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Enterococcus faecalis alters endo-lysosomal trafficking to replicate and persist within mammalian cells

['Ronni A. G. Da Silva', 'Singapore Centre For Environmental Life Sciences Engineering', 'Nanyang Technological University', 'Singapore-Mit Alliance For Research', 'Technology', 'Antimicrobial Drug Resistance Interdisciplinary Research Group', 'Wei Hong Tay', 'Foo Kiong Ho', 'Frederick Reinhart Tanoto', 'Kelvin K. L. Chong']

Date: 2022-06

Enterococcus faecalis is a frequent opportunistic pathogen of wounds, whose infections are associated with biofilm formation, persistence, and recalcitrance toward treatment. We have previously shown that E. faecalis wound infection persists for at least 7 days. Here we report that viable E. faecalis are present within both immune and non-immune cells at the wound site up to 5 days after infection, raising the prospect that intracellular persistence contributes to chronic E. faecalis infection. Using in vitro keratinocyte and macrophage infection models, we show that E. faecalis becomes internalized and a subpopulation of bacteria can survive and replicate intracellularly. E. faecalis are internalized into keratinocytes primarily via macropinocytosis into single membrane-bound compartments and can persist in late endosomes up to 24 h after infection in the absence of colocalization with the lysosomal protease Cathepsin D or apparent fusion with the lysosome, suggesting that E. faecalis blocks endosomal maturation. Indeed, intracellular E. faecalis infection results in heterotypic intracellular trafficking with partial or absent labelling of E. faecalis-containing compartments with Rab5 and Rab7, small GTPases required for the endosome-lysosome trafficking. In addition, E. faecalis infection results in marked reduction of Rab5 and Rab7 protein levels which may also contribute to attenuated Rab incorporation into E. faecalis-containing compartments. Finally, we demonstrate that intracellular E. faecalis derived from infected keratinocytes are significantly more efficient in reinfecting new keratinocytes. Together, these data suggest that intracellular proliferation of E. faecalis may contribute to its persistence in the face of a robust immune response, providing a primed reservoir of bacteria for subsequent reinfection.

Enterococcus faecalis is often isolated from chronic wounds. Prior to this study, E. faecalis has been observed within different cell types, suggesting that it can successfully colonize intracellular spaces. However, to date, little is known about the mechanisms for E. faecalis intracellular survival. Here, we describe key features of the intracellular lifestyle of E. faecalis. We show that E. faecalis exists in an intracellular state within immune cells and non-immune cells during mammalian wound infection. We show that E. faecalis can survive and replicate inside keratinocytes and macrophages, and intracellularly replicating E. faecalis are primed to more efficiently cause reinfection, potentially contributing to chronic or persistent infections. To establish this intracellular lifestyle, E. faecalis is taken up by keratinocytes primarily via macropinocytosis, whereupon it manipulates the endosomal pathway and expression of trafficking molecules required for endo-lysosomal fusion, enabling E. faecalis to avoid lysosomal degradation and consequent death. These results advance our understanding of E. faecalis pathogenesis, demonstrating mechanistically how this classic extracellular pathogen can co-opt host cells for intracellular persistence, and highlight the heterogeneity of mechanisms bacteria can use to avoid host-mediated killing.

Funding: Funding for this work was provided by the National Research Foundation and Ministry of Education Singapore under its Research Centre of Excellence Program, by the National Research Foundation under its Singapore NRF Fellowship program ( https://www.nrf.gov.sg/funding-grants/nrf-fellowship ) to KAK (NRF-NRFF2011-11), by the Ministry of Education Singapore ( https://researchgrant.gov.sg/Pages/GrantCallDetail.aspx?AXID=MOET2EP2-01-2021&CompanyCode=moe ) under its Tier 2 programs to KAK (MOE2014-T2-1–129 and MOE2018-T2-1-127), by the Ministry of Education Singapore Tier 1 grants to A.L. (MOE RG136/17 and MOE RG39/14), and by an NTU Start-up grant to AL. RAGDS is supported by the National Research Foundation, Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program, through core funding of the Singapore-MIT Alliance for Research and Technology (SMART) Antimicrobial Resistance Interdisciplinary Research Group (AMR IRG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we sought to understand how E. faecalis persist within mammalian cells and how intracellularity contributes to pathogenesis. Using a mouse model of wound infection, we found viable E. faecalis within both immune and non-immune cells at the wound site up to 5 days after infection and provide evidence that intracellular E. faecalis is found in an active state of replication in vivo. Using an in vitro model of keratinocyte infection, we show that E. faecalis is taken up into these cells via macropinocytosis into single-membrane bound compartments, whereupon they can persist and manipulate the endosomal pathway. We show that internalized E. faecalis rarely co-localize with Cathepsin D and a subset of intracellular bacteria ultimately undergoes replication. Interestingly, E. faecalis infection results in a marked reduction of Rab5 and Rab7 protein levels, which may explain how E. faecalis prevent endo-lysosomal fusion. Finally, we show that E. faecalis derived from the intracellular niche are primed to more efficiently reinfect new keratinocytes. Together, our data are consistent with a model in which a subpopulation of E. faecalis are taken up into mammalian cells during wound infection, providing immune protection and a replicative niche, which may serve as a nidus for chronically infected wounds.

