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Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. A Nature Research Journal. 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. 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. Together, these results quantify a period of viable persistence and the ultimate fate of B. 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.

In response to the emergence of antimicrobial-resistant bacterial infections as a global health issue, several alternative, non-small molecule measures, are being sought to treat drug resistant bacterial infections 1 , 2 , 3 , 4.

One such approach is the potential use of living predatory bacteria such as Bdellovibrio bacteriovorus 4 , 5 , 6 , 7 , a Gram-negative predatory bacterium, which invades and preys upon a wide range of Gram-negative bacteria in their natural environments that are soil and water 6. In in vitro conditions, B.

Recent studies have verified the apparent safety of predators using in vitro cell culture 12 , 13 , 14 and in vivo animal models 9 , 10 , 11 , 14 , 15 , 16 , 17 , The questions that remain to be addressed are with regard to their interactions as living, but seemingly non-pathogenic bacteria, with the host immune system, which involves evaluation of the mechanisms of uptake and persistence of predatory bacteria within phagocytes and the processes involved in their clearance from these host cells.

Also it is not known how frequently the human immune system encounters predatory bacteria in normal life. All micro-organisms, including bacterial pathogens, encounter professional phagocytic cells such as macrophages and dendritic cells which are the first line of defence and the essential components of the innate immune system 19 , These host cells engulf and ingest internalised micro-organisms through phagocytosis, a process driven by receptor-ligand interactions resulting in cytoskeletal remodelling and engulfment of targets by pseudopods.

Phagocytosis culminates in the formation of sealed intracellular compartments, namely, phagosomes that harbour the ingested bacteria 19 , 20 , The nascent phagosome matures into an organelle with microbiocidal properties through its complex interactions with the endolysosomal network, a process that involves sequential acquisition of different proteins of the endocytic pathway and ultimately results in fusion of phagosomes with lysosomes to form phagolysosomes with an acidic pH facilitating bacterial killing and degradation 19 , Phagosomal maturation also routes antigens for presentation with MHC molecules to the helper T cells resulting in adaptive immune response through T and B cell activation Our previous work in zebrafish model showed that the injected B.

However, in that study, the duration of persistence and fate of B. In the current study, we were interested in understanding the timescale of persistence and dynamics of B. Being mindful of the mechanisms adopted by replicative pathogens to evade or combat phagocytic activities of the innate immune cells to prolong their survival 19 , 20 , 23 , 24 , we wished to investigate whether predatory B.

Although there are helpful studies evaluating the cytotoxicity and cytokine responses induced by B. Such data will not only profile predator availability in vivo , but eventually aid in the assessment of the potential of predatory bacteria to meet and prey on intracellular pathogens, such as Salmonella, Klebsiella and Francisella species, inside cells. There needs to be a better understanding of predator persistence in different host environments and verification of duration of predator availability, in vivo , if employed as a future therapeutic.

This requires reliable enumeration of live B. Even though predator enumeration can be challenging in in vivo studies, recently we have sought ways to quantify predators in our studies in the zebrafish model 10 as well as in the current study.

PMA-differentiated U cells have been used for studying interactions and intracellular trafficking of several Gram-negative pathogens within macrophages 26 , 27 , 28 , 29 and we adopted similar methodology to study the interactions of B. We counted predatory bacteria internalised by the phagocytic cells and assessed their persistence and effects on host cell viability, intracellular trafficking of predators, the role of cytoskeleton in their uptake, and the associated immune responses.

We also assayed B. To test for potential engulfment of B. S1 for protocol scheme. Predatory bacteria engulfed by the macrophages were recovered following experimental lysis of the U cells, and enumerated by viable plaque counts on prey bacterial lawns , In parallel experiments, engulfed predatory bacteria were observed and counted in whole U cells by fluorescence microscopy Fig.

Raw data points comprising Fig. Persistence and survival of B. Images are representative of two independent experiments, each set up in duplicate. We further confirmed the intracellular location of B. Even though we were unable to determine bacterial viability microscopically by live-dead staining of BbHDCFP, we observed persistence with a small decrease in the median B.

Taken together these experimental approaches demonstrate the persistence of viable B. As we were able to detect and recover live B. Pathogenic bacteria often can exploit the actin and microtubules of the host cell cytoskeleton to their advantage using secreted effectors to facilitate their uptake and intracellular survival 30 , 31 , 32 , however, whether non-pathogenic, predatory B.

To investigate th e nature of B. Although uptake was not completely abolished by either treatment, the percentage of BbHDTFP containing cells observed microscopically Fig. These data together strongly indicate that both F-actin and microtubules contribute to B. Role of cytoskeleton in the uptake of B.

The actin filaments of fixed cells were stained with Rhodamine-phalloidin false coloured in cyan , the nuclei were stained with SiR-DNA red and imaged. Shown are the maximum intensity 2D-projections Stacks 1—20 used for both control and cytochalasin D treated cells of the restored z-stack images. Images are representative of two independent experiments.

