Peter Sebo

Complete protection against P. berghei malaria upon heterologous prime/boost immunization against circumsporozoite protein employing Salmonella type III secretion system and Bordetella adenylate cyclase toxoid

Sterile immunity against malaria can be achieved by the induction of IFNgamma-producing CD8(+) T cells that target infected hepatocytes presenting epitopes of the circumsporozoite protein (CSP). In the present study we evaluate the protective efficacy of a heterologous prime/boost immunization protocol based on the delivery of the CD8(+) epitope of Plasmodium berghei CSP into the MHC class I presentation pathway, by either a type III secretion system of live recombinant Salmonella and/or by direct translocation of a recombinant Bordetella adenylate cyclase toxoid fusion (ACT-CSP) into the cytosol of professional antigen-presenting cells (APCs). A single intraperitoneal application of the recombinant ACT-CSP toxoid, as well as a single oral immunization with the Salmonella vaccine, induced a specific CD8(+) T cell response, which however conferred only a partial protection on mice against a subsequent sporozoite challenge. In contrast, a heterologous prime/boost vaccination with the live Salmonella followed by ACT-CSP led to a significant enhancement of the CSP-specific T cell response and induced complete protection in all vaccinated mice.

Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) is a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA targets myeloid phagocytes expressing the complement receptor 3 (CR3, known as αMβ2 integrin CD11b/CD18 or Mac-1) and translocates by a poorly understood mechanism directly across the cytoplasmic membrane into cell cytosol of phagocytes an adenylyl cyclase(AC) enzyme. This binds intracellular calmodulin and catalyzes unregulated conversion of cytosolic ATP into cAMP. Among other effects, this yields activation of the tyrosine phosphatase SHP-1, BimEL accumulation and phagocyte apoptosis induction. In parallel, CyaA acts as a cytolysin that forms cation-selective pores in target membranes. Direct penetration of CyaA into the cytosol of professional antigen-presenting cells allows the use of an enzymatically inactive CyaA toxoid as a tool for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can be exploited for induction of antigen-specific CD8(+) cytotoxic T-lymphocyte immune responses.

Bordetella pertussis Adenylate Cyclase Toxin Blocks Induction of Bactericidal Nitric Oxide in Macrophages through cAMP-Dependent Activation of the SHP-1 Phosphatase

The adenylate cyclase toxin–hemolysin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates complement receptor 3–expressing phagocytes and catalyzes uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by complement-dependent oxidative burst and opsonophagocytic mechanisms. We show that cAMP signaling through the protein kinase A (PKA) pathway activates Src homology domain 2 containing protein tyrosine phosphatase (SHP) 1 and suppresses production of bactericidal NO in macrophage cells. Selective activation of PKA by the cell-permeable analog N6-benzoyladenosine-3′,5′-cyclic monophosphate interfered with LPS-induced inducible NO synthase (iNOS) expression in RAW264.7 macrophages, whereas inhibition of PKA by H-89 largely restored the production of iNOS in CyaA-treated murine macrophages. CyaA/cAMP signaling induced SHP phosphatase–dependent dephosphorylation of the c-Fos subunit of the transcription factor AP-1 and thereby inhibited TLR4-triggered induction of iNOS gene expression. Selective small interfering RNA knockdown of SHP-1, but not of the SHP-2 phosphatase, rescued production of TLR-inducible NO in toxin-treated cells. Finally, inhibition of SHP phosphatase activity by NSC87877 abrogated B. pertussis survival inside murine macrophages. These results reveal that an as yet unknown cAMP-activated signaling pathway controls SHP-1 phosphatase activity and may regulate numerous receptor signaling pathways in leukocytes. Hijacking of SHP-1 by CyaA action then enables B. pertussis to evade NO-mediated killing in sentinel cells of innate immunity.

Nematode-induced interference with the anti-Plasmodium CD8 T-cell response can be overcome by optimizing antigen administration

