PST receptors were purified and characterized in the liver, hepatoma membranes, as well as the signal transduction (55, 62, 63). This receptor appears to mediate the dual signaling mechanism in liver (57). PST stimulation activates pertussis toxin-insensitive G protein (Gαq/11), leading to the activation of phospholipase C b3 isoform (PLC-b3) (69), and therefore mediates the glycogenolytic effect in the liver by increasing cytoplasmic free calcium and stimulating PKC, while the pertussis toxin-sensitive G protein (Gai1,2) leads to the activation of guanylatecyclase (51). In the signaling pathway, hydrolysis of phosphatidylinositol 4,5-bisphosphate by Ca2+-mobilizing hormones leads to the formation of two second messengers i.e., inositol-1,4,5-triphosphate (InsP3) and diacylglycerol (DAG). The primary function of InsP3 is to mobilize Ca2+ from intracellular stores (60), whereas DAG stimulates PKC (58).. Active PST receptors were solubilized from rat liver membranes, and these results support the ...
The quantitative determination of pertussis-toxin-sensitive guanine-nucleotide-binding proteins (G-proteins) in cell membranes is still a problem. Pertussis-toxin-catalysed [32P]ADP-ribosylation strongly relies on the substrate quality of the alpha-subunits and is influenced by the concentration of nucleotides, beta gamma-subunits, the physicochemical properties of the membranes influencing the availability of Gi alpha for pertussis toxin, and covalent modification of Gi alpha. Quantification of immunoreactive material on Western blots can be only imprecisely performed by two-dimensional densitometry. In order to generate a method for quantification of pertussis-toxin-sensitive G-proteins in membranes we have developed a fast and sensitive radioimmunoassay. The C-terminal decapeptide of retinal transducin alpha (KENLKDCGLF) was 125I-labelled and used as tracer. Polyclonal antiserum (DS 4) was raised against this peptide. Gi alpha proteins were determined by competition of solubilized membranes ...
Previous studies on immortalized B lymphoblasts from patients with EH and enhanced Na+-H+ exchanger activity have revealed an enhanced activation of PTX-sensitive G proteins.7 This conclusion was mainly based on two findings. First, HT lymphoblasts displayed enhanced [Ca2+]i signals upon stimulation with platelet-activating factor and somatostatin. Pretreatment with PTX strongly reduced these agonist-evoked Ca2+ signals, and the residual Ca2+ responses were no longer different between NT and HT cell lines. Second, both receptor-mediated stimulation and direct (by mastoparan-7) stimulation of GTPγS binding to PTX-sensitive G proteins were significantly increased in HT lymphoblasts.7 Unfortunately, B lymphoblasts apparently do not express functional receptors that are selectively coupled to PTX-insensitive G proteins, eg, Gq or Gs. Therefore, our proposal of a selective enhancement of signal transduction via PTX-sensitive G proteins in HT cell lines was based on circumstantial evidence but could ...
TY - JOUR. T1 - The effect of pertussis toxin on the growth of vascular smooth muscle cells stimulated by serum or platelet-derived growth factor. AU - Zhang, L. M.. AU - Newman, W. H.. AU - Castresana, Manuel R. AU - Hildebrandt, J. D.. PY - 1994/1/1. Y1 - 1994/1/1. N2 - The involvement of a G(i)- or G(o)-related G-protein as a regulator of the growth of guinea pig thoracic aorta smooth muscle (TASM) cells was studied by investigating the effects of pertussis toxin (PTX) on the growth of these cells. PTX treatment decreased the growth rate of TASM cells by 70-100%. This effect was apparent within 24 h after exposure in the toxin and persisted for at least 10 days after starting the treatment. The effect of the toxin appeared to be the result of the inactivation of a G-protein because 1) TASM cell membranes contained a 40-kilodalton substrate for the toxin in in vitro assays that was absent in membranes prepared from cells pretreated with toxin; and 2) the effect required both the enzymatic ...
