Correction: TFEB and MCOLN1 are important for Coxiella burnetii egress via lysosomal exocytosis

Abstract

Coxiella burnetii is an obligate intracellular zoonotic pathogen, which infects a variety of different vertebrates and non-vertebrates, such as mammals, birds, reptiles and arthropods (Angelakis et al., 2010). Ruminants, like cattle, sheep and goats, are considered its primary reservoir (Maurin et al., 1999, Pouquet et al., 2020). Infected ruminants might develop coxiellosis, which has a quite diverse manifestation, ranging from being asymptomatic to abortion or reproductive disorders (Agerholm, 2013, Bauer et al., 2023). The infected animals shed the bacteria via feces, milk and birthing products. Aerosolized bacteria from these sources are the main cause for human disease (Delsing et al., 2011). However, consumption of raw milk products might also cause infection, but less efficiently (Sobotta et al., 2025, Miller et al., 2021). C. burnetii causes Q fever in humans, which can be either acute or chronic (Maurin et al., 1999). In addition, Q fever can result in the development of the Q fever fatigue syndrome (QFS), which reduces the patient´s quality of life for months or even years (Morroy et al., 2016).The first target cells of C. burnetii are alveolar macrophages, but during the course of infection, other cell types also become infected, including endothelial cells, epithelial cells, fibroblasts and trophoblasts (Maurin et al., 1999). In these cells, C. burnetii establishes a phagolysosomal compartment called the C. burnetii-containing vacuole (CCV) (Howe et al., 2010, Pechstein et al., 2018). The acidic conditions within the CCV lumen activate the type IVB secretion system (T4BSS), which injects effector proteins into the host cell cytoplasm to modulate host cell function (Beare et al., 2011, Carey et al., 2011). Around 150 effector proteins have been predicted (Lührmann et al., 2017). For several effector proteins a function in establishing and maintaining the CCV has been assigned (Bauer et al., 2023). This is in line with the observation that the T4BSS is essential for the maturation of the CCV into a very spacious vacuole permissive for bacterial replication (Carey et al., 2011, Howe et al., 2010). However, which host cell factors are involved in the establishment of the spacious CCV is less understood. It was demonstrated that the interaction with the autophagy pathway (Thomas et al., 2020), the secretory pathway (Campoy et al., 2011) and recycling endosomes (Hall et al., 2024) are important for CCV expansion. In addition, the transcription factor EB (TFEB) seems to be involved in the biogenesis of the spacious CCV (Padmanabhan et al., 2020). TFEB belongs to the microphthalmia-associated transcription factor (MiTF) family of basic helix-loop-helix (bHLH) transcription factors (Kim et al., 2021). They recognize the Coordinated Lysosomal Expression and Regulation (CLEAR) element present within 200 base pairs of the transcriptional start site in many lysosomal (Sardiello et al., 2009) and autophagy-related genes (Settembre et al., 2013). Hence, TFEB is a bona fide master regulator of lysosomal biogenesis and autophagy induction (Raben et al., 2016). There are controversial reports about the function of TFEB during C. burnetii infection. Initially, it was shown that the infection with C. burnetii induces TFEB activation in a T4BSS-dependent manner. Silencing of TFEB resulted in reduced CCV sizes (Padmanabhan et al., 2020), indicating a role of TFEB for CCV expansion. Similar results were obtained in another study, where the authors demonstrated that knockouts of TFEB and TFE3, which also belongs to the MiTF family and functions as a bona fide master regulator of lysosomal biogenesis, in macrophages led to smaller CCVs. Interestingly, TFEB/TFE3 knock-out cells supported increased bacterial replication, indicating that TFEB might have a different function with regard to CCV maturation and bacterial replication (Larson et al., 2019).In the latter study, the C. burnetii-mediated activation of TFE3 was independent of the T4BSS (Larson et al., 2019). In contrast, a recent study showed that C. burnetii actively blocks TFEB activation in a T4BSS-dependent manner. TFEB-deficient cells were characterized by increased CCV size and improved bacterial growth (Kilips et al., 2024). From these three studies it is still not clear whether i) TFEB is activated or inhibited by C. burnetii infection; ii) TFEB activation results in increased or decreased CCV size; iii) the T4BSS is involved in TFEB activation.Hence, we addressed the question which role TFEB plays in maturation of the CCV and