Escherichia coli K1-induced cytopathogenicity of human brain microvascular endothelial cells

Microbial Pathogenesis 53 (2012) 269e275

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Escherichia coli K1-induced cytopathogenicity of human brain microvascular endothelial cells Naveed Ahmed Khan*, Junaid Iqbal, Ruqaiyyah Siddiqui Department of Biological and Biomedical Sciences, Aga Khan University, Stadium Road, Karachi, Pakistan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 May 2012 Received in revised form 2 July 2012 Accepted 3 July 2012 Available online 20 July 2012

Pathophysiology of Escherichia coli sepsis is complex involving circulating bacterial products, cytokine release, and sustained bacteremia resulting in the damage of vascular endothelium. Here, it is shown that E. coli K1 produced cytopathogenicity of human brain microvascular endothelial cells (HBMEC), that constitute the bloodebrain barrier. Whole bacteria or their conditioned medium produced severe HBMEC damage suggesting E. coli K1-cytopathogenicity is a contact-independent process. Using lipopolysaccharide (LPS) inhibitor, polymyxin B, purified LPS extracted from E. coli K1 as well as LPS mutant derived from E. coli K1, we showed that LPS is not the sole determinant of E. coli K1-mediated HBMEC death. Bacterial product(s) for HBMEC cytopathogenicity was heat-labile suggesting LPS-associated proteins. Several isogenic gene-deletion mutants (DompA, DibeA, DibeB, Dcnf1) exhibited HBMEC cytopathogenicity similar to that produced by wild type E. coli K1. E. coli K1-mediated HBMEC death was independent of phosphatidylinositol 3-kinase (PI3K) but dependent partially on focal adhesion kinase (FAK) using HBMEC expressing dominant negative FAK and PI3K. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Sepsis Endothelial cells Lipopolysaccharide Cytopathogenicity

1. Introduction Escherichia coli is a Gram-negative bacterium, a causative agent of septicemia and meningitis with fatal consequences. Although the pathophysiological events in these diseases differ remarkably, a high level of bacteremia is a pre-requisite in both E. coli-mediated sepsis and meningitis [1]. Research in sepsis has demonstrated that pathophysiological events during the course of Gram-negative septic shock are primarily elicited by lipopolysaccharide (LPS) [2], as characterized by high concentrations of endotoxin and excessive cytokines release [3e6]. In vitro data showed that purified LPS from pathogenic E. coli produced cell death in bovine endothelial cells [7,8]. Taken together, these findings signified LPS as a potential candidate to design therapeutic interventions against E. coli infections. However, anti-LPS strategies explored to date have failed to prevent the clinical course of Gram-negative sepsis, [9e11] which raises the question whether LPS is the sole toxic element in Gramnegative sepsis [12]. With regards to meningitis, E. coli traversal of the bloodebrain barrier is shown to be the key step in the development of E. coli K1 meningitis [13,14]. Using human brain microvascular endothelial cells (HBMEC), which constitutes the bloodebrain barrier,

* Corresponding author. Tel.: þ92 (0) 21 3486 4540; fax: þ92 (0) 21 3493 4294. E-mail address: [email protected] (N.A. Khan). 0882-4010/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micpath.2012.07.001

several determinants (such as OmpA, IbeA, IbeB, CNF1) are shown to be important for E. coli K1 binding to and invasion of HBMEC, leading to bacterial crossing of the bloodebrain barrier endothelium. Here, we studied the ability of E. coli K1 to produce HBMEC cytopathogenicity and determined the requirement of several bacterial determinants that are known to play crucial roles in E. coli K1 crossing of the bloodebrain barrier endothelium. In addition, we studied the role of signaling molecules including protein tyrosine kinases, phosphatidylinositol 3-kinase (PI3K) and focal adhesion kinase (FAK) in E. coli K1-mediated HBMEC cytopathogenicity. 2. Materials and methods 2.1. Human brain microvascular endothelial cell (HBMEC) cultures and transfections HBMEC were routinely grown on rat tail collagen-coated dishes in growth medium [RPMI containing 10% heat-inactivated fetal bovine serum, 10% Nu-serum, 2 mM glutamine, 1 mM pyruvate, penicillin (100 U/ml), streptomycin (100 mg/ml), non-essential amino acids and vitamins] as previously described [15,16]. The HBMEC expressing dominant negative forms of PI3K (Dp85 or Dp110) and FAK (Y397F construct in a pcDNA3 plasmid) were kindly provided by K. S. Kim (Johns Hopkins University, USA) and cultured as previously described [17,18].

