What Are Our Defenses?
Puerperal fever is an infection caused by the bacterial contamination of a woman’s reproductive tract during and after giving birth. Today, one in eight women who deliver children will contract this infection, and three in every 100,000 women still die from it. Two hundred years ago, though, the mortality rate from puerperal fever—or childbed fever as it was known then—was about a thousand times greater. In the 1840s, Ignaz Semmelweis, a physician at Vienna General Hospital, noticed that women who gave birth in hospitals and stayed overnight were much more likely to die from childbirth fever than women who gave birth at home. Why was the hospital such a dangerous place to give birth? As Semmelweis commented in his treatise on the disease, “Everything was in question; everything seemed inexplicable; everything was doubtful. Only the large number of deaths was an unquestionable reality.” To Semmelweis, observation was the way to stem the tide of the disease. He first started to observe the hygienic habits of doctors, who at the time refused to wash their hands before surgery; in fact, the stiffer their operating gowns were with grime and blood, the higher respect they garnered. It wasn’t until one of his colleagues died of a terrible infection after cutting himself during a routine autopsy in the hospital’s morgue that Semmelweis’s powers of deduction clicked. His colleague, a Professor Kolletschka, had contracted symptoms very similar to childbed fever. In a burst of inspiration, Semmelweis commented, “I was haunted by the image of Kolletschka’s disease and was forced to recognize, ever more decisively, that the disease from which Kolletschka died was identical to that from which so many maternity patients died.” Semmelweis then started to trace the disease to its common causes. He noticed that there were two clinics where women went to give birth in Vienna: one was at the medical school in Vienna (First Clinic) and the other was a clinic where midwives were trained and delivered babies (Second Clinic). The results of this examination were puzzling to Semmelweis, because the midwife clinic had far less mortality than the medical school clinic (see Figure 5.1, which includes a translation of the original manuscript).
Semmelweis reasoned that surgeons delivering babies were transferring something from the autopsy room to the delivery room that was causing the deaths in the medical school clinic. Sadly, he recognized that “only God knows the number of patients who went prematurely to their graves because of me.” Semmelweis published a paper reporting his observations and was roundly criticized by the medical community of the time. In this paper he recommended that surgeons wash their hands in chlorinated lime solutions before doing any kind of procedure, including the delivery of babies. While his colleagues were not impressed with his ideas (his reappointment at the Vienna General Hospital was denied), Semmelweis could at least feel comfortable that his individual work had saved lives.
Figure 5.1. Data table from Ignaz Semmelweis’s work on puerperal fever. The “first clinic” refers to the medical school of Vienna; data from the “second clinic” came from the midwife training clinic. Semmelweis’s observations led to hand-washing guidelines for doctors, saving countless lives.
Joseph Lister, a British surgeon, did not read Semmelweis’s work when it first came out. He did, however, read in detail the work of Louis Pasteur, which led him to the very same conclusions that Semmelweis had made. Lister noticed that phenol compounds were often placed over rotting, smelly sewage. The phenol eliminated the smell, but Lister noted that the smell had to be coming from microbes. He proceeded to develop a wash solution for surgeons that today bears his name. Listerine is nothing more than a simple compound called carbolic acid. How it worked did not matter to Lister, but it did to a chemist named Emery I. Valko and his colleague, A. S. DuBois. Valko suggested that soaps such as carbolic acid simply “narcotize” bacteria rather than kill them. Valko also showed that narcotized bacterial cells could be revived after treatment with soap. According to Valko, the positively charged carbolic acid has this narcotizing effect and prevents the bacterial cell from growing. If the positive charges are removed or neutralized, then the bacteria come back to life. Chemically what happens is that the bacterial cell wall is made of lipids with positive and negative ends. This allows the bacteria to stick to body parts such as the hands that have different charges because of the dirt, grime, or grease on them. When you wash your hands with soap, the soap both influences the cell membranes of the bacteria and loosens the hold that bacteria have on the surface of the hands, allowing the bacteria to rinse off more thoroughly. This work was so exciting that it was reported in Time magazine in 1942. (Some modern soaps also contain anti microbial chemicals, and these work very differently than the classical soaps. We will return to this issue in due course.)
A much older method of staving off infection had its origins in India and China. This method, perhaps several millennia old, involves treating a person with material from a similar but less dangerous version of a deadly disease in order to inoculate the person against contracting the more dangerous disease later in life. Smallpox vaccination is the poster child for this approach: material from the benign cowpox disease was very successfully used to inoculate people against smallpox, a terrible killer virus. There are several other diseases that have also been treated in this way.
One of the most prolific vaccine developers was an interesting scientist from Montana, Maurice Hilleman. Raised as a strict Lutheran, Hilleman’s sharp intellect even as a child pushed him toward understanding the world in a scientific context. Evolution was an important part of how he understood biology. His desire to understand the natural world in an evolutionary context drove him to read On the Origin of Species in, of all places, his church. He became a microbiologist, and over his illustrious career with drug companies such as E. R. Squibb and Merck, he developed more than forty vaccines. He had a keen understanding of how the human immune system works and how it recognizes microbes when they invade the body. Although the molecular and genetic mechanisms of how the immune system works would not be discovered until the last quarter of the twentieth century, he was the most prolific warrior in the war on microbes of the mid-1900s. Hilleman did not garner as much fame as Jonas Salk and Albert Sabin, who created vaccines against the devastating disease polio, but his understanding of how microbes, and even parts of microbes, kickstart the immune system was well advanced.
