Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin.

Bordetella pertussis causes the severe childhood disease whooping cough, by releasing several toxins, including pertussis toxin (PT) as a major virulence factor. PT is an AB5-type toxin, and consists of the enzymatic A-subunit PTS1 and five B-subunits, which facilitate binding to cells and transport of PTS1 into the cytosol. PTS1 ADP-ribosylates α-subunits of inhibitory G-proteins (Gαi) in the cytosol, which leads to disturbed cAMP signaling. Since PT is crucial for causing severe courses of disease, our aim is to identify new inhibitors against PT, to provide starting points for novel therapeutic approaches. Here, we investigated the effect of human antimicrobial peptides of the defensin family on PT. We demonstrated that PTS1 enzyme activity in vitro was inhibited by α-defensin-1 and -5, but not ß-defensin-1. The amount of ADP-ribosylated Gαi was significantly reduced in PT-treated cells, in the presence of α-defensin-1 and -5. Moreover, both α-defensins decreased PT-mediated effects on cAMP signaling in the living cell-based interference in the Gαi-mediated signal transduction (iGIST) assay. Taken together, we identified the human peptides α-defensin-1 and -5 as inhibitors of PT activity, suggesting that these human peptides bear potential for developing novel therapeutic strategies against whooping cough.

Discovery of Compounds Inhibiting the ADP-Ribosyltransferase Activity of Pertussis Toxin.

The targeted pathogen-selective approach to drug development holds promise to minimize collateral damage to the beneficial microbiome. The AB5-topology pertussis toxin (PtxS1-S5) is a major virulence factor of Bordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates α-subunits of the heterotrimeric Gαi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify milligram-levels of active recombinant B. pertussis PtxS1 from Escherichia coli and an in vitro high throughput-compatible assay to quantify NAD+ consumption during PtxS1-catalyzed ADP-ribosylation of Gαi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC50-values (3.0 µM and 6.8 µM) were identified in the in vitro NAD+ consumption assay that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gαi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD+-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB5 holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells with a low micromolar IC50-value (2.4 µM). NSC228155 and NSC29193 might prove to be useful hit compounds in targeted B. pertussis-selective drug development.

A Novel Role for Somatostatin in the Survival of Mouse Pancreatic Beta Cells.

BACKGROUND/AIMS: Cross-talk between different pancreatic islet cell types regulates islet function and somatostatin (SST) released from pancreatic delta cells inhibits insulin secretion from pancreatic beta cells. In other tissues SST exhibits both protective and pro-apoptotic properties in a tissue-specific manner, but little is known about the impact of the peptide on beta cell survival. Here we investigate the specific role of SST in the regulation of beta cell survival in response to physiologically relevant inducers of cellular stress including palmitate, cytokines and glucose. METHODS: Pancreatic MIN6 beta cells and primary mouse islet cells were pre-treated with SST with or without the Gi/o signalling inhibitor, pertussis toxin, and exposed to different cellular stress factors. Apoptosis and proliferation were assessed by measurement of caspase 3/7 activity, TUNEL and BrdU incorporation, respectively, and expression of target genes was measured by qPCR. RESULTS: SST partly alleviated upregulation of cellular stress markers (Hspa1a and Ddit3) and beta cell apoptosis in response to factors such as lipotoxicity (palmitate), pro-inflammatory cytokines (IL1ß and TNFα) and low glucose levels. This effect was mediated via a Gi/o protein-dependent pathway, but did not modify transcriptional upregulation of the specific NFκB-dependent genes, Nos2 and Ccl2, nor was it associated with transcriptional changes in SST receptor expression. CONCLUSION: Our results suggest an underlying protective effect of SST which modulates the beta cell response to ER stress and apoptosis induced by a range of cellular stressors associated with type 2 diabetes.

Comparison of anti-pertussis toxin ELISA and agglutination assays to assess immune responses to pertussis.

