Reciprocal Regulation of PASTA Kinase Signaling by Differential Modification

Abstract

Transmembrane Ser/Thr kinases containing extracellular PASTA (penicillin-binding protein [PBP] and Ser/Thr-associated) domains are ubiquitous among Actinobacteria and Firmicutes species. Such PASTA kinases regulate critical bacterial processes, including antibiotic resistance, cell division, cell envelope homeostasis, and virulence, and are sometimes essential for viability. Previous studies of purified PASTA kinase fragments revealed they are capable of autophosphorylation in vitro, typically at multiple sites on the kinase domain. Autophosphorylation of a specific structural element of the kinase known as the activation loop is thought to enhance kinase activity in response to stimuli. However, the role of kinase phosphorylation at other sites is largely unknown. Moreover, the mechanisms by which PASTA kinases are deactivated once their stimulus has diminished are poorly understood. Enterococcus faecalis is a Gram-positive intestinal bacterium and a major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, and such antimicrobials trigger enhanced phosphorylation of IreK in vivo. Here we identify multiple sites of phosphorylation on IreK and evaluate their function in vivo and in vitro. While phosphorylation of the IreK activation loop is required for kinase activity, we found that phosphorylation at a site distinct from the activation loop reciprocally modulates IreK activity in vivo, leading to diminished activity (and diminished antimicrobial resistance). Moreover, this site is important for deactivation of IreK in vivo upon removal of an activating stimulus. Our results are consistent with a model in which phosphorylation of IreK at distinct sites reciprocally regulates IreK activity in vivo to promote adaptation to cell wall stresses. IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes species and regulate critical processes, including antibiotic resistance, cell division, and cell envelope homeostasis. Previous studies of PASTA kinase fragments revealed autophosphorylation at multiple sites. However, the functional role of autophosphorylation and the relative impacts of phosphorylation at distinct sites are poorly understood. The PASTA kinase of Enterococcus faecalis, IreK, regulates intrinsic resistance to antimicrobials. Here we identify multiple sites of phosphorylation on IreK and show that modification of IreK at distinct sites reciprocally regulates IreK activity and antimicrobial resistance in vivo. Thus, these results provide new insights into the mechanisms by which PASTA kinases can regulate critical physiological processes in a wide variety of bacterial species.

The enterococcal PASTA kinase: A sentinel for cell envelope stress

“…No specific site on the activation loop was obviously more important than any other, and the effect of phosphorylation at multiple sites appeared to be additive (Labbe et al, 2019).…”

“…However, it remains unclear how, or if, that interaction modulates the activity of FhaA or the PASTA kinase itself. Labbe et al (2019) identified 2 sites of phosphorylation on the juxtamembrane linker of IreK (Labbe et al, 2019). Mutations The cytoplasmic kinase domains of PASTA kinases are the most highly conserved domains and belong to the superfamily of Hanks-type kinases that are widespread in eukaryotic organisms.…”

“…Phosphorylation of the activation loop is a common feature of PASTA kinases and has been thought to be required for kinase activation in vivo (Boitel et al, 2003;Bryant-Hudson et al, 2011;Madec et al, 2002;Nolen et al, 2004;Shakir et al, 2010;Young et al, 2003), although most studies of PASTA kinase phosphorylation focus on the kinase in vitro, with an exception where a surrogate strain is used as an in vivo host (Zheng et al, 2018). Labbe et al (2019) (Kristich et al, 2011;Labbe et al, 2019).…”

Importance of protein Ser/Thr/Tyr phosphorylation for bacterial pathogenesis

“…Several studies have found that antibiotic resistance can be driven by bacterial kinases. Particularly, transmembrane Hanks kinases with PASTA domains are seen to confer resistance to cell-wall-inhibiting b-lactam-type antibiotics in various Gram-positive species [14,19,34,36,[218][219][220][221]. Although this may not be surprising given their role in cell division and cell wall homeostasis as described earlier, the mechanism(s) by which Hanks-type PASTA kinases promote b-lactam resistance are only poorly defined.…”

“…Deleting PhpP augments StkP phosphorylation levels and confers drug resistance, suggesting that StkP inactivation could be a strategy for overcoming S. pneumoniae blactam resistance [218]. Similarly, L. monocytogenes PrkA, Enterococcus faecalis IreK, C. difficile PrkC, and MRSA Stk1 are all required for resistance to different types of b-lactams [19,34,36,[219][220][221]. In S. aureus, b-lactams induce the phosphorylation of the blactam antibiotic-sensing protein BlaR1 and this initiates a cascade leading to expression of antibiotic resistance determinants [223].…”