Abstract
Since the 1960\’s the MRSA bacterium has been a global epidemic. MRSA, methicillin-resistant staphylococcus aureus, is a type of bacterium that resides on and inside humans. Because of the usage of antibiotics to treat this, selection and procreating of mutating methicillin-resistant bacteria remains a continuous threat. An alternative against the plague of resistant bacteria has to be found. The SAATELLITE study within the COMBACTE program5 is researching the development of an antibody that decimates the component inside the bacterium that damages our cells: its alpha toxins. Before this solution can be realised, studying the different naturally occurring mutations of alpha toxin and their cell lysation-efficacy is essential. In this experiment, we studied 3 different mutations of alpha toxin and compared their toxicity to that of the wild type. Using molecular cloning techniques, the MRSA bacterium\’s genetic code for alpha toxin was placed into samples of the E. coli bacterium. From the results of the experiment after, it may be deduced that the mutations need a higher concentration of alpha toxins to be as effective in hemolysis as the wild type is. Apparently, the mutated alpha toxin did not result in the bacteria’s increase of alpha toxin functionality. The different varieties of alpha toxin analyzed were all slightly less toxic than the wild type. Possibly, the mutation provides other advantages for the bacteria, that are not yet known.
Introduction
During World War II it was discovered that penicillin, a group of antibiotics derived from penicillium moulds, was extremely effective against many bacterial infections. Doctors believed that infections would never be a problem ever again and penicillin was prescribed freely and abundantly. However, in the 1960s a type of antibiotic resistant bacteria was found; the Methicillin Resistant Staphylococcus Aureus, or MRSA.3 MRSA resides on about 2% of people. Their skin, nose, mouth and other places where it is about 37 °C (310 °K)4 are locations where MRSA is likely to be found. Usually, man’s immune system is perfectly equipped to handle the bacteria, and most people will hardly experience any symptoms. However, in places with people with a weaker immune system such as hospitals or nursing homes, MRSA can incur serious damage. Once this hospital-acquired infection has taken over, there is almost no exterminating it.
Nowadays, it is known that doctors\’ excessive use of antibiotics for even minor infections has stimulated the development of resistance in bacteria. Where, before, MRSA was an inconvenient hospital-acquired infection, now it has become an international epidemic. In most western countries, there are strict guidelines to confine and prevent resistance. In some however, for example Spain or Portugal, incautious and excessive use of penicillin is still the status quo5, causing an increase in resistant bacterial strains.
Before antibiotics were widely used, it was relatively rare for bacteria to gain resistance, because this specific genetic adaptation or mutation did not create an advantage for them. When antibiotics are used, the susceptible bacteria die, while the rare resistant types survive and procreate, resulting in an increased number of resistant mutants.
The MRSA used in this study is the mutant version of S. aureus, a gram-positive, round bacterium, meaning that its bacterial cell wall does not consist of a peptidoglycan and outer membrane, but a few layers of peptidoglycan instead. This peptidoglycan layer plays, among other things, a role in serotyping. In total, 21 different MRSA mutants exist, all with minor differences in structure and efficacy.
MRSA bacteria produce proteins, among them: alpha toxin, or Hla. This protein punctures red blood cells, causing them to die eventually. MRSA attaches itself to a pure lipid target membrane, where a heptameric complex forms. This complex creates a water-filled channel in the membrane. This channel acts as a tap, all molecules can flow freely into or out of the cell. This causes the cell to either shrink or explode, starting emergency reactions, such as apoptosis. These require a significant amount of energy. The loss of energy is enhanced by mitochondrial failure and ATP leakage. The cell then starts autolysis, self-destruction using its own enzymes. In cells with comparable problems ESCRT-dependent release of cellular substances can postpone programmed cell-death in punctured cells.6 Perhaps this could be a future aid to the research on MRSA-related pores.
This study was performed as part of a COMBACTE-research programme, called SAATELLITE (Human Monoclonal Antibody Against Staphylococcus aureus Alpha Toxin in Mechanically Ventilated Adult Subjects).
SAATELLITE is the first programme conducting tests using anti-infective drugs besides antibiotics. The programme is part of the New Drugs 4 Bad Bugs (ND4BB) organisation. “SAATELLITE is a Phase II, randomized, double-blind, placebo-controlled trial.”7 This trial tests the effects of the potential drugs in terms of safety and its efficacy. The drug is called MEDI4893. MEDI4893 is “a promising monoclonal antibody against S. aureus alpha toxin. […] MEDI4893 uses an antibody – not an antibiotic – against this alpha toxin.”7 This means that resistance is no longer relevant, because the antibody would react either way. Another benefit is that it is unlikely that there will be antibody-resistant organisms, therefore, the drug is sustainable and effective in the long term.
