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Coxiella burnetii as a useful tool to investigate bacteria-friendly host cell compartments
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Coxiella burnetiias a useful tool to investigate bacteria-friendly host cell compartments Julian Pechstein, Jan Schulze-Luehrmann, Anja Lührmann
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Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Wasserturmstraße 3/5, D-91054 Erlangen, Germany
A R T I C L E I N F O
A B S T R AC T
Keywords: Coxiella burnetii Phagolysosome Type IV secretion system Microenvironment
Coxiella burnetii is an obligate intracellular and airborne pathogen which can cause the zoonotic disease Q fever. After inhalation of contaminated aerosols alveolar macrophages are taking up C. burnetii into a phagosome. This phagosome matures to a very large vacuole called the C. burnetii-containing vacuole (CCV). Host endogenous and bacterial driven processes lead to the biogenesis of this unusual compartment, which resembles partially a phagolysosome. However, there are several important differences to the classical phagolysosome, which are crucial for the ability of C. burnetii to replicate intracellularly and depend on a functional type IV secretion system (T4SS). The T4SS delivers effector proteins into the host cell cytoplasm to redirect intracellular processes, leading to the establishment of a microenvironment allowing bacterial replication. This article summarizes the current knowledge of the microenvironment permissive for C. burnetii replication.
1. Introduction Pathogens co-evolve with their hosts, including us humans. As pathogens represent threats to human health, our immune system has established an arsenal of defense mechanisms. Consequently, pathogens have developed virulence factors to prevent clearance by the host immune response. Additional virulence factors modulate host cell pathways to enable bacterial survival and replication. However, the microenvironment present at the site of infection is decisive for the hostpathogen-interaction. This microenvironment affects not only the ability of the host to combat infection, but also the activity of the pathogen. Depending on the conditions existing at the site of infection, the pathogen might escape the unfavorable micro-milieu, induce dormancy, adjust to the conditions at hand or modulate the microenvironment to its own benefit. For obligate intracellular pathogens this microenvironment is represented by the phagosome/vacuole surrounding the pathogen. Importantly, the conditions within the pathogen-containing vacuole can differ, dependent on the host cell type and the immune status of the host. In this review we will discuss our current understanding of a Coxiella burnetii-friendly microenvironment (Fig. 1). 2. The pathogen C. burnetii Coxiella burnetii is a Gram-negative, small (0.4–1.0 μm length,
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0.2–0.4 μm width) and pleomorphic coccoid to rod-shaped bacterium (Maurin and Raoult, 1999). Although C. burnetii is obligate intracellular in nature, a medium has been established that allows axenic culture in the laboratory (Omsland et al., 2011; Omsland et al., 2009). C. burnetii belongs to the gamma subdivision of Proteobacteria (Stein et al., 1993). Its closest relatives are the facultative intracellular human pathogen Legionella pneumophila and the intracellular arthrophod pathogen Rickettsiella grylli (Seshadri et al., 2003). Inside of its eukaryotic host cell, C. burnetii is able to replicate in high numbers in a parasitophorous vacuole (Burton et al., 1978). Depending on the host cell type and the bacterial strain, doubling times in vitro are estimated to range from 15 to 37 h (Boulos et al., 2004). C. burnetii has a biphasic lifestyle (McCaul and Williams, 1981). The small cell variant (SCV) represents the environmentally resistant and metabolically less active form. It allows survival of C. burnetii outside of a host cell (Maurin and Raoult, 1999). Upon infection, SCVs differentiate into the large cell variant (LCV), which is the intracellular and metabolically active form (Coleman et al., 2004). The genome sizes of different C. burnetii strains vary from 1.5 to 2.4 Mb (Willems et al., 1998). In 2003 the C. burnetii isolate Nine Mile I was sequenced. Its genome has a size of about 2 Mb with a GC content of roughly 43% and includes 2134 predicted coding sequences (Seshadri et al., 2003). One virulence determinant of C. burnetii is the lipopolysaccharide (LPS). The virulent form of C. burnetii, which is called phase I, expresses a full-length, smooth LPS with a typical core glycolipid and O-specific polysaccharide chain (Amano and Williams,
Corresponding author. E-mail address: [email protected] (A. Lührmann).
http://dx.doi.org/10.1016/j.ijmm.2017.09.010 Received 29 June 2017; Received in revised form 21 August 2017; Accepted 11 September 2017 1438-4221/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Pechstein, J., International Journal of Medical Microbiology (2017), http://dx.doi.org/10.1016/j.ijmm.2017.09.010
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Fig. 1. Biogenesis and microenvironment of the replicative CCV. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Representative events that favor or hamper CCV biogenesis and C. burnetii replication are shown in this figure. Maturation of the CCV results from fusion events with endosomes, autophagosomes, lysosomes and secretory vesicles. These fusion processes are partially mediated by T4SS effector proteins and provide nutrients for C. burnetii biogenesis. Additionally, C. burnetii possesses several counter measures to prevent host cell mediated killing. The details are described and discussed in the main text. Green boxes represent events in favor of CCV biogenesis and bacterial replication. Red boxes indicate detrimental effects on C. burnetii viability. Dotted lines represent translocation, transport- and fusion processes. Filled lines represent direct interaction or consequences of action. ACP: C. burnetii acid phosphatase; AP2: adapter protein complex 2; ROS: reactive oxygen species; RNS: reactive nitrogen species; iNOS: inducible nitric oxide synthase.
