Autophagy Is Associated with Pathogenesis of Haemophilus parasuis

Haemophilus parasuis (H. parasuis) is a common commensal Gram-negative extracellular bacterium in the upper respiratory tract of swine, which can cause Glässer's disease in stress conditions. Research on the pathogenicity of H. parasuis has mainly focused on immune evasion and bacterial virulence factors, while few studies have examined the interactions of H. parasuis and its host. Autophagy is associated with the replication and proliferation of many pathogenic bacteria, but whether it plays a role during infection by H. parasuis is unknown. In this study, an adenovirus construct expressing GFP, RFP, and LC3 was used to infect H. parasuis. Western blotting, laser confocal microscopy, and electron microscopy showed that Hps5 infection induced obvious autophagy in PK-15 cells. In cells infected with strains of H. parasuis differing in invasiveness, the levels of autophagy were positively correlated with the presence of alive bacteria in PK-15 cells. In addition, autophagy inhibited the invasion of Hps5 in PK-15 cells. Autophagy related genes Beclin, Atg5 and Atg7 were silenced with RNA interference, the results showed that autophagy induced by H. parasuis infection is a classical pathway. Our observations demonstrate that H. parasuis can induce autophagy and that the levels of autophagy are associated with the presence of alive bacteria in cells, which opened novel avenues to further our understanding of H. parasuis-host interplay and pathogenesis.

Bacterial pathogenesis is a complex process, requiring multiple steps to establish infection and cause disease. Infection by H. parasuis requires invasion of host cells and evasion of host barriers such as serum complement (Cerda-Cuellar and Aragon, 2008) and macrophages (Yuan et al., 2012), thus resulting the inflammatory response. Virulent strains of H. parasuis can colonize and initiate infection by adhesion to and invasion of epithelial cells (Vanier et al., 2006;Frandoloso et al., 2012;Zhang et al., 2012Zhang et al., , 2013, indicating that these processes are important first steps in infection. A previous report demonstrated that the ability of biofilm production of virulent strains in vitro is higher than avirulent strains isolated from nasal cavity, which is associated with colonization of H. parasuis (Jin et al., 2006). Adherence and invasion of virulent strains to cells play important roles in the infection of H. parasuis. Though the mechanism for virulent strains entering bloosdstream is poorly understood, the invasion of virulent strains to cells is associated with systemic infection (Olvera et al., 2009). Intracellular bacterial pathogens have evolved many strategies to manipulate host cell functions during infection. For example, autophagy is a fundamental biological process, crucial in maintaining organismal homeostasis. It serves a housekeeping role by removing nonfunctioning proteins and damaged organelles, as well as intercellular pathogens, and has an adaptive role in protecting organisms against diverse pathogens (Cuervo, 2004;Levine and Kroemer, 2008). Autophagy can be induced by many kinds of cellular stresses, including hypoxia (Fang et al., 2015), nutrient  or growth factor deprivation (Ni et al., 2014), DNA damage (Houten et al., 2016), or pathogen infection Winchell et al., 2015). Numerous studies have shown that autophagy plays an important role in the innate immune responses against infection (Yuan et al., 2012;Jo et al., 2013;Wang et al., 2015). Conversely, pathogens have evolved strategies to take advantage of autophagy for infection, replication and survival (Dorn et al., 2002;Campoy and Colombo, 2009;Castrejon-Jimenez et al., 2015).
Most researches confirmed that autophagy functioned as an effective mechanism to compete with intracellular bacteria. H. parasuis is able to adhere to and invade epithelial cells, but whether autophagy is associated with H. parasuis pathogenesis is unknown. In this study, western blotting, transmission electron microscopy, and confocal microscopy were used to detect and monitor autophagy in PK-15 cells infected by H. parasuis. We showed that autophagy induced by H. parasuis infection is associated with the invasiveness of H. parasuis. Our results have advanced our understanding of the interplay between autophagy and the pathogenesis of H. parasuis, which will be help to develop new drugs against H. parssuis infection.

Reagents
Rabbit polyclonal antibody against LC3B was obtained from Sigma-Aldrich (Sigma, St. Louis, MO, USA), goat polyclonal antibodies against Beclin-1, Atg5, and Atg7, and GAPDH antibody were obtained from Santa Cruz Biotechnology (California, USA). Goat anti-mouse and anti-rabbit antibodies labeled with horseradish peroxidase (HRP) were acquired from Boster (Wuhan, China). The enhanced chemiluminescent (ECL) chromogenic kit was purchased from Biouniquer (Nanjing, China). Recombinant adenovirus tandom expressing GFP-RFP-LC3 was purchased from HANBIO (Shanghai, China), which was used to increase the expression of LC3 and facilitate the observation of fluorescence.
Bacteria were grown overnight and then inoculated in fresh TSB medium (1:100). Optical density at 600 nm was measured at 1 h intervals to establish growth curves. Bacteria from exponential phase cultures were serially diluted and plated onto TSA to determine the relationship between OD and viable bacterial counts (colony forming units).