For the producer of this antibody and possibly future medicine, it is vital to study mutations in the part of the DNA that codes for the alpha toxin. Whether the antibody works for all existing mutations we will have to find out by examining the toxic activity of all mutants compared to the wild type.
This study analyses the efficacy of three of the total of 21 mutations of Hla in hemolysis.
While the hypothesis is that the mutations will be more effective than the wild type because the mutants would not survive otherwise, it is possible that the mutated bacteria survive or even thrive because of other advantages that would have to have nothing to do with production and efficacy of alpha toxins.
Method
PCR8
First of all, 3 genes (60, 62 and 63, for genetic codes view appendix) are multiplied using PCR master mix. This mix consists of the forward and the reverse primer to the genomic S. Aureus DNA. These primers hold the genetic code for the beginning or the ending of the part of the nucleotide sequence of the DNA that codes for the alpha toxin production. These 3 substances, now consisting of one type of gene and the complying primer and reverse primer, are placed in the PCR-machine. During the PCR process several copies of the required specific DNA segment are made.
These DNA samples are loaded onto an agarose gel electrophoresis device to determine whether the PCR procedure has worked as it should have. During this process of electrophoresis an electric current through the agarose gel pulls the DNA towards the positive pole, because DNA is negatively charged. Smaller pieces of DNA move faster, which is why the fragments get separated and organised by length. This is the easiest way to make sure the correct fractures are collected and used.
Ligation and transformation
Secondly, the PCR products are purified with a PCR clean-up kit and placed in a centrifuge for further purifying.
The DNA pieces are ligated into plasmids. After incubation, these plasmids are added to E. coli samples, because of E. coli’s rapid reproduction, securing fast alpha toxin production. Then, to make sure the plasmids enter the cell nucleus of the bacteria, a heat shock is applied. The E. coli are able to produce alpha toxins now and to optimize their productivity, the bacteria are placed on a culture medium to incubate overnight.
Colony PCR9
Following a night of incubation, enough bacteria should have multiplied and colony PCR should be conducted. To do that, forward and reverse primers are added again, this time to a colony sample.
After the PCR procedure, the products are loaded onto an agarose gel for a validation check.
Next, the colony samples are centrifuged and using a plasmid purification kit the plasmids are isolated from the colonies. To ensure maximum productivity in the shortest time possible, the plasmids are placed into new E. coli bacteria and incubated overnight on an LB medium with ampicillin. The resistant bacteria, that coincidentally are also the ones producing alpha toxins, will survive this ampicillin treatment. Through a selection method comparable to the principle of survival of the fittest, the alpha toxin-producing bacteria are isolated.
Expression
The bacteria are now technically able to produce alpha toxin, but to get them to start actually doing so, expression has to be started. As soon as the bacteria have reached an optical density of 0.5 at 600 nm, there are enough bacteria. The optical density is a number used to indicate the DNA’s absorption rate. DNA absorbs light, therefore, when the density is high enough, there is enough DNA present to absorb most of the light.
Then, Hla expression is induced in part of the sample using IPTG, which activates the gene assigned to alpha toxin production.
Three hours later, both the samples with and without Hla expression are loaded onto an SDS-page gel to confirm whether or not the Hla expression was successful. In essence, the way an SDS-page gel works is broadly similar to that of an agarose gel, but another gel and medium are used.
Overnight, the bacteria are stored at 253 K.
Protein Purification
Using sonication, the bacteria are completely destroyed. This is necessary, because it is the only way to extract the alpha toxin. However, when a bacterium is destroyed, more than just alpha toxin comes out, so the whole composition has to be purified. Using a centrifuge, proteins are separated from non-proteins according to their weight. The alpha toxins are separated from other proteins using affinity column chromatography10. Using the column chromatography technique, the alpha toxins bind to the stationary phase because it is lined with zinc, which other proteins do not react with. THerefore, only the alpha toxins will stick to the lining. Next, an increasingly high dose of imidazole is added to different samples of the now slightly purer mixture. This molecule reacts even better with the zinc lining than the alpha toxins do, which causes the alpha toxins to eject from the lining and drop. The spectroscopy graphs show when the alpha toxins drop and which samples contain pure alpha toxins in high concentrations.
These samples are pooled and dialysed to PBS overnight in dialysis tubes. Because of this, the last small proteins are filtered from the samples, leaving the purest form of our alpha toxins achievable.