BACKGROUND: The goal of our study was to compare the following two methods of assessment of pertussis post-vaccination immunity: bacterial agglutination test and pertussis toxin enzyme-linked immunosorbent assay (ELISA). METHODS: The study was carried out in Perm Region, Russia. We measured pertussis immunity using two serological methods: ELISA of IgG to pertussis toxin (PT) and the agglutination test (AT) among 135 children, in the age range from 2 months to 17 years old. The immunization schedule included four doses of DTwP: at 3, 4.5 and 6 months of age and a booster at 18 months. All participants were divided into six age groups. RESULTS: The percentage of samples with IgG level less than the detection limit in vaccinated children was 52.2%. The total seropositivity rate (the percent of children with agglutinin titres ≥1:160) in vaccinated children was 47.8%. Only a weak association was observed between agglutinin and anti-PT IgG titres (R = .3). Neither the primary nor the booster vaccination with DTwP influenced the IgG levels in children. Agglutinin titres significantly increased after vaccination and declined 5 years after the booster dose. Significant growth of IgG concentration was observed in 11-year-olds, indicating the presence of B. pertussis circulation in the childhood population. CONCLUSIONS: Based on the obtained results and the results of other authors, we summarize that anti-PT ELISA should be carefully used to assess the population immunity to pertussis. Currently, there is neither a serological test that accurately determines the protection against pertussis nor a distinctive criterion of protection that can be applied in seroepidemiological studies.

Proteome Analysis Is a Valuable Tool to Monitor Antigen Expression during Upstream Processing of Whole-Cell Pertussis Vaccines.

Physicochemical and immunochemical assays were applied to substantiate the relation between upstream processing and the quality of whole-cell pertussis vaccines. Bordetella pertussis bacteria were cultured on a chemically defined medium using a continuous cultivation process in stirred tank reactors to obtain uniform protein expression. Continuous culture favors the consistent production of proteins known as virulence factors. Magnesium sulfate was added during the steady state of the culture in order to diminish the expression of virulence proteins. Changes in gene expression and antigen composition were measured by microarrays, mass spectrometry and ELISA. Transcriptome and proteome data revealed high similarity between the biological triplicates demonstrating consistent cultivation of B. pertussis. The addition of magnesium sulfate resulted in an instant downregulation of the virulence genes in B. pertussis, but a gradual decrease of virulence proteins. The quantity of virulence proteins concurred highly with the potency of the corresponding whole-cell pertussis vaccines, which were determined by the Kendrick test. In conclusion, proteome analysis provided detailed information on the composition and proportion of virulence proteins present in the whole-cell preparations of B. pertussis. Moreover, proteome analysis is a valuable method to monitor the production process of whole-cell biomass and predict the product quality of whole-cell pertussis vaccines.

Reduction of Pertussis Inflammatory Pathology by Therapeutic Treatment With Sphingosine-1-Phosphate Receptor Ligands by a Pertussis Toxin-Insensitive Mechanism.

Recent data have demonstrated the potential of sphingosine 1-phosphate (S1P) receptor (S1PR) agonism in the treatment of infectious diseases. A previous study used a murine model of Bordetella pertussis infection to demonstrate that treatment with the S1PR agonist AAL-R reduces pulmonary inflammation during infection. In the current study, we showed that this effect is mediated via the S1PR1 on LysM+ (myeloid) cells. Signaling via this receptor results in reduced lung inflammation and cellular recruitment as well as reduced morbidity and mortality in a neonatal mouse model of disease. Despite the fact that S1PRs are pertussis toxin-sensitive G protein-coupled receptors, the effects of AAL-R were pertussis toxin insensitive in our model. Furthermore, our data demonstrate that S1PR agonist administration may be effective at therapeutic time points. These results indicate a role for S1P signaling in B. pertussis-mediated pathology and highlight the possibility of host-targeted therapy for pertussis.

Pertussis leukocytosis: mechanisms, clinical relevance and treatment.

