Biofilm formation on human immune cells is a multicellular predation strategy of Vibrio cholerae

Abstract

Biofilm formation is generally recognized as a bacterial defense mechanism against environmental threats, including antibiotics, bacteriophages, and leukocytes of the human immune system. Here, we show that for the human pathogen Vibrio cholerae, biofilm formation is not only a protective trait but also an aggressive trait to collectively predate different immune cells. We find that V. cholerae forms biofilms on the eukaryotic cell surface using an extracellular matrix comprising primarily mannose-sensitive hemagglutinin pili, toxin-coregulated pili, and the secreted colonization factor TcpF, which differs from the matrix composition of biofilms on other surfaces. These biofilms encase immune cells and establish a high local concentration of a secreted hemolysin to kill the immune cells before the biofilms disperse in a c-di-GMP-dependent manner. Together, these results uncover how bacteria employ biofilm formation as a multicellular strategy to invert the typical relationship between human immune cells as the hunters and bacteria as the hunted.

Keywords: cholera infection; enteroid; host-pathogen interaction; immunity; organoid; type IV pili.

Graphical abstract

Figure 1

V. cholerae forms biofilms on human leukocytes, which is followed by immune cell death and biofilm dispersal (A) V. cholerae cells attach to different types of human leukocytes isolated from blood and form biofilms before the leukocytes die and bacteria disperse collectively from the immune cell surface. Confocal microscopy images were acquired with a 40× NA 1.3 objective and show xy slices of V. cholerae (cyan) attached to leukocytes (red) at the time of peak number of attached bacterial cells shown in (B). Dead leukocytes are shown in yellow. (B) Bacterial attachment to leukocytes was measured from confocal images: lines represent the mean ratio of the volume of attached bacteria per volume of an annulus around each immune cell; shaded regions denote the standard deviation of n independent biological replicates (n_macrophages = 17; n_neutrophils = 5; n_{CD4+ T cells} = 3; n_{NK cells} = 3; n_{B cells} = 3). (C) V. cholerae cells also form biofilms on macrophages derived from the monocytic THP-1 cell line, followed by biofilm dispersal and macrophage cell death. Confocal images from different time points show distinct stages of the interaction process. Dead staining (yellow, propidium iodide) reveals an increasing number of dead macrophages over time. Bacterial attachment to macrophages was measured from confocal images: the solid line indicates the mean ratio of the volume of attached bacteria per volume of an annulus around each macrophage; the shaded region corresponds to the standard deviation of n = 41 independent biological replicates. (D) Attachment of V. cholerae to the eukaryotic surface is strongly reduced for THP-1 monocytes, compared with THP-1-derived macrophages, measured after 30 min of co-incubation. Bars denote mean values; error bars denote the standard deviation of n independent biological replicates (n_macrophages = 18; n_monocytes = 6). Statistical significance was calculated using an unpaired t test (^∗∗∗∗ indicates p < 0.001). Similar results were obtained when comparing V. cholerae attachment to macrophages and monocytes derived from human blood (Figure S1A). (E) Quantification of bacterial accumulation on the macrophage surface, which results either from the attachment of new planktonic cells or from the division of bacteria that are already attached to the macrophage surface. Macrophages were derived from THP-1 monocytes. Lines denote mean values; shaded areas are the standard deviation of n = 16 independent biological replicates.

