Comparative Proteomics Analyses Reveal the virB of B. melitensis Affects Expression of Intracellular Survival Related Proteins

BACKGROUND: Brucella melitensis is a facultative, intracellular, pathogenic bacterium that replicates within macrophages. The type IV secretion system encoded by the virB operon (virB) is involved in Brucella intracellular survival. However, the underlying molecular mechanisms, especially the target proteins affected by the virB, remain largely unclear. METHODOLOGY/PRINCIPAL FINDINGS: In order to define the proteins affected by virB, the proteomes of wild-type and the virB mutant were compared under in vitro conditions where virB was highly activated. The differentially expressed proteins were identified by MALDI-TOF-MS. Forty-four down-regulated and eighteen up-regulated proteins which exhibited a 2-fold or greater change were identified. These proteins included those involved in amino acid transport and metabolism, lipid metabolism, energy production, cell membrane biogenesis, translation, post-translational modifications and protein turnover, as well as unknown proteins. Interestingly, several important virulence related proteins involved in intracellular survival, including VjbR, DnaK, HtrA, Omp25, and GntR, were down-regulated in the virB mutant. Transcription analysis of virB and vjbR at different growth phase showed that virB positively affect transcription of vjbR in a growth phase dependent manner. Quantitative RT-PCR showed that transcription of these genes was also affected by virB during macrophage cell infection, consistent with the observed decreased survival of the virB mutant in macrophage. CONCLUSIONS/SIGNIFICANCE: These data indicated that the virB operon may control the intracellular survival of Brucella by affecting the expression of relevant proteins

Overexpression of Mitochondrial Uncoupling Protein 2 Inhibits Inflammatory Cytokines and Activates Cell Survival Factors after Cerebral Ischemia

Mitochondria play a critical role in cell survival and death after cerebral ischemia. Uncoupling proteins (UCPs) are inner mitochondrial membrane proteins that disperse the mitochondrial proton gradient by translocating H+ across the inner membrane in order to stabilize the inner mitochondrial membrane potential (ΔΨm) and reduce the formation of reactive oxygen species. Previous studies have demonstrated that mice transgenically overexpressing UCP2 (UCP2 Tg) in the brain are protected from cerebral ischemia, traumatic brain injury and epileptic challenges. This study seeks to clarify the mechanisms responsible for neuroprotection after transient focal ischemia. Our hypothesis is that UCP2 is neuroprotective by suppressing innate inflammation and regulating cell cycle mediators. PCR gene arrays and protein arrays were used to determine mechanisms of damage and protection after transient focal ischemia. Our results showed that ischemia increased the expression of inflammatory genes and suppressed the expression of anti-apoptotic and cell cycle genes. Overexpression of UCP2 blunted the ischemia-induced increase in IL-6 and decrease in Bcl2. Further, UCP2 increased the expression of cell cycle genes and protein levels of phospho-AKT, PKC and MEK after ischemia. It is concluded that the neuroprotective effects of UCP2 against ischemic brain injury are associated with inhibition of pro-inflammatory cytokines and activation of cell survival factors

Implication of Proteins Containing Tetratricopeptide Repeats in Conditional Virulence Phenotypes of Legionella pneumophila

Legionella pneumophila, the causative agent of Legionnaires' disease, is a ubiquitous freshwater bacterium whose virulence phenotypes require a type IV secretion system (T4SS). L. pneumophila strain JR32 contains two virulence-associated T4SSs, the Dot/Icm and Lvh T4SSs. Defective entry and phagosome acidification phenotypes of dot/icm mutants are conditional and reversed by incubating broth-grown stationary-phase cultures in water (WS treatment) prior to infection, as a mimic of the aquatic environment of Legionella. Reversal of dot/icm virulence defects requires the Lvh T4SS and is associated with a >10-fold induction of LpnE, a tetratricopeptide repeat (TPR)-containing protein. In the current study, we demonstrated that defective entry and phagosome acidification phenotypes of mutants with changes in LpnE and EnhC, another TPR-containing protein, were similarly reversed by WS treatment. In contrast to dot/icm mutants for which the Lvh T4SS was required, reversal for the ΔlpnE or the ΔenhC mutant required that the other TPR-containing protein be present. The single and double ΔlpnE and ΔenhC mutants showed a hypersensitivity to sodium ion, a phenotype associated with dysfunction of the Dot/Icm T4SS. The ΔlpnE single and the ΔlpnE ΔenhC double mutant showed 3- to 9-fold increases in translocation of Dot/Icm T4SS substrates, LegS2/SplY and LepB. Taken together, these data identify TPR-containing proteins in a second mechanism by which the WS mimic of a Legionella environmental niche can reverse virulence defects of broth-grown cultures and implicate LpnE and EnhC directly or indirectly in translocation of Dot/Icm T4SS protein substrates