Dynamic Phosphorylation of NudC by Aurora B in Cytokinesis.

Nuclear distribution protein C (NudC) is a mitotic regulator that plays a role in cytokinesis. However, how NudC is regulated during cytokinesis remains unclear. Here, we show that NudC is phosphorylated by Aurora B, a kinase critical for cell abscission. NudC is co-localized with Aurora B at the midbody and co-immunoprecipitated with Aurora B in mitosis. Inhibition of Aurora B by ZM447439 reduced NudC phosphorylation, suggesting that NudC is an Aurora B substrate in vivo. We identified T40 on NudC as an Aurora B phosphorylation site. NudC depletion resulted in cytokinesis failure with a dramatic elongation of the intercellular bridge between daughter cells, sustained Aurora B activity at the midbody, and reduced cell abscission. These cytokinetic defects can be rescued by the ectopic expression of wild-type NudC. Reconstitution with T40A phospho-defective NudC was found to rescue the cytokinesis defect. In contrast, reconstitution with the T40D phospho-mimetic NudC was inefficient in supporting the completion of cytokinesis. These results suggest that that dynamic phosphorylation of NudC by Aurora B regulates cytokinesis.

Aurora B is regulated by acetylation/deacetylation during mitosis in prostate cancer cells.

Protein acetylation has been implicated in playing an important role during mitotic progression. Aurora B kinase is known to play a critical role in mitosis. However, whether Aurora B is regulated by acetylation is not known. Using IP with an anti-acetyl lysine antibody, we identified Aurora B as an acetylated protein in PC3 prostate cancer cells. Knockdown of HDAC3 or inhibiting HDAC3 deacetylase activity led to a significant increase (P<0.01 and P<0.05, respectively) in Aurora B acetylation as compared to siLuc or vehicle-treated controls. Increased Aurora B acetylation is correlated with a 30% reduction in Aurora B kinase activity in vitro and resulted in significant defects in Aurora B-dependent mitotic processes, including kinetochore-microtubule attachment and chromosome congression. Furthermore, Aurora B transiently interacts with HDAC3 at the kinetochore-microtubule interface of congressing chromosomes during prometaphase. This window of interaction corresponded with a transient but significant reduction (P=0.02) in Aurora B acetylation during early mitosis. Together, these results indicate that Aurora B is more active in its deacetylated state and further suggest a new mechanism by which dynamic acetylation/deacetylation acts as a rheostat to fine-tune Aurora B activity during mitotic progression.

Prophylactic application of CpG oligonucleotides augments the early host response and confers protection in acute melioidosis.

Prophylactic administration of CpG oligodeoxynucleotides (CpG ODNs) is known to confer protection against lethal sepsis caused by Burkholderia pseudomallei in the mouse model. The mechanisms whereby CpG regulates the innate immune response to provide protection against B. pseudomallei, however, are poorly characterized. In the present study, we demonstrate that intranasal treatment of mice with Class C CpG, results in recruitment of inflammatory monocytes and neutrophils to the lung at 48 h post-treatment. Mice infected with B. pseudomallei 48 h post-CpG treatment had reduced organ bacterial load and significantly altered cytokine and chemokine profiles concomitant with protection as compared to control animals. CpG administration reduced the robust production of chemokines and pro-inflammatory cytokines in blood, lung and spleen, observed following infection of non-treated animals. Death of control animals coincided with the time of peak cytokine production (day 1-3), while a moderate; sustained cytokine production in CpG-treated animals was associated with survival. In general, CpG treatment resulted in diminished expression of cytokines and chemokines post-infection, though IL-12p40 was released in larger quantities in CpG treated animals. In contrast to CpG-treated animals, the lungs of infected control animals were infiltrated with leukocytes, especially neutrophils, and large numbers of necrotic lesions were observed in lung sections. Therapeutic treatment of B. pseudomallei-infected animals with CpG at 24 h post-infection did not impact survival compared to control animals. In summary, protection of CpG-treated animals was associated with recruitment of inflammatory monocytes and neutrophils into the lungs prior to infection. These responses correspond with early control of bacterial growth, a dampened inflammatory cytokine/chemokine response, reduced lung pathology, and greatly increased survival. In contrast, a delay in recruitment of inflammatory cell populations, despite a robust production of pro-inflammatory cytokines, was associated with poorly controlled bacterial growth, severe lung pathology, and death of control animals.

Protective antigens against glanders identified by expression library immunization.

Burkholderia are highly evolved Gram-negative bacteria that primarily infect solipeds but are transmitted to humans by ingestion and cutaneous or aerosol exposures. Heightened concern over human infections of Burkholderia mallei and the very closely related species B. pseudomallei is due to the pathogens' proven effectiveness as bioweapons, and to the increased potential for natural opportunistic infections in the growing diabetic and immuno-compromised populations. These Burkholderia species are nearly impervious to antibiotic treatments and no vaccine exists. In this study, the genome of the highly virulent B. mallei ATCC23344 strain was examined by expression library immunization for gene-encoded protective antigens. This protocol for genomic-scale functional screening was customized to accommodate the unusually large complexity of Burkholderia, and yielded 12 new putative vaccine candidates. Five of the candidates were individually tested as protein immunogens and three were found to confer significant partial protection against a lethal pulmonary infection in a murine model of disease. Determinations of peripheral blood cytokine and chemokine profiles following individual protein immunizations show that interleukin-2 (IL-2) and IL-4 are elicited by the three confirmed candidates, but unexpectedly interferon-γ and tumor necrosis factor-α are not. We suggest that these pathogen components, discovered using genetic immunization and confirmed in a conventional protein format, will be useful toward the development of a safe and effective glanders vaccine.