Once internalized, intracellular bacteria can be trafficked via the endo-lysosomal pathway. In this pathway, the small GTPase Rab5 regulates early endosome/macropinosome formation while Rab7, via replacement of Rab5, is required for the maturation of early to late endosomes, as well as for the fusion of late endosomes with lysosomes [ 21 ]. Late endosome-lysosome fusion is a critical step for the formation of an acidic and degradative compartment that eliminates bacteria, yet bacteria have evolved multiple mechanisms to interfere with this process [ 22 ]. For instance, Mycobacterium tuberculosis prevents Rab7 recruitment and, consequently, phagosome maturation, by interfering with Rab5 effectors, which are auxiliary proteins that support Rab5 conversion to Rab7 [ 23 , 24 ]. Listeria monocytogenes also inhibits Rab7 recruitment by inhibiting Rab5 GDP exchange activity in host cells [ 25 ]. Coxiella burnetii can localize to compartments labelled with Rab5 and LAMP1 (a marker of the late endosome/lysosome) but not Rab7 [ 26 , 27 ]. However, very little is known about the intracellular trafficking of E. faecalis and whether it can manipulate this pathway for its survival.

Intracellular bacteria were isolated from infected keratinocytes and used as the inoculum (intracellular in vivo) for reinfection of new monolayers of keratinocytes. Parallel infections at various MOI were performed using E. faecalis not yet exposed to keratinocytes, grown planktonically in vitro as the inoculum. (A) Infections proceeded for 3 h followed by 1 h of antibiotic exposure to kill the extracellular bacteria prior to intracellular CFU enumeration. (B) Infections proceeded for 3 h prior extensive washing to remove non-adhered extracellular bacteria, prior to enumeration of total cell-associated (both extracellular adhered and intracellular) CFU. Each circle represents CFU data averaged from 3 separate wells from a single biological experiment, showing a total of 5–7 independent experiments. Data are represented in the figure as the recovery ratio, which for (A) intracellular is the CFU recovered divided by the inoculum CFU and for (B) cell-associated is the CFU recovered divided by the non-cell-associated extracellular CFU in the same well, to account for cell growth during the assay. Horizontal black line indicates the mean for each condition. *p<0.05, **p<0.01 Kruskal Wallis test with Dunn’s post test.

To investigate whether internalization of E. faecalis into keratinocytes provides an advantage for subsequent reinfection, we harvested intracellular bacteria and measured its ability to reinfect keratinocytes. An initial infection was performed at MOI 50 for 3 h to isolate intracellular bacteria. Intracellular-derived bacteria were then used for reinfection of keratinocytes at an MOI of 0.1, the highest MOI practically attainable given the low intracellular CFU, for another 3 h. 1 h of gentamicin and penicillin treatment was performed after both the initial infection and the second round of infection. Internalization recovery ratios were determined by comparing inoculum CFU to intracellular CFU bacteria during the reinfection assay. Parallel experiments with E. faecalis not yet exposed to keratinocytes at comparable MOI showed that reinfection with intracellular-derived bacteria resulted in significantly higher internalization rates, as shown by the recovery ratio ( Fig 8A ) . Intracellular growth exclusively promoted reinfection, because total cell associated bacteria comprising both adherent and intracellular bacteria, was not significantly different from a planktonically grown inoculum ( Fig 8B ) . These results are similar to observations made in S. pyogenes, where longer periods of internalization in macrophages increased recovered CFU during subsequent reinfections [ 41 ]. Taken together, these data suggest that internalized E. faecalis can more efficiently reinfect host cells.

(A) Spinning disk confocal microscopy and correlative TEM of HaCaTs stably expressing LAMP1-mCherry infected with E. faecalis-GFP at 18 hpi. Confocal images are maximum intensity projections of 4–5 optical sections (~2 μm z-volume). (B and C) Enlarged views of the two areas highlighted in (A); panels B and C show the boxed areas 1 and 2 in (A), respectively. Large arrowheads indicate E. faecalis containing vacuoles, small arrows indicate LAMP1+ (LAMP1+ve) compartments. Note that E. faecalis is present in LAMP1- (LAMP1-ve) vacuoles in (B), and in LAMP1+ (LAMP1+ve) vacuoles in (C). LAMP1+ve compartments appear electron-dense (see TEM panel in C). * indicates a bacterium with altered appearance, possibly due to partial degradation. (D and E) Representative high magnification TEM images of LAMP1-ve (D) and LAMP1+ve E. faecalis containing vacuoles corresponding to data shown in (B) and (C), respectively. The large arrow in (E) indicates an electron-dense LAMP1+ve compartment in close proximity to an E. faecalis containing vacuole. Arrowheads in the lower panels indicate the presence of a single layer membrane surrounding the bacterial vacuole. (F) High magnification view of an E. faecalis containing vacuole. The vacuolar membrane (VM), the bacterial envelope (BE), and the septum are indicated. (F and G) 3D surface rendering of representative E. faecalis containing vacuoles reconstructed from serial TEM sections. An E. faecalis containing vacuole containing a LAMP1+ve multilamellar body (MLB) is shown in (G), while the vacuole shown in (F) is LAMP1-ve and does not contain a MLB. (See S12 Fig for data related to F-H, and S13 Fig for data related to D).

