The enterococcal PASTA kinase: A sentinel for cell envelope stress

Djorić, Dušanka; Minton, Nicole E.; Kristich, Christopher J.

doi:10.1111/omi.12313

Cited by 7 publications

(5 citation statements)

References 159 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Among the various regulatory mechanisms controlling PG production, PASTA (PBP and serine/threonine kinase associated) domain-containing eukaryotic-like serine/threonine protein kinases (PASTA-eSTKs) have an outstanding role in PG biosynthesis regulation of firmicutes and actinobacteria, two major types of Gram-positives [14][15][16]. PASTA-eSTKs sense cell wall damaging conditions by interaction of lipid II with their extracellular PASTA domains, and this leads to the activation of their cytosolic kinase domain [17,18].…”

Section: Introductionmentioning

confidence: 99%

MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling

et al. 2022

View full text Add to dashboard Cite

Gram-positive bacteria are protected by a thick mesh of peptidoglycan (PG) completely engulfing their cells. This PG network is the main component of the bacterial cell wall, it provides rigidity and acts as foundation for the attachment of other surface molecules. Biosynthesis of PG consumes a high amount of cellular resources and therefore requires careful adjustments to environmental conditions. An important switch in the control of PG biosynthesis of Listeria monocytogenes, a Gram-positive pathogen with a high infection fatality rate, is the serine/threonine protein kinase PrkA. A key substrate of this kinase is the small cytosolic protein ReoM. We have shown previously that ReoM phosphorylation regulates PG formation through control of MurA stability. MurA catalyzes the first step in PG biosynthesis and the current model suggests that phosphorylated ReoM prevents MurA degradation by the ClpCP protease. In contrast, conditions leading to ReoM dephosphorylation stimulate MurA degradation. How ReoM controls degradation of MurA and potential other substrates is not understood. Also, the individual contribution of the ~20 other known PrkA targets to PG biosynthesis regulation is unknown. We here present murA mutants which escape proteolytic degradation. The release of MurA from ClpCP-dependent proteolysis was able to activate PG biosynthesis and further enhances the intrinsic cephalosporin resistance of L. monocytogenes. This latter effect required the RodA3/PBP B3 transglycosylase/transpeptidase pair. One murA escape mutation not only fully rescued an otherwise non-viable prkA mutant during growth in batch culture and inside macrophages but also overcompensated cephalosporin hypersensitivity. Our data collectively indicate that the main purpose of PrkA-mediated signaling in L. monocytogenes is control of MurA stability during standard laboratory growth conditions and intracellular growth in macrophages. These findings have important implications for the understanding of PG biosynthesis regulation and β-lactam resistance of L. monocytogenes and related Gram-positive bacteria.

show abstract

Section: Introductionmentioning

confidence: 99%

MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling

et al. 2022

View full text Add to dashboard Cite

show abstract

“…PASTA kinases are widespread among bacterial species and regulate a variety of important cellular phenotypes among human pathogens, such as antibiotic resistance, biofilm formation, and virulence (Pereira et al, 2011). For example, in E. faecalis the PASTA kinase IreK is essential for resistance to the cephalosporin class of antibiotics (Djoric et al, 2021; Kristich et al, 2007; Kristich et al, 2011; Labbe & Kristich, 2017). However, the molecular mechanisms by which PASTA kinases are activated are not well understood.…”

Section: Discussionmentioning

confidence: 99%

Conformational changes in the activation loop of a bacterial PASTA kinase

et al. 2023

Self Cite

View full text Add to dashboard Cite

Many bacterial genomes encode a transmembrane protein kinase belonging to the PASTA kinase family, which controls numerous processes in diverse bacterial pathogens, including antibiotic resistance, cell division, stress resistance, toxin production, and virulence. PASTA kinases share a conserved three-part domain architecture, consisting of an extracellular PASTA domain, proposed to sense the peptidoglycan layer status, a single transmembrane helix, and an intracellular Ser/Thr kinase domain. The crystal structures of the kinase domain from two homologous PASTA kinases reveal a characteristic two-lobed structure typical of eukaryotic protein kinases with a centrally located, but unresolved, activation loop that becomes phosphorylated and regulates downstream signaling pathways. We previously identified three sites of phosphorylation on the activation loop (T163, T166, and T168) of IreK, a PASTA kinase from the pathogen Enterococcus faecalis, as well as a distal phosphorylation site (T218) that each influence IreK activity in vivo. Still, the mechanism by which loop phosphorylation regulates PASTA kinase function is yet unknown. Therefore, we utilized site-directed spin labeling (SDSL) and continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy to assess the E. faecalis IreK kinase activation loop dynamics, including the effects of phosphorylation on activation loop motion, and the IreK-IreB interaction. Our results reveal that the IreK activation loop occupies a more immobile state when dephosphorylated, and that loop autophosphorylation shifts the loop to a more mobile state that can then enable interaction with IreB, a known substrate.

