Involvement of clathrin and β-arrestins in Aggregatibacter actinomycetemcomitans endocytosis of human vascular endothelial cells

Department of Microbiology, School of Public Health and Tropical Medicine, Southern Medical University, Tonghe, Guangzhou, 510515, China. Department of Periodontology, Guangdong Provincial Stomatological Hospital, Southern Medical University , S366 Jiangnan Boulevard, Guangzhou, 510280, China. Department of Vacular, Thyroid and Breast surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080, China.

Phosphorylcholine (PC) has been reported as a viru-lence factor in a number of pathogenic prokaryotes, including Gram-positive bacteria Streptococcus pneumoniae and the Gram-negative species Haemophilus influenzae (Harnett and Harnett, 1999).PC not only plays an important role in S. pneumoniae adherence and invasion of host cells (Cundell et al., 1995), but also enhances H. influenzae biofilm maturation (Hong et al., 2007).Recent studies have identified A. actinomycetemcomitans as Table 1.Plasmids of β-arrestin-1/-2 siRNA.
As typical of G proteins coupled receptors (GPCRs), PAFR activation by PC could be regulated by β-arrestins (McLaughlin et al., 2006;Eckels et al., 2009).β-Arrestins could bind to activated GPCRs and clathrin to promote the clathrin-mediated endocytosis and transcytosis (Shenoy and Lefkowitz, 2011;Gyombolai et al., 2013).The role of β-arrestins in inflammation process has been well known by regulation of chemokine responsiveness (Vroon et al., 2006;Cattaruzza et al., 2013;Porter et al., 2010).It has been reported that clathrin and β-arrestin-1 could mediate S. pneumonia and Acinetobacter baumannii invasion of epithelial cells (Radin et al., 2005;Smani et al., 2012).However, there are no studies about clathrin and β-arrestins in A. actinomycetemcomitans internalizetion by epithelial or endothelial cells.In this work, we aimed to study whether clathrins and β-arrestins participated in endocytosis of A. actinomycetemcomitans.

Bacterial strains and media
A. actinomycetemcomitans strains HK1651 and ATCC29523 were purchased from ATCC Bioresource Center (USA).HK1651 was identified as PC-positive strain and ATCC29523 was PC-negative strain before.The brain heart infusion medium was used as the liquid medium and Tryptic soy agar (TSA) was used as the solid medium (Huankai Microbial Sci.&Tech.Co. Let, Guangdong, China).The A. actinomycetemcomitans strains were grown in humidified 5% CO 2 atmosphere at 37°C.

Cell culture
The human umbilical vein endothelial cells (HUVEC) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China).They were cultured in DMEM medium, supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA).Cells were incubated at 37°C, 5% CO 2 , 95% air humidified atmosphere.

Plasmids and transfection
The double-stranded siRNAs for β -arrestin-1 and β -arrestin-2 were chemically synthesized and sequenced by Shanghai GenePharma Co., Ltd.The sequences of β -arrestin-1 and β -arrestin-2 siRNAs used in this study Are listed in Table 1.HUVEC was transfected with the plasmids using Lipofectamine™ LTX and PLUS™ Reagent (Invitrogen, USA) according to the manufacture's instruction.Briefly, 0.5 μg of DNA in 100 μl of Opti-MEM® I Reduced Serum Medium (Hyclone, USA) without serum were mixed with 1.25 μl of Lipofecta-mine™ LTX and 0.5 μl PLUS™ Reagent.The mixture was then added to the wells containing HUVEC at 37°C for 24 h.After the transfection, the cells were observed under an inverted fluorescence microscope and the real-time reverse-transcription PCR was used to examine the β -arrestin-1 and β -arrestin-2 expression.

Real-time reverse-transcription PCR
The total cellular RNA was extracted with TRIzol® (TaKaRa Biotechnology Co., Ltd.) and reverse transcribed into cDNA using the ExScript RT Reagent Kit (TaKaRa Biotechnology Co., Ltd.).Realtime PCR was performed by employing the SYBR® Premix Ex Taq™ Kit (TaKaRa Biotechnology Co., Ltd.) and was run on the ABI 7500 Real-Time PCR System (Applied Biosystems, CA, USA).The β-actin was used as an internal control.Primers used in this study are listed in Table 2.The thermal cycling was initiated with a first denaturation step of 30 s at 95°C, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s and 72°C for 90 s.Melting curve analysis was performed and concentration values were measured.The detected cycle threshold (Ct) values were interpolated into the standard curves of the plasmid constructs, and the mRNA expression in the samples was calculated by 2 -△△Ct .

Bacterial invasion and adhesion assays
To determine the role of clathrin in A. actinomycetemcomitans internalization by HUVEC, the cells were pretreated with monodansylcadaverine (MDC) or chlorpromazine (CPZ) for 30 min prior to infection with PC positive and negative A. actinomycetemcomitans strains (OD650=0.9)at a multiplicity of infection (MOI) of 100.After 4 h incubation at 37°C, the invasion assay was performed as described by Schenkein et al. (2000).
Briefly, the monolayers were incubated with gentamicin (50 mg/ml) for 2 h to kill bacteria external to the HUVEC.Then the monolayers were lysed and bacteria were plated to enumerate total cell-associated bacteria.For adhesion assay, the number of total cell-associated bacteria was determined as above without the gen-tamicin step.
To study the role of β-arrestins in A. actinomycetemcomitans internalization by HUVEC, PC positive and negative A. actinomycetemcomitans strains were added to the confluent monolayers of HUVEC transfected or not with siRNA of β-arrestin-1, β-arrestin-2 and control.The adhesion and internalization assays were done as described above.