To visualize the association between E. faecalis-containing compartments and endo-lysosomal organelles with greater resolution, we turned to correlative light and electron microscopy (CLEM). We created a HaCaT cell line that stably expresses LAMP1-mCherry and infected these cells with GFP-expressing E. faecalis for 18 h (3 h followed by 15 h antibiotic treatment). Confocal microscopy of fixed cells enabled us to locate E. faecalis and LAMP1+ compartments in infected cells before processing them for serial section transmission electron microscopy (TEM) (Figs 7 , S12 , and S13 ) . These experiments revealed several features of E. faecalis intracellular infection that we could not appreciate using fluorescence microscopy alone. First, we observed E. faecalis in LAMP1+ compartments (8/26 or 30% of the observed intracellular E. faecalis) as well as in vacuoles that appeared to be devoid of LAMP1 (18/26) ( Fig 7A–7C ) , which is in line with our immunofluorescence microscopy data (Figs 4E and 4F , 5 , and S8B ) . Second, and importantly, regardless of the degree of colocalization with LAMP1, E. faecalis-containing vacuoles were invariably bounded by a single membrane ( Fig 7D–7H ) . In addition, most internalized bacteria appeared to be morphologically intact, with a uniform density and a clearly defined septum and bacterial envelope (Figs 7D–7H and S13A ) . Third, we did not find examples of multiple replicating E. faecalis within a single LAMP1+ compartment leading to membrane distension, as predicted by our immunofluorescence imaging (Figs 4E and 4F , 5 and S10 ) . Rather, we observed at most two diplococci within a single compartment (Figs 7D and S13B ) . Finally, although, we observed that LAMP1+ electron dense compartments of unknown nature (potentially lysosomes which are LAMP1+ organelles with dense ultrastructural appearance) were often located in close proximity to E. faecalis containing vacuoles, we did not observe any obvious fusion events between the two compartments ( Fig 7C and 7E ) . In some instances, however, vacuoles harbouring E. faecalis appeared to contain LAMP1 multi-lamellar bodies (MLBs) (Figs 7G , S12 and S13 ). Together, these EM data confirm both that internalized E. faecalis can survive in late endosomal organelles and that there is heterogeneity within the intracellular niches for this organism. Furthermore, E. faecalis-containing vacuoles do not exhibit lysosomal features and do not appear to fuse with lysosomes. These findings raise the possibility that E. faecalis could be hijacking the endo-lysosomal pathway, altering organelle identity to prevent lysosomal recognition, and allowing for intracellular survival, replication and eventual escape.

Rab5 and Rab7 are small GTPases that are critical for the formation of early and late endocytic compartments. To test whether E. faecalis infection affects Rab protein levels, we analyzed Rab5 and Rab7 proteins in the infected keratinocyte population, following infection with either E. faecalis strain OG1RF or strain V583. At 4 hpi, E. faecalis infection with either strain resulted in significantly lower Rab5 protein levels ( Fig 6A and 6B ) . Somewhat unexpectedly, although we observed Rab7 associated with some E. faecalis compartments, we also observed a global reduction in Rab7 protein levels for both strains at 4 hpi. While Rab7 levels were restored by 24 hpi for both strains, Rab5 in V583-infected keratinocytes remained lower at 24 hpi, which may correlate with greater CFU and intracellular survival for V583 compared to OG1RF ( S1F and S1G Fig ) . The expression of other endo-lysosomal proteins, such as Cathepsin D and LAMP1, were unchanged upon E. faecalis infection ( S11 Fig ) , indicating that E. faecalis selectively interferes with the levels of endosomal Rab5 and Rab7 proteins. Taken together, the combined E. faecalis-mediated reduction in Rab expression, coupled with the ability of nearly 70% of E. faecalis-containing compartments to avoid Rab7 recruitment (Figs 4 and S8 ) could explain the lack of colocalization of Cathepsin D with E. faecalis-containing compartments ( Fig 5 ) and is consistent with the conclusion that most E. faecalis-containing compartments do not fuse with lysosomes. Infected and non-infected HaCaT cells were both maintained under the same antibiotic treatment for protein collection, therefore observed differences in protein levels are not a consequence of antibiotic exposure.

(A) CLSM of infected keratinocytes stained for the lysosomal protease Cathepsin D (late endosome/lysosome) and LAMP1 (late endosome/lysosome; polyclonal antibody) at 24 hpi. Bottom panel shows the boxed region above. Images are maximum intensity projections of 4–5 optical sections (~2 μm z-volume) and are representative of 3 independent experiments. Scale bars: top panel: 10 μm; C bottom panel: 5 μm. (B) CLSM of infected keratinocytes stained for the lysosomal protease Cathepsin D (late endosome/lysosome) and LAMP1 (late endosome/lysosome; monoclonal antibody) at 4 hpi and 24 hpi (C) Fluorescence intensity of Cathepsin D (Alexa Fluor 568, blue), LAMP1 (Alexa Fluor 647, red) and E. faecalis (pDasherGFP, green) were assessed over a linear segment (histograms) and scored as labelled by visualization of orthogonal views and 3D projections on Imaris 9.0.2. Arrow indicates points of colocalization. Images are representative of three independent experiments. Measurements (percentages) are derived from a minimum of 8 individual confocal images per time point (2–3 images per biological replicate).