The lipoglycoproteins of the WNT family act on seven transmembrane-spanning Class Frizzled receptors. Here, we show that WNT-5A evokes a proliferative response in a mouse microglia-like cell line (N13), which is sensitive to pertussis toxin, thus implicating the involvement of heterotrimeric G proteins of the G(i/o) family. We continue to show that WNT-5A stimulation of N13 membranes and permeabilized cells evokes the exchange of GDP for GTP of pertussis toxin-sensitive G proteins employing [gamma-(35)S]GTP assay and activity state-specific antibodies to GTP-bound G(i) proteins. Our functional analysis of the PTX-sensitivity of WNT-induced G protein activation and PCR analysis of G protein and FZD expression patterns suggest that WNT-5A stimulation leads to the activation of G(i2/3) proteins in N13 cells possibly mediated by FZD(5), the predominant FZD expressed ...
Chronic stimulation of beta-adrenoceptors leads to increased mRNA and protein levels of pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G proteins) in the heart. In the present study the time course is reported of the effect of isoprenaline infusions on myocardial mRNA levels of Gi alpha-2, Gi alpha-3, G(o) alpha, Gs alpha, and G beta and myocardial levels of PTX-sensitive G proteins. Rats were treated by subcutaneous infusions, with osmotic minipumps, of 0.9% NaCl, isoprenaline (2.4 mg/kg/day), propranolol (9.9 mg/kg/day), or a combination thereof for 1, 2, 3, 4, or 8 days, and two groups were treated with NaCl or isoprenaline for 13 or 26 days. Additional groups of rats were treated with 0.24 or 0.07 mg/kg/day for 4 days to determine the dose dependency of the effects of isoprenaline. mRNA concentrations were determined by standardized slot blotting with 32P-labeled cDNA or cRNA probes. In isoprenaline-treated rats, mRNA levels of all members of the Gi alpha/G(o) alpha ...
GTP-binding regulatory proteins (G proteins) regulate various biological functions, but their participation in controlling coronary microvascular tone has not been established yet. The goal of the present study was to elucidate the role of pertussis toxin (PTX)-sensitive G protein in regulating coronary microvascular tone during autoregulation and ischemia. In 42 open-chest dogs, coronary arterial microvessels on the surface of the left ventricle were directly observed by epi-illuminated fluorescence microangiography using a floating objective system. PTX (300 ng/mL) was superfused onto the surface of the left ventricle for 2 hours to block Gi and G(o) protein in epimyocardial coronary microvessels in vivo. PTX superfusion caused no change in the resting diameters of microvessels and significantly blocked the vasoconstriction induced by BHT 920 (a selective alpha 2-agonist). After pretreatment with PTX or its vehicle, the left anterior descending coronary artery (LAD) was occluded by a hydraulic ...
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The purpose of the present study was to investigate the role of G proteins in α1-adrenergic inhibition of β-adrenergic responses in cardiac myocytes. PTX prevents receptor-dependent activation of the G proteins Gi and Go, and it has been shown to antagonize the ability of α1-adrenergic receptor agonists to inhibit Iso-mediated responses (11). This suggests that α1-adrenergic inhibition of β-adrenergic responses may involve one of these PTX-sensitive G proteins. This conclusion is also consistent with our results demonstrating that PTX increases the sensitivity of the cAMP-regulated Cl− current to activation by NE (Fig. 3). NE is an agonist at both α- and β-adrenergic receptors, and the net response to NE is a balance between the inhibitory and stimulatory effects of α- and β-adrenergic receptor stimulation, respectively (11). Therefore, the increase in NE sensitivity could be explained if PTX were blocking the α1-adrenergic component. However, this interpretation is complicated by ...
Guanine nucleotide-binding proteins (G proteins) are involved as modulators or transducers in various transmembrane signaling systems.
Guanine nucleotide-binding proteins (G proteins) are involved as modulators or transducers in various transmembrane signaling systems.
Guanine nucleotide-binding proteins (G proteins) ares involved as modulators or transducers in various transmembranes signaling ...
