FEMS Microbiology Letters Advance Access published May 25, 2015

TITLE: Listeriolysin O Mediates Cytotoxicity Against Human Brain Microvascular Endothelial Cells Ting Zhang, Dongryeoul Bae, and Chinling Wang* Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University Running title: Listeriolysin facilitates entry into CNS cells *Corresponding author Mailing address: P.O. Box 6100, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762. Phone: (662) 325 1683 Fax: (662) 325 1031 E-mail:[email protected]

Abstract Penetration of the brain microvascular endothelial layer is one of the routes L. monocytogenes cells use to breach the blood-brain barrier. Because host factors in the blood severely limit direct invasion of human brain microvascular endothelial cells (HBMECs) by L. monocytogenes, alternative mechanisms might be used by this bacterium to penetrate the endothelial cell layer. In this study, we evaluated the cytotoxicity of proteins secreted by L. monocytogenes against HBEMCs using a live/dead staining method. Interestingly, the integrity of the plasma membrane of HBMECs was impaired by proteins secreted by the EGD wild type strain but not proteins secreted by

the isogenic ∆prfA strain. Therefore, we investigated the cytotoxicity of proteins secreted by several isogenic mutant strains (∆plcA, ∆mpl, and ∆hly) incapable of producing the

prfA-regulated bacterial products PlcA, Mpl, and LLO, respectively. Results from both fluorescent microscopy and flow cytometry analyses showed that proteins secreted by the ∆hly strain were not cytotoxic to HBMECs, whereas those secreted by the ∆plcA and ∆mpl strains were cytotoxic. These results suggest that LLO-mediated cytotoxicity against brain microvascular endothelial cells enables L. monocytogenes cells to effectively penetrate the brain microvascular endothelial layer.

Key words: Listeria monocytogenes, listeriolysin, cytotoxicity, CNS, HBME Introduction Listeria monocytogenes is a gram-positive pathogen that causes invasive diseases of the central nervous system (CNS) in both humans and ruminants, leading to several severe clinical manifestations (Vazquez-Boland, et al., 2001, Oevermann, et al., 2010, Disson & Lecuit, 2012). Although L. monocytogenes exhibits a low overall incidence of infection, it has the highest fatality rate (18.1%) among the top five bacterial meningitis–causing agents (Thigpen, et al., 2011). There are three major routes through which L. monocytogenes crosses the blood-brain barrier: a neural retrograde migration route; invasion of brain endothelium; and through trafficking of infected leukocytes, which is known as the “Trojan horse” mechanism (Drevets, et al., 2004, Drevets & Bronze, 2008,

Oevermann, et al., 2010). Brain microvascular endothelial cells are closely joined with each other via tight junctions, which form the key component of the blood-brain barrier (Disson & Lecuit, 2012). An electron microscopic study indicated that the vascular endothelium is a possible target of L. monocytogenes in CNS infection (Kirk, 1993). In vitro studies showed that L. monocytogenes can invade transformed immortal human brain microvascular endothelial cells (HBMECs) (Greiffenberg, et al., 1998, Greiffenberg, et al., 2000) and primary cultured HBMECs (Wilson & Drevets, 1998). A more recent study showed that antibodies in human serum effectively block the entry of L. monocytogenes into HBMECs (Hertzig, et al., 2003), suggesting that direct invasion through endothelial cells resulting in CNS infection might be impeded in vivo. PrfA-dependent bacterial products, such as listeriolysin O (LLO), phosphatidylinositolspecific phospholipase C (PI-PLC), phosphatidylcholine-specific phospholipase C (PCPLC), and metalloprotease (Mpl) play critical roles during the process of L. monocytogenes infection (Vazquez-Boland, et al., 2001, Cossart, 2011). LLO, a poreforming toxin produced by L. monocytogenes, is a particularly critical virulence factor that plays multiple roles in ensuring intracellular survival of the pathogen in hosts (Portnoy, et al., 1988, Schnupf & Portnoy, 2007). Emerging evidence also indicates that LLO functions extracellularly in cell invasion (Vadia, et al., 2011, Vadia & Seveau, 2014), host cell function subversion (Hamon, et al., 2007, Ribet, et al., 2010, Stavru, et al., 2011), and immune response modulation (Stavru, et al., 2011). In addition, LLO exhibits a pore-forming effect on the plasma membrane of a variety of cells (Repp, et al., 2002, Richter, et al., 2009, Vadia, et al., 2011, Vadia & Seveau, 2014), has a strong apoptogenic effect on T cells and dendritic cells (Guzman, et al., 1996, Carrero, et al.,

