Intracellular survival and vascular cell-to-cell transmission of Porphyromonas gingivalis

Background Porphyromonas gingivalis is associated with periodontal disease and invades different cell types including epithelial, endothelial and smooth muscle cells. In addition to P. gingivalis DNA, we have previously identified live invasive bacteria in atheromatous tissue. However, the mechanism of persistence of this organism in vascular tissues remains unclear. Therefore, the objective of this study was to analyze the ability of intracellular P. gingivalis to persist for extended periods of time, transmit to and possibly replicate in different cell types. Results Using antibiotic protection assays, immunofluorescent and laser confocal microscopy, we found that after a prolonged intracellular phase, while P. gingivalis can still be detected by immunostaining, the intracellular organisms lose their ability to be recovered in vitro. Surprisingly however, intracellular P. gingivalis could be recovered in vitro upon co incubation with fresh vascular host cells. We then demonstrated that the organism was able to exit the initially infected host cells, then enter and multiply in new host cells. Further, we found that cell-to-cell contact increased the transmission rate but was not required for transmission. Finally, we found that the invasion of new host cells allowed P. gingivalis to increase its numbers. Conclusion Our results suggest that the persistence of vascular tissue-embedded P. gingivalis is due to its ability to transmit among different cell types. This is the first communication demonstrating the intercellular transmission as a likely mechanism converting latent intracellular bacteria from state of dormancy to a viable state allowing for persistence of an inflammatory pathogen in vascular tissue.


Background
P. gingivalis, a gram-negative anaerobe, plays a critical role in the development of adult periodontitis, a chronic inflammatory disease [1]. Epidemiological studies have demonstrated a positive association between periodonti-tis and cardiovascular diseases [2]. The immune response to this organism correlates with atherosclerosis [3,4]. We have detected intracellularly located P. gingivalis in periodontal tissues from patients [5]. We and others have also demonstrated the presence of DNA from periodontal pathogens such as P. gingivalis in atheromatous tissues [6,7]. Most importantly, we recently demonstrated for the first time the presence of viable invasive P. gingivalis in atheromatous plaque [8], thus implicating this chronic inflammatory agent in direct contribution to the development of inflammatory lesions at remote sites. This discovery led us to the legitimate question of the mechanism that would allow this organism to penetrate vascular walls upon dissemination and to persist in human vascular tissue.
Oral epithelia are likely the primary site for P. gingivalis infection but this bacterial species can enter the circulation following tooth brushing and other dental procedures therefore periodontitis is known to cause transient and low-grade bacteremia in patients [9,10]. This makes the periodontal site an "open gate" to circulation.
In addition to numerous other virulence factors, the invasion ability of P. gingivalis allows it to invade multiple cell types including animal cell lines, human vascular and gingival epithelial cell lines in vitro [11][12][13][14][15][16][17]. Tissue invasion is very likely a key virulence factor for this bacterium as it provides 1) a "privileged niche" with access to host protein (nutritional) and iron substrates, 2) a sequestration from the humoral and cellular immune response, and 3) a means for persistence that is essential for a chronic pathogen.
Studies from several groups have demonstrated the ability of P. gingivalis to invade gingival epithelial cells [11][12][13]. Studies of invasion in epithelial cells demonstrated that 1) the invasion is mediated by the interaction between P. gingivalis fimbriae and β1 integrin receptors [18]. 2) host cell cytoskeletal rearrangements are required for the entry [12,19]; 3) while inhibitors of protein kinase activity have no obvious effect on invasion, suppression of protease activity inhibited invasion process [12]; 4) during the first four hours of invasion, P. gingivalis appears to be able to replicate inside cells [12,13], but then recoverable CFU counts decrease [20]. In fact, we have detected intracellular P. gingivalis immunohistochemicallyin periodontal tissues from patients, located in the perinuclear region [5].
Many bacteria capable of invasion have been found to reside in phagosomes. Invasion of host cells is a common strategy for bacteria to escape immunosurvelliance and hostile environment. In phagocytic cells, Mycobacterium tuberculosis persists in phagosomes (review by [21]). It stops the normal maturation of phagosomes into an acidic, hydrolytic active environment, a phenomenon referred to as inhibition of phagosome-lysosome fusion [22]. Similarly, Legionella pneumophila enters its host, amoebae and protozoa [23] through phagocytosis, then rapidly modifies the phagosome to create an environment that supports its replication. Again a critical modification is the prevention of phagosome-lysosome fusion [24,25]. Virulent Brucella abortus also replicates in phagosomes with an increase in intraphagosomal pH [26]. The inhibition of the fusion between phagosomes and lysosomes seems to be a common theme to ensure the survival of invaded bacteria in phagosomes. When the inhibition is overcome, the acidification of the phagosomes eliminates the intracellular microbes [27,28]. In some cases, cytoplasmic bacteria can be ultimately trafficked into the lysosomal compartment for elimination [29,28].
P. gingivalis was found to be able to reside in phagosomes following invasion of endothelial cells [16] and possibly modify the phagosome to delay its fusion with lysosomes [30]. Therefore, host cell invasion by P. gingivalis may play a critical role in the development of vascular inflammatory lesions.
This raises the general question, what is the fate of this intracellular organism? Does P. gingivalis actually persist in tissues while in fact it is uncultivable? Even more important for vascular pathology, can P. gingivalis penetrate the vascular tissue, invading different cell types? The data on the intracellular fate of the organism itself hasn't clarified these broad questions yet while their answer is central for further elucidation of the persistent presence of this organism in atheromatous tissue and for clarification of the impact of that presence on the vascular wall.
Although there is an abundance of studies on P. gingivalis invasion of different cell lines [31], there is only one communication on P. gingivalis persistence that focuses on intercellular spreading [32]. The authors demonstrated that the bacteria can spread cell-to-cell, similarly to Actinobacillus actinomycetemcomitans [33]. However, the study is performed with gingival epithelial cells that are not representative for vascular pathology. Further, only one cell type was studied. Therefore, in the present study we decided to analyze the fate of intracellular P. gingivalis during extended incubation using cell-to-cell transmission among various cell types that exist in the vasculature in vivo. Using intracellular visualization by in situ immunofluorescent microscopy and bacterial recovery on blood agar plates (BAP), our experiments addressed the ability of P. gingivalis to survive over extended periods of time inside epithelial, endothelial (EC) and smooth muscle cells (SMC). In particular, we investigated whether P. gingivalis can leave the infected cells to invade and replicate in new host cells, including cells from different tissue type.