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2.2. E. coli K1 strains E44 and C5 and their derivative mutant strains A serum-resistant E. coli strain possessing the K1 capsular polysaccharide, C5 (serotype O18:K1:H7), a CSF isolate of a newborn infant with meningitis and its rough LPS mutant strain constructed using chemical mutagenesis were used as previously described [19]. For simplicity, the rough LPS mutant is referred to as LPS mutant, even though there were no genetic manipulations. Another E. coli K1 strain E44, a spontaneous rifampin-resistant mutant of a CSF isolate of K1-encapsulated E. coli RS218 (O18:K1:H7) [20] and its derivatives DibeA [21], DompA [22], DibeB [23], and Dcnf1 [24] were used in the present study. All bacteria were grown in LuriaeBertani (LB) broth overnight with appropriate antibiotics. 2.3. Extraction of lipopolysaccharide from E. coli K1 strains E44 and C5

used as a negative control. In addition, supernatants were collected and cytopathogenicity determined by measuring lactate dehydrogenase (LDH) release using Cytotoxicity detection kit (Roche, Indianapolis, IN) and converted into percent cytotoxicity as follows: [(sample value  control value)/(total LDH release  control value)  100 ¼ % cytotoxicity]. Control values were obtained from cells incubated alone or cells incubated with E. coli strain HB101. Total LDH release was determined from HBMEC treated with 5% Triton X-100 for 15 min at 37  C. To determine whether E. coli K1-mediated HBMEC death is contact-dependent or -independent, cytopathogenicity assays were performed using E. coli K1 conditioned medium (CM). To produce CM, E. coli K1 strains C5 or E44 and their mutant strains were incubated at 37  C for 8 h in experimental medium without shaking. Cell-free CM was removed by centrifugation at 10,000 g for 2 min. Next, CM was filtered through 0.2 mm filter and used for cytopathogenicity assays as described above. 2.5. Immunoprecipitation and immunoblotting

Lipopolysaccharide (LPS) was extracted from 1 L overnight E. coli K1 strains E44 and C5 culture through modified phenol water method. Briefly E. coli K1 cell pellet was resuspended in 30 ml lysis buffer (TriseCl; pH 8.0, 2% SDS, 4% 2-mercaptoethanol and 2 mM MgCl2) and sonicated briefly to lyse cells. For protein removal, Proteinase-K was added at 0.1 mg/ml final concentration and incubated first at 65  C for 1 h and then at 37  C for overnight. Next day, LPS was precipitated from the mixture by addition of 4 ml of 3 M sodium acetate (pH 5.2), 80 ml of chilled ethanol and incubated at 20  C for 6 h. LPS pellet was recovered by centrifugation at 10,000 g for 10 min. In order to remove residual SDS from LPS pellet, it was resuspended in 36 ml of distilled water and precipitated as defined above. This step is repeated once again and then the LPS pellet was resuspended in 18 ml of TriseCl (pH 7.4) and 1 ml DNase I (100 mg/ml) and 1 ml RNase A (25 mg/ml) were added and incubated at 37  C for 4 h, in order to remove contaminating nucleic acids. After digestion these nucleases and other contaminating proteins were removed from the mixture by incubation at 65  C for 30 min and addition of an equal volume of 90% aqueous phenol, pre-heated at 65  C. The mixture was vortexed vigorously and incubated further at 65  C for 15 min. Organic and aqueous phases were separated by centrifugation at 4000 g for 25 min and then the LPS containing aqueous phase was collected. The phenol layer was re-extracted with equal volume of distilled water and then both aqueous phases were pooled and dialyzed against deionized water with multiple changes over 2 days in order to remove residual phenol completely. After dialysis LPS solution was lyophilized and analyzed by 15% SDS polyacrylamide gel after silver staining. Additionally, purified LPS from E. coli (serotype O111:B4) was purchased from Sigma Laboratories for comparative analyses. 2.4. Cytopathogenicity assays To determine whether E. coli K1 can produce cytopathic effects on HBMEC, cytopathogenicity assays were performed as previously described [25]. Briefly, HBMEC were grown to monolayers in 24well plates. Once confluent, cultures were washed three times with experimental medium [Medium 199 and Ham-F12 (1:1) containing 5% heat-inactivated fetal bovine serum, 2 mM glutamine]. Next, E. coli K1 strains C5 and E44 and their mutants: LPS mutant, DompA, DibeA, DibeB, or Dcnf1 were incubated (2  107/ml; 1:100 MOI) with HBMEC in experimental medium at 37  C in 5% CO2 incubator and periodically examined under the phase-contrast microscope for the integrity of the monolayers. At the end of the incubation period, the monolayers were stained with Hematoxylin (Sigma, St. Louis, MO). A laboratory E. coli K-12 strain HB101 was

