Activation of Mechanistic Target of Rapamycin (mTOR) in Human Endothelial Cells Infected with Pathogenic Spotted Fever Group Rickettsiae

Abstract

Attributed to the tropism for host microvascular endothelium lining the blood vessels, vascular inflammation and dysfunction represent salient features of rickettsial pathogenesis, yet the details of fundamentally important pathogen interactions with host endothelial cells (ECs) as the primary targets of infection remain poorly appreciated. Mechanistic target of rapamycin (mTOR), a serine/threonine protein kinase of the phosphatidylinositol kinase-related kinase family, assembles into two functionally distinct complexes, namely mTORC1 (Raptor) and mTORC2 (Rictor), implicated in the determination of innate immune responses to intracellular pathogens via transcriptional regulation. In the present study, we investigated activation status of mTOR and its potential contributions to host EC responses during Rickettsia rickettsii and R. conorii infection. Protein lysates from infected ECs were analyzed for threonine 421/serine 424 phosphorylation of p70 S6 kinase (p70 S6K) and that of serine 2448 on mTOR itself as established markers of mTORC1 activation. For mTORC2, we assessed phosphorylation of protein kinase B (PKB or Akt) and protein kinase C (PKC), respectively, on serine 473 and serine 657. The results suggest increased phosphorylation of p70 S6K and mTOR during Rickettsia infection of ECs as early as 3 h and persisting for up to 24 h post-infection. The steady-state levels of phospho-Akt and phospho-PKC were also increased. Infection with pathogenic rickettsiae also resulted in the formation of microtubule-associated protein 1A/1B-light chain 3 (LC3-II) puncta and increased lipidation of LC3-II, a response significantly inhibited by introduction of siRNA targeting mTORC1 into ECs. These findings thus yield first evidence for the activation of both mTORC1 and mTORC2 during EC infection in vitro with Rickettsia species and suggest that early induction of autophagy in response to intracellular infection might be regulated by this important pathway known to function as a central integrator of cellular immunity and inflammation.

Apoptosis and Autophagy: Current Understanding in Tick–Pathogen Interactions

“…Infection with a range of spotted fever group rickettsiae (R. conorii, R. japonica, R. montanensis, R. parkeri and R. rickettsii) results in autophagy induction in mammalian host cells (Uchiyama et al, 2012;Engström et al, 2019;Sahni et al, 2020). Pathogenic rickettsiae appear to be capable of evading this immune response, whilst non-pathogenic species lack this ability (Uchiyama et al, 2012;Engström et al, 2019).…”

“…Pathogenic rickettsiae appear to be capable of evading this immune response, whilst non-pathogenic species lack this ability (Uchiyama et al, 2012;Engström et al, 2019). Infection of human umbilical vein endothelial cells with R. rickettsii or R. conorii results in mTOR activation, potentially as a mechanism by which these rickettsiae limit anti-microbial autophagy (Sahni et al, 2020), whilst Rickettsia parkeri is able to evade autophagy by employing outer membrane protein B (OmpB) to prevent the ubiquitination of surface proteins and their subsequent recognition by autophagic receptors in both human microvascular endothelial cells and mouse bone-marrowderived macrophages (Engström et al, 2019). Even in the same cell type, different Rickettsia species utilize contrasting strategies to evade autophagy.…”

Mediterranean Spotted Fever: Current Knowledge and Recent Advances

“…In addition, infection of endothelial cells with R. conorii in vitro has been associated with the activation of the mechanistic target of rapamycin (mTOR) signaling. It is hypothesized that rickettsiae may exploit this mechanism to evade xenophagy, a form of selective autophagy, that constitutes an important host defense strategy against intracellular bacteria [71].…”

The host mTOR pathway and parasitic diseases pathogenesis

“…Autophagy can be either inhibited or activated upon pathogen infection; to evade pathogen degradation and recognition or to support nutrients for replication, respectively (Le Sage et al 2016;Wang et al 2009a). Recently, it has been discovered that rickettsiae interfere in vitro with the autophagic response of endothelial cells activating mTOR and triggering an initial autophagy response, thus allowing the establishment of an intracellular infection (Sahni et al 2020). Now, we know that many pathogens affect host cell pathways including mTOR and autophagy to develop the infection processes in their hosts (Nouwen and Everts 2020).…”