Search

Objective: Irinotecan is a useful anticancer drug for colorectal cancer treatment. UGT1A1*28 and *6 gene polymorphisms are known risk factors for irinotecan‐associated toxicity. However, severe adverse effects due to irinotecan have been observed even in patients who do not harbor UGT1A1*28 or *6. We investigated gene polymorphisms in the whole exome to identify useful biomarkers for irinotecan toxicity other than UGT1A. Methods: A total of 178 patients with metastatic colorectal cancer (mCRC) and 87 patients with pancreatic cancer were treated with FOLFIRI, FOLFOX, FOLFOXIRI, modified FOLFIRINOX, or gemcitabine plus nab‐paclitaxel. Genome‐wide screening was performed using whole‐exome sequencing (WES), and validation analysis was performed using qPCR with a hydrolysis probe. Results: Using WES after a doublet chemotherapy regimen comprising irinotecan and 5‐fluorouracil (n = 15), seven single nucleotide polymorphisms (SNPs) were identified as candidate biomarkers for irinotecan‐associated toxicity of neutropenia. Among the seven SNPs, an SNP in R3H domain and coiled‐coil containing 1 (R3HCC1; c.919G > A, rs2272761) showed a significant association with neutropenia (>grade 3) after doublet chemotherapy. Patients receiving irinotecan including triplet chemotherapy, FOLFOXIRI for mCRC (n = 23) or modified FOLFIRINOX for pancreatic cancer (n = 40), also showed significant linear trends between R3HCC1 polymorphism and neutropenia (p = 0.017 and 0.046, respectively). No significant association was observed in patients treated with irinotecan‐free regimens, FOLFOX for mCRC (n = 66), and gemcitabine plus nab‐paclitaxel for pancreatic cancer (n = 47). Conclusion: Thus, an SNP in the R3HCC1 gene may be a useful biomarker for the toxicity of irinotecan‐containing chemotherapy for mCRC and pancreatic cancer. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria ubiquitously release membrane vesicles (MVs) into the extracellular milieu. Although MVs are the product of growing bacteria, not of cell lysis or death, the regulatory mechanisms underlying MV formation remained unknown. We have found that MV biogenesis is provoked by the induction of PagC, a Salmonella-specific protein whose expression is activated by conditions that mimic acidified macrophage phagosomes. PagC is a major constituent of Salmonella MVs, and increased expression accelerates vesiculation. Expression of PagC is regulated at the posttranscriptional and/or posttranslational level in a sigmaS (RpoS)-dependent manner. Serial quantitative analysis has demonstrated that MV formation can accelerate when the quantity of the MV constituents, OmpX and PagC, rises. Overproduction of PagC dramatically impacts the difference in the relative amount of vesiculation, but the corresponding overproduction of OmpX was less pronounced. Quantitative examination of the ratios of PagC and OmpX in the periplasm, outer membrane, and MVs demonstrates that PagC is preferentially enriched in MVs released from Salmonella cells. This suggests that specific protein sorting mechanisms operate when MVs are formed. The possible role(s) of PagC-MV in host cells is discussed. doi: 10.1128/JB.00590-10

Glucose metabolism in Legionella pneumophila was studied by focusing on the Entner-Doudorotf (ED) pathway with a combined genetic and biochemical approach. The bacterium utilized exogenous glucose for synthesis of acid-insoluble cell components but manifested no discernible increase in the growth rate. Assays with permeabilized cell preparations revealed the activities of three enzymes involved in the pathway, i.e., glucokinase, phosphogluconate dehydratase, and 2-dehydro-3-deoxy-phosphogluconate aidolase, presumed to be encoded by the glk, edd, and eda genes, respectively. Gene-disrupted mutants for the three genes and the ywt G gene encoding a putative sugar transporter were devoid of the ability to metabolize exogenous glucose, indicating that the pathway is almost exclusively responsible for glucose metabolism and that the ywt G gene product is the glucose transporter. It was also established that these four genes formed part of an operon in which the gene order was edd-glk-eda-ywtG, as predicted by genomic information. Intriguingly, while the mutants exhibited no appreciable change in growth characteristics in vitro, they were defective in multiplication within eukaryotic cells, strongly indicating that the ED pathway must be functional for the intracellular growth of the bacterium to occur. Curiously, while the deficient glucose metabolism of the ywtG mutant was successfully complemented by the ywt [G.sup.+] gene supplied in trans via plasmid, its defect in intracellular growth was not. However, the latter defect was also manifested in wild-type cells when a plasmid carrying the mutant ywtG gene was introduced. This phenomenon, resembling so-called dominant negativity, awaits further investigation. doi: 10.1128/JB.01535-09

Streptococcus pneumoniae was shown to possess lactate oxidase in addition to well-documented pyruvate oxidase. The activities of both [H.sub.2][O.sub.2]-forming oxidases in wild-type cultures were detectable even in the early exponential phase of growth and attained the highest levels in the early stationary phase. For each of these oxidases, a defective mutant was constructed and compared to the parent regarding the dynamics of pyruvate and lactate in aerobic cultures. The results obtained indicated that the energy-yielding metabolism in the wild type could be best described by the following scheme. (i) As long as glucose is available, approximately one-fourth of the pyruvate formed is converted to acetate by the sequential action of pyruvate oxidase and acetate kinase with acquisition of additional ATP; (ii) the rest of the pyruvate is reduced by lactate dehydrogenase to form lactate, with partial achievement of redox balance; (iii) the lactate is oxidized by lactate oxidase back to pyruvate, which is converted to acetate as described above; and (iv) the sequential reactions mentioned above continue to occur as long as lactate is present. As predicted by this model, exogenously added lactate was shown to increase the final growth yield in the presence of both oxidases.