Confluent HBMEC were incubated with low serum medium [RPMI containing 5% heat-inactivated fetal bovine serum, 2 mM glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml) and non-essential amino acids] for 17 h, followed by incubation with serum-free medium for 2 h. Serum starved HBMEC were stimulated with CM from E44 or C5 for the indicated periods. In controls, HBMEC were treated with CM produced by incubating laboratory K-12 strain HB101 in experimental medium. Following this, cells were rinsed with ice-cold phosphate buffered saline (PBS) containing 0.1 mM Na3VO4. The monolayers were lysed in lysis buffer composed of 50 mM TriseHCl (pH 7.4), 0.1% SDS, 0.5% Na deoxycholate, 10 mM Na pyrophosphate, 25 mM b-glycerophosphate, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 1 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 1 mg/ml aprotonin, 1 mg/ml leupeptin and 1 mg/ml pepstatin. Cell lysates were centrifuged at 10,000 g at 4  C and supernatant quantified for protein concentration (Bio-Rad, Hercules, CA). Equal amounts of proteins (500 mg) were incubated with appropriate antibody overnight at 4  C and incubated for 1 h with Protein A-agarose (Roche). The samples were washed four times with lysis buffer without sodium deoxycholate. Samples were eluted by boiling in SDS-sample buffer (Invitrogen) containing 10% b-mercaptoethanol (Sigma) and separated by SDSPAGE. Samples were then electrophoretically transferred onto pure nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The blots were blocked in TBST (25 mM Tris pH 7.4, 150 mM NaCl and 0.1% Tween 20) containing 5% non-fat dry milk at room temperature for 1 h. Blots were probed with specific antibodies overnight at 4  C washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, blots were washed with TBST and immune complexes were visualized through enhanced chemiluminescence (Amersham, La Jolla, CA). 3. Results 3.1. E. coli K1-induced HBMEC cytopathogenicity E. coli K1 strains E44 and C5 induced HBMEC death. Within 8 h of incubation, complete loss of HBMEC monolayer was observed with E. coli K1 strains (Fig. 1A). In contrast, laboratory E. coli K-12 strain HB101 exhibited no effect on HBMEC monolayers (Fig. 1A). In addition, cytopathogenicity was determined by measuring LDH release. More than 60% LDH release was observed with E. coli K1 strains E44 and C5 after 8 h incubation as compared to minimal LDH release with E. coli K-12 strain HB101 (Fig. 1B).

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Fig. 2. E. coli K1-induced cytopathic effects on HBMEC did not require hosteparasite interactions. Confluent HBMEC monolayers grown in 24-well plates were incubated with cell-free conditioned medium (CM) of E. coli K1 strain E44/C5 and HB101 for 8 h. The plates were then stained with hematoxylin (A) and LDH release determined (B) as described in “Materials and methods”. Note that CM from K1 produced severe cytopathic effects. Data represent the mean  SE of three independent experiments.

