Adenylate cyclase toxin from Bordetella pertussis requires posttranslational acylation of lysine 983 for the ability to deliver its catalytic domain to the target cell interior and produce cyclic adenosine monophosphate (cell-invasive activity) and to form transmembrane channels (hemolytic activity). When the toxin is expressed in Escherichia coli, it has reduced hemolytic activity, but comparable cell-invasive activity to that of adenylate cyclase toxin from B. pertussis. In contrast to the native protein from B. pertussis, which is exclusively palmitoylated, recombinant toxin from E. coli is acylated at lysine 983 with about 87% palmitoylated and the remainder myristoylated. Furthermore, the recombinant toxin contains an additional palmitoylation on approximately two-thirds of the lysines at position 860. These observations suggest that the site and nature of posttranslational fatty-acylation can be dictated by the bacterial host used for expression and can have a significant, but selective, effect on protein function.

Adenylate cyclase (AC) toxin from Bordetella pertussis delivers its catalytic domain to the interior of target cells where it converts host ATP to cAMP in a process referred to as intoxication. This toxin also hemolyzes sheep erythrocytes by a mechanism presumed to include pore formation and osmotic lysis. Intoxication and hemolysis appear at strikingly different toxin concentrations and evolve over different time scales, suggesting that different molecular processes may be involved. The present study was designed to test the hypothesis that intoxication and hemolysis occur by distinct mechanisms.Although the hemolytic activity of AC toxin has a lag of >1 h, intoxication starts immediately. Because of this difference, we sought a surrogate or precursor lesion that leads to hemolysis, and potassium efflux has been observed from erythrocytes treated with other poreforming toxins. AC toxin elicits an increase in K ؉ efflux from sheep erythrocytes and Jurkat cells, a human Tcell leukemia line, that begins within minutes of toxin addition. The toxin concentration dependence along with the analysis of the time course suggest that toxin monomers are sufficient to elicit release of K ؉ and to deliver the catalytic domain to the cell interior. Hemolysis, on the other hand, is a highly cooperative event that likely requires a subsequent oligomerization of these individual units. Although induction of K ؉ efflux shares some structural and environmental requirements with both intoxication and hemolysis, it can occur under conditions in which intoxication is reduced or prevented. The data presented here suggest that the transmembrane pathway by which K ؉ is released is separate and distinct from the structure required for intoxication but may be related to, or a precursor of, that which is ultimately responsible for hemolysis.

Acute stress can exert beneficial or detrimental effects on different forms of cognition. In the present study, we assessed the effects of acute restraint stress on different forms of cost/benefit decision-making, and some of the hormonal and neurochemical mechanisms that may underlie these effects. Effort-based decision-making was assessed where rats chose between a low effort/reward (1 press=2 pellets) or high effort/reward option (4 pellets), with the effort requirement increasing over 4 blocks of trials (2, 5, 10, and 20 lever presses). Restraint stress for 1 h decreased preference for the more costly reward and induced longer choice latencies. Control experiments revealed that the effects on decision-making were not mediated by general reductions in motivation or preference for larger rewards. In contrast, acute stress did not affect delay-discounting, when rats chose between a small/immediate vs larger/delayed reward. The effects of stress on decision-making were not mimicked by treatment with physiological doses of corticosterone (1-3 mg/kg). Blockade of dopamine receptors with flupenthixol (0.25 mg/kg) before restraint did not attenuate stress-induced effects on effort-related choice, but abolished effects on choice latencies. These data suggest that acute stress interferes somewhat selectively with cost/benefit evaluations concerning effort costs. These effects do not appear to be mediated solely by enhanced glucocorticoid activity, whereas dopaminergic activation may contribute to increased deliberation times induced by stress. These findings may provide insight into impairments in decision-making and anergia associated with stress-related disorders, such as depression.