The significant and sometimes dramatic rise in the number of circulating white blood cells (leukocytosis) in infants suffering from pertussis (whooping cough) has been recognized for over a century. Although pertussis is a disease that afflicts people of all ages, it can be particularly severe in young infants, and these are the individuals in whom leukocytosis is most pronounced. Very high levels of leukocytosis are associated with poor outcome in infants hospitalized with pertussis and modern treatments are often aimed at reducing the number of leukocytes. Pertussis leukocytosis is caused by pertussis toxin, a soluble protein toxin released by Bordetella pertussis during infection, but the exact mechanisms by which this occurs are still unclear. In this minireview, I discuss the history of clinical and experimental findings on pertussis leukocytosis, possible contributing mechanisms causing this condition and treatments aimed at reducing leukocytosis in hospitalized infants. Since recent studies have detailed significant associations between specific levels of pertussis leukocytosis and fatal outcome, this is a timely review that may stimulate new thinking on how to understand and combat this problem.

Pneumococcal Surface Protein A does not affect the immune responses to a combined diphtheria tetanus and pertussis vaccine in mice.

The Pneumococcal Surface Protein A (PspA) is a promising candidate for the composition of a protein vaccine against Streptococcus pneumoniae. We have previously shown that the whole cell Bordetella pertussis vaccine (wP) is a good adjuvant to PspA, inducing protective responses against pneumococcal infection in mice. In Brazil, wP is administered to children, formulated with diphtheria and tetanus toxoids (DTPw) and aluminum hydroxide (alum) as adjuvant. A single subcutaneous dose of PspA5-DTPlow (a formulation containing PspA from clade 5 and a new generation DTPw, containing low levels of B. pertussis LPS and Alum) induced high levels of systemic anti-PspA5 antibodies in mice and conferred protection against respiratory lethal challenges with two different pneumococcal strains. Here we evaluate the mucosal immune responses against PspA5 as well as the immune responses against the DTP antigens in mice vaccinated with PspA5-DTPlow. Subcutaneous immunization of mice with PspA5-DTPlow induced high levels of anti-PspA5 IgG in the airways but no IgA. In addition, no differences in the influx of cells to the respiratory mucosa, after the challenge, were observed in vaccinated mice, when compared with control mice. The levels of circulating anti-pertussis, -tetanus and -diphtheria antibodies were equivalent in mice vaccinated with DTPlow or PspA5-DTPlow. Antibodies induced by DTPlow or PspA5-DTPlow showed similar ability to neutralize the cytotoxic effects of the diphtheria toxin on Vero cells. Furthermore, combination with PspA5 did not affect protection against B. pertussis and tetanus toxin challenges in mice. Our results support the proposal for a combined PspA-DTP vaccine.

Bis-halogen-anthraniloyl-substituted nucleoside 5'-triphosphates as potent and selective inhibitors of Bordetella pertussis adenylyl cyclase toxin.

Whooping cough is caused by Bordetella pertussis and still constitutes one of the top five causes of death in young children, particularly in developing countries. The calmodulin-activated adenylyl cyclase (AC) toxin CyaA substantially contributes to disease development. Thus, potent and selective CyaA inhibitors would be valuable drugs for the treatment of whooping cough. However, it has been difficult to obtain potent CyaA inhibitors with selectivity relative to mammalian ACs. Selectivity is important for reducing potential toxic effects. In a previous study we serendipitously found that bis-methylanthraniloyl (bis-MANT)-IMP is a more potent CyaA inhibitor than MANT-IMP (Mol Pharmacol 72:526-535, 2007). These data prompted us to study the effects of a series of 32 bulky mono- and bis-anthraniloyl (ANT)-substituted nucleotides on CyaA and mammalian ACs. The novel nucleotides differentially inhibited CyaA and ACs 1, 2, and 5. Bis-ANT nucleotides inhibited CyaA competitively. Most strikingly, bis-Cl-ANT-ATP inhibited CyaA with a potency ≥100-fold higher than ACs 1, 2, and 5. In contrast to MANT-ATP, bis-MANT-ATP exhibited low intrinsic fluorescence, thereby substantially enhancing the signal-to noise ratio for the analysis of nucleotide binding to CyaA. The high sensitivity of the fluorescence assay revealed that bis-MANT-ATP binds to CyaA already in the absence of calmodulin. Molecular modeling showed that the catalytic site of CyaA is sufficiently spacious to accommodate both MANT substituents. Collectively, we have identified the first potent CyaA inhibitor with high selectivity relative to mammalian ACs. The fluorescence properties of bis-ANT nucleotides facilitate development of a high-throughput screening assay.