Fig. 1. Escherichia coli K1-induced host cell death. E. coli K1 strains E44/C5 (2  107/ml) was added to confluent cultures of primary human brain microvascular endothelial cells (HBMEC) grown in 24-well plates. Plates were incubated in a CO2 incubator for 8 h. A laboratory E. coli K-12 strain HB101 was used as a negative control. (A) At the end of the incubation period, the plates were stained with hematoxylin and photographed. Note that monolayers incubated with E44 or C5 resulted in complete loss of cell layer. Data represent the average of three independent experiments. (B) In addition, at the end of the incubation supernatants were tested for LDH release and converted to percentage cell death as described in Materials and methods. Data represent the mean  SE of three independent experiments.

3.2. E. coli K1-induced HBMEC cytopathogenicity is contactindependent To determine whether E. coli K1-induced cytopathogenicity required interactions with the host cells, assays were performed using cell-free conditioned medium (CM) obtained by incubating E. coli K1 strain E44 in experimental medium [Medium 199 and Ham-F12 (1:1) containing 5% heat-inactivated fetal bovine serum, 2 mM glutamine] for 8 h. The results revealed that CM produced HBMEC death similar to that observed with live E. coli K1 strains as shown by loss of HBMEC monolayers (Fig. 2A) and LDH release (Fig. 2B).

antibiotic that binds to the lipid A component of LPS; Sigma). Polymyxin B sulfate had no effect on CM-mediated HBMEC cytopathogenicity (Fig. 4), suggesting that CM-mediated HBMEC death is not entirely due to LPS activity. To further confirm these findings, purified LPS from E. coli (serotype O111:B4; Sigma) or from K1 strains E44 and C5 was incubated with HBMEC and cytopathogenicity determined. The results revealed that the purified LPS did not induce HBMEC death even up to concentrations of 20 mg/ml from any LPS preparations. In addition, CM derived from E. coli K1 strains E44 or C5 was heat-inactivated at 70  C for 30 min (which does not affect LPS). Heat-inactivation abolished the cytopathic activity of CM, suggesting the role of heat-labile factors such as proteins in CM-mediated cytopathogenicity of HBMEC (Fig. 4). Taken together, these findings illustrated that LPS is not the sole determinant contributing to E. coli K1- and CM-mediated cytopathogenicity of HBMEC. 3.5. E. coli K1-mediated HBMEC death is serum-dependent To determine whether E. coli K1-mediated HBMEC cytopathogenicity required the presence of serum, assays were performed

3.3. The rough lipopolysaccharide mutant (LPS mutant) of E. coli K1 and its CM did not exhibit HBMEC death To determine the role of LPS in E. coli K1-mediated HBMEC death, assays were performed using LPS mutant and its CM. The results revealed that neither whole bacterium (i.e., LPS mutant) nor its CM-induced HBMEC cytopathogenicity (Fig. 3), suggesting that LPS plays an important role in E. coli K1-mediated HBMEC cytopathogenicity. 3.4. LPS is not the sole determinant of E. coli K1-mediated cytopathogenicity Cytopathogenicity assays were performed by incubating CM with HBMEC in the presence of polymyxin B sulfate (10 mg/ml, an

Fig. 3. The rough lipopolysaccharide mutant of E. coli K1 and its CM did not exhibit HBMEC cytotoxicity. To determine the role of LPS in E. coli K1-mediated HBMEC death, cytopathogenicity assays were performed using rough LPS mutant and its CM. Neither whole bacterium (i.e., LPS mutant) nor its CM produced HBMEC death. Data represent the mean  SE of three independent experiments.

