No abstract

Although some outer membrane (OM) proteins involved in antibiotic resistance have been previously reported, the OM proteins regulating chlortetracycline (CTC) resistance are largely unknown. In this study, we employed a subproteomics approach to identify altered OM proteins of Escherichia coli in response to CTC exposure. Upregulation of TolC and downregulation of LamB, FadL, OmpC, OmpT, and OmpW were found in E. coli strains exposed to CTC at a high concentration that was increased suddenly and at a half-minimum inhibitory concentration (MIC) that was kept constant in the culture medium. These changes in the level of protein expression were validated using Western blotting. In addition, the possible roles of these altered proteins and their regulation mechanisms in response to CTC exposure were investigated using genetically modified strains with gene deletion of these altered proteins. It was found that deletion of tolC, fadL, ompC, ompT, or ompW resulted in a decrease in the MICs and survival capabilities of the gene-deleted strains, whereas the absence of lamB led to an improvement of the two abilities. The downregulation of LamB expression in the CTC-resistant E. coli strain and the increased antibiotic resistance in its gene-deleted strain suggested a negative regulation mechanism in E. coli in response to CTC exposure. Meanwhile, the direction of the regulation pattern in response to CTC exposure was different from that in E. coli in response to exposure to other antibiotics. These findings uncover a novel antibiotic-resistant mechanism in which bacteria respond to exposure to antibiotics through alteration of the direction of regulation of OM proteins.

Though congenital hydrocephalus is heritable, it has been linked only to eight genes, one of which is MPDZ. Humans and mice that carry a truncated version of MPDZ incur severe hydrocephalus resulting in acute morbidity and lethality. We show by magnetic resonance imaging that contrast medium penetrates into the brain ventricles of mice carrying a Mpdz loss‐of‐function mutation, whereas none is detected in the ventricles of normal mice, implying that the permeability of the choroid plexus epithelial cell monolayer is abnormally high. Comparative proteomic analysis of the cerebrospinal fluid of normal and hydrocephalic mice revealed up to a 53‐fold increase in protein concentration, suggesting that transcytosis through the choroid plexus epithelial cells of Mpdz KO mice is substantially higher than in normal mice. These conclusions are supported by ultrastructural evidence, and by immunohistochemistry and cytology data. Our results provide a straightforward and concise explanation for the pathophysiology of Mpdz‐linked hydrocephalus.

Spontaneous neural differentiation of embryonic stem cells is induced by Noggin-mediated inhibition of bone morphogenetic protein 4 (BMP4) signaling. RhoA is a guanosine triphosphatase (GTPase) that regulates cytoskeletal dynamics and gene expression, both of which control stem cell fate. We found that disruption of Syx, a gene encoding a RhoA-specific guanine nucleotide exchange factor, accelerated retinoic acid–induced neural differentiation in murine embryonic stem cells aggregated into embryoid bodies. Cells from Syx+/+ and Syx−/− embryoid bodies had different abundances of proteins implicated in stem cell pluripotency. The differentiation-promoting proteins Noggin and RARγ (a retinoic acid receptor) were more abundant in cells of Syx−/− embryoid bodies, whereas the differentiation-suppressing proteins SIRT1 (a protein deacetylase) and the phosphorylated form of SMAD1 (the active form of this transcription factor) were more abundant in cells of Syx+/+ embryoid bodies. These differences were blocked by the overexpression of constitutively active RhoA, indicating that the abundance of these proteins was maintained, at least in part, by RhoA activity. The peripheral stress fibers in cells from Syx−/− embryoid bodies were thinner than those in Syx+/+ cells. Furthermore, less Noggin and fewer vesicles containing Rab3d, a GTPase that mediates Noggin trafficking, were detected in cells from Syx−/− embryoid bodies, which could result from increased Noggin exocytosis. These results suggested that, in addition to inhibiting Noggin transcription, RhoA activity in wild-type murine embryonic stem cells also prevented neural differentiation by limiting Noggin secretion.

Escherichia coli growth is a complicated process involved in many factors including the utilization of glucose. It has been reported that E. coli cell growth rate is closely related with glucose concentrations in the cell culture medium. However, the protein regulation networks in response to glucose concentration changes are largely unknown. In the present study, a sub-proteomic methodology has been utilized to characterize alterations of E. coli OM proteins in response to 0.02, 0.2 and 2% concentrations of glucose. In comparison with E. coli cells treated with 0.2% glucose concentration, downregulation of FhuE, FepA, CirA, TolC and OmpX and upregulation of LamB, FadL, OmpF, OmpT and Dps were detected in the E. coli cells treated with 0.02% glucose, and a decrease of TolC, LamB, OmpF, OmpT, OmpX, Dps and elevation of FhuE, FepA, CirA, YncD, FadL and MipA were found in 2% glucose. TolC, LamB and OmpT showed more important roles than other altered OM proteins. Furthermore, the interaction among these altered OM proteins was investigated, and protein interaction networks were characterized. In the networks, all proteins were interacted and regulated by others. TolC, LamB and Dps were the top three proteins that regulated more proteins than others, whereas CirA and OmpT were the top two proteins that were regulated by others. The protein networks could be modified correspondingly with the changes of glucose concentrations. The modifications included the addition of new OM proteins or the change of regulation direction. These findings suggest the important roles of the bacterial OM protein network in E. coli's responses to glucose concentration changes and other environment stresses.