treatment options exist for acute Q fever, these are missing for chronic Q fever. About 1–5% of the infected individuals will develop chronic Q fever years after the primary infection (Kazar, 2005), which is typically characterized by endocarditis and potentially fatal (Maurin and Raoult, 1999). The main source of human Q fever infections are infected ruminants and transmission occurs via aerosols of contaminated dust or of animal excretions like milk, urine, feces and parturient products from infected animals (Angelakis and Raoult, 2010). During parturition more than 109 bacteria per gram of placenta are released into the environment (Maurin and Raoult, 1999). Because infection with less than ten bacteria can result in human disease (Benenson and Tigertt, 1956; Madariaga et al., 2003), birthing products are a tremendous biohazard. Interestingly, the infection routes as well as the clinical symptoms differ between humans and ruminants. Domestic livestock may be infected by tick bites or tick feces (Stoker and Marmion, 1955), while infected ticks do not seem to be involved in human infection. Infected animals perpetuate and maintain the infection within animal populations (Sting et al., 2013) and the infection is mainly unapparent. Interestingly, chronic infections do not result in endocarditis as observed in humans. Instead chronic C. burnetii infection in ruminants is mainly observed in mammary glands and uteri. As a result, chronic infection in ruminants might lead to abortion and reduced reproductive efficiency (Maurin and Raoult, 1999). Similarly, in pregnant woman the illness is likely
1984). After serial passage in cell culture, C. burnetii undergoes a phase variation to phase II (Fiset et al., 1956). C. burnetii phase II is less virulent and characterized by the expression of a truncated, rough LPS (Hoover et al., 2002). It has been suggested that this phase variation is due to several deletion events in the chromosome and intermediate forms have been described (Amano et al., 1987). While phase I is classified as a biosafety level 3 organism, the plaque-isolated C. burnetii Nine Mile phase II clone 4 can be used under biosafety level 2 (Amano and Williams, 1984). Importantly, both phase I and II C. burnetii Nine Mile variants replicate with similar kinetics in human macrophages (Howe et al., 2010). Therefore, C. burnetii Nine Mile phase II is a good and widely used model to investigate the host pathogen interaction. Recently, the sequence of C. burnetii Nine Mile phase II became available (Millar et al., 2017), which will allow to determine which genes are decisive for the pathogenic potential of the bacterium. 3. C. burnetii infection - Q fever Coxiella burnetii is the causative agent of Q fever. With the exception of New Zealand this disease is found worldwide. In humans, Q fever is asymptomatic or might manifest as a mild and self-limited flu-like illness. However, acute Q fever can also develop into an interstitial pneumonia or hepatitis (Maurin and Raoult, 1999). While good 2
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is maintained at pH 4.7–4.8 (Ohkuma and Poole, 1978). The actual pH of the mature CCV that was measured differs between studies. An early study measured the pH of the CCV between 4.5 and 4.9 (Maurin et al., 1992). Our study found the pH of the CCV to be slightly more acidic with values between pH 4.0 and 4.5 (Schulze-Luehrmann et al., 2016). Two recent publications showed that the pH of the CCV is more alkaline at pH 5.7 and pH 5.2, respectively (Mansilla Pareja et al., 2017; Mulye et al., 2017). It can be only speculated about the underlying reasons for these differences. The cell lines used are certainly a factor, as we also obtained more alkaline pH values in CCVs in murine bone marrowderived macrophages (unpublished observation, Fabian Fischer and Anja Lührmann). Additionally, the different studies used different pHsensitive reagents to measure the pH. However, in the last study mentioned, the authors demonstrated that a cholesterol-mediated increased acidification to a pH of 4.4 leads to killing of C. burnetii (Mulye et al., 2017). The reason for the death of C. burnetii under these conditions is unclear, but it is most likely not directly mediated by the pH per se, as the pH optimum for replication is between 4.5 and 4.75 (Vallejo Esquerra et al., 2017). At pH 4.25, C. burnetii still replicates, but no replication is measurable at pH value of 4.0 and 5.0 (Vallejo Esquerra et al., 2017). Similarly, glutamate and glucose metabolism in C. burnetii was observed above pH 3.0 and below pH 5.5 (Hackstadt and Williams, 1981), indicating that C. burnetii not only survives in an acidic environment, but requires it for metabolism and, thus, for replication. Although the maturation of the CCV follows the canonical endocytic pathway, there are several characteristics. Thus, the v-SNAREs Vamp3, Vamp7 and Vamp8 co-localize with the CCV at different time points during infection and are most likely involved in the fusiogenicity of the CCV (Campoy et al., 2013). The SNARE-like protein Syntaxin-17 seems to be important for homotypic fusion of CCVs (McDonough et al., 2013). Components of the retromer complex, which are involved in cargo cycling between the endocytic pathway and the Golgi apparatus, are also required for optimal fusiogenicity of the CCV (McDonough et al., 2013). Additionally, the CCV fuses with autophagosomes. Thus, the nascent CCV is decorated already as early as 5 min after infection with the autophagosomal marker microtubule-associated protein 1A/ 1B-light chain 3 (LC3) (Romano et al., 2007; Schulze-Luehrmann et al., 2016). Interference with the autophagic pathway results in a multivacuolar phenotype, indicating that autophagy is important in the establishment of the large replicative CCV (Newton et al., 2014). The CCV also interacts with the secretory pathway. Thus, the small GTPase rab1b, which regulates transport between the ER and the Golgi apparatus, is recruited to the CCV (Campoy et al., 2011). Overexpression of a GTPase-defective mutant of rab1b results in the inhibition of bacterial replication (Campoy et al., 2011), indicating that the interaction with the secretory pathway is essential for establishing the replicative CCV. Although C. burnetii replicates to high numbers within the CCV, no synchronous egress events seem to exist (Coleman et al., 2004).
asymptomatic, but increases the risk of adverse pregnancy outcomes (Carcopino et al., 2009; Nielsen et al., 2014). 4. Intracellular persistence The fact that chronic Q fever develops years after infection already demonstrates that there must be a latency state, in which the pathogen hides somewhere in the human body. Neither the location of persistent bacteria, nor how latency is regulated is known to date. Additionally, it is unknown whether symptomless latency is the result in all human cases or if only patients with certain preconditions fail to clear the pathogen. In BALB/c and C57BL/6 mice, the pathogen was detected in adipose tissue 4 months post-infection, when the bacteria were undetectable in blood, liver, lung and spleen (Bechah et al., 2014). Thus, adipose tissue might represent a cellular reservoir for C. burnetii persistence. However, in a patient who died due to severe multisystem dysfunction 10 years after a febrile encephalitis-like illness, C. burnetii DNA was detected in the liver, lung, spleen, heart and lymph nodes, but not in the bone marrow (Sukocheva et al., 2016). In contrast, in a cohort of Q fever patients from Australia and England, C. burnetii genomic DNA was detected in 65% and 88%, respectively, of bone marrow aspirates years after primary infection (Marmion et al., 2005). Similarly, the long-term presence of C. burnetii was detected in bone marrow aspirates in patients after primary C. burnetii infection (Harris et al., 2000). Thus, the bone marrow might also represent a place of C. burnetii persistence. More research will be necessary to clearly define the host reservoir(s) and the regulation of bacterial latency. Additionally, the trigger(s) which leads to the development of chronic Q fever have to be determined. 