Bordetella pertussis adenylate cyclase. Penetration into host cells.: Exposure of Chinese hamster ovary, mouse adrenal cortex tumor (Y-1), THP-1 and U-937 cells
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The effect of exogenously added adenylate cyclase from Bordetella pertussis (strain 114) has been investigated in Y-1 mouse adrenal tumor, chinese hamster ovary (CHO) and several other cells. A partially purified adenylate cyclase was found not to enter cells but, nevertheless, produced large amounts of cAMP in the medium. We could show that this resulted from release of ATP (and not larger molecules). The ATP released by the cells could be (1) directly measured and was replenished after each change of medium; (2) was reciprocally related to the cAMP produced; and (3) was competed for by ATPases present in added serum or by hexokinase and, less effectively, by exoenzymes on the cell surface. The extent of ATP leakage varied widely between different cell lines, being marked in CHO and Y-1 adrenal cells but negligible in transformed lymphocyte lines. The uncertainty of the origin of cAMP found in media of cultured cells requires separate analysis of cell and medium cAMP and an assessment of ATP ...
In vivo administration of pertussis toxin is often used to study the involvement of guanine nucleotide binding proteins in signal transduction. Especially when it is administered in the brain the effect is often poor. This could be due to the fact that pertussis toxin does not reach the area of interest. To evaluate the extent to which pertussis toxin is distributed in rat brain after intraventricular injection, different techniques were used. Immunohistochemical studies with an antibody against pertussis toxin showed that immunoreactivity was limited to periventricular brain structures less than 0.5 mm from the lumen. The highest immunoreactivity was seen 16-24 h after injection. After 96 h the labeling was very weak. The proportion of guanine nucleotide binding proteins that were ADP-ribosylated by in vivo injection of pertussis toxin into the ventricles as assessed by in vitro [32P]-back-ADP-ribosylation was very low 48 h after the injection, in all regions studied. Direct injection of pertussis
Regulation of neuronal voltage-activated Ca2+ channels by neurotransmitters and intracellular signaling pathways is an important step in the control of neurotransmitter release, synaptic transmission, and neuronal plasticity. In the present study, we have determined the effect of a novel anti-dementia drug FK960 on voltage-activated Ca2+ channels in isolated rat hippocampal neurons. Our results demonstrate for the first time that FK960 modulates the G protein-mediated inhibitory effect of somatostatin on Ca2+ channels and, furthermore, enhances the basal Ca2+ currents in hippocampal neurons.. It has been suggested that somatostatin receptors inhibit N-type Ca2+ channels via PTX-sensitive G proteins through a direct membrane-delimited model (Shapiro and Hille, 1993; Hille et al., 1995; Zhang et al., 1996). Somatostatin-induced inhibition of Ca2+ currents in these isolated hippocampal neurons is mediated by activation of G proteins because PTX as well as GTPγ S eliminated this inhibition. In ...
In this study, the regulation of striatal cyclic-3,5-adenosine monophosphate (cAMP) formation and GABA release by dopamine D1 and metabotropic glutamate receptors (mGluR) was studied in brain slices. In the absence of adenosine A2 receptor blockade, the mGluR agonist, 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) stimulated cAMP accumulation through a pertussis toxin-insensitive mechanism that could be blocked by L-serine-o-phosphate, but not by L(+)-2-amino-3-phosphonopropionic acid. However, in the presence of the adenosine antagonist, 3-isobutyl-1-methylxanthine, 1S,3R-ACPD had no significant effect on basal cAMP, but it inhibited cAMP formation stimulated by the D1 agonist, SKF 38393. This inhibitory response was prevented by pertussis toxin pretreatment and mimicked by L(+)-2-amino-3-phosphonopropionic acid, but it was unaffected by L-serine-o-phosphate. Thus, 1S,3R-ACPD was determined to activate distinct mGluRs in the striatum that mediate either inhibition or activation of ...
The abilities of cysteine-containing compounds to support growth of and influence pertussis toxin transcription, assembly, and secretion were examined. source of cysteine; however, in the absence of reduced glutathione, pertussis toxin was not efficiently secreted. Addition of the reducing agent dithiothreitol was unable to compensate for the lack of reduced glutathione and did not promote secretion of pertussis toxin. These results suggest that reduced glutathione does not affect the accumulation of assembled active pertussis toxin in the periplasm but plays a role in efficient pertussis toxin secretion by the bacterium. Pertussis toxin is a major virulence factor of heat-labile toxin, and Shiga toxin. Pertussis toxin has the most complex structure of any bacterial toxin (18, 20, 24). It is assembled from six subunits encoded by five genes, to encodes the structural gene for the A-subunit, which is an ADP-ribosyltransferase. S1 modifies mammalian G-proteins, which play a critical role in ...