Figure S1

Colonization and death of macrophages differentiated from CD14⁺ monocytes isolated from human blood by V. cholerae, and attachment of bacteria to primary CD14⁺ monocytes, refers to Figures 1, 2, 3, and 5 (A) V. cholerae WT exhibits strongly reduced attachment to CD14⁺ monocytes compared with macrophages (Mφ) derived from CD14⁺ monocytes. Attachment of V. cholerae to immune cells was quantified from confocal images after 0.5 h of co-incubation and normalized to the mean value for macrophages. Statistical significance was calculated using an unpaired t test (number of independent biological replicates: n_macrophage = 12, n_monocyte = 3; ^∗∗∗∗ indicates p < 0.0001). Error bars denote the standard deviation. (B) Attachment capabilities of different V. cholerae strains to the surface of macrophages derived from CD14⁺ monocytes normalized to the mean value of WT bacteria. For bacteria with a stalled flagellar motor (achieved by the addition of phenamil), cells that lack the polar flagellum (ΔflaA), or cells lacking MSHA pili (ΔmshA), attachment to macrophages is attenuated. Ectopic expression of mshA or flaA under control of the native promoter restores bacterial attachment. Statistical significance was calculated using one-way ANOVA (number of independent biological replicates: n = 3–16; ^∗ indicates p < 0.05; ^∗∗ indicates p < 0.005; ^∗∗∗∗ indicates p < 0.0001). Error bars denote the standard deviation. (C) Representative microscopy images from n = 3–8 independent biological replicates show V. cholerae biofilms (cyan) formed on primary macrophages (red) differentiated from CD14⁺ monocytes isolated from human blood, imaged at peak time of biofilm formation. The V. cholerae biofilm matrix components Bap1, RbmA, RbmC, and Vibrio polysaccharide (VPS) are not required for biofilm formation on human macrophages. Instead, V. cholerae biofilm formation on macrophages derived from CD14⁺ monocytes depends on the production of MSHA pili and TC pili (with the secreted protein TcpF). (D) During co-incubation of macrophages with V. cholerae lacking hemolysin HlyA, macrophage death is significantly reduced compared with WT bacteria. Overexpression of hlyA under control of the inducible P_tac promoter restores and enhances bacteria-induced killing of macrophages. Macrophage death was measured after 7 h of exposure to bacteria. Bars represent the percentage of dead macrophages for different V. cholerae strains, normalized to the WT mean value. Statistical significance was calculated using an unpaired t test (n = 3–11 independent biological replicates; ^∗∗∗∗ indicates p < 0.0001). Error bars denote the standard deviation. (E) Biofilm growth on macrophages supports hemolysin-dependent death of macrophages. During co-incubation, V. cholerae strains deficient in the formation of biofilms on macrophages (strains carrying the ΔmshA and ΔtcpA mutations) cause less death of macrophages compared with biofilm-capable bacteria. Bars represent the percentage of dead macrophages for different V. cholerae strains normalized to the WT. Macrophage death was measured after 7 h of exposure to bacteria. Statistical significance was calculated using an unpaired t test (n = 3–11 independent biological replicates; ^∗∗∗∗ indicates p < 0.0001; ^∗∗ indicates p < 0.007). Error bars denote the standard deviation.