Malaria is still responsible for up to 1 million deaths per year worldwide, highlighting the need for protective malaria vaccines. Helminth infections that are prevalent in malaria endemic areas can modulate immune responses of the host. Here we show that Strongy‐Ioides ratti, a gut‐dwelling nematode that causes transient infections, did not change the efficacy of vaccination against Plasmodium berghei. An ongoing infection with Litomosoides sigmodontis, a tissue‐dwelling filaria that induces chronic infections in BALB/c mice, significantly interfered with vaccination efficacy. The induction of P. berghei circumspor‐ozoite protein (CSP)‐specific CD8+ T cells, achieved by a single immunization with a CSP fusion protein, was diminished in L. sigmodontis‐infected mice. This modulation was reflected by reduced frequencies of CSP‐specific CD8+ T cells, reduced CSP‐specific IFN‐y and TNF‐a production, reduced CSP‐specific cytotoxicity, and reduced protection against P. berghei challenge infection. Implementation of a more potent vaccine regime, by first priming with CSP‐expressing recombinant live Salmonella prior to CSP fusion protein immunization, restored induction of CSP‐specific CD8+ T cells and conferred almost sterile immunity to P. berghei challenge infection also in L. sigmodontis‐infected mice. In summary, we show that appropriate vaccination regimes can overcome helminth‐induced interference with vaccination efficacy.

cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity.

The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in immune evasion and virulence of the whooping cough agent Bordetella pertussis. CyaA penetrates the complement receptor 3–expressing phagocytes and ablates their bactericidal capacities by catalyzing unregulated conversion of cytosolic ATP to the key second messenger molecule cAMP. We show that signaling of CyaA-generated cAMP blocks the oxidative burst capacity of neutrophils by two converging mechanisms. One involves cAMP/protein kinase A–mediated activation of the Src homology region 2 domain–containing phosphatase-1 (SHP-1) and limits the activation of MAPK ERK and p38 that are required for assembly of the NADPH oxidase complex. In parallel, activation of the exchange protein directly activated by cAMP (Epac) provokes inhibition of the phospholipase C by an as yet unknown mechanism. Indeed, selective activation of Epac by the cell-permeable analog 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate counteracted the direct activation of phospholipase C by 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide. Hence, by inhibiting production of the protein kinase C–activating lipid, diacylglycerol, cAMP/Epac signaling blocks the bottleneck step of the converging pathways of oxidative burst triggering. Manipulation of neutrophil membrane composition by CyaA-produced signaling of cAMP thus enables B. pertussis to evade the key innate host defense mechanism of reactive oxygen species–mediated killing of bacteria by neutrophils.

cAMP signalling of Bordetella adenylate cyclase toxin through the SHP-1 phosphatase activates the BimEL-Bax pro-apoptotic cascade in phagocytes

The adenylate cyclase toxin‐hemolysin (CyaA, ACT or AC‐Hly) plays a key role in virulence of Bordetella pertussis. CyaA penetrates myeloid cells expressing the complement receptor 3 (αMβ2 integrin CD11b/CD18) and subverts bactericidal capacities of neutrophils and macrophages by catalysing unregulated conversion of cytosolic ATP to the key signalling molecule adenosine 3,5‐cyclic monophosphate (cAMP). We show that the signalling of CyaA‐produced cAMP hijacks, by an as yet unknown mechanism, the activity of the tyrosine phosphatase SHP‐1 and activates the pro‐apoptotic BimEL‐Bax cascade. Mitochondrial hyperpolarization occurred in human THP‐1 macrophages within 10u2009min of exposure to low CyaA concentrations (e.g. 20u2009ngu2009ml−1) and was accompanied by accumulation of BimEL and association of the pro‐apoptotic factor Bax with mitochondria. BimEL accumulation required cAMP/protein kinase A signalling, depended on SHP‐1 activity and was selectively inhibited upon small interfering RNA knockdown of SHP‐1 but not of the SHP‐2 phosphatase. Moreover, signalling of CyaA‐produced cAMP inhibited the AKT/protein kinase B pro‐survival cascade, enhancing activity of the FoxO3a transcription factor and inducing Bim transcription. Synergy of FoxO3a activation with SHP‐1 hijacking thus enables the toxin to rapidly trigger a persistent accumulation of BimEL, thereby activating the pro‐apoptotic programme of macrophages and subverting the innate immunity of the host.

