Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines.

Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of corresponding mRNAs were determined in leaves of hydroponically grown rice (Oryza sativa) plants during natural senescence. The plants were grown in the greenhouse for 105 days at which time the thirteenth leaf was fully expanded. This was counted as zero time for senescence of the twelfth leaf. The twelfth leaf blade on the main stem was analyzed over a time period of -7 days (98 days after germination) to +42 days (147 days after germination). Total GS activity declined to less than a quarter of its initial level during the senescence for 35 days and this decline was mainly caused by a decrease in the amount of GS2 polypeptide. Immunoblotting analyses showed that contents of other chloroplastic enzymes, such as ribulose-1,5-bisphosphate carboxylase/oxygenase and Fd-glutamate synthase, declined in parallel with GS2. In contrast, the GS1 polypeptide remained constant throughout the senescence period. Translatable mRNA for GS1 increased about fourfold during the senescence for 35 days. During senescence, there was a marked decrease in content of glutamate (to about one-sixth of the zero time value); glutamate is the major form of free amino acid in rice leaves. Glutamine, the major transported amino acid, increased about threefold compared to the early phase of the harvest in the senescing rice leaf blades. These observations suggest that GS1 in senescing leaf blades is responsible for the synthesis of glutamine, which is then transferred to the growing tissues in rice plants.The major source of nitrogen for developing leaves and ears in mature rice plants is the nitrogen released from older senescing leaves. Our previous study with 'sN showed that remobilized nitrogen accounted for 64% of the total nitrogen in the youngest leaf blades (12). Rubisco, the major soluble protein in leaves, decreases with senescence and is the principal source of transported nitrogen (8,12 Glutamate is a major free amino acid in mature leaf blades of rice. Recently, Hayashi and Chino (7) showed that glutamine and asparagine accounted for 42 and 12%, respectively, of the total amino acids in phloem sap of rice plants. These amides are derived from amino acids and ammonia released by the hydrolysis of Rubisco, other leaf proteins, and Chl.GS2 is a candidate for the conversion of glutamate and NH4' to glutamine in senescing leaves and the resultant glutamine could be the precursor for asparagine (18,19).However, GS activity is known to decrease rapidly during either natural senescence of wheat leaves (2, 21) or dark induced senescence of detached Lolium temulentum leaves (24) and radish cotyledons (10). In many plants, there are two isoforms of GS in leaves (17): one located in the cytosol (GS 1) and the other in the chloroplast stroma (GS2). Because the physiological function of GS2 is considered to be the reassimilation of NH4' released during photorespiration (26,27) and because the rate of photosynth...

The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn−) B. pertussis was observed in Japan. The Prn− isolate was first discovered in 1997, and 33 (27%) Prn− isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn− isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn− isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn− isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn− isolates belong to MLVA-186, and 6 and 3 Prn− isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn− clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn− isolates have a higher growth potential than the Prn+ back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn− strains have arisen and that Prn expression is not essential for fitness under these conditions.

Tissue localizations of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), chloroplastic GS (GS2), and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in rice (Oryza sativa L.) leaf blades were investigated using a tissue-print immunoblot method with specific antibodies. The cross-sections of mature and senescent leaf blades from middle and basal regions were used for tissue printing. The anti-GS1 antibody, raised against a synthetic 17-residue peptide corresponding to the deduced N-terminal amino acid sequence of rice GS1, cross-reacted specifically with native GS1 protein, but not with GS2 after transfer onto a nitrocellulose membrane. Tissue-print immunoblots showed that the GS1 protein was located in large and small vascular bundles in all regions of the leaf blade prepared from either stage of maturity. On the other hand, GS2 and Fd-GOGAT proteins were mainly located in mesophyll cells. The intensity of the developed color on the membrane for GSI was similar between the two leaf ages, whereas that for GS2 and Fd-GOGAT decreased during senescence. The tissuespecific localization of GS1 suggests that this GS isoform is important in the synthesis of glutamine, which is a major form of1itrogen exported from the senescing leaf in rice plants. GS2 catalyzes the first step in the assimilation of NH33 in higher plants (12,13). In many plants, there are two isoforms of GS in leaves: one located in the cytosol (GS1) and the other in the chloroplast stroma (GS2) (11,15). GOGAT is a second enzyme active in the transfer of the amide nitrogen of Gln to 2-oxoglutarate and hence in the generation of two Glu molecules (12,13,15 senescing leaves (10). Glu is a major free amino acid in rice (Oryza sativa L.) leaf blades (8), whereas Gln is a major form of the total amino acids in phloem sap of rice plants (6). Therefore, Glu in the blades is probably converted into Gln during the remobilization process. GS is a candidate for this conversion. Because the barley mutant lacking GS2 was able to grow normally under nonphotorespiratory conditions (22), GS1 in leaves could also be important in the synthesis of Gln for normal growth and development. Recently, we showed that the content of GS1 polypeptide remained constant during the natural senescence process in rice leaf blades, whereas that of the GS2 polypeptide declined (8).Although the intracellular distribution of GS isoforms is well established in leaves, there is little information with respect to their tissue localization. If GS1 were truly responsible for export of leaf nitrogen, it would be expected to be localized in close proximity to the phloem in leaf tissues. From molecular-genetic analyses, Edwards et al. (2) recently showed that the promoter for GS1 of pea nodules was expressed within the phloem elements, whereas that for GS2 was expressed within photosynthetic cell types in transgenic tobacco plants. However, direct evidence for tissue localization of GS isoforms in leaves has not yet been described. The same situation is also true for Fd-GO...

SummaryHelicobacter pylori infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis-inducing protein that functions as a leading factor in H. pylori -mediated apoptosis induction. We purified the protein from H. pylori by separating fractions that showed apoptosis-inducing activity. This protein induced apoptosis of AGS cells in a dosedependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N-terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited g g g g -glutamyl transpeptidase activity, the inhibition of which by 6-diazo-5-oxo-L -norleucine resulted in a complete loss of apoptosis-inducing activity. To the best of our knowledge, the apoptosisinducing function is a newly identified physiological role for bacterial g g g g -glutamyl transpeptidase. The apoptosis-inducing activity of the isogenic mutant g g g gglutamyl transpeptidase-deficient strain was significantly lower compared with that of the parent strain, demonstrating that g g g g -glutamyl transpeptidase plays a significant role in H. pylori -mediated apoptosis. Our findings provide new insights into H. pylori pathogenicity and reveal a novel aspect of the bacterial g g g gglutamyl transpeptidase function.