Figure S2

The impact of initial bacterial and macrophage counts on V. cholerae biofilm formation dynamics and macrophage death, the effect of macrophage lysate on V. cholerae growth, the effect of V. cholerae on monocyte death, and responses of V. cholerae and macrophages to each other during co-culture, refers to Figures 1 and 5 (A) Changing the number of bacterial cells added to a constant number of macrophages (2.5 × 10⁴; in a well of a 96-well plate filled with 200 μL of medium) does not change the interaction outcome qualitatively but impacts the dynamics of biofilm growth and the time point of biofilm dispersal. A higher number of bacterial cells added to 2.5 × 10⁴ macrophages at the beginning of co-incubation results in a faster biofilm formation on the surface of the immune cells. Lower bacterial seeding densities lead to longer lag times in biofilm formation. The different bacterial seeding densities correspond to an MOI at the start of infection in the range of 1–256. Lines represent the mean ratio of the volume of attached bacteria per volume of an annulus around each macrophage. Shaded areas indicate the standard deviation of n = 3 independent biological replicates. (B) Changing the number of macrophages present inside a well of a 96-well plate, filled with 200 μL of medium containing initially 3.2 × 10⁶V. cholerae cells, does not change the biofilm formation process temporally. The different bacterial seeding densities correspond to an MOI at the start of infection in the range of 64–640. Lines denote the mean ratio of the volume of attached bacteria per volume of an annulus around each macrophage, and shaded areas are the standard deviation of n = 3 independent biological replicates. (C) Changing the number of bacterial cells added to a constant number of macrophages (2.5 × 10⁴; in a well of a 96-well plate filled with 200 μL of medium) strongly impacts the occurrence of macrophage death. A higher number of bacterial cells added at the beginning of co-incubation results in a higher percentage of dead macrophages after 7 h of co-incubation. Lines represent the mean of dead macrophages for different initial bacterial counts, and shaded areas represent the standard deviation of n = 3 independent biological replicates. (D) Supplementation of growth medium with lysate from macrophages enhances bacterial growth by reducing the lag phase in a concentration-dependent manner. When the number of lysed macrophages that is added to a constant number of V. cholerae cells (3.2 × 10⁶ cells, in a total volume of 200 μL) is increased, the bacterial cells reach exponential growth faster. The effect of lysed macrophages on the growth of V. cholerae was monitored with an automated plate reader at 37°C. Colored lines represent the mean OD₆₀₀ values that reflect the V. cholerae cell density in the culture, and shaded areas represent the standard deviation of n = 4 independent biological replicates. (E) Measurements of the fraction of dead macrophages and monocytes after 3 h and 7 h of co-incubation with V. cholerae WT cells. After 3 h of exposure to V. cholerae, the differences in the fraction of dead macrophages and monocytes are not statistically significant. After 7 h of co-incubation, the occurrence of dead immune cells is significantly increased for macrophages compared to monocytes. Statistical significance was calculated using an unpaired t test (n = 4 independent biological replicates; ^∗∗∗ indicates p = 0.0002). Error bars denote the standard deviation. (F) Principal-component analysis (PCA) of the bacterial RNA-seq data shows global changes in the bacterial transcriptome during the interaction with macrophages. In addition, the four biological replicates (dots with the same color) belonging to each sampled time point (indicated by different colors) cluster together. The PCA was performed based on fold change values that were calculated for each significantly differential expressed gene, relative to the average of all naive bacterial samples at 0 min (immediately prior to exposure to macrophages). Individual data points of n = 4 independent biological replicates are shown. Transcriptome data are available at the Gene Expression Omnibus (GEO: GSE184078). (G) Clustering of the 3,519 V. cholerae genes (rows) shows significant changes in gene expression over the course of the interaction with macrophages (columns). The heatmap displays the Z score calculated from average fold change values for each gene and time point relative to the average of the naive bacterial sample at 0 min. Values are the mean of n = 4 independent biological replicates. (H) ELISA measurements of cytokine abundance in supernatants show that biofilm formation of V. cholerae on the surface of macrophages does not prevent the release of IL-1B, TNFA, or IL-8 into the extracellular environment. The amount of IL-1B, TNFA, and IL-8 in the supernatant were similar for biofilm-producing bacteria (WT) and biofilm-deficient bacteria (ΔmshA ΔtcpA). However, the production of all three cytokines was significantly induced by co-culture with V. cholerae compared to unstimulated macrophages. Statistical significance was calculated using an unpaired t test (n = 3 independent biological replicates). Error bars denote the standard deviation. (I) Macrophage transcriptomes display changes after exposure to V. cholerae as illustrated by the PCA. Transcriptomes were measured by RNA-seq. Bacteria deficient in biofilm formation or hemolysin production and WT bacteria induce similar transcriptional changes in macrophages. The PCA was performed on 296 significantly upregulated macrophage genes. Individual data points of n = 3 independent biological replicates are shown: each data point corresponds to a transcriptome; different time points are indicated by a dot or triangle, and each bacterial strain used for the interaction studies is indicated by a different color. Transcriptome data are available at the Gene Expression Omnibus (GEO: GSE184077).

Figure 2

Attachment of V. cholerae to the surface of macrophages is enabled by type IV pili (A) At different time points during co-incubation of bacteria and macrophages (Mφ), transcriptional changes of V. cholerae attached to macrophages were measured using RNA-seq. The heatmap shows expression dynamics of bacterial factors that are known to be involved in attachment of V. cholerae to different surfaces. Log₂ fold change values for the different time points were calculated relative to the bacterial sample taken at 0 min, prior to macrophage exposure. Values are the mean of n = 4 independent biological replicates. (B) Quantification of bacterial attachment to macrophages after 1 h of co-incubation with V. cholerae deletion mutants that lack genes required for attachment to different surfaces. Bacteria deficient in the assembly of MSHA pili (ΔmshA) or the polar flagellum (ΔflaA), as well as cells with a stalled flagellar rotor (caused by the addition of 100 μM phenamil), exhibit impaired attachment to the macrophage surface. Ectopic expression of mshA or flaA, under the control of the native promoter, restores bacterial attachment. Mutants lacking other surface structures or proteins display attachment levels similar to WT bacteria. Bars represent the mean ratio of the volume of attached bacteria per volume of an annulus around each macrophage, after normalization to the mean value calculated for WT bacteria. Error bars denote the standard deviation of n = 3–39 independent biological replicates. Statistical analysis was performed using a one-way ANOVA (^∗∗∗∗ indicates p < 0.0001; ^∗ indicates p < 0.02). (C) V. cholerae cells unable to produce MSHA pili (ΔmshA) eventually adhere to macrophages after prolonged co-incubation, which is dependent on TC pili and the secreted protein TcpF. The presence of either TC pili (with TcpF) or MSHA pili is sufficient for biofilm formation. Lines represent the mean ratio of the volume of attached bacteria per volume of an annulus around each macrophage at each time point. Shaded areas denote the standard deviation of n independent biological replicates (n_WT = 41; n_ΔmshA = 13; n_ΔmshAΔtcpA = 17; n_ΔmshAΔtcpF = 5). Representative confocal images (xy slices) show bacterial accumulation (cyan) on the surface of macrophages (red) for different bacterial strains at the indicated time point.