2004), and is cytotoxic to mouse PBMCs (Stachowiak, et al., Glomski, et al., 2003). PI-PLC and PC-PLC facilitate vacuolar escape and play a significant role in cell to cell spread (Vazquez-Boland, et al., 1992, Camilli, et al., 1993, Smith, et al., 1995). Mpl, a metalloprotease required for the functional maturation of PC-PLC, is bundled with PCPLC to mediate vacuolar escape in some cultured human cells in which LLO plays a nonessential role (Marquis, et al., 1995, Grundling, et al., 2003, Yeung, et al., 2005). Interestingly, a L. monocytogenes strain in which PC-PLC is constitutively activated was shown to increase the membrane permeability of host cells during intracellular growth, indicating that uncontrolled PC-PLC production leads to damage of the host plasma membrane (Yeung, et al., 2007). In addition, the results of an in vivo experiment involving the intracerebral infection route in mice indicated that PC-PLC is essential for bacterial spread in the brain (Schluter, et al., 1998). In this study, we examined cytotoxicity against HBMECs of proteins secreted by L. monocytogenes and collected from the bacterial culture supernatant. We found that proteins secreted by the ∆prfA and ∆hly strains are not cytotoxic to HBMECs, whereas

proteins secreted by the wild type (WT), ∆plcA, and ∆mpl mutant strains were. Our

results suggest that LLO-mediated perforation of the plasma membrane of HBMECs is a

potential route for L. monocytogenes penetration of the endothelial layer, leading to CNS infection.

Materials and methods

Bacterial strains and cell culture conditions The Listeria strains used in this study are listed in Table 1. The L. monocytogenes EGD strain and the isogenic ∆mpl, ∆plcA, and ∆prfA mutant strains were kindly provided by Dr. Michael Kuhn of the Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften der Universität Würzburg, Germany. Bacteria were cultured in BHI broth (Difco Laboratories, Detroit, MI) at 37°C. Escherichia coli DH5α was cultured in Luria-Bertani (Difco) broth. To culture the deletion mutants, transformed L. monocytogenes cells were selected on BHI agar supplemented with erythromycin (5 μg/ml) or tetracycline (10 μg/ml) when necessary. HBMECs were purchased from Cell Systems Corporation (CSC, Kirkland, WA) and cultured in CSC complete medium supplemented with cell culture boost (CSC). Cells were cultured at 37°C in a 5% CO2 incubator. Construction of an hly mutant The temperature-sensitive shuttle plasmid pMAD (Arnaud, et al., 2004) was used to generate an hly deletion mutant following a previously described method (Bae, et al., 2013). Briefly, using the genomic DNA of L. monocytogenes EGD as the template, the upstream and downstream regions flanking the hly gene were amplified using PCR with the primers listed in Table 2. The PCR products were digested with BamHI/SalI and XhoI/BglII, respectively, and then cloned in tandem into a pMAD tet plasmid. The recombinant plasmid was introduced into L. monocytogenes by electroporation at 2.5 kV, 200 Ω, and 25 µF. Deletion of the hly gene was conducted by allelic exchange. Transformed bacteria were incubated at 43°C for plasmid integration, and colonies were inoculated into antibiotic-free BHI medium at 30°C for homologous recombination.