Cell Culture
Porcine kidney (PK-15) cells were cultured at 37 • C in a humidified 5% CO 2 incubator and maintained in Dulbecco's Modified Eagle Media (DMEM, Gibco), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and antibiotics (100 U/ml of penicillin G and 100 mg/ml of streptomycin, Sigma, USA). Cells were sub-cultured when 90% cell confluence was attained. In 24-well tissue culture plates, cells were diluted in culture medium to a concentration of 10 5 cells per well and incubated to 90% confluence.

Infection Experiments
Bacteria were grown overnight in TSB broth at 37 • C with shaking as previously described (Cue et al., 1998), diluted 1:100 in fresh medium and incubated until reaching an optical density of 0.7 at 600 nm. Bacteria were pelleted by centrifugation at 8000 g for 1min, washed 3 times with PBS, and resuspended in DMEM at 0.25 OD (0.1OD = 1 × 10 8 cells/ml) for the infection experiments (Yuan et al., 2012). PK-15 cells cultivated overnight in serum-containing medium were washed with sterile PBS and transferred to serum-free and antibiotic-free medium. The cells were infected with H. parasuis suspensions at different multiplicities of infection (MOI). At various time points postinfection, cells were treated with different methods as follows.

Adherence and Invasion Assays
Adherence and invasion assays were performed as previously described (Cue et al., 1998;Vanier et al., 2006;Zhang et al., 2013) with modifications. Confluent monolayers of PK-15 cells were grown in 24-well plates and infected with H. parasuis suspensions at an MOI of 100. The plates were centrifuged at 800 × g for 10 min to promote contact of H. parasuis with the cell monolayer surface, incubated for 1 h in 5% CO 2 at 37 • C to allow bacterial adherence, and then vigorously washed five times with PBS to remove non-specifically attached bacteria.
For the adherence assay, after the removal of non-specifically attached bacteria, the plates were incubated for 10 min at 37 • C with 100 µl of 0.025% trypsin. Following incubation, cells removed by scraping were transferred to sterile centrifuge tubes and centrifuged for 1 min at 12,000 rpm. The supernatant was removed and the pellet was resuspended with an equivalent volume of TSB. Serial dilutions of the cell lysate (100 µl) were plated onto Trypticase Soy Agar and incubated for 48 h at 37 • C.
For the invasion assay, after the removal of non-specifically attached bacteria, culture medium containing two antibiotics (100 mg of penicillin G/ml and 5 mg of gentamicin/ml, Sigma) was added to each well, and the plates were incubated for 1 h at 37 • C in 5% CO 2 to kill any remaining extracellular H. parasuis. The monolayers were washed three times and cells were harvested as described above.
Bacterial adhesion/invasion was evaluated as the average number of bacteria per well (CFU, colony forming unit). All assays were performed in triplicate and repeated three times.

Western Blotting
Cell samples were lysed in RIPA buffer (Cell signaling Technologies) on ice for 30 min, then supernatant was collected through centrifugation at 12,000 rpm for 5 min. The lysate mixed with loading buffer was incubated for 8 min in a boiling water bath. Sample was loaded onto a 10% SDSpolyacrylamide mini-gel and subjected to electrophoresis. The proteins were transferred onto NC membranes (BioTrace TM NT, Pall Corporation, United States) and blocked for 2 h at room temperature using 10% non-fat milk blocking buffer. Membranes were incubated overnight at 4 • C with primary antibodies diluted to appropriate concentration in accordance with the instruction with PBST. After three rinses with PBST, the membranes were incubated for 45 min at room temperature with horseradish peroxidase-conjugated secondary antibody diluted 1:10000 (Wu et al., 2002). Bound antibody was visualized using an enhanced chemiluminescence detection kit (Super Signal West Pico; Pierce). For quantification, protein band density was analyzed using the image processing program ImageJ (Schneider et al., 2012).