Figure S3

Impact of TC pili and TcpF on attachment and biofilm formation of ΔmshA cells and localization of RbmA, Bap1, and RbmC, as well as transcription of vps genes inside mature V. cholerae WT biofilms formed on macrophages, refers to Figures 2 and 3 (A and B) Overexpression of the tcp operon was achieved by placing toxT under the control of the arabinose inducible promoter P_BAD. (A) The overexpression of the tcp operon, achieved by the addition of 0.2% arabinose, resulted in strong attachment of ΔmshA cells after only 1 h of co-incubation, which was dependent on the presence of TC pili and TcpF. Bars represent mean values, after normalization to the mean obtained for WT bacteria. Error bars denote the standard deviation of n independent biological replicates (n_{ΔmshA, 0%} = 4; n_{ΔmshA, 0.2%} = 6; n_{ΔmshAΔtcpA, 0%} = 5; n_{ΔmshAΔtcpA, 0.2%} = 8; n_{ΔmshAΔtcpF, 0%} = 5; n_{ΔmshAΔtcpF, 0.2%} = 9). Statistical significance was calculated using an unpaired t test (^∗∗∗∗ indicates p < 0.002). (B) Overexpression of TC pili is not sufficient for biofilm formation on macrophages in a ΔmshA ΔtcpF strain. However, increasing the concentration of exogenously supplied purified TcpF enabled the ΔmshA ΔtcpF strain to form biofilms after 4 h of co-incubation. This indicates that TC pili interact with TcpF in order to bind to the macrophage surface, while secreted TcpF localizes on the macrophage surface (Figure 3D). For these experiments, we initiated the co-culture with 3.2 × 10⁶ bacteria and 2.5 × 10⁴ macrophages in 200 μL of culture medium. Dots denote the mean ratio of the volume of attached bacteria per volume of an annulus around each macrophage, and error bars represent the standard deviation of n = 4–10 independent biological replicates. (C–F) Macrophages are shown in red, V. cholerae cells are shown in cyan, and immunofluorescence or reporter fluorescence is shown in yellow. Each panel shows xy planes at three different z-heights, indicated by the Roman numerals I–III. The Roman numerals in (A)–(D) refer to different z-height layers as indicated in the side view (xz plane) at the top of the figure. For each panel, representative confocal fluorescence microscopy images from n = 3–4 independent biological replicates are shown. (C) The transcription of vps genes is measured via an sfGFP-based transcriptional reporter. Transcription of vps genes occurs throughout the biofilm formed on macrophages. (D) Histidine (His)-tagged RbmA is detected extracellularly using a fluorescent anti-His antibody shown in yellow. (E) Hemagglutinin (HA)-tagged Bap1 is detected extracellularly using a fluorescent anti-HA antibody shown in yellow. (F) Histidine (His)-tagged RbmC is detected using a fluorescent anti-His antibody shown in yellow. (B)–(D) show that RbmA, Bap1, and RbmC are present around some bacterial cells that are part of biofilms on the macrophage surface. RbmA, Bap1, and RbmC encapsulate cells that are located at the outer edge of the biofilms and are not observed on bacterial cells that are closest to the macrophage surface.