Certain microbial toxins are ADP-ribosyltransferases, acting on specific substrate proteins. Although these toxins have been of great utility in studies of cellular regulatory processes, a simple procedure to directly study toxin-catalyzed ADP-ribosy
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G proteins are trimeric (alpha-beta-gamma) membrane-associated proteins that regulate flow of information from cell surface receptors to a variety of internal metabolic effectors. Interaction of a G protein with its activated receptor promotes exchange of GTP for GDP that is bound to the alpha subunit. The alpha-GTP complex dissociates from the beta-gamma heterodimer so that the subunits, in turn, may interact with and regulate effector molecules (Gilman, 1987 [PubMed 3113327]; summary by Ahmad et al., 1995) [PubMed 7606925].[supplied by OMIM, Nov 2010 ...
Both calcitonin and prostaglandin E2 (PGE2) stimulate adenylate cyclase activity in the human breast cancer cell line (T 47D). The maximum cyclic AMP response to calcitonin exceeds that of PGE2. When maximal concentrations of the two hormones were added simultaneously to the cells, the amount of cyclic AMP generated was less than that seen with calcitonin alone. When cells were treated with the protein toxin of Bordetella pertussis (islet-activating protein; IAP) which inactivates the inhibitory regulatory component (Ni) of adenylate cyclase, there was no change in basal or calcitonin-responsive adenylate cyclase in intact cells. However, the PGE2 response was augmented at all dose levels, and this effect was dependent on the concentration of IAP. Moreover, in cells pretreated with IAP, simultaneous addition of PGE2 and calcitonin resulted in additivity rather than in inhibition of cyclic AMP production. The additivity of the response to calcitonin and PGE2 after IAP treatment implies activation ...
Semantic Scholar extracted view of Spermine inhibition of basal and stimulated adenylate cyclase is mediated by the inhibitory GTP-binding protein (Gi). by Carlo Clô et al.
Adenylate cyclase toxin (CyaA) from Bordetella pertussis can subvert host immune responses allowing bacterial colonization. Here we have examined its adjuvant and immunomodulatory properties and the possible contribution of lipopolysaccharide (LPS), known to be present in purified CyaA preparations. CyaA enhanced antigen-specific interleukin-5 (IL-5) and IL-10 production and immunoglobulin G1 antibodies to coadministered antigen in vivo. Antigen-specific CD4+-T-cell clones generated from mice immunized with antigen and CyaA had cytokine profiles characteristic of Th2 or type 1 regulatory T (Tr1) cells. Since innate immune cells direct the induction of T-cell subtypes, we examined the influence of CyaA on activation of dendritic cells (DC) and macrophages. CyaA significantly augmented LPS-induced IL-6 and IL-10 and inhibited LPS-driven tumor necrosis factor alpha and IL-12p70 production from bone marrow-derived DC and macrophages. CyaA also enhanced cell surface expression of CD80, CD86, and ...
Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes one of two cannabinoid receptors. The cannabinoids, principally delta-9-tetrahydrocannabinol and synthetic analogs, are psychoactive ingredients of marijuana. The cannabinoid receptors are members of the guanine-nucleotide-binding protein (G-protein) coupled receptor family, which inhibit adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. The two receptors have been found to be involved in the cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana. Multiple transcript variants encoding two different protein isoforms have been described for this gene. [provided by RefSeq, May 2009 ...
Bordetella pertussis ATCC ® BAA-589D-5™ Designation: Genomic DNA from Bordetella pertussis Strain Tohama 1 TypeStrain=False Application:
Hydrolyzes lysophospholipids to produce lysophosphatidic acid (LPA) in extracellular fluids. Major substrate is lysophosphatidylcholine. Also can act on sphingosylphosphphorylcholine producing sphingosine-1-phosphate, a modulator of cell motility. Can hydrolyze, in vitro, bis-pNPP, to some extent pNP-TMP, and barely ATP. Involved in several motility-related processes such as angiogenesis and neurite outgrowth. Acts as an angiogenic factor by stimulating migration of smooth muscle cells and microtubule formation. Stimulates migration of melanoma cells, probably via a pertussis toxin-sensitive G protein. May have a role in induction of parturition. Possible involvement in cell proliferation and adipose tissue development. Tumor cell motility-stimulating factor ...
