Genetic analysis of anti-amoebae and anti-bacterial activities of the type VI secretion system in Vibrio cholerae

A type VI secretion system (T6SS) was recently shown to be required for full virulence of Vibrio cholerae O37 serogroup strain V52. In this study, we systematically mutagenized each individual gene in T6SS locus and characterized their functions based on expression and secretion of the hemolysin co-regulated protein (Hcp), virulence towards amoebae of Dictyostelium discoideum and killing of Escherichia coli bacterial cells. We group the 17 proteins characterized in the T6SS locus into four categories: twelve (VipA, VipB, VCA0109-VCA0115, ClpV, VCA0119, and VasK) are essential for Hcp secretion and bacterial virulence, and thus likely function as structural components of the apparatus; two (VasH and VCA0122) are regulators that are required for T6SS gene expression and virulence; another two, VCA0121 and valine-glycine repeat protein G 3 (VgrG-3), are not essential for Hcp expression, secretion or bacterial virulence, and their functions are unknown; the last group is represented by VCA0118, which is not required for Hcp expression or secretion but still plays a role in both amoebae and bacterial killing and may therefore be an effector protein. We also showed that the clpV gene product is required for Dictyostelium virulence but is less important for killing E. coli. In addition, one vgrG gene (vgrG-2) outside of the T6SS gene cluster was required for bacterial killing but another (vgrG-1) was not. However, a bacterial killing defect was observed when vgrG-1 and vgrG-3 were both deleted. Several genes encoded in the same putative operon as vgrG-1 and vgrG-2 also contribute to virulence toward Dictyostelium but have a smaller effect on bacterial killing. Our results provide new insights into the functional requirements of V. cholerae's T6SS in the context of secretion as well as killing of bacterial and eukaryotic phagocytic cells.     

Converting visual census data into absolute abundance estimates: a method for calibrating timed counts of a sedentary insect population

Abstract.  1. Visual surveys for small organisms on complex substrates often yield serious underestimates of true counts. When both visual counts (relative estimates of abundance) and absolute counts can be obtained from the same sample, however, the visual counts can be calibrated such that absolute estimates can be obtained in the future from visual surveys alone.
2. A method is presented for converting quick, timed, visual counts of a sedentary insect on a shrub into absolute estimates of abundance.
3. Analogies were drawn from simple, well-known predation theories to develop a two-parameter non-linear model. Parameter estimates were obtained by both inverse prediction and direct estimation methods; the latter were found to yield more accurate predictions of absolute abundance.
4. The calibration model is mechanistic in its approach, and thus has potential for application in other systems in which all individuals are visible, but able to be missed during timed counts.     

Oxidative stress resistance through blocking Hsp60 translocation followed by SAPK/JNK inhibition in aged human diploid fibroblasts

The stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) pathway is a well‐known senescence‐related stress activated protein kinase. Multiple environmental stresses induce programmed cell death, such as apoptosis. Normal human diploid fibroblast (HDF) cells have a limited life span in vitro, halting proliferation after a fixed number of cell divisions. Aged passage HDF showed resistance to oxidative stress involving heat shock proteins (Hsp60) through a mechanism involving the translocation of Hsp60 from the mitochondria to the cytosol. The present study showed that the translocation of Hsp60 from the mitochondria to the cytosol followed by high levels of p‐SAPK/JNK activation as a result of oxidative stress was observed in the young cells only. The inhibition of SAPK/JNK activation by SP600125 under oxidative stress almost completely blocked the translocation of Hsp60 in both young and aged cells. This suggests that aged HDF cells are resistant to oxidative stress by blocking the translocation of Hsp60 from the mitochondria to the cytosol followed by SAPK/JNK inhibition. Overall, the mechanism of resistance by oxidative stress in aged cells is induced by blocked of the translocation of Hsp60 followed by SAPK/JNK inactivation. Copyright © 2008 John Wiley & Sons, Ltd.     

