABC systems, may help bacterial predators adapt to surrounding environmental changes and present different predation strategies for survival in their habitats.

Indole negatively impacts predation by Bdellovibrio bacteriovorus and its release from the bdelloplast.

Bdellovibrio bacteriovorus is a predatory bacterium that attacks a wide range of Gram-negative bacterial pathogens and is proposed to be a potential living antibiotic. In this study, we evaluated the effects of indole, a bacterial signalling molecule commonly produced within the gut, on the predatory ability of B. bacteriovorus HD100. Indole significantly delayed predation on Escherichia coli MG1655 and Salmonella enterica KACC 11595 at physiological concentrations (0.25 to 1 mM) and completely inhibited predation when present at 2 mM. Microscopic analysis revealed that indole blocked the predator from attacking the prey. Furthermore, indole was not toxic to the predator but slowed down its motility. Microarray and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses confirmed that as the gene group showing the greatest downregulation in the presence of indole was flagellar assembly genes. Indole also caused a wide spectrum changes in gene expression including general downregulation of genes involved in ribosome assembly. Furthermore, indole addition to the predatory culture after the entrance of B. bacteriovorus into the prey periplasm slowed down bdelloplast lysis. In conclusion, indole can have significant impacts on the predation efficiency, which should be taken into consideration especially if B. bacteriovorus is to be applied as a probiotic or living antibiotic.

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.

BACKGROUND: Evolution 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 ~1x10(7) 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. RESULTS: To 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. CONCLUSIONS: The 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.

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.

Specialized peptidoglycan hydrolases sculpt the intra-bacterial niche of predatory Bdellovibrio and increase population fitness.

Bdellovibrio are predatory bacteria that have evolved to invade virtually all gram-negative bacteria, including many prominent pathogens. Upon invasion, prey bacteria become rounded up into an osmotically stable niche for the Bdellovibrio, preventing further superinfection and allowing Bdellovibrio to replicate inside without competition, killing the prey bacterium and degrading its contents. Historically, prey rounding was hypothesized to be associated with peptidoglycan (PG) metabolism; we found two Bdellovibrio genes, bd0816 and bd3459, expressed at prey entry and encoding proteins with limited homologies to conventional dacB/PBP4 DD-endo/carboxypeptidases (responsible for peptidoglycan maintenance during growth and division). We tested possible links between Bd0816/3459 activity and predation. Bd3459, but not an active site serine mutant protein, bound ß-lactam, exhibited DD-endo/carboxypeptidase activity against purified peptidoglycan and, importantly, rounded up E. coli cells upon periplasmic expression. A ΔBd0816 ΔBd3459 double mutant invaded prey more slowly than the wild type (with negligible prey cell rounding) and double invasions of single prey by more than one Bdellovibrio became more frequent. We solved the crystal structure of Bd3459 to 1.45 Å and this revealed predation-associated domain differences to conventional PBP4 housekeeping enzymes (loss of the regulatory domain III, alteration of domain II and a more exposed active site). The Bd3459 active site (and by similarity the Bd0816 active site) can thus accommodate and remodel the various bacterial PGs that Bdellovibrio may encounter across its diverse prey range, compared to the more closed active site that "regular" PBP4s have for self cell wall maintenance. Therefore, during evolution, Bdellovibrio peptidoglycan endopeptidases have adapted into secreted predation-specific proteins, preventing wasteful double invasion, and allowing activity upon the diverse prey peptidoglycan structures to sculpt the prey cell into a stable intracellular niche for replication.

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.

Bacterial intein-like domains of predatory bacteria: a new domain type characterized in Bdellovibrio bacteriovorus.

We report a new family of bacterial intein-like domains (BILs) identified in ten proteins of four diverse predatory bacteria. BILs belong to the HINT (Hedgehog/Intein) superfamily of domains that post-translationally self-process their protein molecules by protein splicing and self-cleavage. The new, C-type, BILs appear with other domains, including putative predator-specific domain 1 (PPS-1), a new domain typically appearing immediately upstream of C-type BILs. The Bd2400 protein of the obligate predator Bdellovibrio bacteriovorus includes a C-type BIL and a PPS-1 domains at its C-terminal part, and a signal peptide and two polycystic kidney disease domains at its N-terminal part. We demonstrate the in vivo transcription, translation, secretion, and processing of the B. bacteriovorus protein, and the in vitro autocatalytic N-terminal cleavage activity of its C-type BIL. Interestingly, whereas the Bd2400 gene is constitutively expressed, its protein product is differentially processed throughout the dimorphic life cycle of the B. bacteriovorus predator. The modular structure of the protein, its localization, and complex processing suggest that it may be involved in the interaction between the predator and its prey.

Downregulation of the motA gene delays the escape of the obligate predator Bdellovibrio bacteriovorus 109J from bdelloplasts of bacterial prey cells.