The microtubules of the fixed cells were stained with anti-tubulin primary antibody and Alexa secondary antibody false coloured in magenta , nuclei were stained with SiR-DNA red and imaged. Shown are the maximum intensity 2D-projections Stacks 5—25 for both control and Nocodazole treated cells of the restored images. Data shown, as number of BbHDTFP visualised per cell, are representative of one of the two independent experiments, each set up in duplicate and a minimum of cells were analysed from each experiment.

As our data showed that Gram-negative B. Cytokine responses induced by B. Typhimurium LT2 and K. BbHD, S. U cells alone are known to secrete IL following PMA differentiation 37 and for this reason we display those control results, rather than subtracting them from experimental data. The cytokines measured in these experiments followed a similar trend to that observed in the BbHD synchronous spin-assisted uptake experiments Fig.

We also conducted additional cytokine analyses, from direct uptake experiments, for U cells with enumerated intracellular B. These were to validate the live numbers of intracellular predators at the same time as the cytokine measurements, but experimentally did not allow simultaneous pathogen comparisons due to enumeration complexity at short timepoints.

Many Gram-negative bacteria are known to be cytotoxic and inducers of host cell death Whilst previous studies have assessed the cytotoxic effects of B.

Viability of U cells exposed to B. The images were acquired using 60x lens a. A minimum of cells were counted from each independent experiment at each time point. To this end, two different experiments involving labelling of acidic vacuoles with lysotracker and staining of phagolysosomal markers were performed on separate occasions.

In order to determine whether the engulfed B. Trafficking of B. To further investigate the localisation of B. This co-localisation of B. Colocalisation of B. The magnified images of the merged panel are representative regions showing the predatory bacteria colocalising with the phagosomal markers. A minimum of cells with BbHDCFP were analysed at each time point to study colocalisation of predators with phagosomal markers. Having measured the persistence of viable B.

Ethical approval for generic consent for prospective collections and for the use of Pathology archival material at the Biobank has been granted by the Greater Manchester National Research Ethics Service. Two standard recall antigens to which humans are exposed from vaccination or environmental exposure, Tetanus toxoid and Candida albicans surface antigen, were used as positive controls to confirm the presence of antibodies in the serum samples tested.

As the positive controls were purified antigens, levels of responses to them were not directly comparable to lysed B. For C. All other values were measured within standards. Even though highest concentrations of The concentrations of IgM antibodies detected against both the predators were lower than that of IgG antibodies indicating that the exposure of serum donors to B.

Prevalence of antibodies against B. Data shown are the antibody responses analysed from 25 different serum samples and median values for each antigen are represented as a line. IgA antibodies were also detected against both predators with the highest concentrations being 3. The presence of anti- B. For B. What are the processes involved in their uptake by phagocytic cells? Would these predatory bacteria survive in the host cells and for how long?

What immune response would be induced as these predatory bacteria persist inside host cells? And how ultimately are these non-pathogenic bacteria cleared by the host?

Whilst several recent studies have verified the safety of predators using in vitro cell culture models 12 , 13 , 14 and in vivo animal models 9 , 11 , 15 , 16 , 17 , 18 , the live persistence and fate of predatory bacteria within the cells of the immune system has not yet been investigated.

In this current study, we have tracked B. We postulated that non-pathogenic B. By performing bacterial uptake experiments in the presence of pharmacological inhibitors, we demonstrated a significant role of the host actin cytoskeleton and its rearrangement and microtubule dynamics in the uptake of B.

An inhibitor-associated reduction in uptake of B. Hence disruption of actin cytoskeleton with cytochalasin D or the disassembly of microtubule network with nocodazole, which in turn can influence actin remodelling via Rho GTPases 42 , could have led to further sensing and killing of internalised B.


Associated Content

Bdellovibrio is a genus of Gram-negative , obligate aerobic bacteria. One of the more notable characteristics of this genus is that members can prey upon other Gram-negative bacteria and feed on the biopolymers , e. They have two lifestyles: a host-dependent, highly mobile phase, the "attack phase", in which they form "bdelloplasts" in their host bacteria; and a slow-growing, irregularly shaped, host-independent form. The most well studied of these is Bdellovibrio bacteriovorus , which is found almost exclusively in host dependent growth in nature.


Bdellovibrio bacteriovorus

A Microbial Biorealm page on the genus Bdellovibrio bacteriovorus. Bdellovibrios were discovered by Stolp and Petzhold in , in an attempt to isolate bacteriophage from soil samples. Stolp and Petzhold observed unique plaques in their samples that took several days to develop and continued to grow for over a week, instead of plaques caused by bacteriophages that would appear within hours. A closer inspection of the plaques under a light microscope revealed cells that were small, highly motile, and vibrio-shaped. These cells were Bdellovibrios. After the discovery of Bdellovibrios further observations revealed many interesting and unique properties.

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