25 9 / 2013
To understand how bacteria become resistant to antibiotics on a molecular level, the way antibiotics work must first be explained. In a later post I will explain how resistance spreads but for now we will focus on the molecular mechanisms involved in resistance development.
Antibiotics mainly target and disrupt 3 fundamental processes in bacteria:
1) Cell wall biosynthesis
2) Protein synthesis
3) DNA replication and repair
An important thing to remember is that antibiotics MUST target fundamental processes that are different in bacteria than in our own cells. We wouldn’t want antibiotics to work on our cells because then they would be toxic! Below is an example of the resistance mechanisms that developed to prevent the action of penicillin.
Targetting Cell Walls - The Penicillin Example
How it works: Penicillin is an example of the class of antibiotic that targets bacterial cell walls. It doesn’t affect our own cells because eukaryotic cells don’t have cell walls. Bacterial cell walls are made up of glycan strands which are linked up by peptidoglycan units known as NAG and NAM. A bacterial enzyme, transpeptidase, joins up all the NAM molecules as you can see below. This makes the bacterial wall strong and protective. However, bacteria need to grow so sometimes they unpick the links between NAM molecules so that they can lay down more building blocks and expand. Following expansion, the NAM molecules are joined back up to provide strength once more.
Penicillin binds and modifies the transpeptidase enzyme so that NAM molecules do not get joined up quick enough. However penicillin does not prevent the cell from unpicking NAM links during expansion. So the overall result is that the cell unpicks the NAM links but isn’t able to then link them back together. This causes the cell to burst and die.
How resistance developed: In the case of penicillin, resistance develops quite simply. The gene that encodes the transpeptidase protein is mutated. The transpeptidase enzyme is altered in such a way that penicillin can’t bind and modify it and cell wall building goes on as usual.
Many people have heard of MRSA or methicillin resistant Staphylococcus aureus. It is often described as a superbug, is the leading cause of hospital acquired infections and is responsible for up to 40,000 deaths a year. It is resistant to many antibiotics including penicillin. MRSA has developed resistance to penicillin by creating a completely new transpeptidase enzyme instead of just mutating the old one. Penicillin does not work on this new transpeptidase.
What types of resistance are there?
Aside from preventing the antibiotic from acting on enzymes important to the bacterium there are several other ways that bacteria can become resistant. They can:
- Pump out the antibiotic
- Destroy the antibiotic
- Become impermeable to the antibiotic
Does resistance develop to all antibiotics?
Molecular resistance mechanisms will develop, it is more a question of when and how. The development of resistance mechanisms is a natural part of evolution in which bacteria simply become better at surviving. Resistance develops in the following way:
- You have a pool of bacteria in an infection which are being attacked by an antibiotic
- Mutations occur in bacteria when they are dividing
- Statistically, there is a chance that at least 1 bacterium will mutate in such a way that the antibiotic won’t affect it, like we discussed with penicillin.
- That 1 bacterium now has an increased chance of survival compared to the other bacteria that haven’t mutated, it has become resistant
- That 1 bacterium will divide and produce more bacteria that also have this increased chance of survival
- Eventually, the infection pool will be made up mostly of the bacteria with this resistance
- The pool is now resistant to the antibiotic
In any infection, there are large numbers of bacteria. To make matters worse, bacteria divide and new bacteria are generated very rapidly. If you imagine that there is a mutation rate of 1 in 107 and there are 1010 bacteria in a particular infection pool it is easy to see how the above scenario would occur. So, statistically, resistance will develop at some point in some infection. The problem doesn’t end here though. If this was the only trick up the bacterial sleeve, then resistance could be contained more easily. Bacteria are able to do what is known as horizontal gene transfer. They can pass their genes to other bacteria in their pool, not just to their daughters. So if you have a resistant bacterium lying around on a remote control in a hospital, it can pass its resistance on to other bacteria in the general environment.