SummaryThe cytotoxic effect of adenylate cyclase (AC) toxin from Bordetella pertussis on host cells has been attributed to the production of supraphysiologic levels of cyclic AMP by the toxin. We have tested this hypothesis and show that at least two different mechanisms, cAMP accumulation/ATP depletion and oligomerization/pore formation, contribute, perhaps synergistically, to AC toxin-induced cytotoxicity. Wildtype (WT) AC toxin causes cell death associated with an increase in cAMP, a reduction in ATP, activation of caspases 3/7, and increased annexin V and TUNEL staining. In contrast, a non-acylated, enzymatically active, non-haemolytic form of AC toxin is able to increase cAMP, reduce ATP and elicit annexin V staining, but the decrease in ATP and the annexin staining are transient and there is minimal caspase activation, TUNEL staining and cell death. Mutant AC toxins defective in either enzymatic activity or the ability to deliver their enzymatic domain are able to kill J774 cells, without cAMP production, and with minimal caspase activation and TUNEL staining. Comparison of the potencies of WT toxin and those of mutants that only increase cAMP or only create transmembrane pores establishes that at least two mechanisms are contributory and that simply the production of cAMP is not enough to account for the cytotoxicity produced by AC toxin.

In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were <1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.

BackgroundInjection drug use (IDU) is a growing public health threat in Virginia, though there is limited knowledge of related morbidity. The purpose of this study was to describe the temporal, geographic and clinical trends and characteristics of infective endocarditis associated with IDU (IDU-IE) and to identify opportunities for better-quality care of people who inject drugs (PWID).MethodsWe reviewed charts for all admissions coded for both IE and drug use disorders at the University of Virginia Medical Center (UVA) from January 2000 to July 2016. A random sample of 30 admissions coded for IE per year were reviewed to evaluate temporal trends in the proportion of IDU associated IE cases.ResultsThere were a total of 76 patients with IDU-IE during the study period, 7.54-fold increase (prevalence ratio: 8.54, 95% CI 3.70–19.72) from 2000 to 2016. The proportion of IE that was IDU-associated increased by nearly 10% each year (prevalence ratio of IDU per year: 1.09, 95% CI: 1.05–1.14). Patients with IDU-IE had longer hospital stays [median days (interquartile range); IDU-IE, 17 (10–29); non-IDU-IE, 10 (6–18); p-value = 0.001] with almost twice the cost of admission as those without IDU [median (interquartile range); IDU-IE, $47,899 ($24,578-78,144); non-IDU-IE, $26,460 ($10,220-60,059); p-value = 0.001]. In 52% of cases there was no documentation of any discussion regarding addiction treatment.ConclusionIDU-IE is a severe infection that leads to significant morbidity and healthcare related costs. IDU-IE rates are increasing and will likely continue to do so without targeted interventions to help PWID. The diagnosis and treatment of IDU-IE provides an opportunity for the delivery of addiction treatment, counseling, and harm reduction strategies.Electronic supplementary materialThe online version of this article (10.1186/s12879-018-3408-y) contains supplementary material, which is available to authorized users.

SummaryAdenylate cyclase (AC) toxin from Bordetella pertussis is unusual in that, unlike most other members of the repeats-in-toxin family that are released into the extracellular milieu, it remains associated with the bacterial surface. In this study, we investigated the nature of the association of this toxin with the surface of B. pertussis . AC toxin was extracted from crude outer membrane preparations of B. pertussis with 8 M urea, but only partially with alkaline sodium carbonate and not at all with octylglucoside, suggesting that denaturation of the toxin is necessary for its removal from the membrane. B. pertussis mutants lacking filamentous haemagglutinin (FHA) released significantly more AC toxin into the medium, and AC toxin association with the bacterial surface was partially restored by expression of FHA from a plasmid, suggesting a role for FHA in surface retention of AC toxin. AC toxin distribution was unaffected by the absence of pertactin, or full-length lipopolysaccharide, or a defect in secretion of pertussis toxin. Using overlay and immunoprecipitation, we found that a direct physical association can occur between AC toxin and FHA. Combined, these findings suggest that FHA may play a role in AC toxin retention on the surface of B. pertussis and raise the possibility of an involvement of adherence mediated by FHA in delivery of AC toxin from the bacterium to the target cell.

dWhooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ϳ10 8 CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ϳ10 8 CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ϳ60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.