P. gingivalis W83 invasion ability of KB, EC and SMC differs and can be increased by centrifugation
To investigate the ability of P. gingivalis to invade and survive inside epithelial, endothelial and smooth muscle cells, we first assessed the invasion ability of P. gingivalisW83 to epithelial (KB) cells, EC and SMC at multiplicity of infection (M.O.I.) of 100 using an antibiotic protection assay as described [16]. At three hours post invasion, the host cells were lysed and the number of invaded P. gingivalis was enumerated on BAP. As shown in Figure 1, the number of P. gingivalis colony-forming units (CFU) recovered on BAP varied among cell lines, suggesting differential invasion ability, as previously observed [14]. Among these three cell lines, the P. gingivalis recovery from KB on BAP was the highest while the recovery from SMC was the lowest. This may be due to the differential affinity between P. gingivalis and each cell type. This differential affinity could be overcome by spin inoculation. When increasing P.gingivalis and cell contact by spin inoculation (1000 × g, 10 minutes at room temperature), the bacterial invasion into each cell type increased by orders of magnitude ( Figure 2).

P. gingivalis W83 invasion doesn't affect the viability of the infected host cells
Since infection using the spin inoculation protocol can cause considerable stress to the host cells, we verified their viability using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] cell proliferation assay. Ten thousand cells were seeded per well in a 96-well format, reaching 70% confluency, similarly to the conflu-  C F U ency we used in the invasion assays. MTT assay measures the ability of living cells to convert yellow water soluble MTT into insoluble purple formazan. With uninfected cells as a control, P. gingivalis invasion at MOI of 100 with spin inoculation has no effect on cell proliferation/metabolism in all three cell types using MTT assays at 3 hr, 24 hr and 48 hr post infection (data not shown).

