Protein kinases have a diverse array of functions in bacterial physiology, with a distinct role in the regulation of development, stress responses, and pathogenicity. pknF, one of the 11 kinases of Mycobacterium tuberculosis, encodes an autophosphorylating, transmembrane serine/threonine protein kinase, which is absent in the fast-growing, nonpathogenic Mycobacterium smegmatis. Herein, we investigate the physiological role of PknF using an antisense strategy with M. tuberculosis and expressing PknF and its kinase mutant (K41M) in M. smegmatis. Expression of PknF in M. smegmatis led to reduction in the growth rate and shortening and swelling of cells with constrictions. Interestingly, an antisense strain of M. tuberculosis expressing a low level of PknF displayed fast growth and a deformed cell morphology compared to the wild-type strain. Electron microscopy showed that most of the cells of the antisense strain were of a smaller size with an aberrant septum. Furthermore, nutrient transport analysis of these strains was conducted using 3 H-labeled and 14 C-labeled substrates. A significant increase in the uptake of D-glucose but not of glycerol, leucine, or oleic acid was observed in the antisense strain compared to the wild-type strain. The results suggest that PknF plays a direct/indirect role in the regulation of glucose transport, cell growth, and septum formation in M. tuberculosis.

CD8 T cells resolve intracellular pathogens by responding to pathogen-derived peptides that are presented on the cell surface by MHC class I molecules. Although most pathogens encode a large variety of antigenic peptides, protective CD8 T cell responses target usually only a few of these. To determine the mechanism by which the IFN-γ-inducible proteasome (immuno) subunits enhance the ability of specific pathogen-derived peptides to elicit CD8 T cell responses, we generated a recombinant Listeria monocytogenes strain (rLM-E1) that secretes a model Ag encompassing the immunoproteasome-dependent E1B192–200 and immunoproteasome-independent E1A234–243 epitope. Analyses of Ag presentation showed that infected gene-deficient professional APCs, lacking the immunosubunits LMP7/iβ5 and MECL-1/iβ2, processed and presented the rLM-E1-derived E1B192–200 epitope but with delayed kinetics. E1A epitope processing proceeded normally in these cells. Accordingly, infected gene-deficient mice failed to respond to the otherwise immunodominant E1B192–200 epitope but mounted normal CD8 T cell responses to E1A234–243 which was processed by the same professional APCs, from the same rLM-E1 Ag. The inability of gene-deficient mice to respond to E1B192–200 was not explained by insufficient quantities of antigenic peptide, as splenic APC of 36-h-infected gene-deficient mice that presented the two E1 epitopes at steady state levels elicited responses to both E1B192–200 and E1A234–243 when transferred into LMP7+MECL-1-deficient mice. Taken together, our findings indicate that not absolute epitope quantities but early Ag-processing kinetics determine the ability of pathogen-derived peptides to elicit CD8 T cell responses, which is of importance for rational T cell vaccine design.

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