How vaccinations stimulate our immune system is an interesting story that can only be told by first describing our immune system and the immune systems of other organisms. It is also a story that is relevant to our understanding of how the microbes in our microbiomes coexist with us. It has been pointed out that it is much easier to describe what our immune systems are than what the immune system does. The human immune system is, in one word, “complex,” and in two words, “very complex.” To explain it in a thousand words, or ten thousand or even a hundred thousand words, is indeed a difficult task. So we will only delve into the “tip of the iceberg” of the immune system. But by taking an evolutionary approach, we can make some dent in the complexity. Perhaps the most important message is that immune systems evolved through common ancestors that had very different challenges than humans presently experience.
The first step in immunity is the recognition of self. If you are a cell and you evolve a mechanism to destroy other cells, you had better not destroy your own cells through “friendly fire.” So systems that recognize self and non-self are of first and foremost importance.
How the Immune System Began
One might think that microbes have rather simple lives. With respect to the three rules by which organisms live (I run away from that, I eat that, and I mate with that), they probably spend most of their time dealing with the second rule. It certainly seems as though they have very little capacity to run away or defend themselves. After all, they are single cells with very rudimentary defense systems. But as we learned in Chapter 4, microbes can communicate with members of their own species in order to initiate coordinated responses to the environment using a process known as quorum sensing.
Consider, for example, the incredibly interesting case of bioluminescence in the light-producing organ of the Hawaiian bobtail squid. These squid have evolved a mechanism whereby they produce light from this organ for camouflage—that is, these tiny squid can hide their silhouettes from predators by controlling the amount of light that emanates from their bodies. To control the amount of light emitted, they have coevolved a symbiotic relationship with a species of bacterium called Vibrio fischeri. (Another species of Vibrio, Vibrio cholerae, is a terrible pathogen in humans causing cholera, and the story of how it became pathogenic is fascinating but beyond the scope of this chapter.) In exchange for nutrients supplied by the squid, V. fischeri makes light in organs that the squid has evolved called photophores. For the light to both be produced in the photophores and be bright enough to camouflage the squid, there need to be enough V. fischeri and they need to make the light simultaneously. In fact, only if the concentration of bacteria in the photophore is over 100 billion cells per milliliter will there be enough light produced to fool the predator. So how does V. fischeri count to 100 billion? The V. fischeri in the photophore use quorum sensing to sense the critical population size and, in turn, produce the molecule (called luciferase) that causes the bioluminescence only when it matters. Specifically, the bacteria make small amounts of an autoinducer, and only when the photophore attains a critical population size of 100 billion is a threshold reached. At this point the autoinducer starts to bind to receptors that trigger the synthesis of luciferase by genes in the V. fischeri genome and, voilà, there is light! Light that benefits both the squid and the bacteria.
Bacteria need to communicate with each other, and they do so by using molecules. They are also made up of molecules that have evolved over billions of years, and these molecules look quite specific compared with the molecules we use in our bodies. Like quorumsensing components, these other molecules are designed specifically to do the job the bacteria needs and so they look very foreign to the eukaryotic cell. It is this foreignness that the immune systems of animals, and—believe it or not—plants, use to recognize self from non-self.
The Body’s Defense System: A Bacterium’s Experience
Pretend you are an infectious bacterium. What would you encounter as you moved into a new home in a human body? There are many routes into the body through which you can enter, and some of your infectious colleagues have developed specialized ways of entering the bodies of their hosts. Some will hide out in food or liquids and try to enter when the host eats. Still others lurk around the genitals and in the fluids that are part of their host’s sex act. Others enter through cuts or simply get into the mouth and find a soft tissue to colonize. And some will simply reside in the crevices of the body, such as the belly button, or the armpit, or between the toes. For this exercise, imagine you are one of those airborne bacteria that have specialized ways of entering the body through the respiratory tract. You float in the air, waiting to be sucked in by the inhalation of your victim. Your goal as an airborne bacterium is to get to an appropriate tissue such as the lungs, find a nice place to stick, establish yourself, and divide to produce offspring with your genome in them.
You hope you don’t encounter skin, because in animals it is the first real line of defense. Skin has evolved to be very exclusive as to what it lets in and out. Once you encounter skin, your goal will be thwarted. It is dry and inhospitable. But as you cruise by, you notice there are a lot of other species of bacteria happily living on the skin. You luck out and miss the skin, and are sucked into the nasal passage, where you encounter a bunch of hairs that threaten to knock you away from your goal. You manage to dodge them, then hurtle through the nasal passage and on to the lungs. There you land in one of the many tiny passageways of the lungs called an alveolus, where you encounter a landing strip lined with mucus. You might have defenses against some of the chemicals being secreted into the mucus, and if you get past them you alight on the external tissue of the alveoli. You establish yourself and start to make proteins, an activity that makes it clear to the host cells that your intention is to stay. This is when your host’s body starts to respond at the cellular level—rapidly and with a vengeance—with what is called its innate immune system. The response is vicious and well planned, because it is the product of millions of years of evolution.