Spin-inoculation increases
To independently confirm the result of the MTT assays, invasion assays were also carried out where the host cells were counted using a hemocytometer at each time point (3 hr, 24 hr and 48 hr post invasion). Similarly, there was no difference in cell numbers between invaded cells and uninfected controls in all three cell types (data not shown).

Recovery of intracellular P. gingivalis W83 on blood agar plates (BAP) declines over time after invasion
In order to study the replication kinetics of the intracellular P. gingivalis, we harvested intracellular P. gingivalis using the antibiotic protection assay at various time points post invasion. To our surprise, as shown in Figure  3, we did not detect intracellular P. gingivalis amplification using the antibiotic protection assay at the described infection condition. Instead, a sharp decline in the number of P. gingivalis recovered on BAP at 24 hr post invasion was detected and no bacteria could be recovered at 48 hour time point from EC and SMC (only very low numbers of P. gingivalis could be recovered from KB cells). These experiments were repeated five times to confirm the unexpected data. When facilitating the invasion by spin inoculation, the number of P. gingivalis recovered on BAP at 3 hr time point increased 10 fold as described above ( Figure 2B), but still no P. gingivalis could be recovered on BAP from EC and SMC at 48 hr post invasion. Similarly, only very low number of P. gingivalis was recovered from KB cells at 48 hr time point (Table 1, "medium only" row).

In contrast to the recovery rate on BAP, immunofluorescent microscopy demonstrates the presence of a large number of P. gingivalis at 48 hour post invasion
Surprised by the loss of P. gingivalis recovery over time on BAP, we performed immunofluorescent microscopy to verify the status of intracellular P. gingivalis. P. gingivalis was easily found throughout the cultured cells at each of the time points, including the 48-hr time point. As demonstrated in Figure 4, at 48 hr post-invasion, while no P. gingivalis can be retrieved on BAP, the bacteria could be observed with the immunofluorescent staining and most were perinuclear in all three cell types. This is consistent with the observation made in primary oral epithelial cells [34]. Similarly, under the condition of spin inoculation, while no P. gingvalis was recovered on BAP, copious numbers of the intracellular P. gingivalis could be found by immunofluorescent staining (Figure 4).

P. gingivalis is able to transmit from infected to uninfected cells
Once we found that no P. gingivalis could be recovered on BAP from EC and SMC at 48 hours post invasion, we investigated the possibility of intracellular P. gingivalis leaving the host cells to invade and possibly replicate in new host cells.
Endothelial, SMC, and KB cells were infected with P. gingivalis at MOI of 100 using the spin inoculation protocol. Twenty-four hours post invasion, the invaded host cells were harvested by trypsinization, 2 × 10 4 of these initially infected cells were mixed with 8 × 10 4 fresh uninfected host cells and plated in 24 well plates using the uninfected host cell culture media. After twenty-four hours of co culturing (48 hours post initial invasion), the cells from each well were lysed by sterile water and plated on BAP. The results are shown in Table 1 (upper portion). Surprisingly, the amount of colony forming units (CFU) recovered on BAP increased above the zero or minimal recovery obtained after 48 hour post-infection in the initial cultures alone, indicating that P. gingivalis had transmitted and possibly replicated in the new host cells. Invasion kinetics of P. gingivalis W83   Smooth muscle cells were selected in order to utilize anti-SMC actin antibody that is able to stain intracellularly, therefore can be used to determine if the bacteria are intracellular. As described above, 2 × 10 4 P. gingivalis-invaded EC (iEC) were co cultured with 8 × 10 4 uninfected SMC cells. Twenty-four hours later (48 hours post initial invasion), the cells were fixed and stained using antibodies against P. gingivalis and SMC actin. The result indicated presence of P. gingivalis in the SMC cells by both immunofluorescent ( Figure 5A) and confocal ( Figure 5B) microscope analysis, confirming our hypothesis that P. gingivalis is able to leave primary host and enter new host cells in the co culture system ( Figure 5 and Table 1).

