Ajay Kalanjana Monnappa

Bdellovibrio bacteriovorus Inhibits Staphylococcus aureus Biofilm Formation and Invasion into Human Epithelial Cells

Bdellovibrio bacteriovorus HD100 is a predatory bacterium that attacks many Gram-negative human pathogens. A serious drawback of this strain, however, is its ineffectiveness against Gram-positive strains, such as the human pathogen Staphylococcus aureus. Here we demonstrate that the extracellular proteases produced by a host-independent B. bacteriovorus (HIB) effectively degrade/inhibit the formation of S. aureus biofilms and reduce its virulence. A 10% addition of HIB supernatant caused a 75% or greater reduction in S. aureus biofilm formation as well as 75% dispersal of pre-formed biofilms. LC-MS-MS analyses identified various B. bacteriovorus proteases within the supernatant, including the serine proteases Bd2269 and Bd2321. Tests with AEBSF confirmed that serine proteases were active in the supernatant and that they impacted S. aureus biofilm formation. The supernatant also possessed a slight DNAse activity. Furthermore, treatment of planktonic S. aureus with the supernatant diminished its ability to invade MCF-10a epithelial cells by 5-fold but did not affect the MCF-10a viability. In conclusion, this study illustrates the hitherto unknown ability of B. bacteriovorus to disperse Gram-positive pathogenic biofilms and mitigate their virulence.

Investigating the Responses of Human Epithelial Cells to Predatory Bacteria.

One beguiling alternative to antibiotics for treating multi-drug resistant infections are Bdellovibrio-and-like-organisms (BALOs), predatory bacteria known to attack human pathogens. Consequently, in this study, the responses from four cell lines (three human and one mouse) were characterized during an exposure to different predatory bacteria, Bdellovibrio bacteriovorus HD100, Bacteriovorus BY1 and Bacteriovorax stolpii EB1. TNF-α levels were induced in Raw 264.7 mouse macrophage cultures with each predator, but paled in comparison to those obtained with E. coli. This was true even though the latter strain was added at an 11.1-fold lower concentration (p < 0.01). Likewise, E. coli led to a significant (54%) loss in the Raw 264.7 murine macrophage viability while the predatory strains had no impact. Tests with various epithelial cells, including NuLi-1 airway, Caco2, HT29 and T84 colorectal cells, gave similar results, with E. coli inducing IL-8 production. The viabilities of the NuLi-1 and Caco-2 cells were slightly reduced (8%) when exposed to the predators, while T84 viability remained steady. In no cases did the predatory bacteria induce actin rearrangement. These results clearly demonstrate the gentle natures of predatory bacteria and their impacts on human cells.

Sensing of plant hydrolysate-related phenolics with an aaeXAB::luxCDABE bioreporter strain of Escherichia coli.

A bioluminescent Escherichia coli bioreporter strain to detect hydrolysate related phenolics was developed by cloning the aaeXAB promoter from E. coli upstream of the luxCDABE genes. E. coli str. DH5α carrying this plasmid (pDMA3) was responsive to sub-inhibitory concentrations of plant hydrolysate-related phenolics, such as ferulic and vanillic acids, responding to these compounds at concentrations as low as 9.8 and 4.9 mg/L, respectively. Experiments with a mixture of the compounds showed similar responses as with single compound tests, with a minimum detectable concentration of 19.5mg/L. Finally, tests using rice straw hydrolysates were conducted, with E. coli str. DH5α/pDMA3 showing a maximum induction of 33-fold and a minimum detectable phenolic concentration of 9.3mg/L, based upon Folin-Ciocalteus reagent. These results demonstrate that this bioreporter maintains its sensitivity even with hydrolysate samples and that it can be potentially applied within biofuel industries to detect phenolics present within plant hydrolysates.

Effects of Carbon Dioxide Aerosols on the Viability of Escherichia coli during Biofilm Dispersal.

A periodic jet of carbon dioxide (CO2) aerosols is a very quick and effective mechanical technique to remove biofilms from various substrate surfaces. However, the impact of the aerosols on the viability of bacteria during treatment has never been evaluated. In this study, the effects of high-speed CO2 aerosols, a mixture of solid and gaseous CO2, on bacteria viability was studied. It was found that when CO2 aerosols were used to disperse biofilms of Escherichia coli, they led to a significant loss of viability, with approximately 50% of the dispersed bacteria killed in the process. By comparison, 75.6% of the biofilm-associated bacteria were viable when gently dispersed using Proteinase K and DNase I. Indirect proof that the aerosols are damaging the bacteria was found using a recombinant E. coli expressing the cyan fluorescent protein, as nearly half of the fluorescence was found in the supernatant after CO2 aerosol treatment, while the rest was associated with the bacterial pellet. In comparison, the supernatant fluorescence was only 9% when the enzymes were used to disperse the biofilm. As such, these CO2 aerosols not only remove biofilm-associated bacteria effectively but also significantly impact their viability by disrupting membrane integrity.

