Bacteriophage Therapy with Nanoparticles to Penetrate the Bacterial Cell Body

Introduction:

Antibiotic resistance is an increasing trend among pathogenic bacteria, thus posing a severe threat to human health and well-being^1,2 worldwide. The emergence of antibiotic resistant strains of bacteria is an inevitable phenomenon as it is a result of evolutionary selection³ that selects for genes conferring competing characteristics that arise by mutation in each successive generation and has perennially been taking place in bacterial populations; and since bacteria asexually reproduce at logarithmic rates under favorable conditions, it is possible to have the resistance gene transmitted to the majority of bacterial strain.  However, in more recent years the excessive use (and abuse) of antibiotics⁴ at low concentrations⁵ in food, livestock⁴, hospitals and communities (for the purposes of water disinfection, medicine, food packaging and textile industry) along with unnecessary prescribing of antibiotics by medical professionals⁶ has expedited the emergence of multiple drug resistant bacteria (MDRB)^3,7. The horizontal transfer of antibiotic resistance genes among microbial populations⁸ further exacerbates the problem^2,7. The widespread distribution of antibiotic-resistant genes (that render antibiotic drugs harmless to the bacteria) has made infectious diseases that were once easily treatable deadly again^1,9,10. An increase in multiple drug resistant bacteria (which are informally known as superbugs) population is therefore a severe threat to human health in the future that necessitates the need to develop alternative means to kill bacteria without the use of traditional antibiotics. For diseases caused by Gram-negative bacteria, resistant bacteria are even a bigger problem as the pipeline for the development of new antimicrobials that target Gram-negative bacteria remains empty¹¹, largely due to the presence of an outer membrane that serves as a highly impermeable barrier, thereby acting as a defense mechanism. As the rate of development of novel antibiotics has declined, alternatives to antibiotics are beginning to be considered in both animal agriculture and human medicine. These non-antibiotic approaches include phage therapy, phage lysins, bacteriocins, and predatory bacteria¹², and nanoparticles¹³.

Several classes of antibacterial nanoparticles and nanosized carriers for have proven their effectiveness for treating bacterial infections, including antibiotic-resistant ones, in experimental models and in vivo¹³. Nanoparticles offer improved antibacterial properties compared to organic antibacterial drugs principally due to their high surface area to volume ratio, resulting in appearance of new mechanical, chemical, electrical, optical, magnetic, electro-optical, and magneto-optical properties of the NPs that are different from their bulk properties¹⁴. The mechanisms of NP toxicity depend on composition, surface modification, intrinsic properties, and the bacterial species.^15-17. Nanoparticle toxicity is generally believed to be triggered by the induction of oxidative stress by free radical formation¹⁵ (i.e, ROS generation) but the mechanisms are very complex and depends on the physicochemical properties of the nanoparticles¹⁵. TiO₂ and ZnO NPs are known to have weak mutagenic potential that induces frameshift mutation in Salmonella typhimurium (–)¹⁸.  Metallic and ionic forms of copper in Cu based nanoparticles produce hydroxyl radicals that damage DNA and essential proteins of the bacterial cell ¹⁹. Ag NPs modified with titanium are toxic to E. coli and S. aureus. Ag NPs naturally interact with the membrane of bacteria and disrupt the membrane integrity, and moreover binding to  sulfur, oxygen, and nitrogen atoms of essential biological molecules and thereby inhibiting bacterial growth²⁰. NO NPs are able to change the structure of the bacterial membrane and produce reactive nitrogen species (RNS), which lead to modification of essential proteins of bacteria²¹. However, concerns have been raised on the effects of metal nanoparticle persistence and toxicity effects on the beneficial microbes, microbial communities in ecosystems, and public health²². For example, exposure to soluble silver compounds may produce toxic effects like liver and kidney damage; eye, skin, respiratory, and intestinal tract irritations; and untoward changes in blood cells²³. In addition,  adverse toxic effects have also been reported due to prolonged exposure to some nanoparticles therapy^16,24.

