Difficulty in determining the concentration of a bacterial toxin at its target tissue derives from multiple factors, including the limited number of animal infection models that reflect human disease, poor access to human tissues, and a paucity of information on the kinetics of toxin secretion. In this study, we set out to determine the concentration of ACT at target tissue by using complementary techniques that address these limitations. The baboon model recapitulates the paroxysmal cough and transmissibility of pertussis and permits sampling from the nasopharynx to document the proliferation of bacteria from the time of acquisition to the time of disease resolution. The data from baboons indicate that ACT is detectable in NPW, with a threshold at ~4.5 × 106 CFU/ml, and that the concentration of ACT correlates with the bacterial concentration. Measurements of bacterial concentrations in human samples acquired during acute disease showed similar concentrations of bacteria to those in baboon specimens. Production of ACT by this quantity of B. pertussis
in vitro plateaued at 6 h, at ~60 ng/ml, a concentration similar to that found in the NPW from infant 2 (~20 ng/ml). Considering human and baboon data together with measurements of ACT secretion in vitro, we estimate that ACT reaches levels higher than the 20 ng/ml detected in the airway of infant 2. Given the finding that centrifugation of bacteria onto target cells increases cAMP generation relative to that seen without centrifugation, the concentration of ACT at the bacterium-cell interface likely reaches at least 100 ng/ml and may be ≥100 ng/ml in areas of high bacterial concentration. Given that the effects of ACT are concentration dependent and that the concentration of ACT in vivo has until now been entirely unknown, this estimated concentration range can serve as a reference point for future in vitro studies and suggests that the pathophysiologic relevance of studies of ACT which are performed at concentrations of >1,000 ng/ml should be interpreted with caution.
While the findings in the infants are complementary to the baboon and in vitro
data, they are clearly limited and thus highlight the importance of the baboon model for investigating pertussis pathogenesis. With the infants, bacterial colony counts were not determined for the clinical specimens, so analysis of quantitative PCR values was used to estimate bacterial concentrations. Nakamura et al. used PCR for measurement of bacterial burdens during pertussis and found that the outcome did not depend upon the strains of pertussis detected in their study population (52
), suggesting that variations in the copy number of IS481
), the target sequence for the PCR primers, do not substantially affect estimations of bacterial loads. Although there were comparable bacterial and ACT concentrations between these human infants and baboons, it is interesting that these infants progressed to respiratory distress, whereas the baboons recovered uneventfully. Importantly, the samples available in each of these infant cases were taken at the time of presentation, prior to respiratory decompensation, whereas the baboon samples that showed B. pertussis
levels comparable to those of the infants were taken at the peak of infection. Since only a single sample was available from each of these infant cases, we hypothesize that samples taken later during the course of B. pertussis
infection would show substantially higher bacterial and ACT concentrations. In addition, the samples from both infants and baboons were taken only from the nasopharynx, and the time of onset of B. pertussis
pneumonitis in the infants is not known. For this reason, the baboon model is invaluable for future exploration of host and pathogen factors that contribute to disease progression.
There are limitations to in vitro
studies that are overcome by in vivo
studies, and vice versa
. On the one hand, measurements of in vivo
ACT levels may be reduced by ex vivo
handling of samples, the presence of mucous and inflammatory contents in specimens, or the inability for saline washes to completely remove bacteria and toxin from the airway lining. The dense hyaline and necrotic debris in the lumen of the pathological sample shown in does, in fact, suggest that accessibility of ACT in the airway to saline washes may be limited. On the other hand, in vitro
measurements may be elevated in comparison to in vivo
conditions due to the use of a different bacterial strain (BP338 versus D420, respectively) or due to culture in growth media compared with the airway environment. An important advantage of this study is the use of several different bacterial isolates: the baboon model was developed with the D420 clinical isolate (33
), in vitro
studies were performed with the Tohama I-derived laboratory strain, and human samples are all potentially unique specimens. The consistency from infections with these different strains under various conditions is notable and reassuring.
