My laboratory is interested in how eukaryotic-like kinases function in a pathogenic bacterium Mycobacterium tuberculosis. In particular, I study the function of PknA/PknB kinases and their substrates in regulating cell division
and cell physiology of M. tuberculosis.

Tuberculosis (TB) is a bacterial infectious disease caused by an obligate intracellular human pathogen, Mycobacterium tuberculosis. TB remains a major threat worldwide with approximately eight million new cases a year and nearly two million deaths annually (WHO, 2002). In addition to the current burden of the disease, the ability of M. tuberculosis to remain latent in the human host without causing active disease (latency) is an additional threat. It is estimated that one third of the world’s population has been infected with M. tuberculosis. This means that there is a large potential reservoir for new TB disease even if worldwide implementation of effective treatment and control systems could greatly diminish current TB disease and new infections.

My laboratory’s research has implications both for developing drugs for TB and for a basic understanding of how bacterial cells control cell division. The M. tuberculosis genome contains genes encoding 11 eukaryotic-like serine/threonine kinases. My previous work has been dedicated to the two kinases PknA and PknB since these were thought to be important regulators in cell division or morphology based on their potential essentiality, location of genes near the origin of replication, and the proposed functions of the other genes in the operon. The specific inhibition of PknA/PknB or their in vivo substrates could lead to the development of new, potent and specific anti-TB drugs. In my recent publication (Kang et al., Genes & Development, 2005), I demonstrated that the operon containing pknA and pknB is transcribed predominantly in exponential phase and that the overexpression of these kinases in mycobacterial cells causes major growth and morphological changes that indicate defects in cell wall synthesis, and possibly cell division. Using peptide library screening and proteomic methods, I determined the preferred substrate motifs of PknA and PknB, and identified three in vivo substrates (Wag31, Rv1422, and PknB itself) of these kinases. This is the first report on identifying in vivo substrates of Eukaryoticlike kinases from bacterial cells, and these results clearly demonstrated that the techniques could be used for the study of other kinases also.

Wag31, a substrate of PknA and PknB, is a homolog of the Gram-positive cell division protein DivIVA. Though DivIVA functions in cell division in all Grampositive bacteria, the specific roles it plays appear to vary in a species-specific manner. Originally identified as a mini-cell locus in B. subtilis, DivIVA is known to have dual functions in appropriate septum placement of vegetative cells, and chromosome segregation in sporulating cells. In contrast, in Streptomyces coelicolor, DivIVA is essential for hyphal peptidoglycan synthesis. In our most recent publication, we demonstrated that Wag31 is localized to the cell poles in M. smegmatis (Figure 1; Kang et al, 2008). We further showed that wag31 is an essential gene and that its partial depletion causes a dramatic morphological change in which one end of the cell becomes round rather than rod-shaped. This abnormal morphology appears to be caused by a defect in polar peptidoglycan synthesis.

This observation is further supported by our observation in the wag31 conditional mutant where temporal and spatial correlation between Wag31 and nascent peptidoglycan synthesis in wild-type M. smegmatis was clearly observed. Finally, expression of M. tuberculosis wag31 in the wag31 conditional mutant of M. smegmatis altered the growth rate and nascent peptidoglycan synthesis in a manner that depended on the phospho-acceptor residue encoded by the allele being expressed (unpublished data). Consistent with these observations, a recent report by Nguyen et al (2007) showed that overexpression of wag31 caused M. smegmatis cells longer and more wider cells with a bulbous poles indicating a role of Wag31 in modulating cell shape These data, together with the data showing co-localization of Wag31 and nascent peptidoglycan synthesis at the cell poles in both the wag31 conditional mutant and wild-type strains of M. smegmatis, indicate that Wag31 and its phosphorylation are important for maintaining cell shape and cell wall integrity by controlling peptidoglycan synthesis at the cell poles.

Since we wish to achieve comprehensive understanding of the function of the kinases in mycobacterial physiology, we have begun to identify additional substrates by a proteomic search with a phospho-T antibody and sequence homology search with the known substrates of these kinases (Wag31 and RV1422). Our preliminary studies revealed numerous unknown substrates that can be phosphorylated by PknA and/or PknB in M. tuberculosis. We are currently testing if they are real substrates of these kinases, and biochemical and genetic investigations of the newly identified substrates will be pursued after confirming their phosphorylation.

Fig. 2 Current model of PknA/PknB function. The extracellular domain of PknB, which has four PASTA domain can sense the status of cell wall synthesis or other signals from outside. This will activate PknB, which then phosphorylates its substrates such Rv1422 or SigH. PknB can also activate PknA which then phosphorylate Wag31, a cell division protein.