Statistical analysis
The group data are presented as mean ± S.D. A student t-test was used to determine the differences between means.The difference was considered significant at P < 0.05.The SPSS (version 13.0) statistical package was used (SPSS Inc., Chicago, IL).

Clathrin role in A. actinomycetemcomitans internalization by HUVEC
To investigate whether the clathrin was involved in the A. actinomycetemcomitans endocytosis process during A. actinomycetemcomitans invading HUVEC, the impact of clathrin inhibitors, monodansylcadaverine (MDC) and chlorpromazine (CPZ) were studied in A. actinomycetemcomitans adhesion and invasion of HUVEC process.It was found that MDC and CPZ effectively inhibited the PC positive A. actinomycetemcomitans invasion of HUVEC to 41.33 ± 2.59% and 43.22 ± 2.67% of the control (Figure 1), respectively.
However, there was no significant difference between the MDC or CPZ pretreated and nonpretreated groups on adherence (Figure 2), which showed that the MDC and CPZ inhibition of clathrin could not stop A. actinomycetemcomitans adhesion to HUVEC.

β-Arrestins role in A. actinomycetemcomitans internalization by HUVEC
To study the role of β-arrestins in the process of A. actinomycetemcomitans adhesion and invasion of HUVEC, double-stranded siRNAs for β-arrestin-1, βarrestin-2 and control were chemically synthesized and transfected in HUVEC, and then the effects on A. actinomycetemcomitans adhesion and invasion of HUVEC were investigated.First, it was detected that intracellular level of β-arrestins was targetedly reduced after transfection when compared with untransfected group and negative control group (Figures 3 and 4).Then the invasion of PC positive A. actinomycetemcomitans into HUVEC was tested and it was 53.12 ± 2.81 and 56.00 ± 2.37% of the control (Figure 5), respectively.The invasion was effectively blocked by β-arrestins siRNA.However, there was no significant difference in the number of bacteria adhered to the cells (Figure 6), which indicated that β-arrestins siRNA could not inhibit the PC binding to PAFR on the cell surface.Besides, we also examined the effect of clathrin and βarrestins in PC negative A. actinomycetemcomitans groups, there was no significant difference in the adhesion and invasion of HUVEC between PC negative

DISCUSSION
In the present study, we showed new evidences of the mechanism involved in adherence and invasion in human vascular endothelial cells by A. actinomycetemcomitans bearing PC.We demonstrated here that clathrin and βarrestins participated in A. actinomycetemcomitans invasion of host cells.
Previous data have extensively indicated that A. actinomycetemcomitans interacts with host cells via a mechanism of specific receptors (Meyer et al., 1997).Schenken and coworkers had reported that PAFR had participated in the invading HUVEC by A. actinomycetemcomitans bearing PC (Schenkein et al., 2000;Purkall et al., 2002).Clathrin and β-arrestins have been shown to play important roles in regulating PAFR during bacterial endocytosis.S. pneumoniae required the scaffold function of the β-arrestins to recruit clathrin in PAFR-mediated endocytosis by endothelial cells (Radin et al., 2005).Besides, Smani et al. (2012) reported that clathrin and β-arrestin-1 and 2 are involved in A. baumannii invasion of lung epithelial cells.In this study, we found that clathrin inhibitors MDC and CPZ significantly inhibited A. actinomycetemcomitans bearing PC invasion of HUVEC.Additionally, β-arrestin-1 and -2 siRNA prevented A. actinomycetemcomitans internalizetion of HUVEC.These findings are the first report for clathrin and β-arrestin-1 and -2 that participated in A. actinomycetemcomitans invasion of endothelial cells.

Conclusion
In summary, we proposed that PC-positive A. actinomycetemcomitans may invade endothelial cells via a mechanism dependent upon the involvement of clathrin and β-arrestins in mediating the PAFR activation by bacterial PC.However, β-arrestins-mediated receptor internalization can also regulate signal transduction by activating signaling proteins such as ERK1/2, p38 MAPK and JNK (Vroon et al., 2006), which also take part in A. actinomycetemcomitans invasion of host cells.Therefore, further studies are needed to demonstrate these other mechanisms involved in β-arrestins-mediated infection by A. actinomycetemcomitans bearing PC.

Figure 1 .
Figure 1.Clathrin inhibitors block PC positive A. actinomycetemcomitans invasion of HUVEC.Confluent HUVEC monolayers on the 24-well plate were treated with the indicated concentrations of MDC and CPZ for 30 min before addition of the bacteria.Results are presented as relative invasion of PC positive A. actinomycetemcomitans (%).Data are means ± standard deviation of five independent experiments done in triplicate (*P<0.05).
Figure 2. Relative adhesion of PC positive A. actinomycetemcomitans (%) after clathrin inhibitors were blocked.The adhesion examination was carried out as described earlier.Data are means ± standard deviation (P<0.05).The adhesion showed no significant difference between pretreated and unpretreated groups.

Figure 4 .
Figure 4. Real-time reverse-transcription PCR was used to detect β-arrestin-1 mRNA expression in HUVEC as described in the 'materials and methods' section.Values shown are expressed as percent of level of each siRNA of β-arrestin in control-transfected HUVEC.Error bars indicate standard deviations (*P<0.05).

Table 2 .
Primers used in real time PCR.