P. gingivalis is able to exit the primary host cells, to infiltrate the medium and to invade new host cells. The cell-cell contact is not required for bacterial cell-to-cell transmission
To determine whether cell-cell contact is required for the P. gingivalis transmission to new host cells, a Transwell system was utilized to separate the initially infected host cell from uninfected new host cells as described above. The

The cell contact between infected cells and new host cells increases the transmission rate
Furthermore, after the removal of Transwell inserts, we also lysed the cells at the bottom compartment by sterile H 2 O and plated the lysate on BAP. In concordance with the microscopy observations, P. gingivalis could be recovered from cells that had no direct contact with infected cells (Table 1, bottom). However, based on the number of P. gingivalis CFU recovered on the plates, the cell contact between infected cells and new host cells increases the transmission rate.

Discussion
Periodontitis, one of the most prevalent infectious diseases [35] is caused by a small number of bacteria, including P. gingivalis that we have shown to reside in gingival tissue from periodontal patients [5] and, even more significantly, in atheromatous plaque [8]. The main challenge for tissue-embedded P. gingivalis is to survive and we set out to explore a possible mechanism of its persistence.
Here we present data demonstrating that in our experimental set-up P. gingivalis strain W83 displays an invasive ability that differs by up to an order of magnitude in the tested cell lines and that this difference could be minimized by spin inoculation. The differential invasion efficiency observed for different cell types is likely due to different interactions between P. gingivalis and the types of cell surface receptors present on the surface of the different cell types that are involved in the invasion process. P. gingivalis fimbriae and hemagglutinating adhesins have been reported to serve as ligands for the initial attachment to the host cells [36,37]. Beta integrins [18], CD14 and the β2 integrins CD11b/CD182 [38] have been reported to be host cell receptors for P. gingivalis fimbriae. Achieving higher CFU numbers after spin-inoculation of the tested cell lines is likely due to the increased contact between these P. gingivalis ligands and the host surface receptors.
We also demonstrate that the 48-hr invasion (at M.O.I. of 100) of the studied three different cell types, KB, EC and SMC doesn't affect the viability of the host cells, in contrast to the data for gingival fibroblasts [39]. Again, this observation is most likely due to the different cell type, and in terms of invasion of primary EC is supported by a recent report [30]. This effect is plausibly due to the advantage for the bacterium to prevent the death of the host, thus evading the humoral or cellular immune response and securing a source of nutrients.
We next found that P. gingivalis could be detected intracellularly at 48 hr post invasion in large numbers by immunofluorescent microscopy. While different entry pathways and intracellular trafficking in various cell types have been proposed [40][41][42]16,11] we observed that at a relatively high M.O.I. of 100, virtually all host cells in culture contained several microbes, and that recovery of viable intracellular P. gingivalis was relatively high a few hours postinvasion, which was also expected. Over a period of 48 hr, however, while the level of intracellular localization did not change, the recovery of viable microbes on BAP diminished to virtually zero for SMC and EC (and very low numbers for KB). Most of the bacteria in all three cell types are perinuclear at this stage, similarly to what has been demonstrated in primary oral epithelial cells [34,19].
It is unclear why P. gingivalis could not be recovered on BAP at this stage. The inability to recover P. gingivalis on BAP has been reported before. For example, we found high amount of P. gingivalis genomic DNA in atheromas using Q-PCR [7] but no bacteria could be directly recovered on blood agar plates, as previously observed [8,43].
One of the possibilities is that P. gingivalis may not survive the intracellular environment. However, based on our previous data [8] and on the data presented in this study it can not be ruled out that P. gingivalis may actually enter a latent state to reside intracellularly, resulting in inability to be cultivated on BAP, and that it may leave such a state upon contact with fresh host cells.
Therefore, there are two possible scenarios with these observations. The first scenario is that some of sequestered intracellular P. gingivalis had become non-viable. Several killing mechanisms may be proposed, for example the bacteria may be killed by trafficking into the phagolysosomal or autolysosomal compartment [30]. The trigger for this to occur could be initiated by a high number of intracellular P. gingivalis or by depletion of intracellular nutrients. Such mechanism may be used to control the P. gingivalis population. Previously, another P. gingivalis strain (FDC381) has been reported to persist in KB cells for eight days [13]. Using the antibiotic protection assay, the amount of P. gingivalis FDC381 recovered on BAP from KB cells started to decline at day 2. However, in that case, KB was infected at a M.O.I. of 2, or 50-fold less than what we used in our experimental system. This therefore is in line with the hypothesis that the number of intracellular P. gingivalis may serve as a trigger to trafficking to a lysosomal compartment thus regulating the number of intracellular P. gingivalis.
However, this hypothesis does not rule out another possible scenario, that P. gingivalis may not be killed by host cells and that number of bacteria may be instead con-verted into a stage of dormancy. Such hypothesis, supported by ours and others' data, is that the cell-to-cell transmission converts latent intracellular bacteria from state of dormancy to a cultivable state. It explains the observations that dormant intracellular P. gingivalis could not be recovered in vitro by plating, but only after transmission to fresh host cells. In addition, further support of this hypothesis originates from the global genomic profile data of intracellular P. gingivalis [40]. Using microarray genomics, that study demonstrated that the majority of differentially regulated P. gingivalis genes (52 of 63) were down-regulated upon invasion. Even more interesting, a substantial number of the down-regulated genes (21 of 52) were involved in protein synthesis, transcription, and energy metabolism. Such specificity in the down regulation indicates a reduced growth rate of intracellular bacteria, or a latent state.
Our hypothesis that the cell-to-cell transmission converts latent intracellular bacteria from state of dormancy to a cultivable state was first made possible based on our previous data on identification of viable P. gingivalis in atherosclerotic tissue only after transfer to new host via co incubation with fresh cell culture [8]. Subsequently, cellcell transfer of P. gingivalis has been demonstrated within oral epithelial cells as well [32]. Further confirming our observations of leaving the primary infected cell, extracelullar P. gingivalis has been detected in invaded KB culture supernatants [20]. In the present study, this hypothesis is further supported by our experimental data demonstrating that P. gingivalis can spread intercellularly between the same as well as between different cell types, as a consequence substantially increasing the CFU count number when recovered in vitro. This cell-cell transmission took place even when the new cells were separated from the already invaded cells via Transwell set-up, although the numbers were 6-10 times lower, apparently due to the separation (Table 1). Finally, we demonstrated that cell contact between infected cells and new host cells increases the rate of transmission.
The mechanism of this transmission remains to be determined, but bacterial replication in the medium is unlikely for this strict anaerobe, especially providing its average replication time (5-6 hrs) and the rapid internalization by host cells (~10 min). In addition, we tested whether P. gingivalis can survive and replicate in media for up to 48 hours and the results were negative (data not shown). To differentiate between these two scenarios, intracellular trafficking, viability of intercellular P. gingivalis and its gene expression profile at 48 hours post-invasion in different cell types must be extensively studied in parallel.