You, the bacterium, have made it as far as the lungs. But as you sit in the alveoli trying to establish yourself, a cell about one hundred times larger than you stalks you with every intention of engulfing you. This cell, known as a neutrophil, is incredibly persistent, because it can sense your presence. It is part of the innate immune system of the vertebrate you have infected, and its job is to consume bacteria like you through a process called phagocytosis. Many kinds of cells other than the immune cells in vertebrates can phagocytose. In fact, some single-celled organisms make their living by engulfing other living microbes. As we pointed out in Chapter 1, two of the eukaryotic organelles, mitochondria and chloroplasts, are the result of early eukaryotic cells having engulfed bacterial cells.
The human immune system, however, has a more complicated system for phagocytosing invading material, because the cells involved are very selective about what they eat. The bacterium you represent secretes chemicals called chemokines that leave a trail of “bacterial stink” that a phagocytic cell can follow due to its chemokine receptors. (There are some great videos on the web showing phagocytic cells of the human immune system persistently chasing down invading bacteria; see References and Further Reading.) The chemokine receptors of the neutrophil chasing you, the bacterium, have been produced by genes in the genomes of the host you have invaded. There is a whole family of these receptors, which makes them capable of organizing a lethal response to a wide variety of microbial threats. But how did this innate system we describe in your human host come to be so specialized and successful?
Green Immunity
Plants are our most distant multicellular-organism relatives on the planet. They are even less related to us than fungi (mushrooms and yeast). Yet they have evolved immune systems that can recognize self from non-self, and they too will try to destroy anything they sense as non-self. In addition, plants can mount very specific responses to the many bacterial species that come into contact with them and attempt to invade them, and they have an immune memory of some invaders. One of the major differences between human bodies and the bodies of plants is that plants can communicate with distal parts of their bodies only through a simple vascular system; they don’t have a highly organized circulatory system. A circulatory system allows animals to send molecules all around their bodies and enhances the rapid response to challenges our bodies face from infection. But without a circulatory system how do plants defend themselves?
When a pathogenic microbe encounters a plant, it is first faced with the plant’s tough cell walls, which can be fortified with specific molecules to enhance defense. This initial fortification response happens when the plant cells recognize bacterial molecules such as flagellin (a protein important in bacterial flagella) or other molecules that are specific to bacterial cells. They do this by sensing the bacterial molecules and binding them to receptors called patternrecognition receptors. Plant pattern-recognition receptors are actually similar structurally to animal pattern-recognizing molecules in the animal immune system response, such as Toll-like receptors. But most researchers suggest that the similarity in structure is convergent—that is, evolution in each lineage (plants and animals) has independently resulted in a similar and very efficient molecular plan for these receptors; neither lineage influenced the other.
These bound receptors trigger the production of resistance proteins (also known as R proteins), which plants then use to battle any infectious agents. The whole process is called a hypersensitive response, because it acts by destroying or causing the death of the infected host plant cells and by dispersing molecules that kill any microbes near those infected plant cells. Although this system is quite different in many ways from the processes that animal cells initially use to recognize infections, researchers have also called this an “innate” immune response.
Immunity in Lower Animals
When a neutrophil chases a pathogenic bacterium, it ignores other bacteria that it has identified as harmless. By looking at the immune systems of animals at the very base of the animal branch of the tree of life, we can understand why the neutrophil is selective in its pursuit. Lower animals have innate immunity, too. The common ancestor of multicellular animal life lived in a pretty harsh world 600 to 700 million years ago. Bacteria and other single-celled microbes had been around for a long time, and these new multicellular organisms, while they shared common ancestry with microbes, had to contend with a multitude of invaders and “ne’er-do-wells.” And although multicellular animals shared common ancestry with plants at some point in the evolutionary past, plants evolved their innate system of immunity after diverging from this common animal ancestor, which was left to develop its own strategies.
Although the evolution of the earliest multicellular animals, or metazoans, is controversial, with a search for the “mother” of all metazoa engaging researchers for the past decade, scientists do know which animals are at the base of the animal tree of life. By tracing the evolution of genes that provide extant lower animals’ immunity to infections from microbes, we can reconstruct the steps that were involved in molding our own immune response to microbial invaders.
Hydrozoans are bizarre-looking organisms from this lower part of the tree of life where a lot of research attention has been focused. They are members of a larger group of animals collectively called Cnidaria that also includes corals, jellyfish, and anemones. Hydra has a top end (logically called a head) and a bottom end (equally logically called a foot). It also has two cell layers—an endoderm and an ectoderm—as opposed to most other animals (including us) that have three (Figure 5.2). Because microbes in general colonize cell layers, it is important to keep track of these cell layers. There are also several related species in the genus Hydra that researchers use to study lower animal biology. Hydrozoans are very simple animals without a brain but with a nervous system called a neural net.
Figure 5.2. The two cell layers and neural net of a hydra (left), and the entire organism (right).