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This is despite the fact that LPS mutant strain did not exhibit cytopathogenicity. These findings suggest that E. coli K1-mediated HBMEC death is independent of any hemolytic activity. 3.7. E. coli K1-mediated HBMEC death is independent of known virulence determinants including OmpA, IbeA, IbeB, CNF1

Fig. 4. LPS is not the sole determinant of E. coli K1-mediated HBMEC death. To determine the role of LPS in E. coli K1-mediated HBMEC death, CM were incubated with polymyxin B sulfate, LPS inhibitor. Note that polymyxin B sulfate had no effect on CM-mediated HBMEC death, suggesting that CM-mediated HBMEC cytopathogenicity is not entirely due to LPS activity. To further confirm this, CM were heat-inactivated (which does not effect LPS) and assessed for HBMEC death. Heat-inactivation abolished cytolytic activity of CM, suggesting the role of heat-labile factors such as proteins in CM-mediated cytopathogenicity of HBMEC. In support, purified LPS from E. coli (serotype O111:B4; Sigma) or purified from E. coli K1 strains E44 or C5 did not induce cytopathogenicity of HBMEC even up to concentrations of 20 mg/ml (represented as LPS but similar results were observed for all three E. coli strains). Data represent the mean  SE of three independent experiments.

using E. coli K1 strains and their CM obtained in the presence or absence of serum (i.e., experimental medium or RPMI-1640 respectively). As shown in Fig. 5, HBMEC death did not occur in the absence of serum with E. coli K1. Also, CM obtained in the absence of serum did not exhibit HBMEC cytopathogenicity indicating HBMEC cytopathogenicity in response to E. coli K1 and its CM is serum-dependent. 3.6. E. coli K1-mediated cytopathogenicity is independent of hemolytic activity Hemolysin has been implicated as a major E. coli factor contributing to cytotoxicity [26,27]. To determine whether haemolysin plays a role in E. coli K1-mediated cytopathogenicity, E. coli K1 and its LPS mutant strain were plated on sheep blood agar and incubated at 37  C for up to 48 h. Both E. coli K1 and its LPS mutant strain exhibited similar zone of hemolytic activity (data not shown).

Fig. 5. E. coli K1-mediated HBMEC death is serum-dependent. To determine whether E. coli K1-mediated HBMEC cytopathogenicity required the presence of serum, assays were performed using E. coli K1 strain and its CM obtained in the presence or absence of serum as described in “Materials and methods”. Note that HBMEC cytopathogenicity did not occur in the absence of serum. Data represent the mean  SE of three independent experiments.

Previously, E. coli K1 proteins such as OmpA, IbeA, IbeB and CNF1 are shown to play an important role in bacterial traversal of the bloodebrain barrier [13,14]. For example, OmpA contributes to E. coli K1 binding to HBMEC, while IbeA, IbeB, CNF1 are important in HBMEC invasion. In an attempt to determine their role in E. coli K1-mediated HBMEC death, assays were performed using isogenic gene-deletion mutants including DompA, DibeA, DibeB or Dcnf1. The results revealed that all mutants tested exhibited HBMEC death similar to that mediated by the wild type strain (Fig. 6). In addition, CM were obtained by incubating these strains in experimental medium as described above and tested for HBMEC death. The CM derived from the mutant strains also exhibited cytopathogenicity of HBMEC, suggesting that these proteins are not crucial for E. coli K1mediated HBMEC death (Fig. 6). 3.8. E. coli K1-mediated cytopathogenicity of HBMEC required tyrosine kinase signaling pathways It was next determined whether genistein (a protein tyrosine kinase inhibitor) can interfere with E. coli K1-mediated HBMEC death. HBMEC monolayers were pretreated with genistein (25 mM) before the addition of CM. The CM-induced HBMEC death was inhibited by more than 50% with genistein, suggesting the role of tyrosine kinase signaling pathways in CM-mediated HBMEC cytopathogenicity (Fig. 7A). These findings led us to investigate signaling molecules that are involved in E. coli K1- and CM-induced HBMEC death. The effect of CM on tyrosine phosphorylation of HBMEC was investigated using anti-phosphotyrosine antibody (Upstate Biotech., Lake Placid, NY). Conditioned medium increased the tyrosine phosphorylation of several proteins (approximate molecular weights of 170, 120, 75, 65, 40 kDa) in HBMEC (Fig. 7B). We next assessed the possible signaling molecules involved in CM-induced HBMEC death using dominant negative constructs. Assays were performed using HBMEC expressing dominant negative FAK or PI3K molecules. We observed that CM-induced HBMEC death was partially inhibited in FAK dominant negative cells as compared to HBMEC transfected with pcDNA3 alone (Fig. 8). In contrast, CM-mediated HBMEC cytopathogenicity was not affected in HBMEC expressing either mutant p85 or mutant p110 (PI3K subunits) (Fig. 8). Overall, these results suggest that E. coli K1-