Confocal Laser Scanning Microscopy
PK-15 cells were grown on coverslips in a 24-well plate and infected with adenovirus at an MOI of 1. After 24 h, a subset of the cells were pretreated with the autophagy activator rapamycin for 12 h, or the autophagy inhibitor 3-MA for 3 h, as positive and negative controls, respectively. Adenovirus infected/non-treated cells and adenovirus infected/3-MA treated were infected with H. parasuis at 0.1 MOI. Cells were washed five times with sterile PBS to remove non-specifically attached bacteria. Coverslips were mounted on microscope slides and samples were examined using a Zeiss LSM 700 confocal microscope.

Transmission Electron Microscopy (TEM)
Cells in 6-well plates were infected with adenovirus at an MOI of 1 for 24 h and treated with rapamycin, 3-MA+ H. parasuis, H. parasuis or left untreated, respectively. Cells were harvested, centrifuged for 1 min at 300 × g, resuspended, and fixed in 2.5% glutaraldehyde at 4 • C for 12 h, post-fixed in 1% osmium tetroxide, and finally dehydrated in a graded series of ethanol concentrations. Samples were treated in propylene oxide and embedded in epoxy resin for thin sectioning. Sections were double stained with uranyl acetate and lead citrate before being observed by TEM (Faulkner and Garduno, 2002;Zhang et al., 2015).

Cell Transfection
siRNA fragments specifically targeting Atg5, Atg7, Beclin-1, and one nonspecific random fragment (NC) were synthesized by Invitrogen. All siRNA sequences are listed in Table 1. For optimizing the conditions for gene silencing, PK-15 cells were seeded in 24-well plates at 1 × 10 5 cells per well. At approximately 70% confluence, cells were incubated with several concentrations of siRNA fragments for different lengths of time, following the manufacturer's instructions. 24 h after transfection, cells were harvested and lysed for Western blot analysis. Then, the cells transfected with optimal siRNA fragments for Atg5/Atg7 (0.5 µg/well) and Beclin-1 (0.17 µg/well), were infected with H. parasuis suspensions at 0.1 MOI for 6 h, and then harvested and lysed for Western blot analysis.

Statistics
Three biological replicates were performed for every experiment and results were recorded as mean ± SD and GraphPad Prism 5 was used to analyze difference between groups (one-way ANOVA; Tukey's post-hoc test, * * * p < 0.001, * * p < 0.01, * p < 0.05). ImageJ software was used to confirm the band strength of WB and fold difference compared with control was labeled with number.

H. parasuis Infection Induces Autophagy
To determine whether infection by H. parasuis can induce autophagy, PK-15 cells were infected with adenovirus. After confirming successful expression of GFP-RFP-LC3, the PK-15 cells were infected with a highly virulent strain Hps5, in a dose-and time dependent manner (Figures S1, S2). We observed that Hps5 infection with a bacterial:cell multiplicity of infection (MOI) of 1:10 induced the most significant LC3-II level at 6 h ( Figure S2, p < 0.001). Surprisingly, compared with control group, the level of LC3-II was decreased at 1-2 h and increased at 4-6 h during the infection ( Figure S2). Autophagy induced in PK-15 cells was further confirmed with western blotting, confocal laser scanning and transmission electron microscopy. PK-15 cells were first infected with adenovirus, then cells were either (i) infected with Hps5, or (ii) treated with rapamycin for 12 h, or (iii) treated with 3-MA for 3 h before infected with Hps5, or (iv) left untreated and uninfected. As shown in Figures 1A,B, cells infected with Hps5 or treated with rapamycin exhibited increased LC3 punctuation. Consistent with these results, western blot analysis showed that LC3-II levels was increased significantly in cells infected with H. parasuis (Figure 2).
Transmission electron microscopy results showed that the autophagosome, a double membrane structure with heterogeneous contents, is apparent in cells infected by Hps5 (Figure 3B) or treated with rapamycin ( Figure 3C).
Uninfected/untreated cells (−) (Figure 3A) and cells treated with 3-MA before infection with H. parasuis ( Figure 3D) have no obvious autophagosomes. In summary, these results demonstrate that Hps5 infection induces autophagy in PK-15 cells.

