Abstract
Bdellovibrio bacteriovorus HD100 is a predatory bacterium which lives by invading the periplasm of Gram-negative bacteria and consuming them from within. Although B. bacteriovorus HD100 attacks only Gram-negative bacterial strains, our work here shows attack-phase predatory cells also benefit from interacting with Gram-positive biofilms. Using Staphylococcus aureus biofilms, we show this predator degrades the biofilm matrix, obtains nutrients and uses these to produce and secrete proteolytic enzymes to continue this process. When exposed to S. aureus biofilms, the transcriptome of B. bacteriovorus HD100 was analogous to that seen when present intraperiplasmically, suggesting it is responding similarly as when in a prey. Moreover, two of the induced proteases (Bd2269 and Bd2692) were purified and their activities against S. aureus biofilms verified. In addition, B. bacteriovorus HD100 gained several clear benefits from its interactions with S. aureus biofilms, including increased ATP pools and improved downstream predatory activities when provided prey.
Bdellovibrio bacteriovorus is a Gram-negative obligate predatory bacterium which lives by attacking other Gram-negative bacteria [1], including a very wide range of human pathogens [2, 3] but does not predate on Gram-positive bacterial strains [2, 4,5,6,7]. This predator enters the periplasm of its prey, where it hydrolyzes and consumes the prey cell components, grows and septates before lysing the prey and proceeding to attack another. Although a previous study showed the addition of glutamate or glutamate and other amino acids prevented loss of viability [8], it has long been held that this predator is dependent on prey cells for both its replication and as a source of chemical energy. In addition to attacking planktonic prey, this predator is quite capable of reducing Gram-negative biofilm populations [2, 9, 10], even when two preys were present together [2]. In nature, however, B. bacteriovorus is thought to be mainly associated with biofilms [11, 12] and, as such, encounters non-prey Gram-positive strains within these biofilms.
To evaluate how B. bacteriovorus HD100 responds to Gram-positive biofilms, therefore, Staphylococcus aureus was selected since it is a non-prey bacterium [5] and a well-known human pathogen. Figure 1a illustrates a basic characteristic of predatory bacteria, namely that they only prey on Gram-negative bacteria, leading to an increase in their numbers. We found that when added to pre-formed biofilms, the predatory culture dispersed S. aureus biofilms (Fig. 1b) to the same degree as prey, i.e., Salmonella enterica, biofilms based upon crystal violet (CV) staining (Fig. 1c). Figure 1d shows that, while the CV results were similar, the S. enterica biofilm population was reduced by more than 1-log while that of S. aureus was reduced by only 59%. Though mild in comparison, this loss was significant, proving B. bacteriovorus HD100’s ability to disperse S. aureus biofilms.
Given the susceptibility of S. aureus biofilms to proteases [5, 13,14,15], we hypothesized residual proteases from the previous predation cycle, i.e., when culturing this predator for these tests, were responsible for the dispersions seen. The presence of proteases in the cell-free spent media (HDB Sup) was confirmed using the Azocoll assay, with average residual activities equivalent to 3.2 ± 1.3 ng/ml proteinase K. This was sufficient, even when diluted tenfold, to significantly disperse several different Gram-positive biofilms (Fig. 1e). The use of proteinase K led to similar levels of dispersion while the addition of AEBSF, a potent inhibitor of serine proteases, reduced the activity of the predatory supernatants (Figures S1 and S2). One interesting finding was the treatment of S. enterica biofilms with proteases actually increased the biomass (Figure S2). This increase is thought to be due to loss of the flagella, which causes the cells to adhere to the bottom of the wells as described by Teplitski et al [16]. Similarly, the differences seen between the individual Gram-positive biofilms is likely due to variations in their EPS content, a concept that should be studied in a future work.
In subsequent experiments, we found S. aureus biofilms were still dispersed even after the B. bacteriovorus HD100 cells were pelleted and washed to remove the residual secreted proteases (Fig. 1f and S3), implying de novo protease expression also occurred. As shown in Figure S3, this was also true for other S. aureus biofilms, demonstrating this activity is not limited to a single strain of S. aureus. Figure S4 demonstrates the proteolytic activities were from the predatory strain, and not the S. aureus biofilms, as the resulting proteolytic activity levels were nearly identical and the level of dispersion was comparable regardless if the biofilm was viable or UV-killed.