To put everything into context, it’s useful to look at vancomycin as an example of the inevitable resistance that arises. Vancomycin, discovered in the 50s, is an antibiotic of ‘last-resort’ treatment and is sometimes used to treat serious MRSA cases. It took approximately 30 years for resistance to vancomycin to happen but it did. The resistance that developed is highly complex which is why it has taken so long but this should serve as an example that even really powerful antibiotics will be evolved around eventually. This is why the scientific community must endeavor to discover or create new classes of antibiotic. However, this is not enough. As we approach a public health crisis, we must think of new ways to treat bacterial infections and we must amend policy to slow down the approach of the post-antibiotic era.
17 9 / 2013
Most people are familiar with the story surrounding the accidental discovery of penicillin by Alexander Fleming in 1928. Fleming left a culture plate of Staphylococcus lying around in the lab for two weeks while he was on vacation, only to return and find the plate had become contaminated with Penicillium fungus/mold. The Staphylococcus bacteria seemed unable to grow near the mold and he concluded that Penicillium prevented the growth of these bacteria.
Few are familiar with Howard Florey and Ernst Chain, two of several scientists who would take this observation and change the course of public health. Penicillin first needed to be purified and concentrated efficiently and this process was as painstaking as it was time consuming. Subsequently, penicillin was tested in mice and it was found that all mice that were given penicillin lived whilst untreated mice died. In 1941, penicillin was administered to patients for the first time and it was noted that if the penicillin supply ran out before the completion of treatment the patient would relapse and die. It was clear by this point that pharmaceutical firms were needed to produce penicillin on the scale required.
By this point, World War II was raging on and one of the most important decisions in the history of science was about to be made. Chain wanted to patent penicillin but Florey believed this would have adverse consequences in the war climate of the time. Florey was committed to the idea that this discovery would and should benefit all mankind. Penicillin soon shot to fame as the “magic bullet” for infectious disease and the scientific community entered the golden era of antibiotic discovery (1945-1960) during which numerous new classes of antibiotics were discovered.
Fast forward about 60 years and the picture is somewhat different. Gone are the adverts proclaiming antibiotics cure all and we are instead faced with the prospect of entering the post-antibiotic era or, as many sensationalists like to call it, “pharmageddon”. Discovery of new classes of antibiotics is rare, as can be seen in the timeline above. Equally worrying is the spread of antibiotic resistance which is dismantling the arsenal of protection we have built up over the last decades. These are complex issues which pose a real threat to public health globally. We will look at how bacteria develop resistance to antibiotics, what the implications of this are, proposals to mitigate the issues and alternative solutions for treatment in the future.
(Timeline Source: Gerard Wright, Nature 2007)
16 9 / 2013
Anonymous asked: If radiation causes cancer then why do doctors treat cancer with radiotherapy?
Great question! I hope this answer is detailed enough for you (there’s quite a bit of background information to get through first).
So first of all, I should clarify the term ‘radiation’. Radiation can be ionizing (high energy and harmful) or non-ionizing (low energy and not generally harmful). X-rays and gamma rays are types of ionizing radiation and can be harmful whereas visible light is an example of non-ionizing radiation which isn’t harmful. UV rays can be ionizing and non-ionizing, however most ionizing UV rays get filtered out by our atmosphere before they reach us. Nevertheless, UV rays can cause damage through other means.
Ionizing radiation is so high in energy that it causes electrons to be ejected off atoms. So now you’ve got a negatively charged electron floating around as well as a positively charged atom. As a general rule of thumb, charged things like to react with other things and this is exactly what happens. As a result, you get the production of free radicals. Free radicals are things like hydrogen peroxide and they are super reactive! They can react with and damage the DNA backbone. You definitely do not want to damage your DNA because that contains all the information for how a cell should work and damaged DNA can lead to tumour formation if it is not repaired.
(Random cool fact: Mammalian cells can experience up to 200,000 hits which cause damage per day! Your cells successfully deal with damage all the time!)
