by Daniel Keays

Pertussis, aka Whooping Cough

            Most of us have had a cold that lingers. Two, three weeks, maybe longer, we’re still coughing and blowing our nose. Rarely do we get tested for the organism causing it because it goes away eventually, and chances are high that there is no specific treatment for it anyway. That lack of testing is one reason we don’t know how much pertussis, commonly known as whooping cough, is in the community. It presents as the “cold from hell,” one that makes us sicker than usual and continues to plague us for over a month. Adults rarely have a serious outcome, but it can be a very different story when it affects infants and toddlers.

            The common cold, of course, is a symptomatic disease and can be caused by a couple of hundred different viruses. Sore throat, runny nose (rhinitis), cough, general congestion, maybe a slight fever. It lasts a few days to a week; then, we’re back to normal. Some, either due to the nature of the virus or the patient’s immune status, last longer.  But all are an inconvenience rather than a major worry. Pertussis takes the common cold to a much higher level.

            Pertussis is not caused by a virus but by a bacterium, Bordetella pertussis. Tussis is a Latin word meaning “cough.” Per is also Latin and can have several meanings, but in this case, it means “intensive.” Humans are the only creatures that can harbor or be infected by B. pertussis, and it is passed from person to person through respiratory droplets expressed during coughing. The organism does not deeply invade human cells; it merely colonizes the respiratory tract like many harmless commensal bacteria. The toxins it produces make it pathogenic, and it possesses a sophisticated mechanism to release several toxins over time.

            The first step in the development of pertussis is the organism's attachment to the cells of the airway. The human trachea is not a friendly place if you are a bacterium. A lot of mucus sticks to them, and cilia on the epithelial cells are constantly beating to carry away anything caught up in the mucus. If an organism does get in there and attaches, we have a cough reflex to help propel whatever is there to the outside. To allow for this most inhospitable environment, the pertussis organism has several very large proteins that enable the bug to adhere to the human cell surface. One, called filamentous hemagglutinin (FHA), forms a rather loose attachment to the cilia of the trachea, enabling the organism to escape elimination by ciliary action and remain in proximity to the cell surface. The strongest attachment molecules of the organism are fimbriae, threadlike projections from the bacterial surface. Depending on the strain, several different ones can be produced (FIM2 and FIM3 are the most important). These form a solid adherent to both ciliated and non-ciliated cells of the trachea, and the organism locks on.

          Another attachment mechanism is a protein called pertactin, usually abbreviated PRN. While not a fimbria, it is a protein that provides another solid bond of the bug to the host cell surface. Once attached, they are tough to get rid of, even with the continual beating of the cilia. The word that refers to the firmness of the attachment is avidity, from the Latin word avidus, to “crave.” With FHA, various fimbriae, and pertactin, Bordetella pertussis has a very high avidity to our airway epithelial cells.

            Bordetella pertussis does not invade the inside of the cell to which it attaches, as many pathogens do. Sitting outside the cell in the middle of the airway leaves them vulnerable to the immune system, both mechanical (mucus, cilia, cough) and innate (neutrophils, complement, antibody, macrophages). For the bacterium, this will not do. They have several mechanisms to deter the immune system and remain attached. They aim to reproduce and spread from person to person, continuing the species. Their ability to inhibit the immune system is part of their plan. The high avidity of their attachment is one means of circumventing the mechanical forces designed to eliminate them. They also possess the chemical means to help ward off the innate immune system components.

            The “first responder” to the attached Bordetella pertussis is the complement system, which is, of course, found in serum, but is also abundant in mucus secretions. Complement greatly assists neutrophils and antibodies in capturing and destroying invading bacteria, and it can directly damage and destroy the pathogen.  

            Complement is a powerful weapon against invading microbes, but it’s also potentially destructive to human cells, not a good thing. Thankfully, our cells possess molecules that destroy complement components when they contact the host cell membrane. Incredibly, Bordetella pertussis has a system of molecules, most notably one known as Vag8, that can strip a complement-inhibiting protein from a human cell and use it for its own good. When Vag8 is active, C1, the first member of the complement cascade, cannot be converted into C1q, short-circuiting the entire complement system. Essentially, the bacterium steals a molecule from the surface of our cells and uses it to repel our attack on it. Clever.

            Bordetella pertussis has several other complicated mechanisms designed to thwart complement activity, thereby ensuring its more prolonged survival in the human host. The first line of defense, complement, is much less active than it is designed to be, giving the microbe a leg up.