While E. faecalis can survive in murine macrophages by resisting acidification, which in turn prevents fusion with lysosomes [ 10 ], this has not been previously documented in epithelial cells. Our results showing that E. faecalis can replicate in keratinocytes led us to hypothesize that fusion between late endosomes and lysosomes may be impeded, permitting intracellular survival. To determine if lysosomes fuse with E. faecalis–containing late endosomes, we first immunolabeled infected cells at 24 hpi for LAMP1 (with a polyclonal antibody) and the lysosomal protease Cathepsin D. We found that LAMP1 and Cathepsin D colocalized in infected and non-infected cells, as expected. However, when looking at E. faecalis-containing compartments, while 47% (30/64) of internalized E. faecalis were observed in LAMP1+ compartments, these compartments were conspicuously devoid of Cathepsin D ( Fig 5A , white arrows) . The remainder (53% of internalized bacteria (34/64)) did not colocalise with either LAMP1 or Cathepsin D. We validated this finding using a monoclonal antibody against LAMP1 concomitantly with Cathepsin D immunolabeling. We confirmed that the majority of intracellular E. faecalis escaped lysosomal fusion with only 4% (3/69) and 8% (4/50) of the observed intracellular compartments containing E. faecalis labelled with Cathepsin D and LAMP1 simultaneously at 4 hpi and 24 hpi, respectively (Figs 5B, 5C , and S9 ) . Finally, we also observed a complete lack of colocalization between E. faecalis-containing compartments and M6PR (mannose-6-phosphate receptor, a late endosome/pre-lysosome marker), which delivers lysosomal hydrolases to pre-lysosomal compartments ( S10 Fig ) . Importantly, LAMP1+ compartments containing E. faecalis often appeared distended, particularly at 24 hpi (Figs 5A and S10 ) . Based on these observations, we conclude that E. faecalis escapes lysosomal fusion. Moreover, we propose that intracellular replication occurs within late endosomes until a bacterial threshold is reached, whereupon the compartment is unable to accommodate additional bacteria leading to compartment and/or cell lysis.

(A) CLSM of infected keratinocytes labelled with antibodies against Rab5 (Alexa fluor 568, red, early endosome) at 30 min, 1 hpi and 3 hpi. (B) Representative fluorescence intensity profiles of immunolabeled Rab5 and fluorescent E. faecalis (pDasherGFP, green) was assessed over a linear segment of E. faecalis cells (derived from the white lines shown in panel A). Labeling of individual cells was manually scored using a combination of histogram overlap (where each cell is a peak on the histogram) and visualization of orthogonal views and 3D projections generated using Imaris 9.0.2. Arrows indicate points of colocalization. Images are representative of three independent experiments. Percentage of E. faecalis associated with Rab5 was derived from a minimum of 8 individual confocal images per time point (2–3 images per biological replicate). (C) CLSM of infected keratinocytes labelled with antibodies against Rab5 (Alexa Fluor 568, blue; early endosome) and Rab7 (Alexa Fluor 647, red; late endosome) at 30 min, 1 hpi and 3 hpi. (D) Representative fluorescence intensity profiles of immunolabeled Rab5 immunolabeled Rab7 and E. faecalis (pDasherGFP, green) was assessed over a linear segment (derived from the white lines shown in panel C) and scored as described for panel B. Arrow indicates points of colocalization. Images are representative of three independent experiments. Percentages are derived from a minimum of 8 individual confocal images per time point. CLSM of infected HaCaTs immunolabeled for EEA1 (early endosome) and LAMP1 (late endosome/lysosome) at 4 hpi. (F) Magnified images of boxed areas in (A) showing representative images of E. faecalis containing compartments. Percentages are derived from 10 individual confocal images and a total of 55 E. faecalis diplococci. Images are maximum intensity projections of 4–5 optical sections (~2 μm z-volume) and are representative of 3 independent experiments. Scale bars: A: 10 μm; C: 10 μm; E: 10 μm; F: 2 μm. (See S7 and S8 Figs for additional representative images).

Since we observed large numbers of E. faecalis within keratinocytes in the perinuclear region at 24 hpi, we hypothesized that E. faecalis may be trafficked through the host endo-lysosomal pathway. To interrogate this hypothesis, we infected cells for 30 min, 1 hpi and 3 hpi and subsequently immunolabelled the early endosome Rab5 GTPase in infected cells to visualize early intracellular endo-lysosomal compartments. Fluorescence histograms were generated for individual E. faecalis-containing compartments and were further validated by visualization of orthogonal views and 3D projections. At 30 min, 1 hpi, and 3 hpi we observed that 31% (13/42), 28% (18/64), and 35% (35/111) of intracellular E. faecalis, respectively, were found in Rab5+ labelled compartments (Figs 4A and S6 ) . These data suggest that E. faecalis-containing compartments that are not in association with Rab5 may either traffic quickly through Rab5+ compartments or avoid association with Rab5 entirely. Additionally, observations of single enterococcal chains, for which we expected uniform association with Rab5, revealed instances of non-uniform Rab5 association along the chains, suggesting either incomplete Rab5 immunolabelling, or different Rab5 interaction or immunolabelling efficiency at different parts of the chain ( Fig 4B ) . To investigate this further, we immunolabelled Rab5 at 30 min, 1 hpi and 3 hpi concomitantly with Rab7, a late endosomal GTPase that ultimately replaces Rab5 as the endo-lysosomal pathway progresses from early to late endosomes [ 40 ]. We observed 32% (18/56), 46% (48/104) and 25% (14/54) of intracellular E. faecalis in compartments labelled for both Rab5 and Rab7 at 30 min, 1 hpi, and 3 hpi, respectively (Figs 4C and 4D , and S7 ) . Looking at Rab7 alone (which may include instances of Rab5 proximal labelling), we observed that 67% (44/65), 54% (63/116) and 63% (40/63) of E. faecalis-containing compartments lacked Rab7 labelling at 30 min, 1 hpi, and 3 hpi, respectively. These data support our hypothesis that some intracellular E. faecalis may be escaping Rab5/7 compartments altogether. In addition, at 4 hpi or 24 hpi we also immunolabeled the infected cells to visualize the following intracellular endo-lysosomal compartments: EEA1 (early endosome antigen 1, early endosome), Rab7 and LAMP1 (lysosomal-associated membrane protein, late endosome/pre-lysosome) (Figs 4E, 4F and S8 ). By 4 hpi, although EEA1-labeled early endosomes were often observed in close proximity to E. faecalis-containing compartments, only 5% (3/55) of internalized bacteria compartments showed a clear association with EEA1, and these compartments did not contain LAMP1 (EEA1+/LAMP1-). Instead, 64% (35/55) of internalized E. faecalis were in compartments associated with LAMP1 but not EEA1 (EEA1-/LAMP1+). The remainder (31% of internalized bacteria (17/55)) was neither associated with LAMP1- nor EEA1-labeled compartments (EEA1-/LAMP1-) ( Fig 4E and 4F ) . Furthermore, we observed that Rab7 was associated with 28% (16/58) of E. faecalis-containing compartments at 4 hpi and with 32% (10/31) of E. faecalis-containing compartments at 24 hpi ( S8A Fig) ; however, some but not all LAMP1+ E. faecalis-containing compartments were also Rab7+ ( S8B Fig) , again suggesting a degree of heterogeneity among the intracellular niche of E. faecalis. Altogether, these data suggest that a subset of internalized E. faecalis traffic rapidly through early endosomes and reach late endosomal compartments as early as 30 min post-infection and LAMP1+ late endosomal compartments by 4 hpi. Another pool of E. faecalis may avoid the canonical Rab5/Rab7 endo-lysosomal pathway entirely.