5. Intracellular lifestyle of C. burnetii The main target cells of C. burnetii in humans, as in ruminants, are mononuclear phagocytes (Russell-Lodrigue et al., 2006; Sobotta et al., 2016). However, in pregnant goats, C. burnetii has a strong tropism for placental trophoblasts (Roest et al., 2012). Similarly, C. burnetii antigen was detected within bovine trophoblasts (van Moll et al., 1993). Whether C. burnetii infects trophoblasts in humans during pregnancy is unknown. However, this is most likely, as C. burnetii infects and efficiently replicates in human trophoblast cell lines (Ben Amara et al., 2010) and infected patients might experience adverse pregnancy outcomes (Carcopino et al., 2009; Nielsen et al., 2014). Additionally, in vitro C. burnetii has been shown to infect a large variety of cells, including myeloid cells, epithelial cells, endothelial cells or fibroblasts (Voth and Heinzen, 2007). The uptake of C. burnetii into phagocytes occurs by canonical phagocytosis and is mediated by αvβ3 integrin and complement receptor 3 (CR 3) (Capo et al., 1999). In non-phagocytic cells, αvβ3 and CR 3 are not present on the cell surface. In these cells the C. burnetii outer membrane protein OmpA was shown to be essential for the invasion process (Martinez et al., 2014). Internalization and formation of the C. burnetii-containing vacuole (CCV) involves actin reorganization (Aguilera et al., 2009; Meconi et al., 1998). Cortactin is a major regulator of the actin cytoskeleton and is implicated to be involved in the entry process of C. burnetii into non-phagocytic cells (Rosales et al., 2012). Additionally, C. burnetii uptake into phagocytic and non-phagocytic cells is regulated by GTPases of the Rho family and the RhoA effectors mDia1 and ROCK (Salinas et al., 2015). The CCV matures along the phagocytic pathway. Interaction with early and late endosomes was shown by subsequent acquisition of the small GTPases rab5 and rab7 (Romano et al., 2007). Finally, progression through the pathway and fusion with lysosomes results in the formation of the replicative CCV. The mature CCV is characterized by the presence of a vacuolar proton ATPase, acid phosphatase, 5′-nucleotidase, cathepsin D and LAMPs 1, 2 and 3 (Burton et al., 1971; Ghigo et al., 2002; Heinzen et al., 1996; Howe and Heinzen, 2008; Howe and Mallavia, 2000; Schulze-Luehrmann et al., 2016). The intralysosomal pH in living cells
6. The type IV secretion system and the establishment of the CCV The type IV secretion system (T4SS) is a bacterial multi-protein complex required for translocating bacterial virulence factors, the socalled effector proteins, into the host cell. C. burnetii mutants lacking a functional T4SS are unable to establish a replicative CCV (Beare et al., 2011), because translocation of effector proteins is blocked (Newton et al., 2013). Translocation of T4SS effector proteins requires an acidic pH and, thus, the maturation of the C. burnetii-containing vacuole to a phagolysosomal-like compartment (Newton et al., 2013). T4SS effector proteins are known to modulate host cell pathways and, so far, ∼150 C. burnetii effector proteins have been identified (Larson et al., 2016). Even though the function of most effector proteins is still unknown, some must be involved in the establishment of the replicative CCV (Beare et al., 2011). Indeed, several effector proteins have been identified as being involved in vesicular trafficking. The T4SS effector protein CvpA was found to subvert clathrin-mediated vesicular 3
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CCVs in mouse embryonic fibroblasts (MEFs) lacking the lysosomal membrane proteins LAMP-1 and LAMP-2 (Schulze-Luehrmann et al., 2016). As described above the CCV fuses with several types of vesicles, ranging from endocytic vesicles and autophagosomes to vesicles of the secretory pathway. The reason for this high degree of fusiogenicity is unknown. But, it might enable bacterial access to host cell nutrients (Larson et al., 2016). In vitro, C. burnetii can use glucose as a major carbon substrate for the biogenesis of cell wall components and to generate energy via glycolysis and the TCA cycle (Hauslein et al., 2017; Vallejo Esquerra et al., 2017), despite lacking a classical hexokinase (Omsland and Heinzen, 2011; Seshadri et al., 2003), an enzyme considered to be essential for glucose catabolism. It was suggested that the lack of the hexokinase might be by-passed by enzyme I of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and the histidine phosphocarrier protein (Hauslein et al., 2017). In vivo C. burnetii mainly uses glucose as a carbon source and fatty acids for energy generation, too (Kuley et al., 2015). However, reduction of the glucose level in eukaryotic cells might induce apoptosis (Zhao et al., 2008). Considering that C. burnetii is a slowly replicating pathogen, the usage of host cell glucose might be spread out over a longer time period and thereby might not result in the induction of apoptosis. Alternatively, C. burnetii might actively inhibit apoptosis-induction caused by glucose deprivation. In vitro C. burnetii can also take up and utilize glycerol, which is used mainly in gluconeogenetic reactions to generate peptidoglycan building units (Hauslein et al., 2017; Vallejo Esquerra et al., 2017). During infection, the levels of several amino acid and oligopeptide transporters are up-regulated, indicating that C. burnetii can also metabolize amino acids from the host (Kuley et al., 2015). Indeed, in vitro C. burnetii uses serine for energy generation in the TCA cycle (Hauslein et al., 2017). Importantly, the metabolic requirements vary between isolates, as indicated by the different abilities to grow in the host cell-free medium ACCM-2 (Kersh et al., 2016). C. burnetii seems to adapt instantly to the available resources. This allows C. burnetii to optimize its biosynthesis to fit to its surroundings. The available nutrients, but also the entire microenvironment, might be heterogenous between the different potential host cell types in the various target tissues. For example, trophoblasts in the placenta might provide more nutrients than macrophages in the bone marrow and moreover, the oxygen tension might also be different. C. burnetii is strictly dependent on a reduced oxygen tension and the presence of carbon dioxide (Vallejo Esquerra et al., 2017). During axenic growth C. burnetii requires a combination of low oxygen tension (2.5%) and elevated carbon dioxide levels (5%). Also in vivo C. burnetii colonizes deeper tissues (Russell-Lodrigue et al., 2006) and, thus, microaerobic areas. C. burnetii favors these conditions as susceptibility to oxidants may be a weak point of C. burnetii (Sandoz et al., 2014). The CCV is characterized by a high abundance of cholesterol in its membrane, which is unusual for lysosomal membranes (Kohler and Roy, 2015). C. burnetii seems not to synthesize cholesterol, but instead recruits it from the host cell (Samanta et al., 2017). Whether the host cell cholesterol-binding protein ORP1L is involved in this process is unknown. However, ORP1L, which is recruited to the CCV in a T4SS dependent manner, is required for optimal CCV expansion (Justis et al., 2017). The role of cholesterol in host pathogen interaction is only beginning to be understood. Inhibition of cholesterol synthesis by pharmacological treatment prevents C. burnetii entry and replication (Howe and Heinzen, 2006). However, C. burnetii replication is not affected in cells lacking cholesterol, while entry was significantly reduced in these cells (Gilk et al., 2013). Addition of cholesterol to C. burnetii-infected cholesterol-free cells leads to increased cholesterol in the CCV and altered CCV membrane dynamics, which leads to C. burnetii lysis (Mulye et al., 2017). Cholesterol does not influence growth of C. burnetii in axenic culture (Czyz et al., 2014; Mulye et al., 2017), indicating that cholesterol per se is not toxic for C. burnetii, but instead the cholesterolmediated changes in CCV dynamics. Interestingly, the alarmone (p)ppGpp, an unusual guanosine
trafficking (Larson et al., 2013). Clathrin is also targeted by the effector protein Cig57 which co-opts clathrin-mediated trafficking via interaction with the FER/CIP 4 homology only protein 2 (FCHO2), an accessory protein of clathrin coated pits, to facilitate the biogenesis of the fusiogenic CCV (Latomanski et al., 2016). The GTPase activity of RhoA is stimulated by the effector protein CirA to promote biogenesis of the bacterial vacuole (Weber et al., 2016). Recently, the T4SS effector protein Cig2/CvpB was identified to be important for the interaction of the CCV with autophagosomes. Cig2/CvpB binds to phosphatidylinositol 3 phosphate (PI3P) and phosphatidylserine (PS) on the CCV to mediate fusion with autophagosomes (Kohler et al., 2016; Martinez et al., 2016). Other effector proteins contribute indirectly to the establishment of the replicative CCV by ensuring host cell survival. Thus, the T4SS effector protein IcaA inhibits host cell pyroptosis (Cunha et al., 2015) and the effector proteins AnkG, CaeA and CaeB prevent host cell apoptosis (Berens et al., 2015; Bisle et al., 2016; Eckart et al., 2014; Friedrich et al., 2017; Klingenbeck et al., 2013; Lührmann et al., 2010; Schäfer et al., 2017). However, we can expect to identify additional T4SS effector proteins that regulate interactions of the CCV with the endocytic and secretory pathways. These interactions will mediate the homo- and heterotypic fusion events that are required for the formation of a single large CCV, which fills the majority of the host cytoplasm. 