show abstract

“…Serine/threonine kinases, another important class of bacterial kinase, have recently been demonstrated to be involved in the regulation of critical physiological processes and virulence of several important bacterial pathogens, making them attractive drug targets (Manuse et al, 2016;Djorić et al, 2021). Recently, several inhibitors against STKs have been identified.…”

Section: Discussionmentioning

confidence: 99%

“…In response to stimuli, STK autophosphorylates on its activation loop of the kinase domain by hydrolyzing adenosine triphosphate (ATP) and transfers the phosphate to the serine or threonine residues of its substrate proteins (Ogawara, 2016;Labbe and Kristich, 2017;Nagarajan et al, 2021). STKs are identified as global regulators controlling a wide range of important bacterial physiological processes, including cell wall hemostasis, virulence, metabolism, and cell division in several important bacterial pathogens (Cheung and Duclos, 2012;Chawla et al, 2014;Manuse et al, 2016;Wamp et al, 2020;Djorić et al, 2021). In some bacteria, STKs are even essential or conditional essential for bacterial viability (Fernandez et al, 2006;Hu et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Inhibitors targeting the autophosphorylation of serine/threonine kinase of Streptococcus suis show potent antimicrobial activity

et al. 2022

Front. Microbiol.

View full text Add to dashboard Cite

Antimicrobial resistance (AMR) is a global concern threatening public health. Developing novel antibiotics is one of the effective strategies to tackle AMR. Serine/threonine kinases (STKs) have been recently shown to play critical roles in the physiology and pathogenesis of several important bacterial pathogens which are regarded as a promising antimicrobial drug target. We previously reported the roles of STK in the regulation of bacterial cell division, metabolism, and pathogenesis in Streptococcus suis, an important zoonotic bacterial pathogen. In this study, we firstly identified the Thr167 and Ser175 residues in the activation loop of S. suis STK (ssSTK) as the kinase autophosphorylation sites. Phenotyping results demonstrated that the autophosphorylation deficient strain resembled the stk deletion strain showing essentiality for bacterial growth in minimal medium, abnormal morphology, and decreased virulence when compared with the wild-type S. suis SC19 strain. Based on these findings, we established an ssSTK inhibitor screening approach by measuring the growth of S. suis in a minimal medium and testing the autophosphorylation inhibition by measuring the consumption of ATP in an enzymatic reaction by ssSTK. A series of inhibitors against ssSTK are identified from a commercial kinase inhibitors library, including Staurosporine, K252a, AT9283, and APY29. These inhibitors showed antimicrobial activity in vitro. Moreover, by using Galleria mellonella larvae infection assay, compound APY29 displayed in vivo efficacy against S. suis infection. Additionally, it was predicted by molecular docking that these inhibitors could interact with ssSTK. Collectively, our data illustrated the essential roles of ssSTK autophosphorylation in the physiology and pathogenicity of S. suis and consider these inhibitors as promising antimicrobial lead compounds.

show abstract

The enterococcal PASTA kinase: A sentinel for cell envelope stress

Abstract: Enterococci are Gram-positive, opportunistic pathogens and inhabitants of the gastrointestinal tract of most terrestrial organisms. Enterococci are considered to be commensals, but in immunocompromised or debilitated individuals, they can cause deadly infections

Cited by 7 publications

References 159 publications

MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling

MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling

Conformational changes in the activation loop of a bacterial PASTA kinase

Inhibitors targeting the autophosphorylation of serine/threonine kinase of Streptococcus suis show potent antimicrobial activity

Contact Info

Product

Resources

About