To directly examine intracellular replication of E. faecalis OG1RF, we treated infected HaCaT and RAW264.7 cell lines with BrdU, a nucleotide analogue that is incorporated into replicating DNA; and RADA, a TAMRA-based fluorescent D-amino acid that labels newly synthesized peptidoglycan and has been recently used to assess E. faecalis replication within hepatocytes [ 36 – 38 ]. As a control, E. faecalis treated with a bacteriostatic concentration of the antibiotic ramoplanin to halt replication do not incorporate fluorescent D-amino acids [ 39 ] or BrdU ( S5A and S5B Fig) . Following 3 h infection and 1 h antibiotic treatment, HaCaT and RAW264.7 cells were treated with BrdU or RADA for another 20 h concomitantly with antibiotics, to ensure that only intracellular replicating bacteria could incorporate the compounds. CLSM images of intracellular bacteria in both cell lines confirmed that E. faecalis incorporated both compounds, indicating E. faecalis were in a state of active replication ( Fig 3C ) . We observed 45% (16/35) of the infected HaCaT contained E. faecalis that had incorporated RADA. To determine whether E. faecalis can replicate intracellularly in vivo, we infected mouse excisional wounds with E. faecalis expressing episomally encoded GFP (pDasherGFP) for 24 h. BrdU was injected and applied topically to the infected wound 1.5 h prior to wound collection. CLSM analysis of ex vivo wounds that were dissociated to multicellular clusters and immunolabeled for CD45 expression showed clusters of BrdU positive intracellular bacteria within CD45- cells (Figs 3D and S6 and S1 Video ) . Altogether these observations suggest that E. faecalis can replicate intracellularly within mammalian cells of both immune and epithelial origin.

(A) CLSM orthogonal view of internalized E. faecalis within HaCaT keratinocytes at 4 hpi (3 h infection + 1 h antibiotic treatment). (B) CLSM orthogonal view of internalized E. faecalis within HaCaT keratinocytes at 24 hpi (3 h infection + 21 h antibiotic treatment). (A, B) Blue, dsDNA stained with Hoechst 33342; green, E. faecalis; red, F-actin. Images are representative of 3 independent experiments. Scale bar: 5 μm (C) CLSM view of internalized E. faecalis stained with BrdU and RADA. Examples of replicating E. faecalis are indicated with a white arrow. Blue, dsDNA stained with Hoechst 33342; green, E-GFP, E. faecalis; red, BrdU or RADA; white, F-actin. Images are representative of at least 3 independent experiments. Scale bar: 10 μm. (D) CLSM view of ex vivo murine wound tissue cells following infection and BrdU treatment. Left panel shows examples of potentially replicating E. faecalis clusters, indicated with white arrows. Scale bar: 10 μm. Right panels show magnified areas of interest. The marked areas with dashed lines white square show CD45-negative E. faecalis containing cells. Scale bars: 2 μm and 0.5 μm. Blue, dsDNA stained with Hoechst 33342; green, E. faecalis; red, BrdU; white, CD45. Images are representative of 3 independent experiments. ( S1 Video shows a 3D video projection of this image. Additional images of ex vivo cell experiments are provided in S6 Fig ).

To confirm the presence of E. faecalis within keratinocytes, we imaged keratinocytes infected with E. faecalis expressing chromosomally encoded green fluorescent protein (GFP) [ 35 ] by confocal laser scanning microscopy (CLSM). Images taken at 4 hpi, from cells infected for 3 h followed by 1 h gentamicin and penicillin treatment to kill extracellular bacteria, revealed 1–10 intracellular bacteria within each infected keratinocyte ( Fig 3A ) . This observation suggested either that selected infected keratinocytes can take up many E. faecalis, or that E. faecalis could replicate within these keratinocytes. We extended the period of post-infection antibiotic exposure up to 24 hpi and recovered similar intracellular CFU within the whole population ( S1D Fig ). However, within single infected keratinocytes, we visualized 10–30 E. faecalis, which often clustered in a perinuclear region ( Fig 3B ) . At the same 24 hpi time point, we also detected clusters of fluorescent E. faecalis peripheral to apparently apoptotic keratinocytes ( S4 Fig ), indicative of intracellular bacteria that have either escaped from the keratinocyte or of bacteria derived from lysed keratinocytes, of which the latter may account for the slight decrease in overall intracellular CFU over time.