We next studied how B. bacteriovorus HD100 responds to S. aureus biofilms by sequencing the total cellular RNA. As shown in Figure 2a, the transcriptomic responses during the exposure are highly reminiscent of those seen both during intraperiplasmic (IP) growth [17] and when B. bacteriovorus HD100 was incubated in nutrient broth (NB) [18]. One striking difference between the S aureus biofilm-induced transcriptomic responses and the other conditions was EggNOG (Evolutionary genealogy of genes: Non-supervised Orthologous Groups) category “N” (Fig. 2a), which includes the genes related with cell motility. Whereas most of the genes included in this category were down-regulated in the NB-treated and IP predatory cultures, the majority were up-regulated when B. bacteriovorus HD100 was exposed to S. aureus biofilms. Of the 38 EggNOG category “N” genes up-regulated in this study, 35 are annotated as being involved in flagellar biosynthesis and were located within eight clusters or operons throughout the chromosome. The other three are annotated as a transporter related with gliding motility (Bd1025), a chemotaxis protein (CheX, Bd1823) and a hypothetical protein located within a flagellar operon (Bd3396). The differences between the three studies are thought to be due to the nature of the predator and its environments; the release of amino acids from the S. aureus biofilm through the induced proteolytic activities may act as a chemoattractant for swimming predator cells, as demonstrated by LaMarre et al. [19] in their study with Bacteriovorax stolpii UKi2. In contrast, IP growth requires little or no movement while the media used by Dwidar et al. [18] was homogeneous, precluding the need for the flagella or chemotactic responses. Likewise, the predators exposed to the S. aureus biofilms tended to have fewer EggNOG category “D” genes upregulated than the other published conditions. The reason for this is the variability in the gene expression levels; although many genes had average expression levels that were upregulated several-fold, their values were not significant based upon the Student's t-test. Consequently, these were categorized as being “No Significant Change or Silent”.
When viewed alongside the findings of Dwidar et al. [18], where B. bacteriovorus HD100 altered its transcriptome and secreted proteases in response to nutrients in the form of nutrient broth, the above results imply B. bacteriovorus HD100 also responds to S. aureus biofilms as if they were a source of amino acids and secretes proteases in reply. This was confirmed by both the transcriptomic data, where the expression levels for several different serine protease genes were elevated when B. bacteriovorus HD100 was exposed to the S. aureus biofilms for 4 h (Figure S5), and the increased extracellular proteolytic activities, which paralleled those seen within tests with NB (Fig. 2b). Two of the proteases (Bd2269 and Bd2692) induced in B. bacteriovorus HD100 by the S. aureus biofilms (Figure S5) were cloned, then expressed and purified (Figures S6 and S7). When tested, we found S. aureus biofilms were susceptible to both (Fig. 2c). Their clear activities against S. aureus biofilms, alongside their expression patterns, imply these two proteases are likely involved in the dispersals seen above and that they provide this predatory bacterium with a source of amino acids.
These amino acids not only provided B. bacteriovorus HD100 with the necessary components to express and secrete proteases but also provided it with a source of energy. This is illustrated in Figure 2d where B. bacteriovorus HD100’s ATP pool was significantly higher (1.7-fold) after an exposure to the S. aureus biofilms when compared to predatory cells incubated for the same amount of time in HEPES. The higher ATP levels were slightly less than but statistically comparable to those seen when B. bacteriovorus HD100 was provided nutrients in the form of NB medium. The higher energy content in both predatory cultures correlated with better predatory activities downstream, as illustrated in Figure 2e. In this assay, the prey was a bioluminescent strain of E. coli and the relative loss in bioluminescence was used to compare the predatory activities [20]. The faster and more significant loss in bioluminescence seen with the NB- and S. aureus biofilm-incubated B. bacteriovorus HD100 cells show these cultures were more active than those incubated in HEPES buffer.
To date, this predator was thought to be dependent upon Gram-negative prey cells as both a source of chemical energy and for its replication, restricting many of the studies published to only those interactions. The findings presented here implicate that bdellovibrios may have an even greater impact on bacterial communities than previously thought and that their interactions within nature are not limited to only Gram-negative strains and biofilms. Although B. bacteriovorus does not predate on Gram-positive bacterial strains, despite one study allegedly claiming otherwise [21], at present very little is known about how this predator responds to non-prey strains and, in particular, their biofilms. Here, we demonstrate B. bacteriovorus HD100 benefits from such interactions, obtaining amino acids from non-prey biofilms and using them to produce and secrete proteases as well as augment their ATP pools and predatory activities. These activities in no manner whatsoever suggest predation is occurring, as illustrated by the fact that the predatory numbers do not increase when provided S. aureus (Fig. 1a), but suggest a hitherto unknown aspect on how predatory bacteria inherently survive and thrive within natural environments, particularly in niches where Gram-positive, non-prey cells are present.
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Acknowledgements
Funding for this research was sponsored by the National Research Foundation of Korea within the General Research Program (Grant No. 2016R1D1A1A09919912). We thank them for the financial support. The following reagent was provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH: Staphylococcus aureus subsp. aureus Strain JE2 (NR-46543)
Author contributions
MD and HI designed and carried out the experiments. RJM supervised the experimental work. MD, HI, and RJM evaluated the data. MD, HI, and RJM wrote the manuscript.
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These authors contributed equally: Hansol Im and Mohammed Dwidar
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Im, H., Dwidar, M. & Mitchell, R.J. Bdellovibrio bacteriovorus HD100, a predator of Gram-negative bacteria, benefits energetically from Staphylococcus aureus biofilms without predation. ISME J 12, 2090–2095 (2018). https://doi.org/10.1038/s41396-018-0154-5
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DOI: https://doi.org/10.1038/s41396-018-0154-5
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