Fig. 6. Escherichia coli K1-induced HBMEC death is independent of DompA, DIbeA, DIbeB or Dcnf1. Confluent monolayers of HBMEC were incubated with E. coli K1 strains E44 and its mutants DompA, DibeA, DibeB and Dcnf1 as well as their CM for cytopathogenicity assays. Data represent the mean  SE of three independent experiments.

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Fig. 8. Escherichia coli-induced cytopathic effects are partially dependent on focal adhesion kinase (FAK) tyrosine phosphoryations. Primary HBMEC transfected with pcDNA3 cloned with DFAK (Y397F) or DPI3K (Dp85 and Dp110) constructs as well as pcNDA3 alone were incubated with CM from E. coli K1 for 8 h. At the end of the incubation, supernatants were collected and LDH release was determined. Note that only FAK dominant negative HBMEC were less susceptible to E. coli cytopathogenicity. Data represent the mean  SE of three independent experiments.

Fig. 7. Escherichia coli K1-induced HBMEC death is dependent on host protein tyrosine phosphoryations. (A) Confluent cultures of HBMEC were treated with genistein (25 mM) for 1 h prior to the addition of CM from E. coli K1 and incubated for 8 h. At the end of the incubation, supernatants were collected and LDH release was determined and converted to percentage cell death. Data represent the mean  SE of three independent experiments. (B) HBMEC were stimulated with CM of E. coli K1. Cell lysates were prepared as described in “Materials and methods”. The lysates were immunoprecipitated and immunoblotted with anti-phosphotyrosine antibody (4G10). Protein molecular size standards (in kilodaltons) are indicated on the left. Results are representative of three independent experiments.

mediated HBMEC death involved host cell protein tyrosine kinases, including FAK. 4. Discussion Sepsis and meningitis are among the most serious complications due to E. coli K1. For example, sepsis accounts for more than 30% of intensive care unit admissions with a mortality rate of more than 50%, characterized by capillary leak, hypotension and organ dysfunction. Similarly, the mortality rate due to E. coli K1 meningitis is more than 40%, while half of patients develop developmental disability [13,14]. Vascular endothelium provides a vital barrier between blood and organs. Here for the first time it is shown that E. coli K1-induced cytopathogenicity in HBMEC in a contact-independent manner suggesting that the pathogenicity factors responsible for E. coli-mediated HBMEC death are secreted and/or shed. This is in contrast to previous studies which showed that E. coli-mediated host cell death is dependent on whole bacteria [28,29]. LPS has been shown to be an important pathogenic factor in septic shock [5,30]. For example, during the Gram-negative septic shock, free LPS is detected at high levels in the bloodstream