Bacterial uptake into non-professional phagocytes such as epithelial cells can proceed via a number of different endogenous endocytic pathways [ 28 ]. Previous studies have suggested that E. faecalis uptake into non-professional phagocytic cells is dependent on actin and microtubule polymerization, suggestive of macropinocytosis or receptor (clathrin)-mediated endocytosis [ 14 , 15 ]. To determine whether an intact cytoskeleton is important for E. faecalis entry into keratinocytes, we pre-treated keratinocytes with specific chemical inhibitors, prior to infection and intracellular CFU enumeration. We found that cytochalasin-D and latrunculin A, inhibitors of actin filament polymerization [ 29 , 30 ], did not alter bacterial adhesion to keratinocytes ( Fig 2C ) , but resulted in a significant 100-fold decrease in recoverable intracellular bacteria at 4 hpi, demonstrating that actin polymerization is important for the entry process ( Fig 2D ) . By contrast, colchicine, an inhibitor of microtubule polymerization [ 31 ], did not impede bacterial adhesion and minimally impacted uptake only at 2 hpi, suggesting that E. faecalis may enter keratinocytes via receptor-mediated endocytosis in some cases ( S3A Fig ) . Since many endocytic pathways rely on an intact actin cytoskeleton [ 32 ], a panel of additional selective inhibitors was used to determine the mechanism of E. faecalis entry. Inhibitors of receptor (clathrin)- and caveolae-mediated endocytosis did not meaningfully affect bacterial adhesion or internalization ( S3B and S3C Fig). Although, we observed a small but statistically significant decrease in the number of internalized bacteria at 3 hpi in dynasore-treated cells, it is likely due to the increased cytotoxicity associated with dynasore treatment ( S1 Table ) . To test the role of macropinocytosis in E. faecalis uptake, we pre-treated keratinocytes with the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin [ 33 , 34 ]. Wortmannin did not affect E. faecalis adhesion to keratinocytes ( Fig 2E ) but resulted in a 10-fold decrease in intracellular CFU, as compared to the untreated controls ( Fig 2F ) . All compounds used were non-cytotoxic to mammalian cells (viability ≥80%), except for dynasore which reduced viability to 76% ( S1 Table ) . Together, the strong dependence of E. faecalis internalization on actin polymerization and PI3K and independence of receptor (clathrin)- and caveolae-mediated endocytosis, is consistent with macropinocytosis as a primary means of uptake.

(A,B) 10 6 keratinocytes were infected with E. faecalis OG1RF at the indicated MOI for 1, 2, or 3 h, each followed by another 1 h of antibiotic treatment to eliminate extracellular bacteria. Infected host cells were washed once, lysed, and intracellular CFU enumerated. Solid lines indicate the mean CFU/well from a total of 3 independent experiments. Dashed black line indicates the limit of detection of the assay. (C-F) Keratinocytes were pre-treated with actin inhibitors cytochalasin-D (CytoD 1 μg/ml), latrunculin A (LatA 0.25 μg/ml), or PI3K inhibitor wortmannin (0.1 μg/ml) for 0.5 h, followed by E. faecalis infection at MOI 100 for 1, 2 or 3 h. Following three PBS washes, cells were lysed and associated adhered bacteria enumerated immediately after infection; or, to quantify intracellular CFU, the initial infection period was followed by 1 h antibiotic treatment, for a total of 2, 3 or 4 hpi, prior to lysis and enumeration. (C,E) Adherent or (D,F) intracellular bacteria were enumerated at the indicated time points (only significant differences are indicated). Solid lines indicate the mean for each data set of at least 3 independent experiments. (D) ****p<0.0001 2 way ANOVA, Tukey’s multiple comparisons test. (F) **p<0.01 2 way ANOVA, Sidak’s multiple comparisons test. (See S1 Fig for data related to antibiotic killing efficiency. See S1 Table for data related to drug cytotoxicity).

To investigate the mechanisms by which E. faecalis infect non-immune cells at the wound site, we infected the spontaneously immortalized human keratinocyte cell line (HaCaT) with E. faecalis strain OG1RF at a multiplicity of infection (MOI) of 1, 10 or 100 for a period of up to 3 hours (h), followed by 1 h of gentamicin and penicillin, and quantified viable intracellular bacteria. The gentamicin and penicillin treatment used was sufficient to kill 99.9% of the extracellular bacteria ( S1A and S1B Fig ) . We observed that E. faecalis can adhere to keratinocytes at all MOI and time points ( Fig 2A ) , and intracellular E. faecalis were recovered as early as 1 h post-infection (hpi) at a MOI of 10 and 100 ( Fig 2B ) . Parallel cytotoxicity experiments established that E. faecalis infection does not negatively affect keratinocytes at early time points of <4 hpi, even in the absence of gentamicin and penicillin ( S2 Fig ) . Thus, we chose MOI 100 and no more than 3 h of infection without antibiotics to characterize its intracellular pathogenesis. Since we recovered intracellular E. faecalis from infected mouse wounds in both immune and non-immune compartments, we expected to also detect viable E. faecalis within mouse fibroblasts and macrophages in vitro. Indeed, intracellular E. faecalis were recovered from RAW264.7 murine macrophages and NIH/3T3 murine fibroblasts, indicating that E. faecalis internalization and persistence is not cell type specific ( S1C Fig ) . Similarly, intracellular persistence within keratinocytes was not E. faecalis strain specific, as the vancomycin resistant strain V583 persisted at even higher numbers within HaCaT cells after 24 hpi ( S1D and S1E Fig ) . E. faecalis V583 required 21 h of antibiotic exposure to kill 99.9% of extracellular bacteria, which may extend the effective infection period, but nonetheless supports the conclusion that E. faecalis V583 is present intracellularly at 24 hpi ( S1F and S1G Fig ) .