Global analysis of the genes involved in the thermotolerance mechanism of thermotolerant Acetobacter tropicalis SKU1100.

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.

Protective response to subunit vaccination against intranasal Burkholderia mallei and B. pseudomallei challenge.

Burkholderia mallei and B. pseudomallei are Gram-negative pathogenic bacteria, responsible for the diseases glanders and melioidosis, respectively. Furthermore, there is currently no vaccine available against these Burkholderia species. In this study, we aimed to identify protective proteins against these pathogens. Immunization with recombinant B. mallei Hcp1 (type VI secreted/structural protein), BimA (autotransporter protein), BopA (type III secreted protein), and B. pseudomallei LolC (ABC transporter protein) generated significant protection against lethal inhaled B. mallei ATCC23344 and B. pseudomallei 1026b challenge. Immunization with BopA elicited the greatest protective activity, resulting in 100% and 60% survival against B. mallei and B. pseudomallei challenge, respectively. Moreover, sera from recovered mice demonstrated reactivity with the recombinant proteins. Dendritic cells stimulated with each of the different recombinant proteins showed distinct cytokine patterns. In addition, T cells from immunized mice produced IFN-γ following in vitro re-stimulation. These results indicated therefore that it was possible to elicit cross-protective immunity against both B. mallei and B. pseudomallei by vaccinating animals with one or more novel recombinant proteins identified in B. mallei.

Synthesis and in vitro Efficacy Studies of Silver Carbene Complexes on Biosafety Level 3 Bacteria.

A series of N-heterocyclic carbene silver complexes have been synthesized and tested against the select group of bio-safety level 3 bacteria Burkholderia pseudomallei, Burkholderia mallei, Bacillus anthracis, methicillin-resistant Staphylococcus aureus and Yersinia pestis. Minimal inhibitory concentrations, minimal bactericidal and killing assays demonstrated the exceptional efficacy of the complexes against these potentially weaponizable pathogens.

Conversion of capsular polysaccharide, involved in pellicle formation, to extracellular polysaccharide by galE deletion in Acetobacter tropicalis.

Acetobacter tropicalis SKU1100 produces a pellicle-forming capsular polysaccharide (CPS), consisting of galactose, glucose, and rhamnose. We cloned the galE gene, a UDP-galactose synthesis gene, from A. tropicalis SKU1100 by PCR. A galE-disruptant was prepared and found not to produce CPS and thus not to form a pellicle under the static condition. Instead, the DeltagalE mutant secreted an extracellular polysaccharide (EPS), which was purified and found to have a unique character, different from the original CPS.

Characterization and spontaneous mutation of a novel gene, polE, involved in pellicle formation in Acetobacter tropicalis SKU1100.

Acetobacter tropicalis SKU1100 produces a pellicle polysaccharide, consisting of galactose, glucose and rhamnose, which attaches to the cell surface. This strain forms two types of colony on agar plates: a rough-surfaced colony (R strain) and a mucoid smooth-surfaced colony (S strain). The R strain forms a pellicle, allowing it to float on the medium surface in static culture, while the S strain does not. The pellicle is an assemblage of cells which are tightly associated with capsular polysaccharides (CPS) on the cell surface. In this study, a gene required for pellicle formation by the R strain was investigated by transposon mutagenesis using Tn10. The resulting mutant, designated Pel-, has a smooth-surfaced colony and a defect in pellicle formation, as for the S strain. The mutant produced polysaccharide which was instead secreted into the culture medium as extracellular polysaccharide (EPS). An ORF was identified at the Tn10 insertion site, designated polE, upstream of which polABCD genes were also found. The deduced amino acid sequences of polABCD showed a high level of homology to those of rfbBACD which are involved in dTDP-rhamnose synthesis, whereas polE had a relatively low level of homology to glycosyltransferase. In this study a polB (rfbA) disruptant was also prepared, which lacked both CPS and EPS production. A plasmid harbouring the polE or polB genes could restore pellicle formation in the Pel(-) mutant and S strains, and in the DeltapolB mutant, respectively. Thus both polE and polB are evidently involved in pellicle formation, most likely by anchoring polysaccharide to the cell surface and through the production of dTDP-rhamnose, respectively. The Pel- and DeltapolB mutants were unable to grow in static culture and became more sensitive to acetic acid due to the loss of pellicle formation. Additionally, this study identified the mutation sites of several S strains which were spontaneously isolated from the original culture and found them to be concentrated in a sequence of 7 C residues in the coding sequence of polE, with the deletion or addition of a single C nucleotide.