The specific killing of bacterial cells has also been accomplished using bacteriophages. Bacteriophages are infectious viruses that have a natural tendency to attack specifically target, and kill  bacteria during its own reproductive life cycle^25-27 by injecting its own DNA, a feature that can be manipulated and used for targeted bacterial killing (phage therapy)^27-30. Using bacteriophages as therapeutic agents present a number of advantages; such as the phages being very specific (thus avoiding the chances of developing secondary infections)^28,31, and that side effects have not been reported (in contrast to traditional antibiotics, for which side effects are common). However, compared to nanoparticles, bacteriophages also present disadvantages in that they are very specific and is difficult to be functionalized, have low stability (less shelf life), and can only be taken intravenously³².  There may also be unknown consequences of introducing a biological sample (with its own DNA) to interact with bacterial cells inside the human body. In contrast, unlike biological specimen, nanoparticles can be manufactured and stored for a long time and in many different solvents³³, can be modified on the surface and intrinsically) in ways to make it more suitable for bacteria killing, like ROS generation, hyperthermia, drug loading capacity, etc³³.  They can also be administered in more feasible routes (orally or inhaled), instead of only having to rely only on intravenous injection³³ (unlike bacteriophage treatment).

For this research project, we use a novel concept that combines the ‘art’ of bacteriophage therapy with nanoparticles by enabling our nanoparticle to penetrate into the bacterial cell body. This is a unique approach to bacteria killing, as no nanoparticles have been developed that is able to use a bacteriophage protein for penetration. The bacteria are then treated with magnetic hyperthermia  To achieve this bacteriocidal effect the mesoporous silica nanoparticles are coated with G3P proteins, which are minor coat proteins derived from the bacteriophage³⁴. G3Pis located, most likely in five copies, at one tip of the filamentous phage particle^34,35 , and is known to be involved in the infection of Escherichia coli cells carrying F-pili³⁵ by mediating penetration of the phage into the host (E-coli) cytoplasm. The core contains iron oxide (Fe₂O₃) that makes it susceptible to magnetic  hyperthermia. Hyperthermia involves raising the temperature of local environment of a cell, using magnetic nanoparticles subjected to high frequency magnetic field³⁶.  At a temperature >46°C, cells subjected to hyperthermia treatment undergo direct tissue necrosis, coagulation or carbonization. During moderate hyperthermia, which is traditionally termed as hyperthermia treatment, cells undergo heat stress in the temperature range of 41–46°C resulting in activation and/or initiation of many intra and extracellular degradation mechanisms like protein denaturation, protein folding, aggregation and DNA cross linking. With a single heat treatment, permanent irreversible protein damage can occur resulting in protein aggregation and/or inhibition of many cellular functions³⁷. The other cellular effects of moderate hyperthermia include induction and regulation of apoptosis, signal transduction, multidrug resistance and heat shock protein (HSP) expression^38-40. The effectiveness of any hyperthermia treatment greatly depends on the temperatures generated at the targeted sites of action, duration of exposure and characteristics of particular cells. The increase in temperature results in changing the physiology of the bacterial cells, finally leading to apoptosis16 In this way, therefore, it is possible to get rid of multiple drug resistant bacteria using nanoparticles based on our model. The final aim is to selectively kill E.Coli bacteria by hyperthermia and ROS generation without killing other cells. The use of magnetic fluid hyperthermia has been extensively researched as a supplementary cancer treatment as it is able to selectively kill tumor cells.

Results and Discussion:

Characterization

iii. Size of magnetic core in silica nanoparticles

The factors that affect heating efficiency of  magnetic nanoparticles by magnetic hyperthermia, and the phenomenon of heat generation of MNPs  have been studied extensively, with an aim to develop MNPs for maximum heat generation³⁶.