Lower animals such as hydrozoans have evolved an innate immune system, albeit a limited one. Like plants, they have patternrecognition receptors that sense the presence of molecules made by foreign organisms, mostly microbes. In Hydra these are called microbe-associated molecular patterns, or MAMPs. This system tries to recognize, as a way of sensing danger, molecules that bacteria make that are not made by the host hydrozoan. If a molecule like a bacterial heat-shock protein is floating around because bacteria are near, then one of the MAMP receptors in the hydrozoan innate immune system will bind to the heat-shock protein, and its binding will trigger two responses in the hydrozoan. First, the infected hydrozoan will make antimicrobial molecules that then attack the invader, and second, it will tell the infected cell to die. This latter form of cell suicide is called apoptosis, and while it seems cruel, it performs an important service for the rest of the hydrozoan body. There are two kinds of receptors that perform the innate immunity response in hydrozoa. The first, which we mentioned earlier, are called Toll-like receptors, whereas the second are called NOD-like receptors (for nucleotide-binding oligomerization domain–like receptors). Genes for both of these receptors also exist in higheranimal genomes and indeed are utilized in both the invertebrate and vertebrate innate immune response.
This innate immune system is an efficient way to protect the body from general invasion by microbes. But does it serve as a castle wall to keep out any and all foreigners? Thomas Bosch, a hydrozoan researcher, has suggested that the innate immune system is more than just a great barrier. By looking at the microbes associated with several species in the genus Hydra, he and his colleagues recognized that different species have different communities of microbes associated with them (Figure 5.3). In fact, the species of bacteria in these communities were very specific with respect to the different hydrozoan species. These results are important because they show that the association of microbes with their hydrozoan hosts is very specific. Another way to put this is that the microbes seem to have coevolved with their hosts. If microbes and Hydra have coevolved, then perhaps some of the microbial species are really important to the lifestyle and survival of the hydrozoan.
Figure 5.3. A phylogenetic tree showing the relationships of four hydroids is in the upper panel. The microbial community living in the various hydroid species is shown in the pie diagrams next to the species name in the top panel. The bottom panel shows the phylogenetic tree for the hydroid placed next to a network showing the similarity of the microbiomes from the four species of hydroid. Note that the two networks are identical.
What this implies is that some microbes actually need to be allowed past the innate immune system. In fact, perhaps our innate immune system evolved not to primarily prohibit infections, but rather to keep in proper balance those microbes that are beneficial to the existence of organisms.
Even if an invading bacterium shakes off the neutrophil, there are other molecules that can, and will, mount a defense. A system called the RNAi pathway, discovered in the late 1990s in Drosophila melanogaster (the fruit fly), is an important, innate defense mechanism that other animals and even plants have. Although the specifics of this and the other three pathways in Drosophila are beyond the scope of this book, a closer look at their basic mechanisms can start the discussion of how foreign molecular patterns trigger specific downstream responses.
The RNAi pathway acts like a Veg-O-Matic, one of the first food-processing appliances sold on television in the United States in the 1960s. If a foreign virus is detected by the system, a protein called Dicer cleaves the viral RNA. This RNA is then incorporated into a larger protein complex called a silencing complex, which then binds to viral RNA genomes. Next the silencing compound is recognized by a degrading system that then destroys that particular viral RNA genome (and anything else attached to that silencing compound). The other three pathways—Toll, Imd, and Jak-STAT—are called signaling pathways. When the receptors in these pathways notice molecular patterns specific to invading microbes and bind to those microbes, they also turn on, like a switch, the expression of antimicrobial genes in Drosophila. Some of these genes have names like cecropin A, drosomycin, defensin, drosocin, diptericin, and attacin A. Each of these antimicrobials, and indeed all innate system antimicrobials of animals, have different shapes and will attack different kinds of viruses, bacteria, or fungi. But all in all, their range of attack is relatively nonspecific.
Let’s return to the imaginary bacterial attack. While some of the molecules start to attach to the cell surface of the bacterium, still another defense system, the complement system, is activated. This system is best described as a cascade and is also present in the lower animals and plants we have discussed. It is called a complement system because it works in concert with, or complements, part of the acquired immune system (described later).
The complement system works by using one or more of twenty proteins to bind to the molecules that have attached to the cell surface. (The complement protein can also attach directly to sugary molecules called carbohydrates that stick out of the cell membrane.) Once the complement protein binds to the cell surface, a very rapid and dynamic response is initiated. The complement molecules belong to a family of proteins called proteases that, as their name implies, degrade the bacterial proteins. The protease activity of the complement protein is only activated when it is bound to a carbohydrate or to another immune molecule. In a brutal chain reaction for the bacterium or other microbe, the initial activation of a complement protein induces the activation of another, and another, and another complement molecule. This cascade could eventually lead to the microbe being coated with complement molecules that then guide other immune cells to its location. The complement proteins can also kill the microbes themselves, by poking holes in the microbes’ plasma membranes.
The host cells (neutrophils and other blood-borne immune cells called macrophages) that have been attacking the microbe come from a single progenitor cell type in the host body. The human body—and indeed any vertebrate body—has hundreds of specialized cell types, whereas some of the organisms we have discussed, such as Placozoa and sponges, are very limited in their cell type repertoire (Figure 5.4). How cell types develop or arise from a single fertilized egg is a fascinating story, and how the specialized cells of the immune system arise is simply a subplot of the general way that cells differentiate. Human immune cells, as well as the cells of the vertebrate circulatory system, arise from the same progenitor cell type called a multipotent hematopoietic stem cell. This is just a fancy name for a cell that can turn into any of the fifteen or so cell types in the vertebrate blood system and immune system, by differentiating again and again, in a branching manner, until each of the types is created. The first differentiation determines whether it will be a lymphoid cell or a myeloid cell. The myeloid cells produce a broad variety of cell types, including our red blood cells and some of the cells we have already discussed with respect to the innate immune system.