Autophagy Is Associated with Virulence of H. parasuis
Adherence and invasion assays were performed to determine if the autophagy induced by H. parasuis results from cell adherence or cell invasion. Briefly, PK-15 cells were infected at MOI 100 with strains Hps5 (highly virulent), Hps4 (less virulent), and Hps11 (avirulent) for 1 h. As shown in Figure 4A, Hps5 adhered to PK-15 cells was 6-fold more than Hps4 and 17-fold more than Hps11; In Figure 4B, invasion Hps5 in PK-15 cells was 5-fold more than Hps4 and 8-fold more than Hps11.
To explore the relationship between autophagy and H. parasuis pathogenesis, PK-15 cells preinfected with adenovirus at 0.1 MOI were infected with H. parasuis strains having different virulence: Hps5 (highly virulent), Hps4 (less virulent), and Hps11 (avirulent). Six hours after infection, cells were collected for western blot analysis. Using the mock-infected control as a baseline, the level ratio of LC3 II/GAPDH were calculated. The ratios were 1.50, 1.28, and 1.15 for the infection with Hps 5, Hps 4, and Hps 11, respectively ( Figure 5A).
In another experiment, PK-15 cells pre-reated with adenovirus were infected with viable and UV-inactivated Hps5 at MOI 100, incubated, and then harvested for Western blot analysis to detect LC3-II level. The results showed that viable H. parasuis induces more autophagy than either the mock-infected control or inactivated Hps5 ( Figure 5B). Given these results, we concluded that autophagy is predominately induced in porcine kidney epithelial cells by the presence of alive bacteria in cells.

Autophagy Inhibits H. parasuis Invasion in PK-15 Cells
Although autophagy ordinarily functions as a defense against bacterial invasion, some pathogens subvert the autophagic pathway to obtain nutrition for survival and replication, and ultimately to establish a persistent infection. In order to explore the role of autophagy in Hps5 infection, PK-15 cells were infected with adenovirus. After confirming successful expression of GFP-RFP-LC3, the cells were incubated with the autophagy activator rapamycin (1 µM, 12 h). Having confirmed that autophagy was induced in pretreated cells in comparison with untreated cells (Figure 6A), the treated and untreated cells were infected with FIGURE 4 | Virulence associated with H. parasuis adherence and invasion to cells. PK-15 cells were infected with Hps5 (highly virulent), Hps4 (less virulent), or Hps11 (avirulent) at 100 MOI, adherence (A) and invasion (B) results were recorded (one-way ANOVA; Tukey's post-hoc test, **p < 0.01, ***p < 0.001). Hps5 at MOI 100 and then the invasion assay was performed as described above. As shown in Figure 6B, the number of intracellular bacteria decreased significantly in rapamycin treated cells (p < 0.05). This result suggests that autophagy inhibited the invasion of H. parasuis.

FIGURE 6 | Autophagy inhibits H. parasuis invasion to PK-15 cells. (A)
PK-15 cells were pretreated with adenovirus expressing GFP-RFP-LC3 for 24 h and then autophagy was induced in PK-15 cells by treated cells with rapamycin (1 µM, 12 h), the untreated cells were used as control. The level of LC3 II was identified with western blotting and analyzed using the software ImageJ. GAPDH was used as a loading control. (B) Invasion assay were performed using cells treated with rapamycin or not (one-way ANOVA; Tukey's post-hoc test, * p < 0.05). Data are representative of three individual experiments with similar results.
The concentration and timing of interference effects were firstly determined by western blot analysis, the best interference result was shown in Figure 7A. After transfection using optimal conditions, PK-15 cells were infected by Hps5 (highly virulent), and untransfected cells were infected as controls. The level of LC3-II was significantly reduced by all three specific interfering fragments ( Figure 7B). We conclude that Atg5, Atg7, and Beclin-1 play an important roles in the signaling pathway of the autophagy induced in PK-15 cells by H. parasuis infection.