            If you are a microbe, the next order of business is to eliminate, or at least mitigate, the host cells designed to attack you, macrophages and neutrophils. Macrophages are constantly wandering through the airway lining, ready to pick up any type of debris that comes their way. They also have the mechanisms to signal the immune system to deploy more help to the area, including more neutrophils, complement and other serum molecules, and the various types of T and B lymphocytes. The main toxin of Bordetella pertussis, pertussis toxin (PT), has an inhibitory effect on the macrophages, rendering them much less a threat. The specific mechanism it uses against macrophages effectively quells their activity, both phagocytosis and signaling.

            The activity of neutrophils in the area is also reduced, not directly but by indirect methods. When a pathogen is detected in our tissues, chemical signals are sent to release more neutrophils from the bone marrow and attract them from nearby blood vessels into the affected area. Pertussis toxin reduces both tools by counteracting the chemical signals designed to make this happen, and neutrophil incursion into the infected area is significantly reduced.

            Without our benefit of complement, macrophages, and neutrophils early in the infection, Bordetella pertussis gains a foothold on the cells lining our trachea and can set up a progressive infection.

            Bordetella pertussis makes other toxins besides pertussis toxin. A very important one is adenylate cyclase toxin or ACT. It is a very large toxin with a short half-life, so it doesn’t move very far from the organism once released. Its action is directed mainly toward macrophages and neutrophils that come into the area close to where the bacteria reside. It enters the phagocytic cell, then converts ATP into cyclic AMP. This reaction effectively halts the ability of phagocytic cells to kill the invading bacteria. We can think of the actions of PT and ACT as a one-two punch: The pertussis toxin mitigates the arrival of phagocytic cells, while the adenylate cyclase toxin, along with PT, kills the few that arrive on the scene. Together, the two toxins allow for the persistence of the organism at the infection site, the trachea and bronchi.

            One reason the symptoms of pertussis are so severe is the presence of a toxin called tracheal cytotoxin, or TCT. Unlike the other toxins, which are made inside the bacterial cell and then released, TCT is a permanent structure of the organism’s cell wall. All bacteria have it because it is a vital component of the bacterial peptidoglycan, which forms the wall’s rigid backbone. Most bacteria have an enzyme that firmly attaches TCT to a section of peptidoglycan in the bacterial cell wall. TCT is needed to construct more cell walls as the bacteria grow. Not so Bordetella pertussis. It lacks the controlling enzyme, so its TCT is released into the local environment. Our cells are equipped with sensors to detect the presence of bacterial cell wall material, so this stuff sends the response into full operation. One chemical our cells produce to help kill invading bacteria is nitric oxide (NO). It is probably induced by interleukin 1, produced because of all the TCT floating around. Because of the abundant Bordetella cell wall material, our cells make much more nitric oxide than would normally be produced with other bacterial infections. Unfortunately, nitric oxide kills our cells too. The primary victims of this attack are the ciliated epithelial cells of the airway, especially the trachea. The result is the build-up of mucus, dead cells, and other gunk in our airways. Without the reliable rhythmic beating of cilia, much of the mucus and debris created locally remains in the airway, inhibiting breathing and initiating a cough reflex to remove it. Pertussis toxin then flows down to the underlying tissues, killing many of the cells there. All this local damage results in fluid release from the surrounding blood vessels, causing edema that further constricts the diameter of the airway. The combination of the debris build-up and the edema makes for a perilous situation for young children; the effects are sometimes fatal.

            Infection with Bordetella pertussis gives us a reduced number of macrophages and neutrophils. But it has the opposite effect on our lymphocytes. They are created in great numbers in the bone marrow and flood into the lymph nodes around the trachea and bronchi. In the peripheral blood, the pertussis patient has an elevated white cell count, but, in contrast to most other bacterial infections, the predominant white blood cells are lymphocytes, not neutrophils. In fact, in a patient with a persistent cough and a peripheral count of over 10,000 lymphocytes, a diagnosis of pertussis can be confidently made.