Figure 5.4. A “family tree” of cells in the vertebrate circulatory system, showing in part that all cells in the system come from a progenitor cell called a multipotential hematopoietic stem cell, or hemocytoblast.
Some of the myeloid cells produced by the multipotent hematopoietic stem cells are called leukocytes, which are the phagocytic munchers of the human innate immune system. Most of the leukocytes in our bodies are the neutrophils we encountered earlier. They are voracious eaters of invading cells and foreign debris, and accomplished wanderers to boot. When part of the body is infected—such as when a person has a cut invaded by some bacteria—pain, heat, redness, and swelling occur. The injured cells being attacked by bacteria or viruses also start to produce a variety of chemicals including interleukins (that communicate with macrophages), chemo kines (involved in recognizing chemicals in the blood stream), and interferons (antiviral responses). The interleukins and chemokines signal to the neutrophils that dinner is served. The neutrophils will then move toward the infected sites via the bloodstream. And if a virus has infected a cell, the release of interferons will signal to the infected cell that it is time to die.
If the unwelcome microbe is a virus, a natural killer cell will be summoned by the body. This kind of cell is important in the innate immune response and uses a strange strategy to fend off infection. Whereas it will produce some nasty proteins and enzymes that can knock you out, its major focus is to target sick host cells and induce them to commit suicide (or as an immunologist would say, it induces apoptosis). It is important to note that the natural killer cell leaves uninfected host cells alone. It can do this because the normal, uninfected cells put molecules on their surface called major histocompatibility complex class I (MHCI) markers. These markers signal to the natural killer cell to halt and move onto the next cell. There is a second class of major histocompatibility complex molecules called MHCII that are important in the acquired immune system. But for now, let’s assume that one nasty bacterium has managed to elude all of these innate defenses. What happens next?
The Acquired Immune System
With the evolution of vertebrates in the last 500 million years, along came a second way to combat invading microbes. This more derived immune response is much more specific with respect to what it targets and how it works. Although there is some evidence that the innate immune systems are pretty flexible, the innate system doesn’t really remember which microbes and other foreign bodies it has encountered and destroyed. The innate system starts from scratch pretty much every time it is confronted with something foreign. A more efficient mode of staving off invasion (or, as the Hydra example suggests, letting the good guys in) would involve the immune system’s remembering what it had encountered in the past, and whether the encounter was good or bad. Such a sophisticated system has indeed evolved in vertebrates. Called the acquired or adaptive immune system, it appears to have evolved in the common ancestor of all jawed vertebrates.
Adding a second dimension to the vertebrate immune system introduces even more complexity to the story. If the two immune systems, innate and adaptive, worked independently, then explaining these processes would be pretty easy. We would simply have two different stories. When the adaptive system evolved, however, it did so in concert with the innate system, so there is considerable overlap of the two systems. To understand how this line of defense works, we need to know which cells are involved in the acquired response, and where they come from.
Three cell types that haven’t yet been mentioned but are essential to the acquired or adaptive immune response are B-lymphocytes, killer T cells, and helper T cells. All of these lymphoid cells (a group that also includes natural killer cells, and B and T lymphocytes) are produced in generalized tissue sources in the body called hematopoietic tissue and take up residence in specific parts of the body. The myeloid precursor cells reside mostly in the bone marrow, while the lymphoid tissues reside in the lymph nodes, spleen, and thymus gland, and, surprisingly, in the mucosa of the digestive and respiratory tracts. In a complex dance of interactions with major histocompatibility complex molecules, specialized molecules called immunoglobulins and each of the other three cell types make up the acquired immune response.
The remarkably specific and effective response of B lymphocyte cells demonstrates well this subtle dance. B lymphocyte cells are covered with molecules called antigen receptors, which are ready to attach to an antigen—that is, a foreign molecule or part of a molecule, or cellular debris from something foreign. So far this sounds a little like the innate immune system with its receptors for foreign material. But there’s a twist to this part of the story: when an antigen encounters the antigen receptor on the B cell surface, the B cell starts to produce more receptors and secretes them into the extracellular space. These free-floating receptors are called antibodies or immunoglobulins (Figure 5.5). Moreover, B cells remember previous infections and are able to mount a more robust and deadly attack on antigens that they have encountered earlier. As B cells replicate, they form lineages of antibody genes based in part on the antigens they remember. These antibody genes can then recombine in different ways to produce a large number of differently tinkered final products, each designed to attack a kind of antigen. (T cells, which also use antibodies to recognize foreign proteins or antigens, are important to how the B cell is activated to produce a specific antibody.) The antibody needs to have a conserved aspect to it, but also to cope with the large variety of microbes that invade the host body. They furthermore need a variable region that can specialize in binding to molecules of invading pathogens.
Figure 5.5. A typical antibody. Note that some parts are constant, or conserved, whereas other parts can be changed to adapt to new challenges posed by microbes.
Antibodies stick really well to microbes because they act like a lock and key with some of the proteins on the microbe’s cell surface. Even if you, the microbe, produce a toxin in a last-ditch effort to harm the host organism, other antibodies have the ability to interlock with that toxin, disabling it. At this point you, as the microbe, are completely coated with antibodies, and have become an attractive meal for scavenger cells in the area. Your life as a microbe has ended, you are engulfed by a voracious white blood cell and die.