An unstable type of green fluorescent protein (Gfp) tagged with a C-terminal extension, which is a target for tail-specific protease, was used as a reporter gene in Legionella pneumophila. To analyse Gfp expression in legionellae, transcriptional fusions of unstable gfp with the Legionella-specific icm (intracellular multiplication) promoters ([P.sub.icms], [P.sub.icmT] and [P.sub.icmO]) were constructed. Infection studies using J774.1 macrophages as the host, and L. pneumophila strains carrying [P.sub.icms]-gfP, [P.sub.icmT]-gfP and [P.sub.icmQ]-gfp fusions, indicated that the icmS, icmT and icmQ genes could be expressed intracellularly. Expression of icmS, icmT and icmQ genes in infected cells was examined by flow cytometry. Furthermore, fluorescent intracellular legionellae were detected directly by confocal microscopy. Real-time quantitative RT-PCR revealed the differences in the gene expression of icmS, and that of icmT and icmQ, during infection. Expression of icmS was high in the late stage of infection, while that of icmT and icmQ was high in the early phase only. We show that unstable gfp is a useful reporter gene whose expression in legionellae can be followed in real-time, and that it allows analysis of promoter activities in legionellae and monitoring of the infection process.

Streptococcus pyogenes strains can be divided into two classes, one capable and the other incapable of producing [H.sub.2][O.sub.2] (M. Saito, S. Ohga, M. Endoh, H. Nakayama, Y. Mizunoe, T. Hara, and S. Yoshida, Microbiology 147:2469-2477, 2001). In the present study, this dichotomy was shown to parallel the presence or absence of [H.sub.2][O.sub.2]-producing lactate oxidase activity in permeabilized cells. Both lactate oxidase activity and [H.sub.2][O.sub.2] production under aerobic conditions were detectable only after glucose in the medium was exhausted. Thus, the glucose-repressible lactate oxidase is likely responsible for [H.sub.2][O.sub.2] production in S. pyogenes. Of the other two potential [H.sub.2][O.sub.2]-producing enzymes of this bacterium, NADH and [alpha]-glycerophosphate oxidase, only the former exhibited low but significant activity in either class of strains. This activity was independent of the growth phase, suggesting that the protein may serve in vivo as a subunit of the [H.sub.2][O.sub.2]-scavenging enzyme NAD(P)H-linked alkylhydroperoxide reductase. The activity of lactate oxidase was associated with the membrane while that of NADH oxidase was in the soluble fraction, findings consistent with their respective physiological roles, i.e., the production and scavenging of [H.sub.2][O.sub.2]. Analyses of fermentation end products revealed that the concentration of lactate initially increased with time and decreased on glucose exhaustion, while that of acetate increased during the culture. These results suggest that the lactate oxidase activity of [H.sub.2][O.sub.2]-producing cells oxidizes lactate to pyruvate, which is in turn converted to acetate. This latter process proceeds presumably via acetyl coenzyme A and acetyl phosphate with formation of extra ATP.

In this study, we examined whether virulence conversion occurs in Legionella pneumophila by conjugal transfer of chromosomal DNA. A virulent strain, K6, which has the genes for K[m.sup.r] and Lac[Z.sup.+] transposed in the chromosome of strain Philadelphia-1, which belongs to serogroup 1, was used as one parent, and an avirulent strain, Chicago-2S, which is a spontaneous streptomycin-resistant derivative of strain Chicago-2 belonging to serogroup 6, was used as the other parent. Experiments in which K6 (approximately 2.6 x [10.sup.9] CFU) and Chicago-2S (approximately 8.9 x [10.sup.9] CFU) were mated typically yielded [10.sup.3] K[m.sup.r] S[m.sup.r] Lac[Z.sup.+] transconjugants. Thirty-two (about 2.8%) of 1,152 transconjugants belonging to serogroup 6 acquired the ability to grow intracellularly in Acanthamoeba castellanii and guinea pig macrophages. When guinea pigs were infected with sublethal doses of Legionella aerosols generated from one of these transconjugants (HM1011), they developed a severe pneumonia similar to that caused by donor strain K6. These results show that avirulent strain Chicago-2S changed into virulent strain HM1011 through conjugation with virulent strain K6. Furthermore, showed that Legionella chromosomal virulence genes (icm-dot locus) were horizontally transferred by the conjugation system. The chromosomal conjugation system may play a role(s) in the evolution of L. pneumophila.

Since July 2002, bacteriologically confirmed shigellosis cases have increased, and multidrug-resistant Shigella dysenteriae serotype 1 strains have reemerged in patients hospitalized with diarrhea in Kolkata, India. The isolated strains of [...]

Accumulation of hydrogen peroxide (H2O2) in non-H2O2-producing Streptococcus pyogenes strains, during the late exponential phase, results in the loss of cell viability but can be prevented by catalase, suggesting that H2O2 is the cause of lethality. Furthermore non-H2O2-producing strains exhibit resistance to phagocyte killing in chronic granulomatous disease patients.