Conclusion
In summary, here we present data demonstrating that in our experimental set-up P. gingivalis strain W83: (1) displays an invasive ability that differs by up to an order of magnitude in the tested cell lines and the difference could be minimized by spin inoculation, (2) could be detected intracellularly in large numbers at 48 hr post invasion by immunofluorescent microscopy, but most of the bacteria could not be recovered on BAP; (3) can spread intercellularly between the same as well as between different cell types; (4) the invasion of fresh host cells ends the dormant, uncultivable state allowing for P. gingivalis isolation in vitro, and (5) cell-cell contact between infected cells and new host cells increases the rate of transmission.
Therefore, our working model, supported by our observations is as follows. Upon invasion, P. gingivalis is able to reside/replicate inside the host cells for a limited time. A high number of P. gingivalis or the depletion of nutrients may then initiate the killing (or turning into a dormant stage) of P. gingivalis leading to control of bacterial population numbers, for example by trafficking it to the lysosomal compartments. In addition, facing hostile environment, some P. gingivalis would exit into intracellular space and invade new host cell, thus escaping from the dormant stage, further penetrating the vascular tissue transcellularly and becoming both invasive and cultivable in vitro.
Using such mechanism, P. gingivalis would control its population in vascular tissues yet allow for a persistent infection, similarly to infection and spreading among primary gingival epithelial cells [32]. With the epithelial host, there was a very low level of cell-to-cell transmission during the early stage of invasion (3 hr time point), but the rate of transmission increased substantially at later stages of infection (24 hr and 48 hr time point). The increased transmission rate at the late stage of infection is in agreement with our hypothesis of cell-cell transmission-mediated bacterial survival and persistence. Our observations support a model of atherosclerosis where the inflammation is initiated and/or exacerbated by invasive inflammatory agents that may endure intracellularly in a dormant stage, whose numbers are controlled but that can also escape the host, become invasive and infect new host cells thus persisting in the vascular tissue. This model is currently under further investigation. Studies of the molecular machinery of P. gingivalis cell-cell transmission will further elucidate the mechanisms by which P. gingivalis is able to establish persistent infection of vascular walls and ultimately contribute to vascular inflammations.