The immune system is a pretty amazing evolutionary adaptation. It learns and has a fantastic memory. When it is working well, it allows certain bacteria that are beneficial to us take up residence in and on us, while attacking bacteria that are harmful. It can also be trained to ward off bacteria and viruses even before they infect us. The vaccinations we discussed are a good example of this preemptive strike capability. Maurice Hilleman created his forty or so vaccines by presenting to the human body bits and pieces of microbes that are pathogenic to us when they are alive and whole. He minced up the bacteria or viruses and introduced them to the human immune system, whose lymphocytes recognized them as foreign, and whose ensuing cascade of T cell and B cell interactions activated B cells to produce antibodies. If that pathogenic microbe invaded the body later on, those B cells would remember the pathogen and quickly produce large quantities of the antibody to combat it. The time saved by having the B cells ready for that particular infection could be enough to stop that infection before it replicated to a point where it could start making us sick or get out of control.
More than one strategy has been developed across the animal and plant world to deal with microbial infection. We have already seen that the innate systems of plants and animals both provide the first line of cellular defense but that these organisms use different molecules for this task. In addition, natural-killer cells appear to work differently in mice and men. In the mouse, the natural-killer cells use receptors called lectin receptors, whereas in men (and women), the natural-killer cells use antibodylike receptors. Jawless vertebrates like the hagfish and the lamprey, too, use very different antigenrecognition receptors in their adaptive immune system. The human kind of adaptive immune system is found only in jawed vertebrates.
What Does It Mean to Be Antimicrobial?
Legend has it that the first scientifically developed antimicrobial was the result of slacking off. Alexander Fleming, a Scottish microbiologist, returned to his lab in London from a summer vacation in the country to find that he had forgotten to discard some Petri dishes. Before he left on holiday he had plated out some bacteria in the genus Staphylococcus on an agar plate. He had finished his observations, but then in his hurry to start his vacation, he left the Petri dish on his lab bench without cleaning up. While he was enjoying the countryside, the plates began to get a little moldy. The fungi had most likely gotten there because, as he was observing the staph on the plate before the vacation, he had lifted the cover of the plate and an airborne fungal spore had drifted in. As hard as any good microbiologist might try, this happens frequently.
Fleming coyly described the morning after his vacation: “When I woke up just after dawn on September 28, 1928, I certainly didn’t plan to revolutionize all medicine by discovering the world’s first antibiotic, or bacteria killer, but I suppose that was exactly what I did.” When he examined the plates on his bench he discovered that the staph colonies growing far away from the moldy blotch were nice and robust, but those growing near the mold were either dead or growing very poorly. He reasoned that the mold was somehow killing the staph, but he also knew that the only way it could be happening was through something the mold was secreting into the agar around its colony. After identifying the mold as a species in the genus Penicillium (Figure 5.6), he grew it in pure culture and isolated the stuff it secreted, first calling it “mold juice,” then renaming it penicillin (Figure 5.7). This discovery was the start of learning how antimicrobial substances like penicillin work.
Figure 5.6. A typical Penicillium mold.
Figure 5.7. Molecular structure of penicillin G, a form of the antibiotic administered intravenously.
By purifying and determining the structure of penicillin from the fungal strain that Fleming first isolated, chemists were able to determine its structure and to make it a viable commercial antimicrobial (Figure 5.7). Penicillin is a relatively small molecule that binds to and inactivates an important bacterial protein called a transpeptidase. It does this by slipping into the transpeptidase enzyme’s active site and causing the enzyme to self-destruct. Transpeptidase is vital to the bacterial cell because it is the protein that allows it to form cell walls. Cells that are susceptible to penicillin die quickly because they cannot reproduce. In fact, because penicillin-susceptible bacteria go through all of the motions of dividing but simply can’t apportion the components of the two daughter cells into real cells, they fill up and explode. There are many penicillin derivatives that have small differences in their molecular structure compared to the original antimicrobial, but they all work on the same general principle.
When we are sick, doctors can prescribe antimicrobials for the infections that bother us. There are a lot of different kinds of antimicrobial medications to use. One researcher who focused his career on development of antimicrobial substances was the New Jersey scientist Selman Waksman. He, along with his students and assistants, developed twenty-two different antimicrobials, including streptomycin and neomycin. He won a Nobel Prize for his long and illustrious career. The theme on which he worked so diligently was that certain compounds could interfere with the everyday lives of bacteria. They might impede the construction of the bacterium’s cell wall, as is the case for penicillin; interfere with the way proteins are translated, as with streptomycin; or disrupt the bacterium’s genetranscription processes.
Whichever way these drugs work, they all utilize pretty much the same general mechanisms, which leads to the problem of “resistance.” Bacteria and viruses can evolve to become resistant, that is, to survive the action of antimicrobial and antiviral agents. Although resistance originates as changes in single viruses or bacteria, it is important to keep in mind that antimicrobial resistance and anti viral resistance are population-level phenomena. Such resistance can happen very rapidly, depending on the population of bacteria or virus involved. HIV, for instance, can evolve resistance over a period of weeks, or even days.