Heat generation in superparamagnetic MNPs occurs by two main mechanisms : Brownian relaxation (occurs by nanoparticle rotation leading to the motion of MNPs against the viscous forces in the fluid dispersion) and Neel relaxation (occurs due to re-orientation of the magnetic moment inside the MNP in response to the alternating magnetic field), as described by the linear response theory (LRT)^41,42. LRT has been used to theoretically calculate the amount of heat generated by MNPs, and based on this theory, the power generation of the nanoparticles can be calculated by the equation:

Here, χ0 is the magnetic susceptibility of the particles, H is the field strength of the applied magnetic field, f is magnetic field frequency, and τ is the relaxation time for reorientation of magnetic moments in MNPs [14]. Usually Néel relaxation dominates for smaller MNPs, while larger MNPs generate more heat due to Brownian relaxation [14]. Brownian relaxation time (τB) depends on the viscosity (η) of the medium, hydrodynamic volume of MNPs (VH), absolute temperature (T), and the Boltzmann constant (k).

and it is assumed that, among several other factors, μ0 is the permeability of free space (4π ∗ 10− 7 T-m/A), Md is domain magnetization of magnetic material used, H is the maximum applied field strength, Vm is the magnetic volume of nanoparticles, k is the Boltzmann constant, T is the absolute temperature, and Hk is the anisotropy field.

Based on this theory, we tested our nanoparticles with differently sized magnetic cores. The three cores available in our lab was 5 nm 10 nm and 20nm.

G3P

G3P (Gene 3 protein) is a minor coat protein from the  bacteriophage M13 that facilitates infection of Escherichia coli bearing an F-pilus. Its N-terminal domain (g3p-D1) enables infection by inducing penetration of the phage into the host (E-coli) cytoplasm via interaction with the Tol complex that resides in the E. coli periplasm³⁴.

Filamentous Ff bacteriophages (M13, f1 and fd) infect strains of E.coli carrying an F-episome. Phage infectivity is facilitated by the phage gene 3 protein (g3p), a minor coat protein. Three to five copies of g3p cap one end of the extended filamentous phage particle.^34,43 The g3p protein is divided into three domains separated by glycine-rich peptide linkers and it contains a short C-terminal transmembrane segment in the figure below.

Permission not taken yet

the N-terminal domain (D1) is thought to be responsible for membrane penetration, the middle domain (D2) for adsorption to the F-pilus tip and the sole function assigned to the C-terminal domain (D3) is the anchoring of g3p in the phage particle ⁴⁴. The transmembrane segment mediates incorporation of g3p into the host inner membrane, a prerequisite for incorporation into the nascent phage particle. No function has been assigned to the two glycine-rich sections in g3p, but the region separating D2 and D3 may simply have a tethering function⁴⁵.

For our project, the adopted nanoparticle is conjugated with the g3P protein derived from M13 bacteriophage that normally infects E. coli.  The phage coat protein g3p [13] from filamentous phage M13 was expressed in E. coli by secretion and purified by immobilized metal affinity chromatography (IMAC). Purified g3p (MW= 47.5 kDa) proved to be a highly soluble protein, which exhibited no tendency to aggregate at mM concentrations. hence the name ‘NanoPhage’

M and M:

The E. coli strain BL21DE3] was used for propagation of plasmids and expression of the g3p protein. The g3pwas PCR amplified with primers (5′-CATGCCATGACTCGCGGCCCAGCCGGCCATGGCAGAAACTGTTGAAAGTTGTTTAGCA-3′) and (5′-GAGTCATTCTGCGGCCGCATTTTCAGGGATAGCAAGCCCAAT-3′) using Taq polymerase (HT Biotechnology Ltd.). The PCR fragment was digested with SfiI and NotI, and ligated into SfiI/NotI digested pUC119His6 [28], which provides the protein with the pelB leader sequence resulting in the secretion of a mature g3p-D1 to the periplasm. The correct sequence of the resulting plasmid was confirmed.

As growth media, standard yeast LB broth rich medium was used for protein expression. Media were supplemented with 100 mgl−1 ampicillin and). Bacterial cultures were grown in 5 ml aliquots at 37°C overnight, diluted 1/100 and grown to an OD600 of 0.6 and then induced for approximately 22 h with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C. g3p was purified from the supernatant after buffer exchange into phosphate buffered saline (PBS), pH7, using IMAC.  Protein was washed and concentrated into the appropriate buffer for subsequent analysis using Amicon YM3 membranes. 

Heat treatment of bacteria

Heat treatment is one of the most widely used methods for causing the death of pathogenic bacteria. Mild-heat treatment, known as pasteurization, is widely used for getting rid of bacteria from food items. An understanding of the mechanism and kinetics of thermal death of bacteria has been studied in the past⁴⁶ by various methods⁴⁷.