In general, ionizing radiation causes double-stranded breaks, single-stranded breaks and cross-linking within a DNA molecule. You can see what this damage looks like in the image below. Double and single stranded breaks occur when the DNA backbone is disrupted whilst cross-linking is when the bases within the DNA form covalent bonds with each other (as indicated by the red line, here only what is known as intrastrand cross-linking is shown for simplicity).
In “normal” cells, there are a number of different repair mechanisms to fix all these types of DNA damage (though sometimes these can fail and lead to cancer). In cancer cells, repair mechanisms are often impaired. The purpose of radiotherapy is to try to target only cancer cells which do not have the capability of repairing the damage, thereby killing them or preventing them from growing further. This is why cancers which have heightened repair mechanisms such as gliomas are often resistant to radiation treatment. Chemotherapy works similarly, the aim is again to damage the DNA of cancer cells.
19 8 / 2013
Anonymous asked: My biology teacher told us about antibiotic resistance today. Can you explain how this affects countries and what is going on?
Antibiotics, the way they are used, problems in their availability to developing nations and resistance are actually topics I’m really interested in. I don’t think a brief answer will do this topic justice but I’ve been planning an in depth series of posts on this for a while so keep an eye out and I promise you’ll hear something soon!
12 8 / 2013
Genetic testing is a powerful technique which often involves taking a blood or tissue sample and examining the DNA for a known mutation that causes a particular disease (eg a mutated BRCA1 gene).
How does a person know if they should have a genetic test for BRCA1/BRCA2 mutations?
Depending on where you live, genetic tests can be expensive and they may also be a source of emotional stress. Therefore, a genetic counsellor can advise whether a test would be beneficial or not. Selection of individuals for genetic testing is based on a risk assessment, so those who are calculated to have a high risk will likely be advised to be tested and vice versa. There are actually several types of risk assessment and each has strengths and limitations. Below are two examples of different types of risk assessment which may be used:
Claus model – This assessment decides what the individual’s risk is based on factors including:
- How many first and second degree relatives have had breast cancer and at what age they were diagnosed (this is because if there are many close relatives that suffered breast cancer at an early age, there is greater likelihood that there is an inherited mutation at play).
The limitations with this model are that it doesn’t take into account more than two relatives and that it doesn’t include tumours in other areas of the body. The reason the latter is important is that as we discussed previously, mutations in BRCA1 and BRCA2 have been associated with other cancers too.
Gail model – This assessment decides what the individual’s risk is based on factors including:
- The age at which a woman experienced her first period (formally known as menarche)
- The age at which the woman had her first live birth
- Results of any previous biopsies
- Presence of any typical hyperplasia (increase in number of cells)
- First degree relatives diagnosed with breast cancer
The limitations of this model are that it excludes second degree relatives, paternal relatives (as discussed, men can get breast cancer too!) and age of diagnosis. As a result, it could underestimate the risk despite someone having a strong family history. However, a professional has a full understanding of the limitations and strengths of different models and so will be able to advise an individual as to their risk.
In a previous post, you said mutations in BRCA genes have been associated with colon cancer. Are people with relatives that have had colon cancer screened for BRCA mutations?
There is a bit controversy over this! One study showed that a BRCA1 mutation can give rise to a 2-fold increase in risk of colon cancer. However, it is important to note that the colon is in anatomical proximity to the ovaries and some professionals have questioned whether the results of this study are due to a misdiagnosis of ovarian cancer. Other studies have shown that colon cancer is not associated with BRCA mutations.
How is the genetic test actually performed?
The DNA is sequenced and scientists receive a sensitive readout of the nucleotides in the DNA. From this information, they can determine if your BRCA genes are “normal” or if they have a mutation associated with breast cancer risk. The technology for sequencing is booming and there are many techniques which can be used. However, it is still expensive and still not as fast as we want it to be! Developing fast, effective and cheap technologies is paramount. Aside from this, we also need to develop the capability to store all this information in order for this technology to truly take off.
Once you’ve had the genetic test, how is the lifetime risk of breast cancer calculated?