Proteome analysis of Bordetella pertussis isolated from human macrophages

UNLABELLEDnPrevious studies have shown that B. pertussis survives inside human macrophages in non-acidic compartments with characteristics of early endosomes. In order to gain new insight into the biology of B. pertussis survival in host cells, we have analyzed the adaptation of the bacterial proteome during intracellular infection. The proteome of B. pertussis 3 h and 48 h after infection of human macrophage-like THP-1 cells was examined by nano-liquid chromatography combined with tandem MS and compared to the protein profile of extracellular B. pertussis growing in the same cell culture medium. Compared with extracellular bacteria, almost 300 proteins out of 762 identified proteins displayed altered levels in intracellular B. pertussis. Functional analyses of the proteins displaying altered abundance revealed enrichment of proteins involved in stress response, iron uptake, cellular metabolism, transcriptional regulation, and virulence. To our knowledge, this is the first analysis of the B. pertussis proteome during adaptation to the intramacrophage environment and the data provide new clues for understanding B. pertussis adaptation and pathogenesis.nnnBIOLOGICAL SIGNIFICANCEnB. pertussis is a respiratory pathogen that has adapted exclusively to the human host. Despite high vaccination rates, whooping cough remains a serious threat to human health and its incidence has been increasing in recent years in vaccinated populations. The mechanisms that allow this pathogen to evade immune clearance, persist in the host, and cause a prolonged paroxysmal cough are still poorly understood. Recent studies regarding B. pertussis survival inside host cells and the cellular response to this bacterial infection indicate that B. pertussis may have an intracellular phase during infection which probably contributes to persistence and vaccine failure. In this study we provide the first global proteome profile of B. pertussis within macrophages. The data provide novel insights into the adaptive responses elicited by these bacteria for physiological adaptation to the host environment.

Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8+ memory T cells

Protection against malaria can be achieved by induction of a strong CD8+ T‐cell response against the Plasmodium circumsporozoite protein (CSP), but most subunit vaccines suffer from insufficient memory responses. In the present study, we analyzed the impact of postimmunization sporozoite challenge on the development of long‐lasting immunity. BALB/c mice were immunized by a heterologous prime/boost regimen against Plasmodium berghei CSP that induces a strong CD8+ T‐cell response and sterile protection, which is short‐lived. Here, we show that protective immunity is prolonged by a sporozoite challenge after immunization. Repeated challenges induced sporozoite‐specific antibodies that showed protective capacity. The numbers of CSP‐specific CD8+ T cells were not substantially enhanced by sporozoite infections; however, CSP‐specific memory CD8+ T cells of challenged mice displayed a higher cytotoxic activity than memory T cells of immunized‐only mice. CD4+ T cells contributed to protection as well; but CD8+ memory T cells were found to be the central mediator of sterile protection. Based on these data, we suggest that prolonged protective immunity observed after immunization and infection is composed of different antiparasitic mechanisms including CD8+ effector‐memory T cells with increased cytotoxic activity as well as CD4+ memory T cells and neutralizing antibodies.

Comparative genomics of Czech vaccine strains of Bordetella pertussis

Bordetella pertussis is a strictly human pathogen causing the respiratory infectious disease called whooping cough or pertussis. B. pertussis adaptation to acellular pertussis vaccine pressure has been repeatedly highlighted, but recent data indicate that adaptation of circulating strains started already in the era of the whole cell pertussis vaccine (wP) use. We sequenced the genomes of five B. pertussis wP vaccine strains isolated in the former Czechoslovakia in the pre-wP (1954-1957) and early wP (1958-1965) eras, when only limited population travel into and out of the country was possible. Four isolates exhibit a similar genome organization and form a distinct phylogenetic cluster with a geographic signature. The fifth strain is rather distinct, both in genome organization and SNP-based phylogeny. Surprisingly, despite isolation of this strain before 1966, its closest sequenced relative appears to be a recent isolate from the US. On the genome content level, the five vaccine strains contained both new and already described regions of difference. One of the new regions contains duplicated genes potentially associated with transport across the membrane. The prevalence of this region in recent isolates indicates that its spread might be associated with selective advantage leading to increased strain fitness.

Bordetella pertussis and Bordetella bronchiseptica filamentous hemagglutinins are processed at different sites

Filamentous hemagglutinin (FHA) mediates adherence and plays an important role in lower respiratory tract infections by pathogenic Bordetellae. The mature FHA proteins of B. pertussis (Bp‐FHA) and the B. bronchiseptica (Bb‐FHA) are generated by processing of the respective FhaB precursors by the autotransporter subtilisin‐type protease SphB1. We have used bottom‐up proteomics with differential 16O/18O labeling and show that despite high‐sequence conservation of the corresponding FhaB segments, the mature Bp‐FHA (~ 230 kDa) and Bb‐FHA (~ 243 kDa) proteins are processed at different sites of FhaB, after the Ala‐2348 and Lys‐2479 residues, respectively. Moreover, protease surface accessibility probing by on‐column (on‐line) digestion of the Bp‐FHA and Bb‐FHA proteins yielded different peptide patterns, revealing structural differences in the N‐terminal and C‐terminal domains of the Bp‐FHA and Bb‐FHA proteins. These data indicate specific structural variations between the highly homologous FHA proteins.