Figure 3

The extracellular matrix of biofilms formed on macrophages consists of MSHA pili, TC pili, and TcpF, which provide different biofilm functions (A) V. cholerae biofilm growth on the surface of macrophages requires the production of MSHA pili or TC pili (together with TcpF) but does not depend on matrix components that are essential for biofilms formed on abiotic surfaces: Bap1, RbmA, RbmC, and VPS. Bars represent the mean ratio of the volume of attached bacteria per volume of an annulus around each macrophage at the time of peak biofilm accumulation. Attachment values are normalized to the WT mean value. Error bars indicate the standard deviation of n = 3–41 independent biological replicates. Statistical significance was calculated using one-way ANOVA (^∗∗∗∗ indicates p < 0.0001). (B–D) Localization and abundance of MSHA pili (B), TC pili (C), and TcpF (D) inside the biofilm formed on macrophages, measured after 4 h of co-incubation. Microscopy images show V. cholerae biofilms (cyan) on macrophages (red) and a specific biofilm matrix component (yellow, immunofluorescence) at the peak time of bacterial biomass accumulation. Graphs show the spatial distribution of MSHA pili, TC pili, and TcpF inside the biofilm. MSHA pili are present throughout the biofilm, but TC pili and the secreted protein TcpF predominantly localize close to the surface of macrophages. Lines represent the mean abundance of a matrix component inside biofilms; shaded areas are the standard deviation of biofilms formed on a number of X macrophage cells (X_{WT;MSHA pili} = 41; X_{ΔmshA;MSHA pili} = 15; X_{WT;TC pili} = 19; X_{ΔtcpA;TC pili} = 31; X_{TcpF-His;TcpF} = 26; X_TcpF;TcpF = 36), from n = 3 independent biological replicates. (E) Production of TC pili provides mechanical stability to biofilms: repeated medium exchange during the V. cholerae-macrophage interaction dynamics resulted in continuous removal of biofilms that lack TC pili from the macrophage surface. “Δ4” denotes the mutations Δbap1 ΔrbmA ΔrbmC ΔvpsL. Lines denote the mean values; shaded areas are the standard deviation of n independent biological replicates (n_Δ4 = 3; n_{Δ4 ΔtcpA} = 3; n_{Δ4 ΔmshA} = 3). (F) Production of TC pili affects biofilm compactness: single-cell resolution analysis of the biofilm architecture revealed that ΔtcpA mutants produce biofilms with a lower cell-cell alignment, measured in terms of the distribution of the nematic order parameter. WT and ΔmshA biofilms display a similar cell-cell alignment. Representative confocal microscopy images show biofilms of different bacterial strains on macrophages (macrophages in red, bacteria in cyan). Lines denote mean values, and shaded areas are the standard deviation of biofilms formed on a number of X macrophages (X_WT = 10; X_ΔmshA = 7; X_ΔtcpA = 9) from n = 3 independent biological replicates.

Figure 4

Biofilm dispersal is determined by intracellular c-di-GMP levels and the presence of TC pili (A) The presence of TC pili in the biofilm matrix of V. cholerae biofilms impacts biofilm dispersal from the macrophage surface. Biofilm dispersal from macrophages was quantified as the difference between the attached bacterial biovolume at the time of peak biofilm formation and 1 h later. The attached bacterial biovolume was measured as the ratio of the volume of attached bacteria per volume of an annulus around each macrophage. Biofilm dispersal is significantly lower for WT biofilms compared with biofilms formed by ΔtcpA cells. Overexpression of toxT under control of the arabinose-inducible promoter P_BAD further reduces the degree of biofilm dispersal, which is dependent on the presence of TC pili. For biofilms lacking MSHA pili, the level of dispersal was comparable to the respective parental strain. Bars represent the mean values. Error bars denote the standard deviation of n = 5 independent biological replicates. Statistical significance was calculated using one-way ANOVA or an unpaired t test (^∗∗∗∗ indicates p < 0.0001; ^∗∗ indicates p < 0.01). (B) Analysis of the V. cholerae transcriptome dynamics during the interaction with macrophages reveals that genes encoding TC pili are upregulated during biofilm growth. Initiation of biofilm dispersal coincides with the downregulation of the tcp operon. Genes encoding MSHA pili are not differentially expressed in the transition between biofilm growth and dispersal. For the two time points that included dispersed bacteria (t = 265 min and t = 285 min), RNA-seq was performed for planktonic cells and bacteria still residing in biofilms separately. Log₂ fold change values for the different time points were calculated relative to the bacterial sample taken at 0 min. Values are the mean of n = 4 independent biological replicates. (C) Measurement of intracellular c-di-GMP levels using a fluorescent reporter (based on an unstable GFP, green line) during biofilm growth and dispersal and simultaneous measurement of the attached bacteria per macrophage (purple line). Biofilm dispersal coincides with a decrease in c-di-GMP reporter fluorescence. Lines represent mean values, and shaded areas indicate the standard deviation of n = 3 independent biological replicates. (D) Induced overexpression of the two c-di-GMP-degrading phosphodiesterases RocS and CdgJ under control of the IPTG-inducible P_tac promoter causes biofilm dispersal (orange line). V. cholerae biofilms harboring the empty vector (negative control; blue line) continue to grow on the macrophage surface despite the addition of IPTG until bacteria naturally disperse from the macrophage surface. Lines denote the mean level of attached bacteria per macrophage; shaded areas indicate the standard deviation of n = 3 independent biological replicates.

