ABSTRACT
The Sinorhizobium meliloti BacA ABC transporter protein plays an important role in its nodulating symbiosis with the legume alfalfa (Medicago sativa). The Mycobacterium tuberculosis BacA homolog was found to be important for the maintenance of chronic murine infections, yet its in vivo function is unknown. In the legume plant as well as in the mammalian host, bacteria encounter host antimicrobial peptides (AMPs). We found that the M. tuberculosis BacA protein was able to partially complement the symbiotic defect of an S. meliloti BacA-deficient mutant on alfalfa plants and to protect this mutant in vitro from the antimicrobial activity of a synthetic legume peptide, NCR247, and a recombinant human ß-defensin 2 (HBD2). This finding was also confirmed using an M. tuberculosis insertion mutant. Furthermore, M. tuberculosis BacA-mediated protection of the legume symbiont S. meliloti against legume defensins as well as HBD2 is dependent on its attached ATPase domain. In addition, we show that M. tuberculosis BacA mediates peptide uptake of the truncated bovine AMP, Bac7(1-16). This process required a functional ATPase domain. We therefore suggest that M. tuberculosis BacA is important for the transport of peptides across the cytoplasmic membrane and is part of a complete ABC transporter. Hence, BacA-mediated protection against host AMPs might be important for the maintenance of latent infections.
Subject(s)
Bacterial Proteins/metabolism , Genetic Complementation Test , Membrane Transport Proteins/deficiency , Membrane Transport Proteins/metabolism , Mycobacterium tuberculosis/genetics , Sinorhizobium meliloti/physiology , Symbiosis , Anti-Infective Agents/pharmacology , Bacterial Proteins/genetics , Medicago sativa/microbiology , Medicago sativa/physiology , Membrane Transport Proteins/genetics , Sinorhizobium meliloti/drug effects , Sinorhizobium meliloti/genetics , beta-Defensins/pharmacologyABSTRACT
Over sub-culturing a cell line generates a selective pressure which can result in key cellular functions being altered such as gene and protein expression. The present study set out to determine whether serial sub-culturing affects the antiviral state of the Salmon Head Kidney (SHK-1) cell line. Cells were cultured under constant conditions and real-time PCR was performed to measure the level of interferon (IFN) and Mx gene expression over different passage numbers. A significant increase in the basal level of IFN and Mx gene expression was recorded at passage number 58 (3 and 14-fold increase versus passage number 53), suggesting a sub-culturing effect on the type I IFN response in SHK-1 cells. Passage dependent variations in morphology and cell sub-populations have been previously observed in SHK-1 cells. Such variations in cell sub-types were suspected to be responsible for the fluctuations in IFN and Mx gene expression recorded in this study.