During this process of heating, the cell proceeds from a living state to a death state. The use of heat is an effective technique by which to control the growth of microorganisms, and it has, Heat can be applied as moist or dry heat. Dry heat (i.e. where water is not a medium of heat transfer) is known to kill microbes by oxidizing the molecules.

Moist heat transfers heat energy to the microbial cells more efficiently and kills microbial cells by denaturing enzymes.⁴⁷ The moist heat method includes boiling, pasteurization and autoclaving.Boiling ~100 °C! for 10 min kills most vegetative fungi, bacterial cells, virus and their spores.

Heat damages cellular proteins by covalent and H-bonds that link the adjoining portions of amino acid chains and maintain the three-dimensional shapes of globular proteins, (like enzymes) that is necessary for its function⁴⁷.

Heat treatment also said to cause inactivation of ‘critical sites’ in the cells membrane of bacteria.

The damage to the cell membrane results in the leakage of potassium, amino acids, and nucleotideso r nucleic acids, and the loss of salt tolerance. Damage to the cell membrane would  also allow diffusion of toxic agents which cannot pass through an undamaged cell membrane.

Conjugation of Protein molecules into mesoporous silica nanoparticles

Killing cells by hyperthermia

Killing cells using magnetic hyperthermia is a well researched method for the past few decades. Papers published in more recent years focuses on detailed research on  (1)

hyperthermia induced cell killing, (2) mathematical

models of cell killing, (3) mechanisms of thermal

effects in the hyperthermia temperature range and (4) effects on proteins that contribute to resistance to other stresses, for example, DNA damage, among many others.

Cell killing

It has been long recognized that hyperthermia in

the 40–47C temperature range kills cells in a

reproducible time and temperature dependent

manner. Survival curves for temperatures in the

43–47C range typically show a shoulder with an

exponential reduction in clonogenic survival as a

function of time at a given temperature [2–4].

In contrast, cell survival curves for temperatures of42.5 degrees C and below, depending on the cell line,

will show a shoulder, an exponential portion of cell

killing followed by a plateau in cell killing, due to the

development of chronic thermal tolerance.

The principal conclusion from these studies is that

for hyperthermia, thermal dose is a combination of

time and temperature.

Mechanisms of thermal effects in the hyperthermia temperature range

In the hyperthermia temperature range (40–47C), the main effects at the cellular level that are of relevance to cancer therapy are cell killing and those effects that alter the resistance of cells to radiation and/or chemotherapeutic agents. While it is acknowledged that at the tissue, organ and whole body level there are numerous effects that can contribute to the clinical uses of hyperthermia; this review focuses on effects at the cellular level. Thus, we will discuss cell killing and those effects that alter resistance to other cellular stresses. Prior to discussing the mechanisms of cell death it is important to consider the types of cell death that are induced by hyperthermia.

The types of cell death induced by hyperthermia include heat induced apoptosis [9],

mitotic catastrophe secondary to alterations in the proteins that support DNA metabolism [10–11].

The type of cell death induced by heat shock is highly cell-type and temperature dependent [12].

In fact, most cell lines die of a combination of death processes. The goal of hyperthermia research is to find the molecular mechanisms by which heat kills tumour cells or any cells and the mechanisms by which hyperthermia radiosensitizes cells to radiation or chemotherapeutic agents. A major problem is that hyperthermia causes a large number of macromolecular changes and affects functions in all cellular compartments at temperatures above 43C. Although significantly fewer macromolecular changes occur in the 40.5 to 42C range, these changes are still numerous and occur in multiple cellular compartments. The challenge is to determine which molecular changes are critical for the relevant endpoint such as tumour cell killing and sensitization to chemo and radiation therapy. By the late 1980s there was a significant body of literature describing heat effects on DNA, proteins, lipids and other cellular components [13]. Information on the effects of hyperthermia on cellular macromolecules has been growing rapidly since that time, making a comprehensive review almost impossible. However, no clear, detailed mechanism for cell killing or sensitization to other stresses has been established. Nevertheless, the main consensus that has emerged is that the mechanism by which hyperthermia kills cells and/or sensitizes cells to radiation involves the unfolding and subsequent aggregation of proteins [14]. Therefore, one approach would be to focus on how heat affects those proteins that contribute to resistance to other stresses.