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Pertactin is a virulence factor for B. pertussis which is an adaptation of the organism that enables it to colonise its host. Pertactin is an adhesin that promotes adhesion of B. pertussis bacteria to the tracheal epithelium of humans. Pertactin (Prn) is one of the antigens in the acellular vaccine along with inactivated pertussis toxin (PT), filamentous hemagglutinin (FHA) and fimbriae types 2 and 3 (Fim). The U.S. group, as reported in NEJM, found both insertions and stop codons that prevented the B. pertussis bacterium from expressing pertactin although the virulence of the pertactin negative strains was the same as pertactin positive strains ...
Bordetella pertussis toxin IgG for suspected cases over 12 months of age with cough ,14 days duration and who have not been immunised against pertussis in the past year. The IgG test can not be used to determine immunity as there are currently no agreed correlated of protection. Bordetella pertussis DNA for suspected cases with cough ,21 days duration and within 48 hours of antibiotic therapy.. ...
Pertussis (also known as whooping cough) continues to be a global health problem with an estimated 45 million cases annually and 300,00 deaths, which occur most...
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pep:known chromosome:VEGA66:3:146499807:146505572:1 gene:OTTMUSG00000029572 transcript:OTTMUST00000073335 gene_biotype:protein_coding transcript_biotype:protein_coding gene_symbol:Gng5 description:guanine nucleotide binding protein (G protein), gamma 5 subunit ...
GNG12 overexpression lysate, 0.1 mg. Transient overexpression lysate of guanine nucleotide binding protein (G protein), gamma 12 (GNG12)
Homo sapiens cDNA FLJ78228 complete cds, highly similar to Homo sapiens guanine nucleotide binding protein (G protein), beta 5(GNB5), transcript variant 1, mRNA ...
The drug brand named Pentact-HIB contains generic salt-Bordetella Pertussis inactivated and is manufactured by Sanofi-Aventis ...
Bordetella pertussis is the causative agent of human whooping cough (pertussis) and is particularly severe in infants. Despite worldwide vaccinations, whooping cough remains a public health problem. A significant increase in the incidence of whooping cough has been observed in many countries since the 1990s. Several reasons for the re-emergence of this highly contagious disease have been suggested. A particularly intriguing possibility is based on evidence indicating that pathogen adaptation may play a role in this process. In an attempt to gain insight into the genomic make-up of B. pertussis over the last 60 years, we used an oligonucleotide DNA microarray to compare the genomic contents of a collection of 171 strains of B. pertussis isolates from different countries. The CGH microarray analysis estimated the core genome of B. pertussis, to consist of 3,281 CDSs that are conserved among all B. pertussis strains, and represent 84.8% of all CDSs found in the 171 B. pertussis strains. A total of 64
TY - JOUR. T1 - Signal transduction by guanine nucleotide binding proteins. AU - Spiegel, Allen M.. PY - 1987/1. Y1 - 1987/1. N2 - High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes S.M. (1981) FEBS Lett. 164, 1-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the ras gene products (Ellis R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effectors (E). A similar function has been suggested but not proven for the ras gene products. Known G-proteins include Gs and Gi, the G-proteins ...
In order to achieve batch-to-batch consistency of whole-cell pertussis vaccines, properties relevant for protection and safety should be characterised. Therefore, ELISAs to quantify pertussis toxin (PT), filamentous haemagglutinin (FHA), 92 kD outer membrane protein (92 kD-OMP) and pertactin (PRN) in Bordetella pertussis (B. pertussis) suspensions were developed. In this paper the influence of the bacterial growth stage on antigen production and antigen release into the supernatant was studied for pertussis strains 134, 509 and CS. The levels of cell-associated and free antigens during growth were strongly strain and antigen dependent. Because of this, the proportion of cell-associated antigens changed during cultivation for all three strains. Substantial amounts of PT and PRN were released into the supernatant, while little free FHA and 92 kD-OMP were found. The amount of cell-associated FHA declined rapidly during growth, whereas cell-associated 92 kD-OMP contents increased. These findings ...