7. The CCV The CCV is a very unique compartment, because it has phagolysosomal properties, which normally lead to bacterial killing. How C. burnetii withstands this harsh environment is not fully understood. Cardiolipin production, which is highly induced during infection, might enable the pathogen to survive in this acidic compartment (Kuley et al., 2015). Additionally, roughly 46% of the C. burnetii proteins are basic (Seshadri et al., 2003) and, thus, may mediate resistance to the acidic environment within the CCV. The maturation of the CCV is delayed, which might provide enough time for the bacterium to adapt to the harsh environment (Beron et al., 2002; Gutierrez et al., 2005; Romano et al., 2007). How this delay is induced and regulated is unknown. It was suggested that it might be caused by the interaction with the autophagic pathway (Romano et al., 2007) or that it might be regulated by the activity of LAMP-1 and LAMP-2 (Schulze-Luehrmann et al., 2016). If this delay has a role in C. burnetii survival and replication is uncertain and it might not even be required for optimal replication (Schulze-Luehrmann et al., 2016). Despite this delayed maturation, the CCV eventually matures to a phagolysosomal-like compartment. This is important, because the acidification of the CCV is necessary for triggering the translocation of T4SS effector proteins, which is essential for bacterial survival and replication (Newton et al., 2013). The pathogen has adapted to this harsh environment, in which it is not only exposed to an acidic pH, but also to reactive oxygen and nitrogen species (Brennan et al., 2004). These can attack different components of the DNA resulting in DNA lesions (Jena, 2012), which might lead to bacterial death. However, C. burnetii encodes genes to combat reactive species. Thus, C. burnetii suppresses reactive oxygen species production by secreting an acid phosphatase (Hill and Samuel, 2011). Furthermore, C. burnetii encodes for two superoxide dismutases and two alkyl hydroperoxide reductases, which are most likely involved in detoxifying superoxide radicals and hydrogen peroxides (Akporiaye and Baca, 1983; Brennan et al., 2004; Heinzen et al., 1990). Additionally, genes that initiate and mediate DNA repair were identified and were shown to be functionally active (Mertens et al., 2008). The CCV is characterized by its tremendous size. At 48 h post-infection, the vacuole can exceed 10 μm in diameter (Larson et al., 2013; Schulze-Luehrmann et al., 2016). The formation of this huge vacuole precedes bacterial replication (Zamboni et al., 2003). This demonstrates that C. burnetii activates multiple fusion events before replication of the pathogen starts. However, this does not seem to be a requisite for bacterial replication, as C. burnetii establishes very small, but replicative 4
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Friedrich, A., Pechstein, J., Berens, C., Lührmann, A., 2017. Modulation of host cell apoptotic pathways by intracellular pathogens. Curr. Opin. Microbiol. 35, 88–99. Gaca, A.O., Colomer-Winter, C., Lemos, J.A., 2015. Many means to a common end: the intricacies of (p)ppGpp metabolism and its control of bacterial homeostasis. J. Bacteriol. 197, 1146–1156. Ghigo, E., Capo, C., Tung, C.H., Raoult, D., Gorvel, J.P., Mege, J.L., 2002. Coxiella burnetii survival in THP-1 monocytes involves the impairment of phagosome maturation: IFNgamma mediates its restoration and bacterial killing. J. Immunol. 169, 4488–4495. Gilk, S.D., Cockrell, D.C., Luterbach, C., Hansen, B., Knodler, L.A., Ibarra, J.A., SteeleMortimer, O., Heinzen, R.A., 2013. Bacterial colonization of host cells in the absence of cholesterol. PLoS Pathog. 9, e1003107. Gutierrez, M.G., Vazquez, C.L., Munafo, D.B., Zoppino, F.C., Beron, W., Rabinovitch, M., Colombo, M.I., 2005. 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nucleotide, accumulates in C. burnetii during infection (Kuley et al., 2015). These hyperphosphorylated guanosine derivatives induce largescale transcriptional alterations and directly inhibit the activity of several enzymes resulting in the induction of the stringent response (Kanjee et al., 2012). The stringent response leads towards adaption to a semi-dormant state, but might also be involved in persister cell formation (Gaca et al., 2015). What triggers this accumulation and if it results in dormancy of C. burnetii is unknown. 8. Conclusions and perspective C. burnetii is an obligate intracellular pathogen, which requires a finely balanced interaction with the host cell. In nature, C. burnetii only replicates within the host cell. Although C. burnetii replicates to high numbers within the host cell, no obvious influence on host cell viability is detectable. These characteristics make C. burnetii a perfect model to study host-pathogen interactions. C. burnetii remodels its host cellular compartment, the CCV, for its own well-being without grossly disturbing the host cell. For intracellular survival and replication C. burnetii strictly depends on an acidic pH, reduced oxygen tension and carbon dioxide (Vallejo Esquerra et al., 2017). The CCV is highly fusiogenic and fuses with vesicles of the endocytic, secretory and autophagic pathways (Kohler and Roy, 2015), which might provide the bacteria directly or indirectly with the required nutrients. C. burnetii expresses transporters for the uptake of organic nutrients from the host (Kuley et al., 2015). In case of specific nutrient limitations, C. burnetii alters the expression of genes involved in nutrient transport and catabolism, indicating that C. burnetii can adjust to the micro-environment. Under unfavorable conditions, C. burnetii shows a stringent response (Kuley et al., 2015), which might enable the pathogen to survive intracellularly for extended periods. A subsequent change in the microenvironment might enable C. burnetii to replicate again and cause chronic Q fever years after the primary infection. The influence of the microenvironment on pathogenesis has only begun to be understood. It will be essential to determine how the microenvironment influences bacterial transcription and translation, as this affects the ability of the pathogen to manipulate the host cell. This will open new routes to combat infection or prevent bacterial spreading. In detail, it will be essential to compare the microenvironments in the different host cell types and species. Thus, it will be important to identify differences in the composition of the CCV in e.g. human macrophages compared to ovine trophoblasts. This might help to understand how the available nutrients influence bacterial activity. Furthermore, it will be important to determine the reservoir(s) of C. burnetii latency/dormancy and to identify what drives this process. This will be important also in the context of the infection in small ruminants, as these are the prime sources of human infections. Understanding this process will enable us to develop strategies to curb bacterial spreading and to prevent chronic infections. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Priority Programme SPP1580 and the Collaborative Research InitiativesCRC796 - B08 and CRC1181 - A06 to AL. We thank Dr. Christian Berens for critical reading of the manuscript. The authors wish to apologize to all colleagues whose work was not referenced in this article due to space limitations and/or only cited indirectly by referring to previous reviews. References Aguilera, M., Salinas, R., Rosales, E., Carminati, S., Colombo, M.I., Beron, W., 2009. Actin dynamics and Rho GTPases regulate the size and formation of parasitophorous vacuoles containing Coxiella burnetii. Infect. Immun. 77, 4609–4620. Akporiaye, E.T., Baca, O.G., 1983. Superoxide anion production and superoxide
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Coxiella burnetiias a useful tool to investigate bacteria-friendly host cell compartments Julian Pechstein, Jan Schulze-Luehrmann, Anja Lührmann
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Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Wasserturmstraße 3/5, D-91054 Erlangen, Germany
A R T I C L E I N F O
A B S T R AC T
Keywords: Coxiella burnetii Phagolysosome Type IV secretion system Microenvironment
Coxiella burnetii is an obligate intracellular and airborne pathogen which can cause the zoonotic disease Q fever. After inhalation of contaminated aerosols alveolar macrophages are taking up C. burnetii into a phagosome. This phagosome matures to a very large vacuole called the C. burnetii-containing vacuole (CCV). Host endogenous and bacterial driven processes lead to the biogenesis of this unusual compartment, which resembles partially a phagolysosome. However, there are several important differences to the classical phagolysosome, which are crucial for the ability of C. burnetii to replicate intracellularly and depend on a functional type IV secretion system (T4SS). The T4SS delivers effector proteins into the host cell cytoplasm to redirect intracellular processes, leading to the establishment of a microenvironment allowing bacterial replication. This article summarizes the current knowledge of the microenvironment permissive for C. burnetii replication.