Pertussis toxin is superior to TLR ligands in enhancing pathogenic autoimmunity, targeted at a neo-self antigen, by triggering robust expansion of Th1 cells and their cytokine production.

Microbial products are assumed to play a major role in triggering pathogenic autoimmunity. Recently accumulated data have shown that these products stimulate the immune system by interacting with TLRs, expressed on APCs. To examine the capacity of various TLR ligands to trigger pathogenic autoimmunity, we used a system in which naive CD4 cells, specific against hen egg lysozyme (HEL), are injected into recipient mice expressing HEL in their eyes. Only when stimulated, the naive cells acquire pathogenic capacity and induce ocular inflammation. Seven TLR ligands were tested in this system: lipoteichoic acid/peptidoglycan, zymosan, poly (I:C), LPS, pertussis toxin (PTX), flagellin, and CpG oligodeoxynucleotide. Treatment of recipient mice with HEL alone stimulated proliferation of the transferred cells, but no disease, whereas ocular inflammation did develop in recipient mice coinjected with HEL and any one of the seven TLR ligands. Inflammation induced by PTX surpassed by its severity those induced by all other tested TLR ligands and was accompanied by a dramatic increase in number of the transferred cells that acquired features of effector Th1 lymphocytes. Ocular inflammation and number of transferred cells in recipients injected with PTX and HEL were substantially reduced by treatment with Abs against IFN-gamma or IL-12, thus indicating the role of these cytokines in the PTX effect. Overall, our observations demonstrate that various TLR ligands are capable of triggering pathogenic autoimmunity and that PTX surpasses other microbial products in this activity, by stimulating excessive proliferation and polarization toward Th1 of naive T cells.

Reversal of ongoing thermal hyperalgesia in mice by a recombinant herpesvirus that encodes human preproenkephalin.

Herpesvirus-mediated transfer of the human preproenkephalin gene to primary afferent nociceptors prevents phasic thermal allodynia/hyperalgesia in mice. It is not known, however, whether similar viral treatments would reverse ongoing or chronic pain and allodynia/hyperalgesia. To this end, mice were given intrathecal injections of pertussis toxin (PTX), which produces a weeks-long thermal hyperalgesia apparently by uncoupling certain G proteins from inhibitory neurotransmitter receptors. This treatment produced profound thermal hyperalgesia in both Adelta and C-fiber thermonociceptive tests lasting at least 6 weeks. However, treatment of skin surfaces with an enkephalin-encoding herpesvirus, but not control virus or vehicle, completely reversed this hyperalgesia. This profound anti-hyperalgesia was observed for both Adelta- and C-fiber-mediated responses. Interestingly, however, while the anti-hyperalgesic effect of the enkephalin-encoding virus on C-fiber-mediated responses was reversed by intrathecal application of micro or delta opioid antagonists, only delta antagonists reversed the effect of this virus on Adelta hyperalgesia. Thus, virus-mediated delivery of the proenkephalin cDNA reverses thermal hyperalgesia produced by PTX-induced ribosylation of inhibitory G proteins by an opioid-mediated mechanism. These results suggest that herpesvirus vectors encoding analgesic peptides may be useful in attenuating centrally mediated, ongoing neuropathic pain and/or hyperalgesia.

