Replication of plasmids derived from Shiga toxin-converting bacteriophages in starved Escherichia coli.

The pathogenicity of Shiga toxin-producing Escherichia coli (STEC) depends on the expression of stx genes that are located on lambdoid prophages. Effective toxin production occurs only after prophage induction, and one may presume that replication of the phage genome is important for an increase in the dosage of stx genes, positively influencing their expression. We investigated the replication of plasmids derived from Shiga toxin (Stx)-converting bacteriophages in starved E. coli cells, as starvation conditions may be common in the intestine of infected humans. We found that, unlike plasmids derived from bacteriophage λ, the Shiga toxin phage-derived replicons did not replicate in amino acid-starved relA(+) and relA(-) cells (showing the stringent and relaxed responses to starvation, respectively). The presence of the stable fraction of the replication initiator O protein was detected in all tested replicons. However, while ppGpp, the stringent response effector, inhibited the activities of the λ P(R) promoter and its homologues from Shiga toxin-converting bacteriophages, these promoters, except for λ P(R), were only weakly stimulated by the DksA protein. We suggest that this less efficient (relative to λ) positive regulation of transcription responsible for transcriptional activation of the origin contributes to the inhibition of DNA replication initiation of Shiga toxin-converting bacteriophages in starved host cells, even in the absence of ppGpp (as in starved relA(-) hosts). Possible clinical implications of these results are discussed.

Plasmids derived from lambdoid bacteriophages as models for studying replication of mobile genetic elements responsible for the production of Shiga toxins by pathogenic Escherichia coli strains.

BACKGROUND: Genes encoding Shiga toxins in pathogenic Escherichia coli strains (Shiga toxin-producing E. coli, STEC) are located on lambdoid prophages. However, studies on the replication of these phages were not reported to date. METHODS: Plasmids derived from Shiga toxin-converting phages were constructed, introduced into wild-type E. coli strain and studied using methods of molecular genetics. RESULTS: The studied replicons behaved similarly to plasmids derived from bacteriophage lambda, and DNA sequence analysis revealed their high level of homology to the lambda replication region. Nevertheless, contrary to lambda and two of six tested plasmids, four tested replicons were able to replicate in the E. colidnaA46 mutant. Specific nucleotide differences, causing amino acid substitutions relative to wild-type lambda O and P proteins, appeared to be responsible for this phenotype. CONCLUSIONS: We conclude that replicons of phages bearing Shiga toxin genes are functionally similar to that of bacteriophage lambda, however, some of them have important differences which influence replication regulation. Furthermore, our results may suggest that there are newly discovered molecular interactions in the DNA replication regulation of lambda and lambdoid phages. Hence, plasmids derived from lambdoid phages appear to be convenient models to study the replication of Shiga toxin-converting bacteriophages.

Receptor for activated C-kinase 1, a novel interaction partner of type II bone morphogenetic protein receptor, regulates smooth muscle cell proliferation in pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by selective elevation of pulmonary arterial pressure. The pathological hallmark of PAH is the narrowing of pulmonary arterioles secondary to endothelial cell dysfunction and smooth muscle cell proliferation. Heterozygous mutations in BMPR2, encoding the type II bone morphogenetic protein receptor (BMPRII), were identified in PAH, suggesting that alterations to BMPRII function are involved in disease onset and/or progression. METHODS AND RESULTS: We identified the receptor for activated C-kinase (RACK1) as a novel interaction partner of BMPRII by yeast 2-hybrid analyses using the kinase domain of BMPRII as a bait. Glutathione-S-transferase pull-down and coimmunoprecipitation confirmed the interaction of RACK1 with BMPRII in vitro and in vivo. RACK1-BMPRII interaction was reduced when kinase domain mutations occurring in patients with PAH were introduced to BMPRII. Immunohistochemistry of lung sections from PAH and control patients and immunofluorescence analysis of primary pulmonary arterial smooth muscle cells demonstrated colocalization of BMPRII and RACK1 in vivo. Quantitative reverse-transcription polymerase chain reaction and Western blot analysis showed significant downregulation of RACK1 expression in the rat model of monocrotaline-induced PAH but not in pulmonary arterial smooth muscle cells from PAH patients. Abrogation of RACK1 expression in pulmonary arterial smooth muscle cells led to decreased Smad1 phosphorylation and increased proliferation, whereas overexpression of RACK1 led to increased Smad1 phosphorylation and decreased proliferation. CONCLUSIONS: RACK1, a novel interaction partner of BMPRII, constitutes a new negative regulator of pulmonary arterial smooth muscle cell proliferation, suggesting a potential role for RACK1 in the pathogenesis of PAH.

Dysregulated bone morphogenetic protein signaling in monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Mutations in the bmpr2 gene, encoding the type II bone morphogenetic protein (BMP) receptor, have been identified in patients with pulmonary arterial hypertension (PAH), implicating BMP signaling in PAH. The aim of this study was to assess BMP signaling and its physiological effects in a monocrotaline (MCT) model of PAH. METHODS AND RESULTS: Expression of BMP receptors Ib and II, and Smads 4, 5, 6, and 8, was downregulated in lungs but not kidneys of MCT-treated rats. Smad1 phosphorylation and expression of BMP/Smad target genes id1 and id3 was also reduced, although ERK1/2 and p38(MAPK) phosphorylation remained unaffected. BMP receptor and Smad expression, Smad1 phosphorylation, and induction of the BMP/Smad-responsive element of the id1 promoter were reduced in pulmonary artery smooth muscle cells (PASMCs) from MCT-treated rats. As a consequence of impaired BMP/Smad signaling, PASMCs from MCT-treated rats were resistant to apoptosis induced by BMP-4 and BMP-7, and were also resistant to BMP-4 antagonism of proliferation induced by platelet-derived growth factor. CONCLUSION: BMP signaling and BMP-regulated physiological phenomena are perturbed in MCT-treated rats, lending solid support to the proposed roles for BMP signaling in the pathogenesis of human PAH.