1. Introduction Pathogens co-evolve with their hosts, including us humans. As pathogens represent threats to human health, our immune system has established an arsenal of defense mechanisms. Consequently, pathogens have developed virulence factors to prevent clearance by the host immune response. Additional virulence factors modulate host cell pathways to enable bacterial survival and replication. However, the microenvironment present at the site of infection is decisive for the hostpathogen-interaction. This microenvironment affects not only the ability of the host to combat infection, but also the activity of the pathogen. Depending on the conditions existing at the site of infection, the pathogen might escape the unfavorable micro-milieu, induce dormancy, adjust to the conditions at hand or modulate the microenvironment to its own benefit. For obligate intracellular pathogens this microenvironment is represented by the phagosome/vacuole surrounding the pathogen. Importantly, the conditions within the pathogen-containing vacuole can differ, dependent on the host cell type and the immune status of the host. In this review we will discuss our current understanding of a Coxiella burnetii-friendly microenvironment (Fig. 1). 2. The pathogen C. burnetii Coxiella burnetii is a Gram-negative, small (0.4–1.0 μm length,
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0.2–0.4 μm width) and pleomorphic coccoid to rod-shaped bacterium (Maurin and Raoult, 1999). Although C. burnetii is obligate intracellular in nature, a medium has been established that allows axenic culture in the laboratory (Omsland et al., 2011; Omsland et al., 2009). C. burnetii belongs to the gamma subdivision of Proteobacteria (Stein et al., 1993). Its closest relatives are the facultative intracellular human pathogen Legionella pneumophila and the intracellular arthrophod pathogen Rickettsiella grylli (Seshadri et al., 2003). Inside of its eukaryotic host cell, C. burnetii is able to replicate in high numbers in a parasitophorous vacuole (Burton et al., 1978). Depending on the host cell type and the bacterial strain, doubling times in vitro are estimated to range from 15 to 37 h (Boulos et al., 2004). C. burnetii has a biphasic lifestyle (McCaul and Williams, 1981). The small cell variant (SCV) represents the environmentally resistant and metabolically less active form. It allows survival of C. burnetii outside of a host cell (Maurin and Raoult, 1999). Upon infection, SCVs differentiate into the large cell variant (LCV), which is the intracellular and metabolically active form (Coleman et al., 2004). The genome sizes of different C. burnetii strains vary from 1.5 to 2.4 Mb (Willems et al., 1998). In 2003 the C. burnetii isolate Nine Mile I was sequenced. Its genome has a size of about 2 Mb with a GC content of roughly 43% and includes 2134 predicted coding sequences (Seshadri et al., 2003). One virulence determinant of C. burnetii is the lipopolysaccharide (LPS). The virulent form of C. burnetii, which is called phase I, expresses a full-length, smooth LPS with a typical core glycolipid and O-specific polysaccharide chain (Amano and Williams,
Corresponding author. E-mail address: [email protected] (A. Lührmann).
http://dx.doi.org/10.1016/j.ijmm.2017.09.010 Received 29 June 2017; Received in revised form 21 August 2017; Accepted 11 September 2017 1438-4221/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Pechstein, J., International Journal of Medical Microbiology (2017), http://dx.doi.org/10.1016/j.ijmm.2017.09.010
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Fig. 1. Biogenesis and microenvironment of the replicative CCV. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Representative events that favor or hamper CCV biogenesis and C. burnetii replication are shown in this figure. Maturation of the CCV results from fusion events with endosomes, autophagosomes, lysosomes and secretory vesicles. These fusion processes are partially mediated by T4SS effector proteins and provide nutrients for C. burnetii biogenesis. Additionally, C. burnetii possesses several counter measures to prevent host cell mediated killing. The details are described and discussed in the main text. Green boxes represent events in favor of CCV biogenesis and bacterial replication. Red boxes indicate detrimental effects on C. burnetii viability. Dotted lines represent translocation, transport- and fusion processes. Filled lines represent direct interaction or consequences of action. ACP: C. burnetii acid phosphatase; AP2: adapter protein complex 2; ROS: reactive oxygen species; RNS: reactive nitrogen species; iNOS: inducible nitric oxide synthase.