The Importance of Variation
Charles Darwin was infatuated with variation. In his On the Origin of Species, he expounds on the astonishing variety of organisms in the natural world, using more than three thousand words to describe variation in pigeons alone. His obsession with variation, however, makes sense given how central it is to his idea that rocked the world—natural selection. He made it clear that, without variation, natural selection would not proceed. He tried very hard to connect the idea of variation with inheritance, which was appropriate. Although he did not succeed in this effort during his lifetime, when the work of Gregor Mendel was rediscovered and the two great laws of genetics were reformulated, inheritance was indeed tied to natural selection.
Where does this variation come from? At the most basic level, mutation of the genetic material is at work. In fact, part of the reason that microbial resistance in populations occurs so rapidly has to do with how frequently the genetic material mutates to produce new variants for natural selection. The other part concerns how intense the selective pressure is for or against the new variants produced by that particular mutation. RNA viruses mutate very rapidly—at a rate about 100,000 times that of eukaryotes and single-celled microbes. And for HIV, about one base in every ten mutates in its genome each generation. In a population of millions of HIVs, this means that there is ample variation for natural selection to work, especially because some variants are more successful at reproducing than others. The reason one variant might be more successful than another with respect to selection can be understood by thinking about what an antiviral or antimicrobial does. If an antimicrobial is sprayed onto a growing population of bacteria, and all of the cells there are susceptible except for one with a mutation, then the only cell left after the antimicrobial treatment will be the one with the variant.
This means, too, that the rate of resistance also depends on how well the antimicrobial or antiviral works. In a population of thousands of viruses where the efficiency for the selection of a variant is one in a thousand, the frequency of the favorable variant will increase very slowly, but steadily. In a population where the selection for a variant is one in ten, or an even larger proportion, the frequency of the variant will increase rapidly. And if the variant is the only genotype that survives, then the frequency of that variant will explode.
For an organism or a virus to respond rapidly to an environmental challenge, genetic variation is needed. Likewise, for multicellular organisms to be able to respond as a population to the threat of an antimicrobial agent, genetic variation has to be present, because it can lead to natural selection. Several mechanisms have evolved to answer this need for variation. Bacteria have mutation rates that are about the same as eukaryotes, which have a rather efficient mechanism for increasing genetic variation—sex. With eukaryotic sex, two genomes come together, and when new sperm or eggs are formed during meiosis, the two genomes can recombine by means of “crossing over,” a very complex molecular mechanism whereby two strands of DNA exchange genetic material. Eukaryotic sex at the molecular level is simply the mixing of genes from two different organisms via a fertilization process. Using sex, eukaryotes can generate pretty variable genomes, which enable populations to respond to natural selection. Bacteria do not have sex, and hence they have evolved other mechanisms, in addition to mutation, for generating variability, all of which can be placed under the general umbrella term “horizontal gene transfer” (Figure 5.8).
There are three major mechanisms by which horizontal gene transfer can occur. The first is through transformation. Because there are DNA strands floating around everywhere, this exogenous DNA can sometimes come into contact with intact bacterial cells. The process of transformation is a relatively sloppy one, whereby the exogenous DNA is sucked into a bacterial cell and then either coexists with the bacteria or gets integrated into the bacterial chromosome. Another mechanism is transduction, that is implemented through the infection of bacterial cells with viruses called phages. In this process, foreign, viral DNA is transduced to a bacterial cell via a phage that binds itself to the cell’s surface. This DNA then becomes incorporated into the bacterial chromosome, where it rests in what is called a lysogenic state. The virus does not have the genes for making the cellular machinery with which it will replicate, so it relies on the machinery of the bacterial host. When the time is right for the virus to replicate, it extracts itself from the bacterial chromosome and then “goes viral” by replicating, reconstituting its coat and tail, and then moving on, in the process possibly even killing the host cell by blowing it apart or lysing it. Often the extraction of the phage from the host bacteria is not exact and some of the host bacterial DNA comes along for the ride. When the virus infects a new bacterial host and integrates into its genome, these genes, or even entire suites of genes, can be a “hostess gift” to that cell. If the genes convey a reproductive advantage to the new bacterial host, that host will keep and use them.
Figure 5.8. Three of the major modes of horizontal gene transfer—transformation, conjugation, and transduction.
The third mechanism, called conjugation, involves the transfer of small, endogenous, circular pieces of DNA from one cell to another. The transfer is made by what is called a pilus, which is simply a structural conduit through which DNA is shuttled. Circular DNAs, called plasmids, can carry genes on them, and they act like internal parasites (much like phages), but they do not integrate into the bacterial chromosome. They are parasitic though, because they rely entirely on the host’s replication, transcription, and translation machinery to make copies of themselves. Sometimes the parasitic relationship turns “mutualistic,” so that the plasmid and the host cell both benefit from the interaction. This occurs when the plasmid carries a gene or set of genes that increases the reproductive success of the bacterium, like an antimicrobial resistance gene. The plasmid benefits, because it can make copies of itself, while the microbe’s situation is also improved, because it gains resistance to some antimicrobial compound.