suggesting its role in the disease [3e6,31]. This is consistent with our findings, which demonstrated that the LPS mutant strain did not induce HBMEC cytopathogenicity. Given that LPS mutant is produced using chemical mutagenesis and that it exhibited different outer membrane protein complexes compared to the wild type strain [32], we confirmed these findings by inhibiting LPS. We anticipated that blocking LPS would inhibit E. coli-mediated cytolysis of HBMEC. To our surprise, polymyxin B sulfate was unable to inhibit E. coli CM-induced cytopathogenicity of HBMEC, suggesting that LPS is not the sole determinant in E. coli-mediated HBMEC death. Furthermore purified LPS had no effect on HBMEC death (from 20 ng/ml up to 20 mg/ml, which is well above its physiological concentration during sepsis). This is not a surprising finding. For example, research in Gram-positive bacterium has shown that nonLPS components play important role(s) in bacterial pathogenesis as indicated by excessive cytokine production [12,33e37]. Also, as the LPS mutant strain used in this study was generated by chemical mutagenesis, the observed effect could be due to secondary mutations introduced during mutagenesis affecting non-LPS components. These non-LPS components include peptideglycans [38e40], lipopeptides [35], lipoproteins [35], lipoteichoic acid [41] and capsular polysaccharides [42]. Furthermore, previous studies showed that lipoproteins (isolated from E. coli) induced cell death in human embryonic kidney 293 cells [43]. In addition, other bacterial components such as chaperonins [44], serine proteases [45] and bacterial DNA [46] have been shown to induce proinflammatory response. Next, it was determined whether pathogenicity factors in E. coli K1-mediated HBMEC death are proteinaceous in nature. The CM was heated at 65  C for 30 min. Heat-inactivation of CM completely abolished its cytolytic ability indicating that E. coli K1-mediated pathogenicity factors are protein(s). Given the requirement of serum in E. coli K1-mediated HBMEC death, we hypothesized that source of protein(s) may be serum. To determine this, CM were produced using heat-inactivated experimental medium. However, this had no effect on E. coli K1 CM-induced HBMEC cytotoxicity (data not shown). This concluded that E. coli K1-mediated pathogenicity factors are protein(s), source of which is bacterial. From these data, we concluded that (i) LPS is not the sole critical determinant for E. coli K1-mediated HBMEC death and (ii) E. coli K1 may require LPS-related and/or associated proteins for its cytopathogenicity on HBMEC. Previously, E. coli secretion of three outer membrane proteins complexed with LPS has been identified using an in vivo model [47,48], however, their role in bacterial pathogenesis remains unknown. One of the proteins was OmpA. Using

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ompA-deletion mutant of E44 in cytopathogenicity assays, it was observed that DompA produced HBMEC cytopathogenicity similar to its parent strain suggesting that OmpA is not crucial for E. coli K1mediated HBMEC death. Similar results were observed using other E. coli K1 mutants including DibeA, DibeB or Dcnf1. Recently, Zhou et al., [49], have identified a cluster of E. coli virulence factors encoding a putative type VI secretion system (T6SS) in the genome of the meningitis-causing E. coli K1 strain RS218. Using deletion mutants of Hcp family proteins (the hallmark of T6SS), it was shown that Hcp2 is involved in E. coli K1 strain RS218 binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SSdependent manner and induced HBMEC death. Future studies will be directed towards the identification and characterization of protein(s) including T6SS-associated Hcp family proteins required for E. coli K1 strain E44-mediated HBMEC death. To determine whether CM-induced HBMEC death required PTK signaling pathways, HBMEC were treated with genistein, prior to the addition of CM. It was interesting to note that PTK inhibition blocked E. coli K1-mediated cytopathogenicity in an LPSindependent manner, as polymyxin B sulfate had no effect on CM-induced HBMEC death (data not shown). It is worth noting that genistein is a broad PTK inhibitor, thus may block several pathways. Cytopathogenicity assays using FAK or PI3K dominant negative HBMEC revealed that E. coli K1-mediated HBMEC death was partially blocked in FAK dominant negative HBMEC but is independent of PI3K. Similar findings by Crane et al., [50] have shown that enteropathogenic E. coli (EPEC) produced cytopathic effects on epithelial cells and these effects were independent of PI3K. However, these findings are in contrast to E. coli K1 invasion of HBMEC, which is dependent on PI3K [18] suggesting that invasion and cytotoxicity involve distinct signaling pathways. Further identification and characterization of both the bacterial determinants and the host factors should help identify molecules that are common (such as FAK) in the invasion as well as cytotoxicity of HBMEC and may provide potential targets for the rationale development of therapeutic interventions against sepsis and meningitis due to E. coli K1.

[5]

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Authors’ contributions [20]

NK conceived the study. JI designed and conducted LPS-related experiments, while RS designed and conducted all other experiments under the supervision of NAK. All authors contributed to the writing of the manuscript and approved the final manuscript.

[21]

[22]

Competing financial interests The author(s) declare no competing financial interests. Acknowledgements The authors are grateful to Kwang Sik Kim and Monique Stins (Johns Hopkins University) for providing HBMEC and bacterial strains used in this study.

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