The first thing to highlight here is that such a test predicts your risk. It does not spell out your fate. Angelina Jolie had an 87% risk of developing breast cancer. This percentage may vary for different people. This is because the development of breast cancer is under the influence of multiple factors. Here are some points to help you understand why risk is different for different people:
- There are many different types of mutations of the BRCA1 and BRCA2 genes
- There are many variants of the BRCA1 and BRCA2 genes. The BRCA2 gene has at least 1,893 variants!
- Each individual has a different genetic background. Think of this as the BRCA1 and BRCA2 genes being like well known actors and all your other genes like different movies. There are a limited number of actors but a huge abundance of different films. The actors act differently depending on the film they’re in. Different BRCA1 and BRCA2 genes will affect cancer risk differently depending on what your other genetic material looks like and that varies greatly between people!
- Everyone is exposed to different risk factors in their environment. This can be due to lifestyle, socioeconomic status, geographical location etc.
- If you have a mutation in the central region of the gene then your risk is likely higher than if it is at the end of the gene (if you’d like to know the molecular details of why, don’t be afraid to ask!)
What are the outcomes of a genetic test?
There are three main outcomes. These are:
Positive – A mutation has been found which is associated with an increased risk of developing the disease (note again, this does not indicate complete certainty of developing the disease). This outcome is actually able to tell you about more people than just the person tested. Relatives of the “positive” individual can be classified as carriers. It also tells you that if you have children, they MAY inherit the mutation you have. There are ethical considerations here which are a source of debate. If your relatives are likely carriers, should this information be shared with them? Not all people want to know they are carriers of a mutation. Other ethical considerations include whether insurance companies should have the right to know the results of such tests and whether they should have the right to prevent positive individuals from buying health insurance.
Negative – This is slightly less straightforward. If an individual is positive, but the relatives are found to be negative then this means that the negative relative has a risk like the rest of the population. If an individual has no BRCA mutations but there IS a family history then there is a chance that other genes are responsible or there is a shared environmental factor. Alternatively, it could just be chance that there is a family history.
Inconclusive – This means a mutation has been found that is unknown or which shouldn’t result in an increase in disease risk. This requires the clinician to examine how significant the mutation is. One way of doing this is to test all members of the family that have developed breast cancer and see if they all have this previously unknown mutation. If they all have it, the clinician may conclude that there is an association between cancer developing and this particular mutation. This would then need to be followed with larger studies to test whether this seems to be the case in the general population. Lab experiments would also be needed to test the structure and function of the protein resulting from this unknown mutation.
What happens if you test positive?
There are a number of options available to reduce the risk of developing cancer, including:
- Prophylactic surgery in which breasts or ovaries are removed
- Prophylactic medication such as tamoxifen which can be given to women at high risk
- Prophylactic lifestyle changes
- Regular screening to ensure that any cancer is caught as early as possible in order to increase the chances of successfully treating it
Finally, it is important to ensure that the public has access to information about genetic testing in order to encourage individuals that may be at increased risk to seek medical advice. In parallel to this, we must push for more affordable genetic screening and greater provision of such services globally.
09 7 / 2013
Two decades ago, Mary-Claire King showed that a gene on chromosome 17 was associated with breast and ovarian cancer. This gene is now known as BRCA1. Since then, BRCA2 on chromosome 13 has also been identified. A few years after Mary-Claire King’s discovery, it was observed that alterations in the BRCA1 gene were present in approximately 1% of the Jewish population. Alterations in other populations such as the general US population can be as low as 0.1%. Jewish populations were therefore of great importance in determining the cancer risk associated with particular alterations.
