Bordetella pertussis uniquely colonizes the cilia of the mammalian respiratory epithelium (Figures 1 and 2); it does not invade tissues. The bacterium is a pathogen for humans and possibly for higher primates, and no other reservoir is known. Whooping cough is a relatively mild disease in adults but has a significant mortality rate in infants. Until immunization was introduced in the 1930s, whooping cough was one of the most frequent and severe diseases of infants in the United States.
Pathogenesis
The disease pertussis has two stages. The first stage, colonization, is an upper respiratory disease with fever, malaise and coughing, which increases in intensity over about a 10-day period. During this stage the organism can be recovered in large numbers from pharyngeal cultures, and the severity and duration of the disease can be reduced by antimicrobial treatment. Adherence mechanisms of B. pertussis involve a "filamentous hemagglutinin" (FHA), which is a fimbrial-like structure on the bacterial surface, and cell-bound pertussis toxin (PT). Short range effects of soluble toxins play a role as well in invasion during the colonization stage.
Figure 1. Colonization of tracheal epithelial cells by Bordetella pertussis
The second or toxemic stage of pertussis follows relatively nonspecific symptoms of the colonizaton stage. It begins gradually with prolonged and paroxysmal coughing that often ends in a characteristic inspiratory gasp (whoop). During the second stage, B. pertussis can rarely be recovered, and antimicrobial agents have no effect on the progress of the disease. This stage is mediated by a variety of soluble toxins.
Colonization
Studies of B. pertussis and its adhesins have focused on cultured mammalian cells that lack most of the features of ciliated epithelial cells. However, some generalities have been drawn. The two most important colonization factors are the filamentous hemagglutinin (FHA) and the pertussis toxin (PT). Filamentous hemagglutinin is a large (220 kDa) protein that forms filamentous structures on the cell surface. FHA binds to galactose residues on a sulfated glycolipid called sulfatide which is very common on the surface of ciliated cells. Mutations in the FHA structural gene reduce the ability of the organism to colonize, and antibodies against FHA provide protection against infection. However, it is unlikely that FHA is the only adhesin involved in colonization. The structural gene for FHA has been cloned and expressed in E. coli, raising the possibility of its production for use in a component vaccine.
One of the toxins of B. pertussis, the pertussis toxin (PT), is also involved in adherence to the tracheal epithelium. Pertussis toxin is a 105 kDa protein composed of six subunits: S1, S2, S3, (2)S4, and S5 (Figure 3). The toxin is both secreted into the extracellular fluid and cell bound. Some components of the cell-bound toxin (S2 and S3) function as adhesins, and appear to bind the bacteria to host cells. S2 and S3 utilize different receptors on host cells. S2 binds specifically to a glycolipid called lactosylceramide, which is found primarily on the ciliated epithelial cells. S3 binds to a glycoprotein found mainly on phagocytic cells.
The S1 subunit of pertussis toxin is the A component with ADP ribosylating activity, and the function of S2 and S3 is presumed to be involved in binding the intact (extracellular) toxin to its target cell surface. Antibodies against PT components prevent colonization of ciliated cells by the bacteria and provide effective protection against infection. Thus, pertussis toxin is clearly an important virulence factor in the initial colonization stage of the infection.
Since the S3 subunit of pertussis toxin is able to bind to the surface of phagocytes, and since FHA will attach to integrin CR3 on phagocyte surfaces (the receptor for complement C3b), it has been speculated that the bacterium might bind preferentially to phagocytes in order to facilitate its own engulfment. The role of such self-initiated phagocytosis is not clear. Bacteria taken up by this abnormal route may avoid stimulating the oxidative burst that normally accompanies phagocytic uptake of bacterial cells which are opsonized by antibodies or complement C3b. Once inside of cells the bacteria might utilize other toxins (i.e. adenylate cyclase toxin) to compromise the bactericidal activities of phagocytes. In any case, there is some evidence that Bordetella pertussis can use this mechanism to get into and to persist in phagocytes as an intracellular parasite. If B. pertussis is an intracellular parasite it would explain why immunity to pertussis correlates better with the presence of specific cytotoxic T cells than it does with the presence of antibodies to bacterial products.
B. pertussis produces at least two other types of adhesins, two types of fimbriae and a nonfimbrial surface protein called pertactin, but their role in adherence and pathogenesis is not well established.
Toxins Produced by B. pertussis
B. pertussis produces a variety of substances with toxic activity in the class of exotoxins and endotoxins. (see Figure 6).
- It secretes its own invasive adenylate cyclase which enters
mammalian cells (Bacillus anthracis produces a similar enzyme, EF).