Although hyperthermia is not believed to cause direct DNA damage, it is assumed that but increase in temperature is believed to cause heat effects on proteins involved in

DNA replication, chromosome segregation and

DNA repai – thus making it much less likely for cells to reproduce or transcribe proteins necessary for survival.

 

 

 

 

 

 

 

 

 

 

 

Materials and Methods:

Making G3P

Cell Culture

1. Get appropriate cell culture in glycerol from -80°C freezer. Or, use a single colony from a streaked agar plate.
2. Get appropriate selecting antibiotic from IPTG/antibiotics box in -20°C freezer.
3. Place items in the fume hood.
4. While frozen agents are thawing, get autoclaved LB broth and sterile pre-culture tube (from oven).
5. Prepare pre-culture in fume hood:

5 ml LB broth
2.5 ul kanamycin (final concentration 50 ug/ml)	5.0 ul ampicillin (or carbenicillin) (final concentration 100 ug/ml)
5-10 ul of cells

6. Grow the pre-culture overnight in incubating shaker at 37°C, 250 rpm.
7. Wait at least 8 hours.
8. In the fume hood, prepare cell culture in 1000 ml culture flask:

250 ml LB broth
125 ul kanamycin (final concentration	250 ul ampicillin/carbenicillin
5 ml of cell pre-culture

9. Place on incubation shaker at 37°C, 250 rpm.
10. Wait 3-4 hours.
11. Check to see if the OD of the cell culture is 0.5-0.6.
12. Add IPTG (0.5M)  to induce:

250 ul IPTG (0.5 M so final concentration of solution is ~ 0.5 mM)

255 ml cell culture

13. Place on room temperature shaker during induction.
14. Wait 16+ hours.
15. Centrifuge down cells at 4°C, 7500 rpm, 5 min and washing with phosphate buffer 3 times.
16. (Optional) Freeze pellet in -20°C freezer.

Lysing Cells

17. Fill foam box with ice and water; place cell sample on ice (ice in R506).
18. Add 1x PBS buffer to cell sample (5 ml per gram of wet-weight cells). Use pipet to help break up pellet.

1 g cell pellet

5 ml 1x PBS

19. Vortex the cells to resuspend.
20. Add PMSF (in Freezer 1, #108, 50ml tube, covered with foil) to prevent protease degradation of the proteins:

5+ ml cell sample

50 ul PMSF (final concentration 1 mM)

21. Sonicate cells to lyse (7 watts, 10 x 30 sec. rounds with 30 sec. breaks (6 minutes total), amplitude 50%).
22. Add lysozyme (20 mg/ml) to cells to facilitate lysis (lysozyme in Freezer 1, #108):

5+ ml cell sample

50 ul lysozyme (20 mg/ml)

23. Incubate on ice.
24. Wait 1 hour.
25. Sonicate again (7 watts, 10 x 30 sec. rounds with 30 sec. breaks (9 minutes total), amplitude 50%).

Protein Extraction

26. Centrifuge lysate (4°C, 7500 rpm, 10 min.).
27. Transfer supernatant to high-speed centrifuge tube.
28. Centrifuge supernatant (4°C, 12000, 30 min.).
29. Wait 30 minutes.
30. Syringe out supernatant, and filter into new tube.

Column Chromatography

31. Make sure that binding buffer is connected, not the nickel stripping solution.

Conjugation of proteins with Nanoparticles:

1. Prepare 200ul G3P solution (0.6mg/ml)

2. Prepare 300ul PBS and add 0.458mg NHS, 0.62mg EDC in PBS

3. Mix 1 and 2 and put it on shaker for 30 min (room temp)

4. Add 500ul MCNP@mSI-NH2 into 3 solution.

(concentration of original particle solution: 1 mg/ml)

5. Incubate it for 12 h at room temp, without light (cover it with Al foil)

6. Centrifuge (?? rpm, ??min) and aspirate supernatant.

7. Fill up with PBS to make 1ml solution

8. Repeat step 6 and 7 3 times

9. Storage the particle at Fridge (4’C) without light

 

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