Plasmids have been used for decades now in molecular genetics experiments, because they are easy to isolate and to manipulate in bacterial cells. But although plasmids are in general rather small and carry simple genetic repertoires, they can also carry loads of genes that have various functions. The smallest are on the order of a thousand nucleotides and carry genes for resistance to kanamycin and other antimicrobials. Some of the largest plasmids, by contrast, are nearly half the size of an average bacterial chromosome and carry hundreds of genes. Because they are an efficient way to generate variation in microbial populations, some plasmids carrying antimicrobial resistance genes have become quite a challenge to those trying to control modern infectious diseases like Salmonellaentericaserotypetyphimurium (S.typhimurium), an intestinal pathogen that is facing off with a plasmid that confers resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline, all commonly prescribed remedies. Another particularly nasty set of resistances conferred by a plasmid is methicillinresistant Staphylococcus aureus, or MRSA. This plasmid confers resistance to a number of antimicrobial agents and is particularly worrisome because it was one of the first to show resistance to vancomycin, the “go-to” drug for many infections (Figure 5.9). The plasmid of MRSA seems to have stacked the deck by collecting genes that ensure its survival and in turn the survival of staph strains with the plasmid.
Figure 5.9. The plasmid pLW1043, of Staphylococcus aureus, which can confer resistance to many different antibiotics.
The dangers of bacterial resistance and adaptation have prompted a great deal of research on why bacteria are so successful at moving genes around. One major discovery is that groups of genes can act a lot like floating islands. These so-called pathogenicity islands (PGIs), which are sometimes composed of large chunks of DNA, can be carried by phage or by plasmids, or they can be autonomous. They usually have suites of genes that enhance their ability to infect organisms or that improve their odds of survival. One example is the “TAd island,” which is pretty widespread across a lot of bacteria. TAd stands for “tight adherence,” and the TAd PGI is chock-full of genes that assist in making biofilms. We will return to this and other resistance mechanisms in Chapter 6, because they are extremely important for understanding the dynamics of how to maintain human health in populations that use antimicrobial agents.
So far we have looked at our immune system, vaccines, and antimicrobials. But there is yet another way to defend against pathogenic microbes: altering our own ecology.
The Great Camel Dung Mystery
When the Nazi army invaded North Africa in 1941, the German tank drivers thought it was good luck to run over piles of camel dung. Little did they know that camel dung would become a life and death issue. The Allies started to make fake camel dung piles and connect them to explosives that would detonate when run over by any luckseeking tank. The deceit was so well planned that the Allies even put tire track marks in their fake dung piles to trick the tank drivers into plowing over them. But camel dung in its true form would hold a life-saving secret. The soldiers were suffering greatly from dysentery and the Nazi medical corps was brought in to attempt to alleviate the outbreaks by figuring out which indigenous microbe from water or food was causing the problem. Early on the local nomads were thought to hold a key to the solution, because dysentery was very rarely a factor in their morbidity. In fact, when an outbreak of dysentery occurred, or even when slight diarrhea was experienced, the nomads would diligently follow their camels around. When a camel defecated, the nomad would quickly scoop up the dung and ingest some while it was still steaming. Only recently defecated dung would work, because cold dung would not prevent the dysentery. The medical corps knew that the dysentery they were dealing with was more than likely caused by a microbe, and after close scrutiny of the dung, the corps discovered that the dung was loaded with the bacterium Bacillus subtilis. (This species of bacterium is in the same genus as a terribly pathogenic species, Bacillus anthracis, which causes anthrax, an often lethal respiratory disease. Bacillus subtilis, however, has since become one of those bacterial species considered “good” for humans.) What is it about B. subtilis that makes it so useful that Arab nomads would ingest camel dung? This species is a voracious eater of viruses and other bacteria, and it essentially clears out any and nearly all bacteria in the gut once it gets there. By ingesting the warm camel dung, the nomads were essentially altering their gut ecology to get rid of the pathogen causing the dysentery. The B. subtilis was present only in the warm dung; it would die out when the dung cooled. Not wanting the troops to ingest camel dung, the German high command and medical corps instead cultured large amounts of B. subtilis in vats and fed the broth from the cultures to the troops, stopping the outbreaks of dysentery. The Nazi medical corps even developed a way to dry out the B. subtilis and put it into powder form for their troops. Since the Nazi experience with camel dung, B. subtilis has been used in much the same way as an antidysenteric agent.
Many animals also eat their own dung. Baby rabbits are notorious for imbibing their mother’s fecal pellets, and this behavior has been described as adaptive for the babies, because it is supposed to be the way they obtain their core gut microbiome. Specifically, baby rabbits need to get rid of the bacteria from the family Bacteroidaceae that they obtain shortly after birth and replace it in their digestive tracts with bacteria from the phylum Firmicutes, and the families Lachnospiraceae and Ruminococcaceae. If baby rabbits are prevented from eating their mothers’ poo, their digestive tract microbiomes are disrupted, which most assuredly has an effect on their ability to digest food.
The literature abounds with examples of humans ingesting various items to alter the ecology of their guts to enhance digestion, rid themselves of pathogens, or unknowingly to stimulate their immune systems. Although many of these practices are unproven, or unsafe, and so should be avoided, there have been some surprising initial discoveries in recent years that may eventually upend our ideas about what we ingest for good health. For example, although eating clay or dirt is considered an eating disorder in developed countries, its utility in altering gut diversity in the human stomach, at least in some cases, has been shown. And although it might seem silly to recommend giving patients certain elements of fecal matter, or a slurry of particular microbes, these strategies are thought to adjust the overall microbiomes of humans and so have become a major focus of immunological research.
