RESUMEN
Acinetobacter baumannii is associated with multidrug resistant (MDR) infections in healthcare settings, with fluoroquinolones such as ciprofloxacin being currently ineffective. Clinical isolates largely harbor mutations in the GyrA and TopoIV fluoroquinolone targets, as well as mutations that increase expression of drug resistance-nodulation-division (RND) efflux pumps. Factors critical for maintaining fitness levels of pump overproducers are uncharacterized despite their prevalence in clinical isolates. We here identify proteins that contribute to the fitness of FQR strains overexpressing three known RND systems using high-density insertion mutagenesis. Overproduction of the AdeFGH efflux pump caused hypersensitization to defects in outer membrane homeostatic regulation, including lesions that reduced LOS biosynthesis and blocked production of the major A. baumannii porin. In contrast, AdeAB pump overproduction, which does not affect the outer membrane pump component, was relatively tolerant to loss of these functions, consistent with outer membrane protein overproduction being the primary disruptive component. Surprisingly, overproduction of proton-transporting efflux pumps had little impact on cytosolic pH, consistent with a compensatory response to pump activity. The most striking transcriptional changes were associated with AdeFGH pump overproduction, resulting in activation of the phenylacetate (PAA) degradation regulon. Disruption of the PAA pathway resulted in cytosolic acidification and defective expression of genes involved in protection from peroxide stress. These results indicate that the RND outer membrane protein overproduction is compensated by cytoplasmic buffering and maintenance of outer membrane integrity in A. baumannii to facilitate fitness of FQR isolates.
RESUMEN
To investigate if differences in imprinting at tropho-microRNA (miRNA) genomic clusters can distinguish between pre-gestational trophoblastic neoplasia cases (pre-GTN) and benign complete hydatidiform mole (CHM) cases at the time of initial uterine evacuation. miRNA sequencing was performed on frozen tissue from 39 CHM cases including 9 GTN cases. DIO3, DLK1, RTL1, and MEG 3 mRNA levels were assessed by qRT-PCR. Protein abundance was assessed by Western blot for DIO3, DLK1, and RTL1. qRT-PCR and Western blot were performed for selenoproteins and markers of oxidative stress. Immunohistochemistry (IHC) was performed for DIO3 on an independent validation set of clinical samples (n = 42) and compared to normal placenta controls across gestational ages. Relative expression of the 14q32 miRNA cluster was lower in pre-GTN cases. There were no differences in protein abundance of DLK1 or RTL1. Notably, there was lower protein expression of DIO3 in pre-GTN cases (5-fold, p < 0.03). There were no differences in mRNA levels of DIO3, DLK1, RTL1 or MEG 3. mRNA levels were higher in all CHM cases compared to normal placenta. IHC showed syncytiotrophoblast-specific DIO3 immunostaining in benign CHM cases and normal placenta, while pre-GTN cases of CHM lacked DIO3 expression. We describe two new biomarkers of pre-GTN CHM cases: decreased 14q32 miRNA expression and loss of DIO3 expression by IHC. Differences in imprinting between benign CHM and pre-GTN cases may provide insight into the fundamental development of CHM.
