Bacterial resistance to antibiotics has been studied extensively, and in most cases there is a reasonably good understanding of the underlying molecular mechanisms (23
). In contrast, tolerance to antimicrobials, which was discovered at about the same time as resistance, is poorly understood, in spite of its considerable importance to human health. According to the CDC, 65% of all infections in developed countries are caused by biofilms, which exhibit multidrug tolerance largely due to the presence of persister cells (9
Persisters are phenotypic variants of the wild type, and their dormancy allows them to escape killing by bactericidal antibiotics. Several candidate genes have been implicated in persister formation. Isolation of persisters by lysing a culture with ampicillin or by physically sorting cells with decreased expression of green fluorescent protein enabled expression profiling, which pointed to overproduction of TA modules. Expression of several toxins, such as RelE (17
), MazF (35
), YgiU (31
), and HipA (12
), has been shown to increase persister formation. TA modules are highly redundant, for example, there are at least 10 in E. coli
) and >60 in Mycobacterium tuberculosis
). Apart from TA modules, GlpD (glycerol-3-phosphate dehydrogenase) was recently identified as a potential persister component in E. coli
The focus of the present study is the HipA toxin; we found it to belong to the phosphatidylinositol 3/4-kinase superfamily and examined its kinase properties.
Alignment of HipA with other members of the PI 3/4-kinase superfamily reveals conservation of all structural elements of the core catalytic domain and the key amino acids that contribute to the active-site formation and Mg2+ binding. This strongly suggests that HipA is an active kinase, but beyond this general prediction, it is hard to predict the substrate specificity of HipA. The members of the PI 3/4-kinase superfamily that are most similar to HipA are the PI4KII kinases that appear to phosphorylate exclusively phosphatidylinositol, a lipid molecule that has not been found in bacteria. Given the diversity of substrates phosphorylated by members of the PI 3/4-kinase superfamily, it is conceivable that HipA family proteins are either lipid kinases with a distinct specificity or protein kinases.
HipA underwent autophosphorylation in vitro in the presence of ATP, and the purified HipA appeared to carry a single phosphate on Ser150. This showed that the protein is a serine kinase and autophosphorylates in vivo as well. Attempts to find trans-kinase activity with universal, artificial kinase substrates casein and histone were unsuccessful, suggesting that HipA is a specific protein kinase or phosphorylates nonprotein substrates. Importantly, replacement of the active-site amino acids, Mg2+-binding residues, and autophosphorylated Ser150 resulted in the loss of the ability of HipA to stop cell growth upon overexpression. This result shows that the kinase activity and phosphorylation of HipA are both required for its function in growth arrest.
HipA proteins with abrogated kinase activity were also found to be defective in their ability to produce persisters. Expression of wild-type HipA strongly protected cells from ofloxacin, a fluoroquinolone that has the ability to kill nongrowing cells, whereas the mutant proteins had no effect. Interestingly, HipA overexpression caused complete protection from mitomycin C, which forms DNA adducts. This was somewhat unexpected because dormancy alone probably would not prevent the chemical reaction of the drug with DNA. In this case, mutant HipA proteins also produced some degree of resistance to mitomycin, although the protection level was 10-fold lower than that with the wild type. Apparently, HipA might have some activity independent of its kinase activity and/or phosphorylation. Overexpression of HipA provided no protection from tobramycin, an aminoglycoside antibiotic. This is surprising, given that activity of this aminoglycoside antibiotic depends on growth rate and cells expressing HipA are essentially nongrowing. Moreover, HipA has been reported to cause inhibition of DNA, RNA, and protein synthesis (20
). Tobramycin, like other aminoglycosides, kills cells by interrupting translation, which yields truncated toxic peptides (other protein synthesis inhibitors, such as chloramphenicol, that simply stop translation are bacteriostatic). Conceivably, HipA-expressing cells have sufficient residual translation, and the inability of HipA to protect cells from tobramycin suggests that protein synthesis is not the target of this toxin.
TA modules are present on plasmids, where they constitute the maintenance mechanism, and on the chromosomes of most bacterial species. Toxins have been shown to inhibit translation by cleaving mRNA (RelE and MazF), inhibiting topoisomerase II (CcdB), and making holes in the membrane (29
). To our knowledge, HipA is the first toxin that has been shown to possess protein kinase activity. Structural data indicated that the ζ toxin of the ζ
TA module encoded by plasmid pSM19035 is a P-loop-fold kinase, which is unrelated to the PI 3/4-kinase superfamily (26
). Thus, the mechanism of HipA, which requires an active kinase domain and, apparently, autophosphorylation, seems to represent a novel principle of bacterial toxin action.
Furthermore, HipA is the first bacterial member of the PI 3/4-kinase superfamily for which the phosphotransferase activity was experimentally demonstrated.