The CB1 receptor is a pre-synaptic heteroreceptor that modulates neurotransmitter release when activated in a dose-dependent, stereoselective and pertussis toxin-sensitive manner.[13] The CB1 receptor is activated by cannabinoids, generated naturally inside the body (endocannabinoids) or introduced into the body as cannabis or a related synthetic compound. Research suggests that the majority of CB1 receptors are coupled through Gi/o proteins. Upon activation, CB1 receptor exhibits its effects mainly through activation of Gi, which decreases intracellular cAMP concentration by inhibiting its production enzyme, adenylate cyclase, and increases mitogen-activated protein kinase (MAP kinase) concentration. Alternatively, in some rare cases CB1 receptor activation may be coupled to Gs proteins, which stimulate adenylate cyclase.[10] cAMP is known to serve as a second messenger coupled to a variety of ion channels, including the positively influenced inwardly rectifying potassium channels (=Kir or ...
Acellular vaccines against Bordetella pertussis were introduced in Australia in 1997. By 2000, these vaccines had replaced whole-cell vaccines. During 2008-2012, a large outbreak of pertussis occurred. During this period, 30% (96/320) of B. pertussis isolates did not express the vaccine antigen pertactin (prn). Multiple mechanisms of prn inactivation were documented, including IS481 and IS1002 disruptions, a variation within a homopolymeric tract, and deletion of the prn gene. The mechanism of lack of expression of prn in 16 (17%) isolates could not be determined at the sequence level. These findings suggest that B. pertussis not expressing prn arose independently multiple times since 2008, rather than by expansion of a single prn-negative clone. All but 1 isolate had ptxA1, prn2, and ptxP3, the alleles representative of currently circulating strains in Australia. This pattern is consistent with continuing evolution of B. pertussis in response to vaccine selection pressure.
Pertussis, also known as whooping cough, is an acute respiratory infectious disease caused by a bacteria called Bordetella Pertussis. The characteristic symptoms are paroxysmal cough, inspiratory wheezing and post-tussive vomiting. Following the inhalation of respiratory secretions from an infected individual, bacteria enter the upper respiratory tract and adhere to epithelial cells. Several adhesion factors have been implicated: the filamentous hemagglutinin (FHA), fimbriae, and pertactin (Prn). Pertussis toxin (Ptx) and adenylate cyclase toxin (ACT) have been identified so far as major protein toxins of B. pertussis. PTX is a hexameric AB5-type exotoxin. Catalytic A subunit catalyzes the ADP-ribosylation of the Gi subunits of the heterotrimeric G protein, then inhibits multiple downstream pathways. ACT is able to penetrate the cytoplasmic membrane of host cells and becomes activated through the cleavage and the binding of calmodulin (CaM). Activated ACT converts ATP to cyclic AMP and subverts ...
Mooi FR; van Loo IHM; van Gent M; He Q; Bart MJ; Heuvelman KJ; de Greeff SC; Diavatopoulos D; Teunis P; Nagelkerke N; Mertsola J (2009 ...
What is pertussis (whooping cough)? Pertussis (also called whooping cough) is a disease caused by the bacteria Bordetella pertussis that spreads from person-to-person with close contact. It may cause severe coughing fits which can affect breathing. Pertussis is often milder in older children and adults, but can cause serious problems in infants. Pertussis can lead to pneumonia, convulsions, inflammation of the brain and sometimes death. Most of these serious problems occur in infants who are less than one year old. Who can get pertussis? Pertussis can occur in any age group; however, pertussis is more common among infants since they are too young to have full protection from the vaccine. Pertussis is also more common in adolescents and adults who have lost the protection they got from vaccination or illness in childhood. How is pertussis spread? Pertussis is spread from one person to another through respiratory droplets from the nose or throat of an infected person by coughing or sneezing, and ...