treatment options exist for acute Q fever, these are missing for chronic Q fever. About 1–5% of the infected individuals will develop chronic Q fever years after the primary infection (Kazar, 2005), which is typically characterized by endocarditis and potentially fatal (Maurin and Raoult, 1999). The main source of human Q fever infections are infected ruminants and transmission occurs via aerosols of contaminated dust or of animal excretions like milk, urine, feces and parturient products from infected animals (Angelakis and Raoult, 2010). During parturition more than 109 bacteria per gram of placenta are released into the environment (Maurin and Raoult, 1999). Because infection with less than ten bacteria can result in human disease (Benenson and Tigertt, 1956; Madariaga et al., 2003), birthing products are a tremendous biohazard. Interestingly, the infection routes as well as the clinical symptoms differ between humans and ruminants. Domestic livestock may be infected by tick bites or tick feces (Stoker and Marmion, 1955), while infected ticks do not seem to be involved in human infection. Infected animals perpetuate and maintain the infection within animal populations (Sting et al., 2013) and the infection is mainly unapparent. Interestingly, chronic infections do not result in endocarditis as observed in humans. Instead chronic C. burnetii infection in ruminants is mainly observed in mammary glands and uteri. As a result, chronic infection in ruminants might lead to abortion and reduced reproductive efficiency (Maurin and Raoult, 1999). Similarly, in pregnant woman the illness is likely
1984). After serial passage in cell culture, C. burnetii undergoes a phase variation to phase II (Fiset et al., 1956). C. burnetii phase II is less virulent and characterized by the expression of a truncated, rough LPS (Hoover et al., 2002). It has been suggested that this phase variation is due to several deletion events in the chromosome and intermediate forms have been described (Amano et al., 1987). While phase I is classified as a biosafety level 3 organism, the plaque-isolated C. burnetii Nine Mile phase II clone 4 can be used under biosafety level 2 (Amano and Williams, 1984). Importantly, both phase I and II C. burnetii Nine Mile variants replicate with similar kinetics in human macrophages (Howe et al., 2010). Therefore, C. burnetii Nine Mile phase II is a good and widely used model to investigate the host pathogen interaction. Recently, the sequence of C. burnetii Nine Mile phase II became available (Millar et al., 2017), which will allow to determine which genes are decisive for the pathogenic potential of the bacterium. 3. C. burnetii infection - Q fever Coxiella burnetii is the causative agent of Q fever. With the exception of New Zealand this disease is found worldwide. In humans, Q fever is asymptomatic or might manifest as a mild and self-limited flu-like illness. However, acute Q fever can also develop into an interstitial pneumonia or hepatitis (Maurin and Raoult, 1999). While good 2
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is maintained at pH 4.7–4.8 (Ohkuma and Poole, 1978). The actual pH of the mature CCV that was measured differs between studies. An early study measured the pH of the CCV between 4.5 and 4.9 (Maurin et al., 1992). Our study found the pH of the CCV to be slightly more acidic with values between pH 4.0 and 4.5 (Schulze-Luehrmann et al., 2016). Two recent publications showed that the pH of the CCV is more alkaline at pH 5.7 and pH 5.2, respectively (Mansilla Pareja et al., 2017; Mulye et al., 2017). It can be only speculated about the underlying reasons for these differences. The cell lines used are certainly a factor, as we also obtained more alkaline pH values in CCVs in murine bone marrowderived macrophages (unpublished observation, Fabian Fischer and Anja Lührmann). Additionally, the different studies used different pHsensitive reagents to measure the pH. However, in the last study mentioned, the authors demonstrated that a cholesterol-mediated increased acidification to a pH of 4.4 leads to killing of C. burnetii (Mulye et al., 2017). The reason for the death of C. burnetii under these conditions is unclear, but it is most likely not directly mediated by the pH per se, as the pH optimum for replication is between 4.5 and 4.75 (Vallejo Esquerra et al., 2017). At pH 4.25, C. burnetii still replicates, but no replication is measurable at pH value of 4.0 and 5.0 (Vallejo Esquerra et al., 2017). Similarly, glutamate and glucose metabolism in C. burnetii was observed above pH 3.0 and below pH 5.5 (Hackstadt and Williams, 1981), indicating that C. burnetii not only survives in an acidic environment, but requires it for metabolism and, thus, for replication. Although the maturation of the CCV follows the canonical endocytic pathway, there are several characteristics. Thus, the v-SNAREs Vamp3, Vamp7 and Vamp8 co-localize with the CCV at different time points during infection and are most likely involved in the fusiogenicity of the CCV (Campoy et al., 2013). The SNARE-like protein Syntaxin-17 seems to be important for homotypic fusion of CCVs (McDonough et al., 2013). Components of the retromer complex, which are involved in cargo cycling between the endocytic pathway and the Golgi apparatus, are also required for optimal fusiogenicity of the CCV (McDonough et al., 2013). Additionally, the CCV fuses with autophagosomes. Thus, the nascent CCV is decorated already as early as 5 min after infection with the autophagosomal marker microtubule-associated protein 1A/ 1B-light chain 3 (LC3) (Romano et al., 2007; Schulze-Luehrmann et al., 2016). Interference with the autophagic pathway results in a multivacuolar phenotype, indicating that autophagy is important in the establishment of the large replicative CCV (Newton et al., 2014). The CCV also interacts with the secretory pathway. Thus, the small GTPase rab1b, which regulates transport between the ER and the Golgi apparatus, is recruited to the CCV (Campoy et al., 2011). Overexpression of a GTPase-defective mutant of rab1b results in the inhibition of bacterial replication (Campoy et al., 2011), indicating that the interaction with the secretory pathway is essential for establishing the replicative CCV. Although C. burnetii replicates to high numbers within the CCV, no synchronous egress events seem to exist (Coleman et al., 2004).
asymptomatic, but increases the risk of adverse pregnancy outcomes (Carcopino et al., 2009; Nielsen et al., 2014). 4. Intracellular persistence The fact that chronic Q fever develops years after infection already demonstrates that there must be a latency state, in which the pathogen hides somewhere in the human body. Neither the location of persistent bacteria, nor how latency is regulated is known to date. Additionally, it is unknown whether symptomless latency is the result in all human cases or if only patients with certain preconditions fail to clear the pathogen. In BALB/c and C57BL/6 mice, the pathogen was detected in adipose tissue 4 months post-infection, when the bacteria were undetectable in blood, liver, lung and spleen (Bechah et al., 2014). Thus, adipose tissue might represent a cellular reservoir for C. burnetii persistence. However, in a patient who died due to severe multisystem dysfunction 10 years after a febrile encephalitis-like illness, C. burnetii DNA was detected in the liver, lung, spleen, heart and lymph nodes, but not in the bone marrow (Sukocheva et al., 2016). In contrast, in a cohort of Q fever patients from Australia and England, C. burnetii genomic DNA was detected in 65% and 88%, respectively, of bone marrow aspirates years after primary infection (Marmion et al., 2005). Similarly, the long-term presence of C. burnetii was detected in bone marrow aspirates in patients after primary C. burnetii infection (Harris et al., 2000). Thus, the bone marrow might also represent a place of C. burnetii persistence. More research will be necessary to clearly define the host reservoir(s) and the regulation of bacterial latency. Additionally, the trigger(s) which leads to the development of chronic Q fever have to be determined. 