Functional assay
Finally, a result check is in order. To start off, the protein isolation must be checked using an SDS-page gel again. Samples from every step of the purification are loaded onto this gel. Furthermore, the Hla concentrations are determined by measuring an optical density of 280 nm, and the samples from the three mutations and the wild type are diluted from 300 ng/mL to 0.14 ng/mL on a well plate (see figure A).
Following that, rabbit blood is purified, to let only the red blood cells remain. The red blood cells and the alpha toxins are mixed together and put into the well plate and incubated for two hours on a shaker. Then the samples are centrifuged and lastly the optical density of 415 nm is measured.
Figure A: dilution of Hla with rabbit blood. From left to right, containing a lower concentration Hla.
Analysis
Figure B: Different steps of the purification on an SDS-page gel. From left to right the solution became purer.
From Figure B, the SDS-page gel showing the purification process, it may be concluded that the purification of the samples and the stimulation of the Hla production was successful, mainly in the SDS-sample added (sample 62). This is because the lines matching the Hla mass in comparison to the marker (location 1), have become much thicker and clearer, while the other lines, belonging to proteins with different masses and other solutions are not visible in the final samples tested (location 4, 6 and 8).
Figure C: plot of the lysation of the Rabbit blood cells (rabbit RBC’s) versus the concentration of alpha toxin 
This assay curve is the plot of the lysation of the rabbit red blood cells versus the concentration of the alpha toxin added in different stages of dilution as the centrifusion progresses.
As Figure C shows, while in smaller concentrations the difference between the mutants and the wild type (ATWT) is minimal, the less diluted the alpha toxins are, the more lysis takes place. Overall, all of the mutants tested needed to be present in notably higher concentrations to be equally as effective as the wild type.
Discussion
As the numbers for mutant S. aureus infections are not yet known, it is too soon to draw a definitive conclusion. Nevertheless, based solely on this evidence, it can be stated that the issue of finding a solution to control these specific mutants is less pressing than to find one for the wild type.
While there is not a lot of relevant data available about this subject, the result of the wild type being the most effective is reasonable, although remarkable. Because, from the final RBC hemolysis assay, all of the used mutants appear to be rather ineffectual in comparison to the wild type. This does not conform to the notion that mutants, to be able to exist, should be at least as effective as the wild type.
A possible explanation for this phenomenon could be that the hemolysis of the MRSA bacterium does not contribute to its survival. If that is indeed the case, the low efficacy of the mutants is no reason for them to become extinct and coexistence with the wild type would be the result.
Some limitations should be noted. First, there has been no repetition of the study. Therefore, we do not know the margin of error in our research and it might be possible to repeat the used protocol exactly and find minor or major differences in the results. However, the mutation-to-mutation ratio in the graph is quite certain.
And second, due to a vast time shortage, there was some rushing involved in the execution of the protocol which may have resulted in a deficient bacterial growth and protein production for the used techniques further on in the experiment.
To preserve accuracy, we performed the ligation and the transformation immediately in the morning, to let the bacteria rest during the day. This had to be rushed, to be able to complete the protocol, but it resulted in a relatively small amount of bacteria.
Now that the efficacy of the different mutants has been defined, the next step would be to test the difference in their effect compared to when the existing antibody is added. The designed MEDI4893 is now in research circulation and defuses the alpha toxins and with it the most harmful feature of the MRSA bacterium. Hereafter it is to be tested whether the antibody also binds with the mutants to result in the wanted deactivation.
Conclusion
The multi-resistant Staphylococcus Aureus is a type of bacterium that is resistant to several of the most common antibiotics. This bacterium uses its alpha toxins to target, among other things, red blood cells and to break down and puncture their membranes, effectively destroying the cell. These alpha toxins exist in 21 different types that we now know of: the wild type and its 20 mutants.
In this project different mutants of the same alpha toxin were tested on how effectively they could lyse rabbit RBC’s in different stages of dilution.
The results of this experiment indicate that, contrary to our initial hypothesis, the wild type lyses more effectively than all three of the tested mutants, because it starts its lysation in smaller concentrations than the mutations do.
Acknowledgements
UMC Utrecht, Medical microbiology
Frank Coenjaerts
Remy Muts
Femke Mulder
U-Talent Academy
Garmt de Vries-Uiterweerd
Sources
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(view appendix)
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Appendix
Protocol exp U-talent
Chromatography Scan 1-3
Genetic Code Mutants
Staphylococcal alpha toxin
Biography
Femke, Marit and Hilde are three Dutch high school seniors, who have a passion for the beta sciences; especially biomedical sciences. This lead them to the U-Talent Academy programme, which allowed them to do research at the Utrecht Medical Centre with the department of Microbiology. Next, Femke and Marit want to pursue a career in Medicine, and Hilde in the biomedical field.