Deletion mutants were selected on BHI agar supplemented with erythromycin (5 μg/ml) or tetracycline (10 μg/ml) and confirmed by PCR using primers harboring the deleted region of the hly gene. Isolation of secreted proteins WT L. monocytogenes EGD and isogenic mutant strains were inoculated into BHI broth and cultured in 37°C for 16 hours, after which 5 ml of each bacterial culture was centrifuged at 7,500g for 10 minutes to remove the cells. The resulting supernatants were mixed with ethanol (1/4 [vol/vol]) and centrifuged at 20,000g to pellet the secreted proteins. The pellets were washed three times with 80% ethanol, air dried, and resuspended with 0.5 ml of sterile water. The resuspended supernatant (10-fold concentrated) was stored or used for the cytotoxicity test. Live/dead cell analysis by fluorescent microscopy HBMECs were seeded into the wells of a 24-well plate and cultured as described above. For the cytotoxicity test, 0.1 ml of concentrated bacterial culture supernatant from L. monocytogenes EGD WT strain or the isogenic ∆prfA, ∆hly, ∆plcA, or ∆mpl strains was added to the HBMEC-containing wells with 0.3 ml of CSC minimum medium (CSC). Cells were incubated at 37°C in a 5% CO2 incubator for 20 minutes. For fluorescent microscopic analysis, 0.4 μM calcein AM and 2 μM ethidium homodimer-1 working solution was added to the cells according to the manufacturer’s instructions (Invitrogen, Grand Island, CA). After 20 minutes of incubation at room temperature, fluoresceinlabeled samples were examined under a fluorescence microscope (Nickon, Tokyo, Japan).

Flow cytometry HBMECs were incubated with the secreted proteins isolated as described above, dissociated from the plate by trypsin, and then pelleted by centrifugation at 400g. The cells were washed once with PBS and then incubated with 0.4 μM calcein AM and 2 μM ethidium homodimer-1 working solution (Invitrogen) for 20 minutes at room temperature. Cells were pelleted, washed once with PBS containing 5 mM EDTA to remove free dye, and then resuspended in PBS containing 0.2% BSA. Samples were analyzed using a BD flow cytometer (BD Biosciences, San Jose, CA) to determine the percentages of dead and live cells.

Results Cytotoxicity of listerial secreted proteins against HBMECs is PrfA dependent To assess the cytotoxicity of proteins secreted by L. monocytogenes against cells of the brain microvascular endothelium, we incubated HBMECs with proteins secreted by the EGD WT and ∆prfA strains. The extent of cytotoxicity against HBMECs was reflected in the signal intensity of two fluorescent dyes: calcein AM, which binds to the cell membrane; and ethidium homodimer-1, a DNA-labeling dye that does not penetrate the membrane. After a 20-minute incubation with proteins secreted by WT L. monocytogenes EGD, the permeability of the plasma membrane of HBMECs was significantly increased compared with HBMECs incubated with a medium control (Figure 1). In contrast, the plasma membrane was not perforated when HBMECs were incubated with proteins secreted by the ∆prfA strain (Figure 1). The proportion of cells with a damaged plasma membrane and that of cells with an intact plasma membrane was evaluated using flow

cytometry. Consistent with fluorescent microscopy observations, the mean fluorescence intensity of the calcein AM channel, which reflects the integrity the plasma membrane, was not changed following exposure to proteins secreted by the ∆prfA strain compared with control cells treated with medium alone (Figure 2). In contrast, the percentage of HBMECs with an intact plasma membrane was dramatically decreased when the cells were incubated with proteins secreted by WT L. monocytogenes EGD (Figure 2). These results suggest that PrfA-dependent secreted bacterial products are responsible for cytotoxicity against HBMECs. LLO mediates cytotoxicity against HBMECs In order to characterize the virulence factors that contribute to the cytotoxicity against HBMECs, we investigated the cytotoxicity of proteins secreted by isogenic mutant strains (∆plcA, ∆mpl, and ∆hly) that are incapable of producing the PrfA-regulated bacterial products (PlcA, Mpl, and LLO, respectively). Interestingly, proteins secreted by all of the mutant strains except ∆hly showed strong cytotoxicity against HBMECs (Figure 3); these results were further confirmed by flow cytometry analysis (Figure 2). Incubation with proteins secreted by the ∆plcA and ∆mpl strains resulted in a dramatic decrease in the percentage of cells with an intact plasma membrane, whereas a high percentage of undamaged cells was observed upon incubation with proteins secreted by the ∆hly strain or cell culture medium (Figure 2). These data indicate that LLO, but not the two phospholipases, is the key player in the listerial secretome that contributes to cytotoxicity against HBMECs. Discussion

