Bordetella pertussiscauses 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 343B. pertussisstrains 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 whichB. pertussisand the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines.IMPORTANCEWhooping cough is mainly caused byBordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343B. pertussisisolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population ofB. pertussisis evolving in response to vaccine introduction, potentially enabling vaccine escape.

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 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 GS1 was similar between the two leaf ages, whereas that for GS2 and Fd-GOGAT decreased during senescence. The tissue-specific localization of GS1 suggests that this GS isoform is important in the synthesis of glutamine, which is a major form of nitrogen exported from the senescing leaf in rice plants.

Summary: Helicobacter 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 dose‐dependent 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 γ‐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 apoptosis‐inducing function is a newly identified physiological role for bacterial γ‐glutamyl transpeptidase. The apoptosis‐inducing activity of the isogenic mutant γ‐glutamyl transpeptidase‐deficient strain was significantly lower compared with that of the parent strain, demonstrating that γ‐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 γ‐glutamyl transpeptidase function.

Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of their corresponding mRNAs have been investigated in segments of the 13th leaf of hydroponically grown rice (Oryza sativa L.) plants during natural senescence. The leaf blade on the main stem at early (0 day), middle (15 days), and late (25 days) stages of senescence was harvested and cut into 18 or 19 segments, 2 centimeters in length from the base to the tip. The amount of GS1 polypeptide, detected with specific antibody for the GS1, was greatest near the middle of the leaf blade (segments 11-13). There was little difference in the GS1 content between corresponding leaf segments obtained at the early and middle stages of senescence. At the late senescence stage, all segments had lost some GS1 polypeptide, but more than 50% of GS1 detected at both the early and middle stages was still detectable in segments. The relative content of mRNA for GS1 in the total RNA in all segments was very low during early senescence but increased in all leaf segments during later senescence. At the late stage of senescence, GS1 mRNA in the total RNA increased about 4.2- to 4.6-fold in segments 12 to 16 in the day-25 samples compared with those in the early stage. The content of the GS2 polypeptide, as well as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein, was highest in segment 17 in the 0-day samples. During senescence, this peak became lower and broader, and finally disappeared, i.e. approximately 80% of GS2 polypeptide and Rubisco protein in segment 17 were lost by day 25. In contrast with GS2 polypeptide, the relative level of GS2 mRNA increased 1.8- to 2.9-fold in individual segments at the middle stage of senescence. Even at the late stage, the transcript signals remained slightly higher than those at the early stage in all segments. Thus, GS1 and GS2 polypeptides and corresponding mRNAs responded in a different manner within an attached rice leaf during natural senescence. The contents of GS1 and GS2 polypeptides were not simply determined by the abundance of their corresponding mRNAs in the rice leaf blades during natural senescence.