secretes multiple virulence factors, most importantly pertussis toxin (PTX) and filamentous hemagglutinin (FHA). FHA interacts with ACT in solution, on the bacterial surface, or indirectly at the target cell (61
). PTX catalyzes ADP ribosylation of Giα
, eliminating inhibitory input to cellular adenylyl cyclase, but only in the setting of ongoing inhibition does this result in an increase in cAMP. This point is illustrated by the fact that PTX alone does not increase cAMP in J774 cells. In addition, treatment of cells with PTX for 2 h prior to or in combination with ACT did not affect ACT-induced cAMP levels or cytotoxicity in J774 cells (data not shown). Similarly, ACT-induced cAMP and cytotoxicity in J774 cells were not affected by addition of FHA (data not shown). Furthermore, the adenylate cyclase enzyme assay is specific for measurement of AC enzyme and has been used to quantify ACT activity on the surfaces of whole bacteria (49
). For these reasons, we believe that measurements of ACT activity and concentrations in vitro
or in vivo
are not altered by the other virulence factors. In fact, shows that purified recombinant ACT increased cAMP in J774 cells equivalently to B. pertussis
when B. pertussis
was incubated with cells for the amount of time required to secrete the same concentration of ACT. This finding also supports previously published data showing that recombinant ACT produced in E. coli
retains catalytic and invasive activity equivalent to that of ACT produced by B. pertussis
These in vitro
studies not only contribute to our understanding of how much ACT may be present in vivo
but also highlight several points that are important for future studies of B. pertussis
. First, because newly secreted ACT is responsible for intoxication of target cells (49
), the effects of secreted ACT on cells in culture will depend on how long a target cell is exposed to bacteria. Mouallem et al. have also reported a delay in ACT-induced cAMP generation in Chinese hamster ovary cells exposed to live B. pertussis
). Second, the concentration of ACT at the bacterium-cell interface is higher than that in the medium, and effects of secreted ACT may be missed if bacteria are not closely apposed to target cells during incubation. For example, phagocytosis of B. pertussis
by neutrophils is very inefficient, with <1% of bacteria in suspension phagocytized by neutrophils in 1 h, but phagocytosis is increased to ~10% by centrifugation (63
). Studies of neutrophil phagocytosis using centrifugation to bring bacteria into close apposition with the target cell have shown that secreted ACT antagonizes phagocytosis under some conditions, an effect of ACT that would have been missed without concentration of ACT at the bacterium-cell interface by centrifugation.
More broadly, knowledge of bacterial protein toxin concentrations at target tissues is limited, and the techniques presented here may be applicable to other toxins, although there are specific problems associated with individual toxins. Diarrheagenic toxins have well-characterized target tissues and easily accessible human specimens. For instance, the concentration of both cholera toxin and Clostridium difficile
toxin in stool may be >100 ng/ml during disease caused by their bacteria (67
). However, there is still difficulty in estimating the amount of toxin at the site of infection. The volume of diarrhea and presence of mucins, in the case of cholera toxin (69
), may reduce the measured concentration of toxin in liquid stool. Some diarrheagenic bacteria are also attached closely to epithelial cells, and the local concentration at the bacterium-cell interface may be different than the concentration measured in the lumen. Unlike most infections with Vibrio cholerae
and Clostridium difficile
, infection with Bacillus anthracis
is characterized by toxemia. Anthrax lethal factor has been measured in the blood from human cases of cutaneous anthrax, at concentrations up to 1.26 ng/ml (70
), and in the blood and pleural fluid of humans and monkeys with inhalational anthrax, at concentrations of >500 ng/ml (71
). Measurement of anthrax toxin levels in target organ tissues has not been reported and requires knowledge of the relevant target tissue and a tissue-specific method for toxin quantification.
In summary, estimation of the concentration of a secreted toxin at the site of bacterial infection has been difficult. The combination of an animal model, human specimens, and in vitro data used for these studies illustrates how this may be accomplished. The determination of ACT concentration and the understanding of the kinetics of secretion provide reference points for in vitro studies. In addition, we have presented the first immunohistochemical imaging of ACT produced by B. pertussis during human infection. These human data further validate the baboon model of pertussis as an invaluable tool for studying pertussis pathogenesis and vaccine efficacy.