Targeting and direct invasion of brain microvascular endothelial cells by L. monocytogenes has been documented by electron microscopic observation and in vitro experiments (Kirk, 1993, Greiffenberg, et al., 1998, Wilson & Drevets, 1998, Greiffenberg, et al., 2000) and is thus considered a route for CNS infection. However, later observations by Hertzig et al. brought into question the accessibility of this route in vivo because the presence of host antibodies could effectively block bacterial invasion (Hertzig, et al., 2003). Results from our experiments showed that invasion of HBMECs by L. monocytogenes was extremely low (less than 0.01% of cells, data not shown), leading us to speculate upon the existence of alternative mechanisms other than direct invasion that enable L. monocytogenes to penetrate the endothelial layer. One possible mechanism is through the cytotoxic action of soluble bacterial proteins, which has been documented as a strategy used by other bacterial pathogens (Kugler, et al., 2007, Tripathi, et al., 2007, Kim, 2008). To assess the possibility of cytotoxicity-mediated penetration of the endothelial layer by L. monocytogenes, the first question to ask is whether the presence of LLO in the blood contributes to penetration. Due to the strong immunogenic properties of LLO, the concentration of LLO that is required to be presented by APCs to trigger the immune response is much lower than the concentration needed to cause cytotoxicity (Carrero, et al., 2012). Indeed, a mutant strain that constitutively expresses LLO is avirulent, as it is not able to evade the host immune response (Glomski, et al., 2003). However, several lines of evidence have indicated that LLO could be present in the blood during infection with L. monocytogenes. First, the expression of the hly gene is higher in the blood

compared with rich BHI medium and the small intestine (Toledo-Arana, et al., 2009), indicating that blood is a favorable environment for L. monocytogenes to produce LLO. Second, antibodies against LLO have been detected in the serum of listeriosis patients, and detection of antibodies against LLO in human serum is considered a diagnostic criterion for Listeria infection (Berche, et al., 1990). It has been shown that patients with the CNS symptoms often have poor immune status (Disson & Lecuit, 2012), and bacteremia was found to be required for CNS infection in a mouse model (Berche, 1995). It is possible that during the late stage of infection, large numbers of bacteria circulate in the blood, allowing L. monocytogenes to produce enough of the secreted form of LLO to increase the permeability of the endothelial layer and achieve endothelial layer penetration. Previous in vitro experiments showed that LLO can be detected in the secretome when L. monocytogenes is grown in BHI broth (Moors, et al., 1999, Cabrita, et al., 2010). Our results showed that the presence of LLO (but not the two phospholipases examined) in the bacterial culture supernatant is responsible for the damage to the plasma membrane of HBMECs. Indeed, this type of pore-forming effect of LLO on the plasma membrane has been observed in a variety of other cells, including epithelial cells (Hep-G2), human embryonic kidney cells (HEK293), and colon epithelial cells (HT-29/B6) (Repp, et al., 2002, Richter, et al., 2009, Vadia, et al., 2011, Vadia & Seveau, 2014), indicating the universal role of LLO with respect to cell membrane perforation. Although the pores made by LLO are transient (Repp, et al., 2002, Dramsi & Cossart, 2003) and can probably be repaired by host factors such as caspase-7 (Cassidy, et al., 2012), it has been