Bdellovibrio bacteriovorus is a Gram-negative bacterium that preys on other Gram-negative bacteria. The lifecycle of B. bacteriovorus alternates between an extracellular flagellated and highly motile non-replicative attack-phase cell and a periplasmic non-flagellated growth-phase cell. The prey bacterium containing periplasmic bdellovibrios becomes spherical but osmotically stable, forming a structure known as the bdelloplast. After completing the growth phase, newly formed bdellovibrios regain their flagellum and escape the bdelloplast into the environment, where they encounter more prey bacteria. The obligate predatory nature of B. bacteriovorus imposes a major difficulty to introducing mutations in genes directly involved in predation, since these mutants could be non-viable. This work reports the cloning of the B. bacteriovorus 109J motAB operon, encoding proteins from the flagellar motor complex, and a genetic approach based on the expression of a motA antisense RNA fragment to downregulate motility. Periplasmic bdellovibrios carrying the plasmid expressing antisense RNA displayed a marked delay in escaping from bdelloplasts, while the released attack-phase cells showed altered motility. These observations suggest that a functionally intact flagellar motor is required for the predatory lifecycle of B. bacteriovorus. Also, the use of antisense RNA expression may be a useful genetic tool to study the Bdellovibrio developmental cycle.

Investigations into the life cycle of the bacterial predator Bdellovibrio bacteriovorus 109J at an interface by atomic force microscopy.

Atomic force microscopy was used to image Bdellovibrio bacteriovorus 109J, a gram-negative bacterial predator that consumes a variety of other gram-negative bacteria. In predator-prey communities grown on filters at hydrated air-solid interfaces, repeated cycles of hunting, invasion, growth, and lysis occurred readily even though the cells were limited to near two-dimensional movement. This system allowed us to image the bacteria directly without extensive preparation or modification, and many of the cells remained alive during imaging. Presented are images of the life cycle in two species of prey organisms, both Escherichia coli (a small prey bacterium that grows two-dimensionally on a surface) and Aquaspirillum serpens (a large prey bacterium that grows three-dimensionally on a surface), including high-resolution images of invaded prey cells called bdelloplasts. We obtained evidence for multiple invasions per prey cell, as well as significant heterogeneity in morphology of bdellovibrios. Mutant host-independent bdellovibrios were observed to have flagella and to excrete a coating that causes the predators to clump together on a surface. Most interestingly, changes in the texture of the cell surface membranes were measured during the course of the invasion cycle. Thus, coupled with our preparation method, atomic force microscopy allowed new observations to be made about Bdellovibrio at an interface. These studies raise important questions about the ways in which bacterial predation at interfaces (air-solid or liquid-solid) may be similar to or different from predation in solution.

Effect of Bdellovibrio bacteriovorus infection on the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli: evidence for activation of cytoplasmic proteolysis.

Intact cells of Bdellovibrio bacteriovorus strain 109J were found to be incapable of taking up 14C-methyl alpha-glucoside, mannitol or fructose, and extracts derived from these cells exhibited negligible activities of the protein components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Escherichia coli strain ML35 cells exhibited high in vivo sugar uptake activities that were progressively lost over a period of 2 h at 30 degrees C following the entry of B. bacteriovorus into the periplasm of E. coli. In vitro complementation assays revealed that the E. coli PTS enzymes, enzyme I, HPr, and the glucose- and mannitol-specific enzymes II, were all lost almost in parallel with the disappearance of uptake activity. Thus, loss of activity in vivo was not due to membrane leakiness, energy depletion, or preferential inhibition or inactivation of any one protein component of the PTS. Instead, loss of PTS activity was attributed to digestion of the protein constituents of the system by proteases present in the cytoplasm of the host cell after bdellovibrio entry. Both ethylenediaminetetraacetate and phenylmethylsulphonyl fluoride partially protected against inactivation in vitro, and the two inhibitors together gave full protection, suggesting that both metallo- and seryl-proteases were responsible for the inactivation. Protease activity increased progressively with time following bdellovibrio entry and appeared to degrade the E. coli PTS enzymes in vivo. Preliminary evidence suggested that the proteases responsible for PTS enzyme degradation may be encoded by the B. bacteriovorus chromosome.

Bdellovibrio and the intestinal flora of vertebrates.

Bdellovibrio strain MS7 force-fed to fish and frogs via an intragastric tube did not become an integral component of the intestinal microflora. Strain MS7 fed to mice in drinking water was not recovered from the intestinal tract of mice. However, in vitro, the organism multiplied in intestinal contents of frogs and mice. Bdellovibrio inoculated into rabbit ileal loops was greatly reduced in number within 24 h. It was concluded that strains MS7 could be considered nonpathogenic to animals, at least when introduced into the digestive tract, and that it is not feasible at the present time to lyse pathogenic, gram-negative bacteria in the alimentary tract with Bdellovibrio.