            Bordetella pertussis differs considerably from other bacteria that commonly infect the human respiratory tract. Three important ones, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus, are readily visualized under a microscope in a Gram-stained preparation from a representative sample. They also grow readily on culture plates commonly used in clinical microbiology laboratories, taking a little less than a day for typical colonies to be observable. Bordetella pertussis is much different. That organism, a tiny Gram-negative coccobacillus, is not easily seen directly in clinical material, especially if the disease has progressed for several days, as it usually does before medical attention is sought. It doesn’t grow on common laboratory media, requiring its own unique blend, and it takes almost a week to grow out, not just a single day. Bordetella pertussis is not seen on a routine laboratory bacterial culture.

 

            Scientists looked in vain for the causative organism of pertussis throughout the 1890s. The first to recognize the organism were two Frenchmen, Jules Bordet and Octave Gengou, in 1900. They were somewhat lucky because they saw it on a Gram-stain preparation from some sputum from a 5-month-old girl sick with the disease, which is not an easy accomplishment. It took them another six years to develop a culture medium that would support the organism's growth, using the starch of potatoes as the medium base, supplemented with blood.  They didn’t have to look far for clinical material to test the culture plate; Jules Bordet’s son Paul was ill with the disease. 

            Bordet and Gengou didn’t stop with the description of the pathogen. They attempted to produce a vaccine, but it didn’t work very well.  It would be another 43 years before a successful one was developed. But they did describe a toxin produced by the organism, one they called dermonecrotic toxin (DNT). When pathogenic strains of the organism are grown in culture, the crude culture broth, when injected into a laboratory animal, produces skin lesions. We now know this dermonecrotic toxin is similar in its action to the toxin produced by the intestinal pathogen Clostridium difficile. Its action disrupts the formation of structural proteins inside the target cell (mainly the so-called rho proteins), leading to cell death. Just which cells and tissues DNT specifically targets is not known, but when acting in concert with the organism’s other toxins, it adds to the bug’s disruptive force.

 

            It’s somewhat unfortunate that pertussis is commonly called “whooping cough.” In some ways, that may trivialize it; the word “whoop” has several connotations. But there is nothing trivial in how the organism infects and injures patients, especially the young. In pertussis, the word “whoop” comes from the sound made on inspiration during a coughing fit, a pernicious feature of the disease. The most malicious symptom of the disease is called a paroxysm (from the Greek paroxysmos, meaning “irritation” or “exasperation”). It is a coughing fit that is a hallmark of the disease. Paroxysms can last for several minutes. Some people get it so bad that coughing may mechanically injure them, such as a pulled back muscle or rib injury. Most of the cilia lining our airways are non-functional, so the only way we can clear the debris clogging it is to cough it out. And the cough can reach epic proportions.  

            Besides the organism's several exotoxins, each bacterial cell contains the classic endotoxin typical of all Gram-negative bacteria. Our immune reaction to the endotoxin gives rise to fever and muscle aches. We feel just plain lousy for the duration of the disease, which, unfortunately, takes several weeks, sometimes months. The Chinese once described it as the “cough of a hundred days.”  

            It would be nice if we could just take an antibiotic for a few days and be done with it. After all, it is caused by a bacterium, and the organism is exquisitely susceptible to the common antibiotic erythromycin in the test tube. Unfortunately, this is another area where Bordetella pertussis differs from other bacteria. Unless treated very early in the disease, antibiotics have little effect on the patient being treated. They may reduce the patient’s ability to spread the disease to others, but there is only minor, if any, relief for the one afflicted.  

            The diagnosis of pertussis is enigmatic as well. A simple Gram stain of expectorated or aspirated material from the airway rarely shows the organism. It does not grow on ordinary laboratory culture media, so the physician ordering the culture must designate a pertussis culture using culture plates like Bordet-Gengou or Regan-Lowe agar. The organism is killed if the swab obtaining the specimen is cotton rather than dacron. The pertussis organism takes about a week to grow, so other bacteria that grow more quickly can overwhelm the culture media. All this means that pertussis is not easy to diagnose, and in many cases, it is missed.

            Today's diagnostic method of choice is a molecular genetics test that detects a portion of the organism’s DNA. Known as polymerase chain reaction (PCR), the test can detect the organism's presence with greater sensitivity than culture. The results are usually available in a day or two rather than a week. 