DISCUSSION
Autophagy, a crucial homeostasis mechanism, is associated with innate immunity and the inflammatory response. In recent years, the roles of autophagy in intracellular bacterial infections have attracted increasing interest. Pathogenic microorganisms such as Pseudomonas aeruginosa (Yuan et al., 2012) and Salmonella (Birmingham and Brumell, 2006) activate autophagy in host cells, while others have evolved mechanisms to subvert this host cell defense mechanism and replicate successfully. For example, some pathogens escape into the cytoplasm to avoid lysosomal killing (Pizarro-Cerda and Cossart, 2006), others remain inside vacuolar phagosomes and hamper their maturation into lysosomes, and some utilize the host's defense mechanisms to their advantage (Colombo, 2005), such as Brucella (Brumell, 2012). Most studies of bacterial autophagy have focused on intracellular pathogens, such as Mycobacterium tuberculosis (Kimmey et al., 2015) and Brucella, but extracellular pathogens such as Pseudomonas aeruginosa (Yuan et al., 2012) and Acinetobacter baumannii (Rumbo et al., 2014) are also known to induce autophagy through cell invasion.
H. parasuis, a Gram-negative extracellular bacterium, causes significant losses on the swine industry throughout the world. Research on the pathogenicity of H. parasuis has focused mainly on immune evasion and bacterial virulence factors, while few studies have examined interactions between H. parasuis and its host, including the relationship between autophagy and H. parasuis infection. Previous studies have demonstrated that the ability of H. parasuis to invade PK-15 cells is correlated with virulence (Frandoloso et al., 2012). In this study we examined the relationship between autophagy and H. parasuis infection. We showed that H. parasuis can induce obvious autophagy in PK-15 cells at MOI of 0.1 ( Figure S1). Interestingly, H. parasuis can decrease autophagy in the early phase (1-2 h) of H. parasuis infection, but which increased in 4-6 h after infection, which may suggest an evolve mechanism for the interplay between H. parasuis and host. Other pathogens are also known to depress autophagy; for example, the human cytomegalovirus (HCMV) inhibits autophagy early in infection by increasing the expression of the rapamycin target protein (Chaumorcel et al., 2008). We also demonstrated a positive correlation between H. parasuis invasion and the level of induced autophagy. Consequently, there is a need to further explore the factors that influence the invasive ability of H. parasuis.
The invasion ability of H. parasuis is positively correlated with the virulence (Frandoloso et al., 2012). In our study, different virulent strains were used to adhere to and invade PK-15 cells with different MOI, the results showed that the infection with 100 MOI of H. parasuis gived best adherence and invasion (data not shown). So, we can conclude that the ability of adherence and invasiveness of H. parasuis is positively correlated with the virulence of bacteria. In addition, we also found different virulent strains of H. parasuis induced different level of autophagy in PK-15 cells. The highly virulent Hps5 induced stronger autophagy level than less virulent Hps4 and avirulent Hps11. Compared with live Hps5 infection, UV-inactivation Hps5 induced weak autophagy. Combining these results, we can draw a conclusion that autophagy induced by H. parasuis is positively associated with the presence of alive bacteria in cells (invasion).
Pre-treat PK-15 cells with rapamycin, the invasion result showed that autophagy effectively inhibited the invasion of Hps5, which suggested that autophagy is a defense mechanism of PK-15 cells to inhibit the invasion of H. parasuis.
To date, more than 30 autophagy related genes (Atg) and their proteins have been identified. These proteins take an active part in the shaping of isolation membranes and autophagosomes, and participate in the fusion process of autophagosome and lysosome, constituting the core mechanisms of autophagy (Yamaguchi and Otsu, 2012). Among them, 15 genes (Atg1-10, Atg12-14, Atg16, Atg18) are essential for all types of autophagy (Longatti and Tooze, 2009). Atg7-Atg4-Atg8, Atg12-Atg7-Atg5 are two classical signaling pathways of autophagy, which are both dependent on Atg6 (Beclin-1 in mammals). In our study, three autophagy related genes Beclin-1, Atg5, and Atg7 were silenced with siRNA fragments. We found that proteins expressed by these three genes played important roles in the autophagy process induced by H. parasuis. Thus, our data identified the classical Beclin-1-Atg5-Atg7 autophagy pathway in PK-15 cells induced by H. parasuis.
In order to effectively invade PK-15 cells, H. parasuis decreased autophagy level at early stage of infection. With the increasing of alive bacteria in cells, high level of autophagy was induced to inhibit the invasion of H. parasuis. So, autophagy is a defense mechanism of PK-15 cells to inhibit the infection of H. parasuis. Our study is helpful to elucidate the signal pathways induced by H. parasuis infection and development of novel drugs against this bacteria.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.01423 Figure S1 | Optimization of MOI of H. parasuis infection associated with autophagy. PK-15 cells pretreated with adenovirus expressing GFP-RFP-LC3 for 24 h were infected with Hps5 at different MOIs. Prior to infection, cells were treated with rapamycin (1 µM) for 12 h. The level of LC3 II was detected by western blotting (A) and quantitated using the ImageJ software package (B) (one-way ANOVA; Tukey's post-hoc test, * * p < 0.01, * * * P < 0.001). GAPDH was used as a loading control. Figure S2 | Optimization of time of H. parasuis infection associated with autophagy. PK-15 cells pretreated with adenovirus expressing GFP-RFP-LC3 for 24 h were infected with Hps5 (MOI = 0.1) or mock infected and analyzed at 1, 2, 4, and 6 h post-infection. The level of LC3 II was detected by western blotting and quantitated using the ImageJ software package (one-way ANOVA; Tukey's post-hoc test, * * p < 0.01, * * * P < 0.001).