This toxin acts locally to reduce phagocytic activity and probably
helps the organism initiate infection. This toxin is a 45 kDa protein
that may be cell-associated or released into the environment. Mutants
of B. pertussis in the adenylate cyclase gene have reduced
virulence in mouse models. The organisms can still colonize but cannot
produce the lethal disease. The adenylate cyclase toxin is a single
polypeptide with an enzymatic domain (i.e., adenylate cyclase
activity) and a binding domain that will attach to host cell surfaces.
The adenylate cyclase was originally identified as a hemolysin because
it will lyse red blood cells. In fact, it is responsible for hemolytic
zones around colonies of Bordetella pertussis growing on blood
agar. Probably it inserts into the erythrocyte membrane which causes
hemolysis. An interesting feature of the adenylate cyclase toxin is
that it is active only in the presence of a eukaryotic regulatory
molecule called calmodulin, which up-regulates the activity of the
eukaryotic adenylate cyclase. The adenylate cyclase toxin is only
active in the eukaryotic cell since no similar regulatory molecule
exists in procaryotes. Thus, the molecule seems to have evolved
specifically to parasitize eukaryotic cells. Anthrax EF (edema factor)
is also a calmodulin-dependent adenylate cyclase.
- It produces a highly lethal toxin (formerly called dermonecrotic
toxin) which causes inflammation and local necrosis adjacent to sites
where B. pertussis is located. The lethal toxin is a 102 kDa
protein composed of four subunits, two with a mw of 24kDa and two with
mw of 30 kDa. It causes necrotic skin lesions when low doses are
injected subcutaneosly in mice and is lethal in high doses. The role
of the toxin in whooping cough is not known.
- It produces a substance called the tracheal cytotoxin which is toxic
for ciliated respiratory epithelium and which will stop the ciliated
cells from beating. This substance is not a classic bacterial exotoxin
since it is not composed of protein. The tracheal cytotoxin is a
peptidoglycan fragment, which appears in the extracellular fluid where
the bacteria are actively growing. The toxin kills ciliated cells and
causes their extrusion from the mucosa. It also stimulates release of
cytokine IL-1, and so causes fever.
- It produces the pertussis toxin, PT, a protein that mediates both
the colonization and toxemic stages of the disease. PT is a two
component, A+B bacterial exotoxin. The A subunit (S1) is an ADP
ribosyl transferase. The B component, composed of five polypeptide
subunits (S2 through S5), binds to specific carbohydrates on cell
surfaces (Figure 3). The role of PT in invasion has already been
discussed. PT is transported from the site of growth of the Bordetella
to various susceptible cells and tissues of the host. Following
binding of the B component to host cells, the A subunit is inserted
through the membrane and released into the cytoplasm in a mechanism of
direct entry (Figure 5). The A subunit gains enzymatic activity and
transfers the ADP ribosyl moiety of NAD to the membrane-bound
regulatory protein Gi that normally inhibits the eukaryotic adenylate
cyclase. The Gi protein is inactivated and cannot perform its normal
function to inhibit adenylate cyclase. The conversion of ATP to cyclic
AMP cannot be stopped and intracellular levels of cAMP increase
(Figure 4). This has the effect to disrupt cellular function, and in
the case of phagocytes, to decrease their phagocytic activities such
as chemotaxis, engulfment, the oxidative burst, and bacteridcidal
killing. Systemic effects of the toxin include lymphocytosis and
alteration of hormonal activities that are regulated by cAMP, such as
increased insulin production (resulting in hypoglycemia) and increased
sensitivity to histamine (resulting in increased capillary
permeability, hypotension and shock). PT also affects the immune
system in experimental animals. B cells and T cells that leave the
lymphatics show an inability to return. This alters both AMI and CMI
responses and may explain the high freqency of secondary infections
that accompany pertussis (the most frequent secondary infections
during whooping cough are pneumomia and otitis media).
Although the effects of the pertussis toxin are dependent on ADP ribosylation, it has been shown that mere binding of the B oligomer can elicit a response on the cell surface such as lymphocyte mitogenicity, platelet activation, and production of insulin effects.
The pertussis toxin gene has been cloned and sequenced and the subunits expressed in E. coli. The toxin can be inactivated and converted to toxoid for use in component vaccines.
Figure 2. Comparison between cholera toxin and pertussis toxin (ptx) in their ability to interfere with the regulation of the eukaryotic adenylate cyclase complex.
Normal regulation of adenylate cyclase activity in mammalian cells (Adenylate cyclase (AC) is activated normally by a stimulatory regulatory protein (Gs) and guanosine triphosphate (GTP); however the activation is normally brief because an inhibitory regulatory protein (Gi) hydrolyzes the GTP.