5. Intracellular lifestyle of C. burnetii The main target cells of C. burnetii in humans, as in ruminants, are mononuclear phagocytes (Russell-Lodrigue et al., 2006; Sobotta et al., 2016). However, in pregnant goats, C. burnetii has a strong tropism for placental trophoblasts (Roest et al., 2012). Similarly, C. burnetii antigen was detected within bovine trophoblasts (van Moll et al., 1993). Whether C. burnetii infects trophoblasts in humans during pregnancy is unknown. However, this is most likely, as C. burnetii infects and efficiently replicates in human trophoblast cell lines (Ben Amara et al., 2010) and infected patients might experience adverse pregnancy outcomes (Carcopino et al., 2009; Nielsen et al., 2014). Additionally, in vitro C. burnetii has been shown to infect a large variety of cells, including myeloid cells, epithelial cells, endothelial cells or fibroblasts (Voth and Heinzen, 2007). The uptake of C. burnetii into phagocytes occurs by canonical phagocytosis and is mediated by αvβ3 integrin and complement receptor 3 (CR 3) (Capo et al., 1999). In non-phagocytic cells, αvβ3 and CR 3 are not present on the cell surface. In these cells the C. burnetii outer membrane protein OmpA was shown to be essential for the invasion process (Martinez et al., 2014). Internalization and formation of the C. burnetii-containing vacuole (CCV) involves actin reorganization (Aguilera et al., 2009; Meconi et al., 1998). Cortactin is a major regulator of the actin cytoskeleton and is implicated to be involved in the entry process of C. burnetii into non-phagocytic cells (Rosales et al., 2012). Additionally, C. burnetii uptake into phagocytic and non-phagocytic cells is regulated by GTPases of the Rho family and the RhoA effectors mDia1 and ROCK (Salinas et al., 2015). The CCV matures along the phagocytic pathway. Interaction with early and late endosomes was shown by subsequent acquisition of the small GTPases rab5 and rab7 (Romano et al., 2007). Finally, progression through the pathway and fusion with lysosomes results in the formation of the replicative CCV. The mature CCV is characterized by the presence of a vacuolar proton ATPase, acid phosphatase, 5′-nucleotidase, cathepsin D and LAMPs 1, 2 and 3 (Burton et al., 1971; Ghigo et al., 2002; Heinzen et al., 1996; Howe and Heinzen, 2008; Howe and Mallavia, 2000; Schulze-Luehrmann et al., 2016). The intralysosomal pH in living cells
6. The type IV secretion system and the establishment of the CCV The type IV secretion system (T4SS) is a bacterial multi-protein complex required for translocating bacterial virulence factors, the socalled effector proteins, into the host cell. C. burnetii mutants lacking a functional T4SS are unable to establish a replicative CCV (Beare et al., 2011), because translocation of effector proteins is blocked (Newton et al., 2013). Translocation of T4SS effector proteins requires an acidic pH and, thus, the maturation of the C. burnetii-containing vacuole to a phagolysosomal-like compartment (Newton et al., 2013). T4SS effector proteins are known to modulate host cell pathways and, so far, ∼150 C. burnetii effector proteins have been identified (Larson et al., 2016). Even though the function of most effector proteins is still unknown, some must be involved in the establishment of the replicative CCV (Beare et al., 2011). Indeed, several effector proteins have been identified as being involved in vesicular trafficking. The T4SS effector protein CvpA was found to subvert clathrin-mediated vesicular 3
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CCVs in mouse embryonic fibroblasts (MEFs) lacking the lysosomal membrane proteins LAMP-1 and LAMP-2 (Schulze-Luehrmann et al., 2016). As described above the CCV fuses with several types of vesicles, ranging from endocytic vesicles and autophagosomes to vesicles of the secretory pathway. The reason for this high degree of fusiogenicity is unknown. But, it might enable bacterial access to host cell nutrients (Larson et al., 2016). In vitro, C. burnetii can use glucose as a major carbon substrate for the biogenesis of cell wall components and to generate energy via glycolysis and the TCA cycle (Hauslein et al., 2017; Vallejo Esquerra et al., 2017), despite lacking a classical hexokinase (Omsland and Heinzen, 2011; Seshadri et al., 2003), an enzyme considered to be essential for glucose catabolism. It was suggested that the lack of the hexokinase might be by-passed by enzyme I of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and the histidine phosphocarrier protein (Hauslein et al., 2017). In vivo C. burnetii mainly uses glucose as a carbon source and fatty acids for energy generation, too (Kuley et al., 2015). However, reduction of the glucose level in eukaryotic cells might induce apoptosis (Zhao et al., 2008). Considering that C. burnetii is a slowly replicating pathogen, the usage of host cell glucose might be spread out over a longer time period and thereby might not result in the induction of apoptosis. Alternatively, C. burnetii might actively inhibit apoptosis-induction caused by glucose deprivation. In vitro C. burnetii can also take up and utilize glycerol, which is used mainly in gluconeogenetic reactions to generate peptidoglycan building units (Hauslein et al., 2017; Vallejo Esquerra et al., 2017). During infection, the levels of several amino acid and oligopeptide transporters are up-regulated, indicating that C. burnetii can also metabolize amino acids from the host (Kuley et al., 2015). Indeed, in vitro C. burnetii uses serine for energy generation in the TCA cycle (Hauslein et al., 2017). Importantly, the metabolic requirements vary between isolates, as indicated by the different abilities to grow in the host cell-free medium ACCM-2 (Kersh et al., 2016). C. burnetii seems to adapt instantly to the available resources. This allows C. burnetii to optimize its biosynthesis to fit to its surroundings. The available nutrients, but also the entire microenvironment, might be heterogenous between the different potential host cell types in the various target tissues. For example, trophoblasts in the placenta might provide more nutrients than macrophages in the bone marrow and moreover, the oxygen tension might also be different. C. burnetii is strictly dependent on a reduced oxygen tension and the presence of carbon dioxide (Vallejo Esquerra et al., 2017). During axenic growth C. burnetii requires a combination of low oxygen tension (2.5%) and elevated carbon dioxide levels (5%). Also in vivo C. burnetii colonizes deeper tissues (Russell-Lodrigue et al., 2006) and, thus, microaerobic areas. C. burnetii favors these conditions as susceptibility to oxidants may be a weak point of C. burnetii (Sandoz et al., 2014). The CCV is characterized by a high abundance of cholesterol in its membrane, which is unusual for lysosomal membranes (Kohler and Roy, 2015). C. burnetii seems not to synthesize cholesterol, but instead recruits it from the host cell (Samanta et al., 2017). Whether the host cell cholesterol-binding protein ORP1L is involved in this process is unknown. However, ORP1L, which is recruited to the CCV in a T4SS dependent manner, is required for optimal CCV expansion (Justis et al., 2017). The role of cholesterol in host pathogen interaction is only beginning to be understood. Inhibition of cholesterol synthesis by pharmacological treatment prevents C. burnetii entry and replication (Howe and Heinzen, 2006). However, C. burnetii replication is not affected in cells lacking cholesterol, while entry was significantly reduced in these cells (Gilk et al., 2013). Addition of cholesterol to C. burnetii-infected cholesterol-free cells leads to increased cholesterol in the CCV and altered CCV membrane dynamics, which leads to C. burnetii lysis (Mulye et al., 2017). Cholesterol does not influence growth of C. burnetii in axenic culture (Czyz et al., 2014; Mulye et al., 2017), indicating that cholesterol per se is not toxic for C. burnetii, but instead the cholesterolmediated changes in CCV dynamics. Interestingly, the alarmone (p)ppGpp, an unusual guanosine