Scientists now think that women who have inherited a harmful alteration/mutation in BRCA1 or BRCA2 are five times more likely to develop breast cancer in their lifetime than those without a harmful mutation. The risk of ovarian cancer is thought to be anywhere between 10 and 30 times higher. It is important to note that these results were acquired by studying families which carry such alterations and who likely share the same environment. Further data from studies of the general population over a long period of time may change these figures. However, the fact remains that harmful mutations in BRCA1 and BRCA2 increase the risk of breast and ovarian cancer. Harmful mutations are not only thought to increase the risk of breast and ovarian cancers in women, the diagram below shows what other types of cancer may be associated with these mutations in both genders:
Molecular Roles of BRCA1 and BRCA2
Human or mouse cells that are deficient in BRCA1 and BRCA2 appear to accumulate DNA damage in the form of chromomal breaks or aneuploidy (an abnormal number of chromosomes). Mice that are engineered so that BRCA1 and BRCA2 can be knocked out appear to form tumours in their mammary glands. This suggests a link between breast cancer, DNA damage and mutated BRCA genes.
However, tumours in these mice also have mutations in other genes such as the p53 gene, which expresses a protein fondly known as the guardian of the genome. The reason this is important is that when our DNA becomes damaged (for example, by UV light), the BRCA proteins are involved in repairing this damage and preventing events such as aneuploidy. If the BRCA genes are mutated, the proteins produced are not functional and cannot repair DNA. In this case, p53 detects that the damage is not being repaired and so it can initiate cellular programmes which result in apoptosis (cell suicide). This removes the DNA-damaged cell from the tissue. However, if p53 is also mutated (as is seen in mice with mammary gland tumours), then the cell does not commit suicide and instead it continues to merrily divide. In this process, it passes on its damaged DNA to its progeny which acquire further mutations and the story quickly spins out of control, giving rise to a tumour.
Because of their molecular role in repairing DNA damage, the BRCA genes are known as tumour suppressors. This means that when they are in their normal form, the act to prevent the formation of tumours by ensuring that any damage is quickly repaired. When they are harmfully mutated, they lose this tumour suppressing activity. There are many other tumour suppressor genes which when lost, play a role in tumourigenesis. Some of these have been implicated in breast and ovarian cancers.
The precise molecular details regarding how BRCA1 and BRCA2 operate in the cell are still being investigated. BRCA1 and BRCA2 mutations are only associated with an increased risk in a small subset of cancers in particular organs. One would expect that two genes which play such an integral role in DNA damage repair should predispose to cancers of all types when mutated. This is perhaps one of the most intriguing questions in the field of cancer biology. In essence, why do particular mutations cause particular types of cancer? In the next post, I will discuss the importance of genetic testing.
11 6 / 2013
Breast cancer is the most common cancer in the UK and in many other countries. It can be classed as ‘hereditary/familial’ or ‘sporadic’, with the latter being most common and arising spontaneously with no prior family history. The term ‘hereditary’ here is highly misleading and one that is not often explained in the media. It implies that you inherit a faulty gene and that you will therefore, at some point in your life, develop cancer. This is not the case, it is actually that there is an increased risk of developing cancer. Cancer is a result of many deregulated processes and failed safeguards, not just a single faulty gene. The term ‘familial breast cancer’ is perhaps better suited.
Approximately 10-15% of breast cancer cases are ‘familial’. This might seem like a small number but it is in fact quite large considering that breast cancer is the most common cancer in many countries. The genes BRCA1 and BRCA2 function in normal cells to ensure that DNA is not damaged and mutation of these genes is responsible for most familial breast cancers. When BRCA1 and/or BRCA2 are mutated, the cell loses some of its ability to repair damaged DNA. This leads to an increase in genetic mutations and this in turn can lead to deregulated cellular processes and loss of safeguards as described earlier. This loss of genomic integrity is a classic hallmark of all cancers. Mutations in these genes also play a role in ovarian cancer as well as male breast cancer.
A few weeks ago, Angelina Jolie released a statement describing her decision to undergo a complete mastectomy following a genetic test which showed she was at increased risk of developing breast cancer. In this series of posts I will describe why the BRCA genes are important, how they can increase the risk of cancer, what general factors increase the chance of developing breast cancer regardless of genetic predisposition and finally the importance of gene testing and intervention at early stages.