The Cytotoxic Necrotizing Factor of Yersinia pseudotuberculosis (CNFy) is Carried on Extracellular Membrane Vesicles to Host Cells

In this study we show Yersinia pseudotuberculosis secretes membrane vesicles (MVs) that contain different proteins and virulence factors depending on the strain. Although MVs from Y. pseudotuberculosis YPIII and ATCC 29833 had many proteins in common (68.8% of all the proteins identified), those located in the outer membrane fraction differed significantly. For instance, the MVs from Y. pseudotuberculosis YPIII harbored numerous Yersinia outer proteins (Yops) while they were absent in the ATCC 29833 MVs. Another virulence factor found solely in the YPIII MVs was the cytotoxic necrotizing factor (CNFy), a toxin that leads to multinucleation of host cells. The ability of YPIII MVs to transport this toxin and its activity to host cells was verified using HeLa cells, which responded in a dose-dependent manner; nearly 70% of the culture was multinucleated after addition of 5 µg/ml of the purified YPIII MVs. In contrast, less than 10% were multinucleated when the ATCC 29833 MVs were added. Semi-quantification of CNFy within the YPIII MVs found this toxin is present at concentrations of 5 ~ 10 ng per µg of total MV protein, a concentration that accounts for the cellular responses seen.

Cyanide Production by Chromobacterium piscinae Shields It from Bdellovibrio bacteriovorus HD100 Predation

ABSTRACT Predation of Chromobacterium piscinae by Bdellovibrio bacteriovorus HD100 was inhibited in dilute nutrient broth (DNB) but not in HEPES. Experiments showed that the effector responsible was present in the medium, as cell-free supernatants retained the ability to inhibit predation, and that the effector was not toxic to B. bacteriovorus. Violacein, a bisindole secondary metabolite produced by C. piscinae, was not responsible. Further characterization of C. piscinae found that this species produces sufficient concentrations of cyanide (202 µM) when grown in DNB to inhibit the predatory activity of B. bacteriovorus, but that in HEPES, the cyanide concentrations were negligible (19 µM). The antagonistic role of cyanide was further confirmed, as the addition of hydroxocobalamin, which chelates cyanide, allowed predation to proceed. The activity of cyanide against B. bacteriovorus was found to be twofold, depending on the life cycle stage of this predator. For the attack-phase predatory cells, cyanide caused the cells to lose motility and tumble, while for intraperiplasmic predators, development and lysis of the prey cell were halted. These findings suggest that cyanogenesis in nature may be employed by the bacterial strains that produce this compound to prevent and reduce their predation by B. bacteriovorus. IMPORTANCE Bacterial predators actively attack, kill, and enter the periplasm of susceptible Gram-negative bacteria, where they consume the prey cell components. To date, the activity of B. bacteriovorus HD100 has been demonstrated against more than 100 human pathogens. As such, this strain and others are being considered as potential alternatives or supplements to conventional antibiotics. However, the production of secondary metabolites by prey bacteria is known to mitigate, and even abolish, predation by bacterivorous nematodes and protists. With the exception of indole, which was shown to inhibit predation, the effects of bacterial secondary metabolites on B. bacteriovorus and its activities have not been considered. Consequently, we undertook this study to better understand the mechanisms that bacterial strains employ to inhibit predation by B. bacteriovorus HD100. We report here that cyanogenic bacterial strains can inhibit predation and show that cyanide affects both attack-phase predators and those within prey, i.e., in the bdelloplast. IMPORTANCE Bacterial predators actively attack, kill, and enter the periplasm of susceptible Gram-negative bacteria, where they consume the prey cell components. To date, the activity of B. bacteriovorus HD100 has been demonstrated against more than 100 human pathogens. As such, this strain and others are being considered as potential alternatives or supplements to conventional antibiotics. However, the production of secondary metabolites by prey bacteria is known to mitigate, and even abolish, predation by bacterivorous nematodes and protists. With the exception of indole, which was shown to inhibit predation, the effects of bacterial secondary metabolites on B. bacteriovorus and its activities have not been considered. Consequently, we undertook this study to better understand the mechanisms that bacterial strains employ to inhibit predation by B. bacteriovorus HD100. We report here that cyanogenic bacterial strains can inhibit predation and show that cyanide affects both attack-phase predators and those within prey, i.e., in the bdelloplast.