Endothelial cell isolation and culture conditions
The primary endothelial cells (EC) were provided by Hong Yu (University of Miami) and grown as described [44]. EC were isolated as follows. Control staining with anti-smooth muscle actin (SMA) antibody and anti-P. gingivalis antibody was negative (data not shown). The isolation protocol was approved by the Institutional Review Board at University of Miami.

Smooth muscle cell isolation and culture conditions
The primary smooth muscle cells (SMC) were also isolated from human saphenous veins and provided by Hong Yu (University of Miami  20 and still expresse smooth muscle actin. Control staining with anti-VWF antibody and anti-P. gingivalis antibody was negative (data not shown).

Antibiotic protection assay
The antibiotic protection assay was performed as described in [16]. In short, 10 5 cells were seeded per well in 24-well plates. Next day, the cell medium was removed, and 10 7 log-phase P. gingivalis suspended in 1.0 ml antibiotic-free cell culture medium were added (MOI of 100) and the cells were incubated for 90 minutes at 37°C, 5% CO 2 . The number of P. gingivalis was determined spectrophotometrically. For spin inoculation, the 24-well plate was spun at 1000 × g for 10 minutes prior to the 90minute incubation. The cells were then washed gently three times with HBSS (Invitrogen, Carlsbad, Calif.) and media containing gentamicin (300 μg/ml) and metronidazole (200 μg/ml) was added followed by incubation for another 60 minutes to kill extracellular P. gingivalis. The cells were then washed again three times with HBSS and maintained in their culture medium. At each time point (3 hours, 24 hours and 48 hours after the initial contact between the bacteria and host cells), 1 ml sterile ddH 2 O was added and cells were lysed for 20 minutes at room temperature. The lysate was collected and P. gingivalis was pelleted at 16,000 × g for 2 minutes before plating in quadruplicate on blood agar plates (BAP).

Cell proliferation assays
The cell proliferation assays were performed using a Vybrant MTT cell proliferation assay kit (Invitrogen, Carlsbad, Calif.) and cell counting. The MTT assay was performed in a 96 well format according to manufacturer's recommendation. Ten-thousand cells were seeded per well, reaching 70% confluency and then 1 × 10 6 P. gingivalis in 100 μl culture media were added per well. The plate was spun at 1000 × g for 10 minutes and incubated at 37°C, 5% CO 2 for 90 minutes. After gentamicin (300 μg/ml) and metronidazole (200 μg/ml) treatment, the cells were washed with HBSS and maintained in their culture medium. At each time point, the medium was replaced with 100 μl fresh medium, 10 μl of 12 mM MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide stock solution was added to each well and cells were incubated for 4 hours at 37°C. The reaction was terminated by the addition of 100 μl of SDS.HCl solution and further incubation for 12 hours at 37°C. Finally, each sample was mixed and absorbance at 570 nm was measured.
For manual counting, 1 × 10 5 cells were seeded per well in 24-well plates and infected with P. gingivalis at MOI of 100 with or without spin inoculation. After gentamicin (300 μg/ml) and metronidazole (200 μg/ml) treatment to kill the extracelullar bacteria, the cells were washed with HBSS and maintained in their culture media. At each time point, cells were trypsinized, pelleted and suspended in 10 μl tissue culture medium. The cells were then mixed with trypan blue and counted using a hemocytometer.