trafficking (Larson et al., 2013). Clathrin is also targeted by the effector protein Cig57 which co-opts clathrin-mediated trafficking via interaction with the FER/CIP 4 homology only protein 2 (FCHO2), an accessory protein of clathrin coated pits, to facilitate the biogenesis of the fusiogenic CCV (Latomanski et al., 2016). The GTPase activity of RhoA is stimulated by the effector protein CirA to promote biogenesis of the bacterial vacuole (Weber et al., 2016). Recently, the T4SS effector protein Cig2/CvpB was identified to be important for the interaction of the CCV with autophagosomes. Cig2/CvpB binds to phosphatidylinositol 3 phosphate (PI3P) and phosphatidylserine (PS) on the CCV to mediate fusion with autophagosomes (Kohler et al., 2016; Martinez et al., 2016). Other effector proteins contribute indirectly to the establishment of the replicative CCV by ensuring host cell survival. Thus, the T4SS effector protein IcaA inhibits host cell pyroptosis (Cunha et al., 2015) and the effector proteins AnkG, CaeA and CaeB prevent host cell apoptosis (Berens et al., 2015; Bisle et al., 2016; Eckart et al., 2014; Friedrich et al., 2017; Klingenbeck et al., 2013; Lührmann et al., 2010; Schäfer et al., 2017). However, we can expect to identify additional T4SS effector proteins that regulate interactions of the CCV with the endocytic and secretory pathways. These interactions will mediate the homo- and heterotypic fusion events that are required for the formation of a single large CCV, which fills the majority of the host cytoplasm. 7. The CCV The CCV is a very unique compartment, because it has phagolysosomal properties, which normally lead to bacterial killing. How C. burnetii withstands this harsh environment is not fully understood. Cardiolipin production, which is highly induced during infection, might enable the pathogen to survive in this acidic compartment (Kuley et al., 2015). Additionally, roughly 46% of the C. burnetii proteins are basic (Seshadri et al., 2003) and, thus, may mediate resistance to the acidic environment within the CCV. The maturation of the CCV is delayed, which might provide enough time for the bacterium to adapt to the harsh environment (Beron et al., 2002; Gutierrez et al., 2005; Romano et al., 2007). How this delay is induced and regulated is unknown. It was suggested that it might be caused by the interaction with the autophagic pathway (Romano et al., 2007) or that it might be regulated by the activity of LAMP-1 and LAMP-2 (Schulze-Luehrmann et al., 2016). If this delay has a role in C. burnetii survival and replication is uncertain and it might not even be required for optimal replication (Schulze-Luehrmann et al., 2016). Despite this delayed maturation, the CCV eventually matures to a phagolysosomal-like compartment. This is important, because the acidification of the CCV is necessary for triggering the translocation of T4SS effector proteins, which is essential for bacterial survival and replication (Newton et al., 2013). The pathogen has adapted to this harsh environment, in which it is not only exposed to an acidic pH, but also to reactive oxygen and nitrogen species (Brennan et al., 2004). These can attack different components of the DNA resulting in DNA lesions (Jena, 2012), which might lead to bacterial death. However, C. burnetii encodes genes to combat reactive species. Thus, C. burnetii suppresses reactive oxygen species production by secreting an acid phosphatase (Hill and Samuel, 2011). Furthermore, C. burnetii encodes for two superoxide dismutases and two alkyl hydroperoxide reductases, which are most likely involved in detoxifying superoxide radicals and hydrogen peroxides (Akporiaye and Baca, 1983; Brennan et al., 2004; Heinzen et al., 1990). Additionally, genes that initiate and mediate DNA repair were identified and were shown to be functionally active (Mertens et al., 2008). The CCV is characterized by its tremendous size. At 48 h post-infection, the vacuole can exceed 10 μm in diameter (Larson et al., 2013; Schulze-Luehrmann et al., 2016). The formation of this huge vacuole precedes bacterial replication (Zamboni et al., 2003). This demonstrates that C. burnetii activates multiple fusion events before replication of the pathogen starts. However, this does not seem to be a requisite for bacterial replication, as C. burnetii establishes very small, but replicative 4
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nucleotide, accumulates in C. burnetii during infection (Kuley et al., 2015). These hyperphosphorylated guanosine derivatives induce largescale transcriptional alterations and directly inhibit the activity of several enzymes resulting in the induction of the stringent response (Kanjee et al., 2012). The stringent response leads towards adaption to a semi-dormant state, but might also be involved in persister cell formation (Gaca et al., 2015). What triggers this accumulation and if it results in dormancy of C. burnetii is unknown. 8. Conclusions and perspective C. burnetii is an obligate intracellular pathogen, which requires a finely balanced interaction with the host cell. In nature, C. burnetii only replicates within the host cell. Although C. burnetii replicates to high numbers within the host cell, no obvious influence on host cell viability is detectable. These characteristics make C. burnetii a perfect model to study host-pathogen interactions. C. burnetii remodels its host cellular compartment, the CCV, for its own well-being without grossly disturbing the host cell. For intracellular survival and replication C. burnetii strictly depends on an acidic pH, reduced oxygen tension and carbon dioxide (Vallejo Esquerra et al., 2017). The CCV is highly fusiogenic and fuses with vesicles of the endocytic, secretory and autophagic pathways (Kohler and Roy, 2015), which might provide the bacteria directly or indirectly with the required nutrients. C. burnetii expresses transporters for the uptake of organic nutrients from the host (Kuley et al., 2015). In case of specific nutrient limitations, C. burnetii alters the expression of genes involved in nutrient transport and catabolism, indicating that C. burnetii can adjust to the micro-environment. Under unfavorable conditions, C. burnetii shows a stringent response (Kuley et al., 2015), which might enable the pathogen to survive intracellularly for extended periods. A subsequent change in the microenvironment might enable C. burnetii to replicate again and cause chronic Q fever years after the primary infection. The influence of the microenvironment on pathogenesis has only begun to be understood. It will be essential to determine how the microenvironment influences bacterial transcription and translation, as this affects the ability of the pathogen to manipulate the host cell. This will open new routes to combat infection or prevent bacterial spreading. In detail, it will be essential to compare the microenvironments in the different host cell types and species. Thus, it will be important to identify differences in the composition of the CCV in e.g. human macrophages compared to ovine trophoblasts. This might help to understand how the available nutrients influence bacterial activity. Furthermore, it will be important to determine the reservoir(s) of C. burnetii latency/dormancy and to identify what drives this process. This will be important also in the context of the infection in small ruminants, as these are the prime sources of human infections. Understanding this process will enable us to develop strategies to curb bacterial spreading and to prevent chronic infections. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Priority Programme SPP1580 and the Collaborative Research InitiativesCRC796 - B08 and CRC1181 - A06 to AL. We thank Dr. Christian Berens for critical reading of the manuscript. The authors wish to apologize to all colleagues whose work was not referenced in this article due to space limitations and/or only cited indirectly by referring to previous reviews. References Aguilera, M., Salinas, R., Rosales, E., Carminati, S., Colombo, M.I., Beron, W., 2009. Actin dynamics and Rho GTPases regulate the size and formation of parasitophorous vacuoles containing Coxiella burnetii. Infect. Immun. 77, 4609–4620. Akporiaye, E.T., Baca, O.G., 1983. Superoxide anion production and superoxide
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