suggested that transient changes in the integrity and function of the plasma membrane might be sufficient for the pathogen to pass through the endothelial layer (Kugler, et al., 2007). The pore-formation activity of LLO is activated under acidic conditions to achieve phagosome escape and is inhibited at neutral pH, preventing host cell damage (Schuerch, et al., 2005). However, recent in vitro data showed that when attached to cholesterol, LLO at least partially retains its fore-forming ability at neutral and even alkaline pH, which is similar to physiologic conditions (Bavdek, et al., 2007). Our results indicate that the disruption of the HBMEC plasma membrane by secreted bacterial proteins can occur in cell culture medium (~pH 7.4), which is consistent with observations from experiments involving direct bacterial infection (Vadia & Seveau, 2014). The aggregation of LLO at neutral pH has been reported and is considered a means of LLO inactivation (Bavdek, et al., 2012); however, our data suggest that LLO retains at least a portion of its poreforming activity under physiologic pH conditions.

Acknowledgements This work was supported by grants from the USDA/ARS (#58-6402-7-230) and the Mississippi Agriculture and Forestry Experiment Station. We want to thank Mr. John Stokes for his help with flow cytometry experiments.

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Table 1. Bacterial strains and plasmids Bacterial strains/Plasmids Listeria monocytogenes EGD EGD_∆PrfA EGD_∆Mpl

Description wild type EGD prfA deletion mutant, Ermr EGD mpl deletion mutant


EGD plcA deletion mutant, Ermr

EGD_∆LLO Plasmid pMAD

EGD hly deletion mutant, Tetr

pMAD tet pMAD hly

Shuttle vector for constructing deletion mutant, Ermr pMAD vector containing Tet cassette, Tetr, Ermr pMAD_tet vector containing hly flanking regions, Tetr, Ermr

Reference this study (Chakraborty, et al., 1992) Böckmann R and Goebel W, unpublished) Domann, Ph.D Thesis, Universität Würzburg, 1992 this study (Arnaud, et al., 2004) this study this study

Table 2 Primer list for hly deletion mutagenesis Lmohly upstream forward Lmohly upstream reverse Lmohly downstream forward Lmohly downstream reverse


Figure legends

Figure 1. Cytotoxicity of secreted proteins against HBMECs is PrfA dependent. Cytotoxicity against HBMECs was accessed using the cell culture medium (control), BHI medium (BHI), and supernatant from the EGD wild type (WT) or ∆prfA (∆prfA) strains. HBMECs were incubated with cell culture medium, BHI medium, and concentrated bacterial culture supernatant at 37°C in a 5% CO2 incubator for 20 minutes. Cells were labeled with calcein AM and EthD-1 to visualize the plasma membrane and nucleic acids. Images are repre

Figure 2. Flow cytometry analysis of cytotoxicity of secreted proteins against HBMECs. Cytotoxicity against HBMECs was assessed using the secreted proteins collected from A) BHI medium, B) EGD wild type, C) ∆prfA, D) ∆plcA, E) ∆mpl, or F) ∆hly or ∆LLO strain. Cell culture and BHI media were used as controls. HBMECs were incubated with secreted proteins or a control at 37°C in a 5% CO2 incubator for 20 minutes. To label the plasma membrane and nuclei, cells were incubated with calcein AM and EthD-1 for 20 minutes. Cells were then analyzed using flow cytometry. Images are representative results of two biological replicates with two technical replicates.

Figure 3. LLO (but not phospholipases) is the major contributor to cytotoxicity against HBMECs. Cytotoxicity against HBMECs was accessed using the cell culture medium (control), or supernatant from the EGD wild type (WT), ∆plcA (∆PlcA), ∆mpl (∆Mpl), or ∆hly (∆LLO) strains. Deletion of LLO but not of the two phospholipases abrogated the HBMEC membrane perforation by secreted proteins. Images are representative results of two biological replicates with two technical replicates.

Listeriolysin O mediates cytotoxicity against human brain microvascular endothelial cells.

Penetration of the brain microvascular endothelial layer is one of the routes Listeria monocytogenes use to breach the blood-brain barrier. Because ho...
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