Interactions between the West Nile virus capsid protein and the host cell-encoded phosphatase inhibitor, I2PP2A.

The West Nile virus (WNV) capsid protein functions in virus assembly to package genomic RNA into nucleocapsid structures. It is becoming clear, that in addition to their structural roles, capsid proteins of RNA viruses have non-structural functions. For example, the WNV capsid protein has been implicated as a pathogenic determinant. Presumably, many, if not all, of the non-structural functions of this protein involve interactions with host cell-encoded proteins. In the present study, we used affinity purification to isolate human proteins that bind to the WNV capsid protein. One of the capsid binding proteins is I(2)(PP2A), a previously characterized inhibitor of the serine/threonine phosphatase PP2A. Mapping studies revealed that capsid binding site overlaps with the region of I(2)(PP2A) that is required for inhibition of PP2A activity. Moreover, expression of the WNV capsid protein resulted in significantly increased PP2A activity and expected downstream events, such as inhibition of AP1-dependent transcription. Infected cells treated with I(2)(PP2A)-specific siRNAs produced less infectious virus than control siRNA-transfected cells, but this difference was minimal. Together, our data indicate that interactions between WNV capsid and I(2)(PP2A) result in increased PP2A activity. Given the central role of this phosphatase in cellular physiology, capsid/I(2)(PP2A) interactions may yet prove to be important for viral pathogenesis.

Reconstitution of O-specific lipopolysaccharide expression in Burkholderia cenocepacia strain J2315, which is associated with transmissible infections in patients with cystic fibrosis.

Burkholderia cenocepacia is an opportunistic bacterium that infects patients with cystic fibrosis. B. cenocepacia strains J2315, K56-2, C5424, and BC7 belong to the ET12 epidemic clone, which is transmissible among patients. We have previously shown that transposon mutants with insertions within the O antigen cluster of strain K56-2 are attenuated for survival in a rat model of lung infection. From the genomic DNA sequence of the O antigen-deficient strain J2315, we have identified an O antigen lipopolysaccharide (LPS) biosynthesis gene cluster that has an IS402 interrupting a predicted glycosyltransferase gene. A comparison with the other clonal isolates revealed that only strain K56-2, which produced O antigen and displayed serum resistance, lacked the insertion element inserted within the putative glycosyltransferase gene. We cloned the uninterrupted gene and additional flanking sequences from K56-2 and conjugated this plasmid into strains J2315, C5424, and BC7. All the exconjugants recovered the ability to form LPS O antigen. We also determined that the structure of the strain K56-2 O antigen repeat, which was absent from the LPS of strain J2315, consisted of a trisaccharide unit made of rhamnose and two N-acetylgalactosamine residues. The complexity of the gene organization of the K56-2 O antigen cluster was also investigated by reverse transcription-PCR, revealing several transcriptional units, one of which also contains genes involved in lipid A-core oligosaccharide biosynthesis.

The Pseudomonas aeruginosa alternative sigma factor PvdS controls exotoxin A expression and is expressed in lung infections associated with cystic fibrosis.

PvdS is an alternative sigma factor regulated by the global iron regulator Fur. It has been demonstrated that PvdS plays a role in the iron-dependent regulation of exotoxin A (ETA) in Pseudomonas aeruginosa strain PAO1. The goals of this research were to determine if pvdS was transcribed by the bacteria in the chronic lung infections associated with cystic fibrosis (CF) and to determine how PvdS interacts with the regAB promoters of the hyper-toxigenic strain PA103. It was found that pvdS is transcribed in the lungs of patients with CF and that it appears to be involved with the regulation of toxA in this environment. This correlated with the finding that in strain PA103, a mutation in pvdS reduced ETA activity while the same mutation in strain PAO1 abrogated ETA production. It was also shown that in strain PA103, pvdS was absolutely required for activation of the regAB P2 promoter. The effect of PvdS on the P2 promoter may be direct or indirect; however, in support of a direct role, an eight-out-of-nine base-pair match to the consensus sequence for PvdS binding was identified at the transcriptional start site for the P2 promoter. The effect of PvdS on the PA103 regAB P1 promoter under aerobic growth conditions was also examined. The results show that PvdS does modulate the expression from this promoter but that both the regAB operon and PvdS are required for optimal P1 promoter activity. These studies demonstrate that the alternative sigma factor PvdS acts as a regulator of ETA expression in P. aeruginosa strain PA103 through the regAB operon and that PvdS is expressed in lung infections associated with CF.

Identification of Burkholderia cenocepacia genes required for bacterial survival in vivo.

Burkholderia cenocepacia (formerly Burkholderia cepacia complex genomovar III) causes chronic lung infections in patients with cystic fibrosis. In this work, we used a modified signature-tagged mutagenesis (STM) strategy for the isolation of B. cenocepacia mutants that cannot survive in vivo. Thirty-seven specialized plasposons, each carrying a unique oligonucleotide tag signature, were constructed and used to examine the survival of 2,627 B. cenocepacia transposon mutants, arranged in pools of 37 unique mutants, after a 10-day lung infection in rats by using the agar bead model. The recovered mutants were screened by real-time PCR, resulting in the identification of 260 mutants which presumably did not survive within the lungs. These mutants were repooled into smaller pools, and the infections were repeated. After a second screen, we isolated 102 mutants unable to survive in the rat model. The location of the transposon in each of these mutants was mapped within the B. cenocepacia chromosomes. We identified mutations in genes involved in cellular metabolism, global regulation, DNA replication and repair, and those encoding bacterial surface structures, including transmembrane proteins and cell surface polysaccharides. Also, we found 18 genes of unknown function, which are conserved in other bacteria. A subset of 12 representative mutants that were individually examined using the rat model in competition with the wild-type strain displayed reduced survival, confirming the predictive value of our STM screen. This study provides a blueprint to investigate at the molecular level the basis for survival and persistence of B. cenocepacia within the airways.