Male C57BL/6 mice were wounded and infected with 10 6 CFU of E. faecalis OG1RF. Wounds were harvested at 1, 3, or 5 days post-infection (dpi), dissociated to single cell suspension, treated with antibiotics to kill extracellular bacteria, labeled, and sorted into (A) CD45+ (immune) or (B) CD45- (non-immune) populations. CD45+ and CD45- cell populations were then lysed and plated for bacterial CFU. Each data point indicates the CFU within the sorted subpopulation from one mouse. Data shown represent at least 3 independent experiments, each of which included at least 4 mice per time point. Horizontal black lines indicate the mean for each group.

To determine whether E. faecalis persist intracellularly within infected wounds, we infected wounded mice with 10 6 CFU of E. faecalis for 1, 3 and 5 days. Infected wounds were dissociated to a single cell suspension, treated with gentamicin and penicillin G to kill extracellular bacteria, immunolabeled with anti-CD45 antibody, and sorted into CD45+ immune cells and CD45- non-immune cells. These sorted cells were then lysed for the enumeration of intracellular bacteria. Consistent with literature reporting the ability of E. faecalis to persist within phagocytic immune cells, we recovered viable E. faecalis from CD45+ cells ( Fig 1A ) . In addition, intracellular E. faecalis was also recovered from the CD45- population, up to 5 days post infection (dpi) ( Fig 1B ) . Compared to the approximately 10 5 CFU total recoverable E. faecalis (both extracellular and intracellular) within wounds at 3 and 5 dpi [ 6 ], we can estimate that approximately 1–10% of the total recovered bacterial population are intracellular at these time points. These data demonstrate that E. faecalis can exist intracellularly during wound infection, implying it is not an exclusive extracellular pathogen.

Discussion

E. faecalis is among the commonly isolated microbial species cultured from chronic wound infections. The ability of E. faecalis to persist in the face of a robust immune response and antibiotic therapy is frequently attributed to its ability to form biofilms during these infections. However, a number of bacterial pathogens undertake an intracellular pathway during infection that can contribute to persistent and or recurrent infection. This is well-described for uropathogenic E. coli (UPEC), particularly in animal models in which UPEC can replicate to high numbers within urothelial cells as intracellular bacterial communities or can persist in a quiescent intracellular state within LAMP1+ compartments for long periods of time, promoting recurrent and chronic infection [42–45]. While there are numerous reports of intracellular E. faecalis within a variety of non-immune cells [13–20], the contribution of an intracellular lifecycle to E. faecalis infection has been minimally investigated. Here, we report that, in vitro, E. faecalis become internalized into keratinocytes primarily via macropinocytosis, whereupon they undergo heterotypic trafficking through the endosomal pathway, which enables their replication and survival. These findings raise the possibility that this intracellular lifecycle may be linked to persistent and chronic infections, such as those that occur in wounds. Further, we demonstrate that intracellularity may be physiologically relevant in a mouse model of wound infection, where E. faecalis exists within both immune and non-immune cells for at least 5 days after infection. Importantly, E. faecalis recovered from within keratinocytes are primed to more efficiently infect new keratinocytes to seed another round of infection.

Previous studies using either professional or non-professional phagocytic cell lines have reported the internalization, but not the replication of intracellular E. faecalis [9,13,15] as these studies used only antibiotic protection assays coupled with TEM at single time points. Here, we performed antibiotic protection assays coupled with imaging across multiple time points, and our results similarly show that E. faecalis can enter and survive intracellularly up to 72 hpi. Importantly, we show with BrdU labelling that E. faecalis can be found in a state of active replication in cells harvested from infected wounds. This finding is further supported by in vitro analyses of intracellular E. faecalis from infected keratinocytes and macrophages that were incubated with BrdU and RADA. This is the first reported evidence, to our knowledge, of E. faecalis intracellular replication within epithelial cells or macrophages. Consistent with our observation, E. faecalis has also been shown to replicate within human hepatocytes in vitro and has been observed as clusters in association with hepatocytes in a mouse model of intravenous infection [38]. Together these data suggest that once E. faecalis enters mammalian cells, at least some of the bacteria are able to replicate intracellularly. Other “classical” extracellular bacteria including S. aureus and P. aeruginosa are also able to replicate intracellularly [46–51]. Similar studies have also shown that S. pyogenes can be taken up by both immune and non-immune cells, where it can replicate, survive host defenses and disseminate to distant sites [52,53].

In this work, we show that E. faecalis enters keratinocytes in a process that is dependent on actin polymerization and PI3K signalling, and independent of receptor (clathrin)- or caveolae-mediated endocytosis. Chemical inhibition of actin polymerization by cytochalasin D and PI3K signaling by wortmannin specifically affects macropinocytosis but not receptor (clathrin)-mediated endocytosis [54–56]. These findings suggest that E. faecalis strain OG1RF enters keratinocytes primarily in a macropinocytotic process. A previous study suggested that clinical isolates of E. faecalis enter HeLa (human epithelioid carcinoma) cells via either macropinocytosis or clathrin-mediated endocytosis, supported by inhibitors of microtubule polymerization and cytosolic acidification that reduced intracellular CFU [15]. We also observe that E. faecalis uptake is transiently diminished when receptor (clathrin)-mediated endocytosis is inhibited, suggesting that E. faecalis may also take advantage of this uptake pathway in some instances. Thus, it may be that different strains of E. faecalis favor entry into mammalian cells by different mechanisms, and E. faecalis OG1RF used in this study preferentially enters via macropinocytosis. However, another study reported that E. faecalis OG1 strain derivatives, closely related to OG1RF, entered human umbilical vein endothelial cells (HUVEC) cells via receptor (clathrin)-mediated endocytosis, in a cytocholasin D- and colchicine-dependent manner [14]. Because Millan et al used similar drug concentrations as we did, we suggest that OG1-related strains may enter epithelial cells primarily via macropinocytosis and endothelial cells primarily via receptor (clathrin)-mediated endocytosis.