            

            The first half of the 20^th century had to be very frustrating to doctors when it came to pertussis. The cause of the disease was known, and it should have been a relatively simple matter to develop a vaccine to prevent it. Just grow the bacterium in culture broth, kill it, then inject some of it into the child to be vaccinated. Sounds simple.  But, alas, it wasn’t to be. Pertussis makes several toxins, but they are denatured with the demise of the bug. Bordetella pertussis is a Gram-negative organism, and therefore contains the hallmark constituent of all such bacteria:  lipopolysaccharide (LPS).  Present in all cell walls of Gram-negative bacteria, LPS contains Lipid A, a very potent toxin, and it is not denatured by killing the organism. Our immune cells are primed to detect and combat Lipid A whenever it is encountered. Lipid A attaches to Toll-like receptor 4, which is present in all our cells. Macrophages are specially equipped to handle it. Once detected, a chain reaction occurs within the cell, enabling nuclear factor k B (NFkB), signaling macrophages and other cells to turn on their cytokine-producing machinery. The result is a potent release of cytokines such as tumor necrosis factor-a, Interleukin-1b, Interleukin-6, and others, collectively making us feel sick. Fever, muscle aches, listlessness, and maybe a headache. Giving someone an injection of killed Bordetella pertussis also gives them a small dose of Lipid A, along with the repercussions.  

            Small quantities of Lipid A are usually tolerable and don’t make us very sick. A little discomfort of a day or two isn’t too high a price to pay for long-lasting immunity to a wretched disease like pertussis. But just how much is a “small dose”? Unfortunately, a small dose of Lipid A also means a small dose of the killed organism, and here again, Bordetella pertussis proves to be a conundrum. It takes many organisms to initiate an immune response, even when an adjuvant is given. The more organisms you give, the more Lipid A you give, along with its symptomatic sequelae. It took several decades to figure out the proper dosage, but administering whole cells of Gram-negative bacteria was never the ideal option.

            The first step toward developing a vaccine for pertussis that didn’t have the nagging side effects of the whole cell preparation came from the Japanese researcher Yugi Sato and colleagues. In the early 1980s, they prepared cell wall fragments that lacked Lipid A but contained pertussis toxin. It worked. Japan went to it as the vaccine of choice for pertussis, and other countries followed. Since then, other preparations have been developed, all based on parts of the organism but lacking Lipid A and its toxic effects. The most important fractions of the vaccine are the organism’s attachment structures, plus pertussis toxin. If it can’t attach and hold on, it can’t infect, and antibodies the vaccine induces block the attachment points. It is known as the “acellular” preparation. Since the pertussis vaccine is usually administered in combination with diphtheria and tetanus in the DPT formula, it is now known as DTaP, with the lower case “a” standing for acellular, concerning the pertussis component.

            

            Before the use of an effective vaccine, pertussis showed a cycle of epidemic outbreaks. The disease would come around every three to five years, mainly striking young children and babies.  Presumably, older patients had previously encountered the organism and were much less susceptible. Pertussis still shows this cyclic pattern, but the affected patients include the young and the older. The acellular vaccine has done much to reduce the side effects of vaccination. Still, it may be that the immunity it imparts doesn’t last as long as the whole cell preparation or natural infection.  

            Bordetella pertussis isn’t known to naturally inhabit animals besides humans. Just where it goes between epidemics is anybody’s guess. There could be a carrier state of some magnitude, but it is very difficult to detect. It’s hard enough isolating and identifying the organism during acute disease; it’s impossible to detect a few relatively dormant critters just hanging on, not causing any trouble. The toxins of B. pertussis are contained in a segment of its DNA strand known as the Bvg (Bordetella virulence gene). The organism can turn off all its virulence factors as environmental conditions change and enter a non-virulent phase. It happens all the time to organisms in laboratory culture. Organisms that are continually passed on culture media go from a silver, hemolytic colony (often described as “drops of mercury”) to a white, chalky non-virulent phase. Finding itself in a human airway, conditions are right for it to activate its Bvg genes and become virulent again. Exactly what this has to do with the 3-5 year period of epidemics is unknown.

 

            Pertussis is a serious disease. Because of vaccination, we in developed countries no longer fear it, as did past generations. But it used to be a serious killer. In the pre-vaccination era, it killed more young children than measles, scarlet fever, diphtheria, polio, and meningitis combined. Worldwide, it is estimated that there are over 20 million cases of pertussis every year, with at least 160,000 deaths, most of them young children. In the U.S., there are reportedly about 50,000 cases per year, but given the difficulties of diagnosis, especially in adults, there are undoubtedly many more.  

Gram stain of Bordetella pertussis, taken from a laboratory cultuire, as seen under the microscope, magnified 1000x  (PHIL)

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