Overexpression of glucose-6-phosphate dehydrogenase is associated with lipid dysregulation and insulin resistance in obesity

Glucose-6-phosphate dehydrogenase (G6PD) produces cellular NADPH, which is required for the biosynthesis of fatty acids and cholesterol. Although G6PD is required for lipogenesis, it is poorly understood whether G6PD in adipocytes is involved in energy homeostasis, such as lipid and glucose metabolism. We report here that G6PD plays a role in adipogenesis and that its increase is tightly associated with the dysregulation of lipid metabolism and insulin resistance in obesity. We observed that the enzymatic activity and expression levels of G6PD were significantly elevated in white adipose tissues of obese models, including db/db, ob/ob, and diet-induced obesity mice. In 3T3-L1 cells, G6PD overexpression stimulated the expression of most adipocyte marker genes and elevated the levels of cellular free fatty acids, triglyceride, and FFA release. Consistently, G6PD knockdown via small interfering RNA attenuated adipocyte differentiation with less lipid droplet accumulation. Surprisingly, the expression of certain adipocytokines such as tumor necrosis factor alpha and resistin was increased, whereas that of adiponectin was decreased in G6PD overexpressed adipocytes. In accordance with these results, overexpression of G6PD impaired insulin signaling and suppressed insulin-dependent glucose uptake in adipocytes. Taken together, these data strongly suggest that aberrant increase of G6PD in obese and/or diabetic subjects would alter lipid metabolism and adipocytokine expression, thereby resulting in failure of lipid homeostasis and insulin resistance in adipocytes.     

Pythium vexans causing patch canker of rubber trees on Hainan Island,China

This is the first report of patch canker disease of rubber trees (clone RRIM600) in China. It is characterized by discrete irregular patches of rotted, discolored bark and wood, accompanied by a decrease in latex flow. A total of seven isolates of Pythium vexans were obtained from the diseased bark of the trunks and roots of rubber trees. Inoculating these isolates into healthy, mature rubber trees resulted in symptoms similar to patch canker and the same fungal species was re-isolated from the diseased tissues. This is also the first record of Py. vexans in Hainan.     

Headloop suppression PCR and its application to selective amplification of methylated DNA sequences

Selective amplification in PCR is principally determined by the sequence of the primers and the temperature of the annealing step. We have developed a new PCR technique for distinguishing related sequences in which additional selectivity is dependent on sequences within the amplicon. A 5′ extension is included in one (or both) primer(s) that corresponds to sequences within one of the related amplicons. After copying and incorporation into the PCR product this sequence is then able to loop back, anneal to the internal sequences and prime to form a hairpin structure—this structure is then refractory to further amplification. Thus, amplification of sequences containing a perfect match to the 5′ extension is suppressed while amplification of sequences containing mismatches or lacking the sequence is unaffected. We have applied Headloop PCR to DNA that had been bisulphite-treated for the selective amplification of methylated sequences of the human GSTP1 gene in the presence of up to a 10⁵-fold excess of unmethylated sequences. Headloop PCR has a potential for clinical application in the detection of differently methylated DNAs following bisulphite treatment as well as for selective amplification of sequence variants or mutants in the presence of an excess of closely related DNA sequences.     

Genomic blueprint of Hahella chejuensis, a marine microbe producing an algicidal agent

Harmful algal blooms, caused by rapid growth and accumulation of certain microalgae in the ocean, pose considerable impacts on marine environments, aquatic industries and even public health. Here, we present the 7.2-megabase genome of the marine bacterium Hahella chejuensis including genes responsible for the biosynthesis of a pigment which has the lytic activity against a red-tide dinoflagellate. H.chejuensis is the first sequenced species in the Oceanospiralles clade, and sequence analysis revealed its distant relationship to the Pseudomonas group. The genome was well equipped with genes for basic metabolic capabilities and contained a large number of genes involved in regulation or transport as well as with characteristics as a marine heterotroph. Sequence analysis also revealed a multitude of genes of functional equivalence or of possible foreign origin. Functions encoded in the genomic islands include biosynthesis of exopolysacchrides, toxins, polyketides or non-ribosomal peptides, iron utilization, motility, type III protein secretion and pigmentation. Molecular structure of the algicidal pigment, which was determined through LC-ESI-MS/MS and NMR analyses, indicated that it is prodigiosin. In conclusion, our work provides new insights into mitigating algal blooms in addition to genetic make-up, physiology, biotic interactions and biological roles in the community of a marine bacterium.