Additionally, once inside keratinocytes, at least some E. faecalis commence trafficking through the endosomal pathway. As soon as 30 min after infection, most E. faecalis-containing compartments lacked the early endosome marker Rab5. Moreover, we did not observe any labelling with Rab7 in nearly 70% of the intracellular compartments containing internalized enterococci at time points <3 hpi. At 4 hpi, the majority (60–70%) of internalized E. faecalis were in compartments that were heterogeneously positive for the late endosomal markers LAMP1 and/or Rab7. These data indicate that Rab5 labelling may be incomplete or that E. faecalis associate with Rab5-containing compartments quickly and transiently, or only in a subset of infected cells, in either case with no subsequent or delayed Rab7 labelling. At the same time, since we observed some Rab5+/Rab7+ compartments at all early time points, traditional Rab5/Rab7 conversion dynamics from early to late endosome also happens in a subset of infected cells. Taken together, these data point to the possibility that at least a subset of internalized E. faecalis enter Rab5+/EEA1+ early endosomes/macropinosomes, and rapidly transit into late endosomal compartments. In parallel, Rab5 and Rab7 protein levels in infected keratinocytes were markedly decreased in comparison to non-infected keratinocytes. We predict that E. faecalis infection-driven reduction in Rab expression is crucial to determine the outcome of E. faecalis intracellular survival since Rab5 and Rab7 control important fusion events between early and late endosomes and late endosomes and lysosomes [22]. Rab GTPases are commonly hijacked by bacteria to promote their survival [57]. For comparison, intracellular microbes such as M. tuberculosis and L. monocytogenes distinctively modify the Rab5 machinery arresting phagosome maturation [22]. C. burnetii prevents Rab7 recruitment [58] and B. cenocepacia affects Rab7 activation [59]. However, to the best of our knowledge, our data are the first to show differences in overall Rab5 and Rab7 protein levels as a potential bacterial subversion mechanism for the macropinosome. Studies are underway to determine the bacterial factors and mechanisms by which E. faecalis affects Rab protein levels. While E. faecalis-containing LAMP1+ compartments appeared distended at 24 hpi, many E. faecalis were not tightly associated with LAMP1 or Rab7. Furthermore, we rarely observed Cathepsin D in E. faecalis-containing compartment, suggesting that late endosomes containing internalized bacteria could be missing markers or that these late endosomal compartments have been modified, making lysosomal fusion a rare event. Other intracellular pathogens such as C. burnetii and Francisella tularensis also reside in compartments devoid of Cathepsin D, or in compartments with very low levels of Cathepsin D [60,61]. The authors suggest that this was achieved by escaping fusion with lysosomes. In support of this view, TEM revealed that all membrane-bound E. faecalis were spatially separated from LAMP1+ electron dense compartments of unknown nature (potentially a lysosome, which is a LAMP1+ organelle), and there was no indication of membrane fusion between E. faecalis-containing compartments and lysosomes. Notably, we observed some E. faecalis in association with LAMP1 and Rab7, suggesting that some internalized E. faecalis cells may transit via the normal endocytic pathway and fuse with lysosomes. Collectively our results support a model in which late endosomes containing E. faecalis are modified, preventing the expected destruction of intracellular E. faecalis by lysosomal fusion and allowing them to replicate from within.

We propose three potential fates for different subsets of internalized E. faecalis (Fig 9). 1) Macropinosome maturation into Rab7+/LAMP1+ late endosomes and fusion with the lysosome, leading to the degradation of E. faecalis (Fig 9B-I). 2) Macropinosome maturation into LAMP1+ but Rab7- compartments, leading to E. faecalis survival because Rab7 presence is required for lysosome fusion (Fig 9B-II). 3) Macropinosome maturation into compartments lacking both Rab7 and LAMP1, which would also lead to E. faecalis survival (Fig 9B-III). Additionally, we cannot exclude the possibility that E. faecalis-containing compartments initially contain both Rab7 and LAMP1 late endosomal markers, but are subsequently modified by E. faecalis to increase its survival. In other words, there could be a transition from I into II and/or III mediated by downregulation of Rab5 and Rab7. Furthermore, intracellular E. faecalis that do not associate with any examined endo-lysosomal markers could reflect a cytosolic state. Finally, we have also shown that infected host cells can eventually die, releasing E. faecalis into the periphery of dead host cells. Based on reinfection studies, we propose that those bacteria released from dead cells may be primed to infect other cells, resulting in an enhanced cycle of reinfection. Altogether, our work has demonstrated that E. faecalis can enter, survive, replicate and escape from keratinocytes in vitro. If this intracellular lifecycle also exists in vivo, and extends to other cell types such as macrophages as our data suggest, these findings may allow for an abundant protective niche for bacterial persistence that could contribute to the chronic persistent infections associated with E. faecalis.

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010434

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