MmpL3, Wag31 and PlrA are involved in coordinating polar growth with peptidoglycan metabolism and nutrient availability.

Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of Mycobacterium smegmatis, we identify a connection between wag31 and trehalose monomycolate (TMM) transporter mmpl3 in a suppressor screen, and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 are responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism, but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth, but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation, and in which the connection between Wag31, PlrA and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate synthesis of the different layers of the mycobacterial cell wall in changing conditions.

Ceragenin mediated selectivity of antimicrobial silver nanoparticles.

The understanding that common broad-spectrum antimicrobials disrupt natural microbial flora important in acquiring nutrients and preventing infection has resulted in a paradigm shift favoring more selective antimicrobials. This work explores silver nanoparticles conjugated with ceragenin, or cationic antimicrobials (CSA-SNPs), as a potential Gram-positive selective antimicrobial. Herein, CSA-SNPs are characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential, and high-performance liquid chromatography-electrospray time-of-flight mass spectrometry (HPLC-ESI-TOF-MS). The antimicrobial properties are determined through minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) and time-kill studies. Spatial selectivity of the conjugate nanoparticle was evaluated using confocal imaging, MATLAB statistical analysis, and video monitored interactions between bacteria and CSA-SNPs via laser trapping techniques. Cytotoxicity was also determined by live/dead staining and flow cytometry. Average particle size, as determined through TEM analysis, and hydrodynamic diameter, as determined via DLS, are 63.5 ± 38.8 and 102.23 ± 2.3 nm, respectively. The zeta potential of the SNP before and after CSA attachment is -18.23 and -8.34 mV, respectively. MIC/MBC data suggest that CSA-SNPs are 8 times more effective against Staphylococcus aureus than SNPs alone. Furthermore, MATLAB analysis of confocal imaging found that 70% of CSA-SNPs are within 2 µm of S. aureus, whereas this percentage falls to below 40% with respect to Escherichia coli. These results are bolstered further by laser trapping experiments demonstrating selective adherence of CSA-SNPs conjugates with bacterial strains. Cytotoxicity studies of CSA-SNPs against 3T3 fibroblasts indicate 50% cell viability at 50 ppm.