D-2-amino-5-phosphonopentanoic acid inhibits intrathecal pertussis toxin-induced thermal hyperalgesia and protein kinase Cgamma up-regulation.

The aim of the present study was to examine the effect of intrathecal (i.t.) injection of pertussis toxin (PTX) on the nociceptive threshold and protein kinase C (PKC) expression in the rat spinal cord. The role of N-methyl-D-aspartic acid (NMDA) receptors in these changes was also examined. Male Wistar rats were implanted with two i.t. catheters, one of which was connected to a mini-osmotic pump and used to infuse saline or D-2-amino-5-phosphonopentanoic acid (D-AP5) (2 microg/h) starting on day 3 after i.t. catheter insertion. Two days later, a single injection of saline or PTX (2 microg) was given via the other catheter, followed by a flush with 10 microl of saline. On day 4 after PTX or saline injection, the thermal paw withdrawal latency was measured, then the rats were sacrificed by decapitation, and the dorsal part of the lumbosacral spinal segments was removed for PKC Western blotting assays. In PTX-treated rats, thermal hyperalgesia was observed, and the PKCgamma content of both the synaptosomal membrane and cytosolic fractions was significantly increased. The levels of alpha-, betaI-, or betaII-PKC isozymes in these fractions were unaffected by PTX treatment. Infusion of the NMDA antagonist, D-AP5, prevented both the thermal hyperalgesia and the increase in PKCgamma isoform expression in PTX-treated rats, and had no effect on these values in nai;ve rats. Intrathecal injection of the PKC inhibitor, chelerythrine (10 microg), significantly inhibited the thermal hyperalgesia observed in PTX-treated rats. These results show that i.t. injection of PTX induced thermal hyperalgesia accompanied by a selective increase in PKCgamma expression in both the synaptosomal membrane and cytosolic fractions of the dorsal horn of the rat lumbar spinal cord, and both effects were inhibited by the NMDA receptor antagonist, D-AP5.

Inhibition of adenylate cyclase activity in the goldfish melanophore is mediated by α2-adrenoceptors and a pertussis toxin-sensitive GTP-binding protein.

The signal-transduction system that mediates the melanosome-aggregating response in melanophores of the black-moor goldfish, Carassius auratus, was investigated by examining the inhibition of adenylate cyclase activity mediated by α-adrenoceptors in cultured cells. When the melanophores were incubated with 1 mmol·1⁻¹ 3-isobutyl-1-methylxanthine for 5 min, the intracellular level of cyclic adenosine-3',5'-monophosphate increased two- to three-fold. Norepinephrine at 100 nmol· 1⁻¹ and naphazoline at 1 µmol· 1⁻¹ inhibited the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3',5'-monophosphate in the cells in both the presence and the absence of isoproterenol, a beta-adrenergic agonist. Methoxamine and phenylephrine also reduced the extent of accumulation of cyclic adenosine-3',5'-monophosphate, but only when they were present at relatively high concentrations (above 100 µmol·1⁻¹). The range of concentrations at which norepinephrine inhibited the accumulation of cyclic adenosine-3',5'-monophosphate was consistent with the range at which it induced the aggregation of melanosomes. Pretreatment of the cells with pertussis toxin (1 µg·m1⁻¹) for 15 h or treatment with 100 nmol·1⁻¹ yohimbine (an α2-adrenergic antagonist) inhibited the effects of the α-adrenergic agonists on both the aggregation of melanosomes and the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3',5'-monophosphate, but prazosin (an α1-adrenergic antagonist) at 100 nmol·1⁻¹ was not inhibitory. These results indicate that the melanosome-aggregating response of the goldfish melanophore is induced mainly via inhibition of the activity of adenylate cyclase,which occurs as a result of stimulation of a pathway that involves α2-adrenoceptors and a inhibitory GTP-binding protein.