vice versa. In addition, we determined how C. burnetii egresses after the completion of its replication cycle within the spacious CCV. We had demonstrated recently, that during later stages of infection, when the spacious CCV has been generated, cells might undergo apoptosis, which allows release of infectious particles and the dissemination of the infection (Schulze-Luehrmann et al., 2024). However, egress also occurs in cells lacking a functional intrinsic apoptosis cascade, suggesting additional egress strategies.Unless otherwise stated, chemicals were purchased from Merck (Darmstadt, Germany) or Carl Roth (Karlsruhe, Germany). The following inhibitors were used: Torin 1 (Cell Signaling, Leiden, Netherlands) and ML-SA1 (Biomol, Hamburg, Germany). The LAMP1 (anti-mouse 1D4B; anti-human H4A3) and LAMP2 (anti-mouse ABL-93; anti-human H4B4) specific primary antibodies were developed by J.T. August and obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Department of Biology, Iowa City, IA, USA).The following primary antibodies against TFEB (#4240, for IF 1:100; for WB 1:1000), pTFEB S122 (#86843, for WB 1:1000), pTFEB S211 (#37681, for WB 1:1000), mTOR (#2983, for WB 1:1000, for IF 1:100), pmTOR S2448 (#5536, for WB 1:1000), pRaptor S792 (#2083, for WB 1:1000), pPRAS40 T246 (#2997, for WB 1:1000), 4E-BP1 (#9452, for WB 1:1000), p4E-BP1 T37/46 (#2855, for WB 1:1000), PP2A C subunit (#2038, for WB 1:1000), Mucolipin-1 (#92176, for WB 1:1000), Pan-Calcineurin A (#2614, for WB 1:1000), Histone H3 (D1H2, #4499, for WB 1:1000), GAPDH (#2188, for WB 1:1000) were supplied by Cell Signaling.Primary antibodies against Neuraminidase-1 (#67032-1-Ig, for WB 1:1000) and Galectin-3 (#60207-1-Ig, WB 1:1000) were from Proteintech (Planegg, Germany). The anti-Galectin-3 antibody (#AF1154-SP, for IF 1:50) were from Bio-techne (Wiesbaden, Germany), and the antibody against Actin was purchased from Sigma Aldrich (Darmstadt, Germany). Secondary antibodies for immunofluorescence staining conjugated with Alexa Fluor-488, -594 or -647 as well as HRP-conjugated secondary antibodies for immunoblots were from Dianova (Hamburg, Germany). The Cell Trace CFSE Cell Proliferation kit was obtained from Thermo Fisher Scientific/ Invitrogen (Darmstadt, Germany). To analyze membrane integrity Fluorescein diacetate (FDA) and Propidium iodide solution were purchased from Cayman Chemical (Ann Arbor, MI, USA).Coxiella burnetii strain Nile Mile Phase II clone 4 (RSA 439), its ∆dotA derivative strain (Schäfer et al., 2020) and a recently created AnkG deficient strain -∆ankG (Cordsmeier et al., 2022) were employed for infection experiments. The C. burnetii wild-type strain expressing GFP (Tn1832) and a strain lacking a functional type IV secretion system ∆dotA GFP (Tn514) for microscopy experiments in fixed samples were generously donated by Dr. Matteo Bonazzi (CNRS, University of Montpellier, France) (Martinez et al., et al., For cell C. burnetii expressing (Schulze-Luehrmann et al., 2024) and for C. burnetii expressing were (Schulze-Luehrmann et al., 2016). were and in for conditions and For was to the et al., for were for to endothelial were purchased from epithelial were by University cell were in Germany), Germany) and fibroblasts and by et al., and in and cells with cells were in Fisher and and et al., Pechstein et al., cells were infected with C. burnetii for The cells were several with supplied with Torin 1 (Cell or ML-SA1 and for and of the and infected cells were as samples for of mTOR and were by a Fisher was by a membrane other proteins were by a Fisher for and via to a membrane or to a membrane Germany). were the specific primary and HRP-conjugated secondary antibodies and by a system Fisher cells were in cells well infection with either wild-type C. an AnkG deficient strain or a deficient was to and to the with the Cell from Cell Leiden, Netherlands) or infected cells, in a well were fixed with in for in the with for which was for the staining in and cells, the were by a with in Thermo Fisher The were in for with the primary antibodies three with and with the secondary antibodies in for three with the were with Fisher staining with TFEB was in for In following first and secondary antibody was was the Carl with a and cell cells infected for 4 with C. burnetii were in a in was in cell and different of the infected cells were for with a was created in to the of TFEB For the cell of an the was was The was and in a The TFEB was for in of a The of to TFEB was to with antibodies and was as and were with the was the for a of the were the cells were infected with C. burnetii expressing 200 for 4 infected cells were in