Figure 5

Encapsulation of macrophages by bacterial biofilms increases cell death of macrophages (A) The fraction of dead macrophages increases with increased V. cholerae WT biofilm amount on the immune cell surface. As a metric for the amount of biofilm, we measured the volume of attached bacteria per volume of an annulus around macrophages after 3 h of co-incubation, and we counted dead macrophages after 7 h of co-incubation (n = 3–52 independent biological replicates, error bars denote the standard deviation). (B) RNA-seq measurements were performed at different time points during the co-incubation of V. cholerae with THP-1-derived macrophages (Mφ). Transcriptomic analysis shows that genes encoding known and putative toxins and the type VI secretion system (T6SS) in V. cholerae were differentially expressed during bacterial interaction with macrophages, particularly after longer co-incubation. At the two time points of co-incubation that included dispersing cells (t = 265 min, t = 285 min), RNA-seq was performed separately on the planktonic cells and the cells that remained in biofilms attached to macrophages. Log₂ fold change values for the different time points were calculated relative to the bacterial sample taken at 0 min. Values are the mean of n = 4 independent biological replicates. (C) Macrophage death was measured using microscopy and propidium iodide staining after 7 h of co-incubation with V. cholerae strains that lack genes required for the production of known or putative toxins. In the presence of ΔhlyA bacteria, the occurrence of macrophage cell death is decreased compared to WT bacteria. Ectopic expression of hlyA under control of the P_tac promoter restored and even increased killing of macrophages. Bars represent the percentage of dead macrophages for different V. cholerae strains, normalized to the WT (n = 4–52 independent biological replicates, error bars denote the standard deviation). Statistical significance was calculated using one-way ANOVA (^∗∗∗∗ indicates p < 0.0001). Data for V. cholerae toxin double mutants are shown in Figure S4. (D) Biofilm formation contributes to V. cholerae-induced macrophage death. Representative microscopy images show dead macrophages (yellow, propidium iodide staining) after 7 h exposure to particular V. cholerae strains. Bars represent the percentage of dead macrophages for different V. cholerae strains, normalized to the WT (n = 10–52 independent biological replicates, error bars denote the standard deviation). Statistical significance was calculated using a one-way ANOVA or an unpaired t test (^∗∗∗∗ indicates p < 0.0001; ^∗ indicates p < 0.05). (E) Immunofluorescence staining of HlyA (cyan in representative confocal images) shows that biofilms establish a high HlyA toxin abundance near the macrophage surface compared to non-biofilm producing bacteria. Bars represent the probability for a particular HlyA abundance per macrophage for different conditions (described with the same color code in the table). Antibody treatments: “a,” addition of mouse anti-HlyA antibody; “b,” addition of anti-mouse Alexa Fluor 647 antibody. Number of imaged field of views that were analyzed: N_ΔhlyA, _{Ptac-hlyA,a+b} = 38, N_ΔhlyA,_{ΔmshAΔtcpAPtac-hlyA,a+b} = 30, N_ΔhlyA, _Ptac-hlyA,b = 30 from n = 3 independent biological replicates.

Figure S4

The toxin HlyA is primarily responsible for causing macrophage death, refers to Figure 5 The extent of cell death of THP-1-derived macrophages was determined after 7 h of co-incubation with V. cholerae strains carrying the ΔhlyA deletion and the deletion of one additional (putative) toxin. The results were normalized to the mean of the ΔhlyA strain. Compared with the ΔhlyA strain, V. cholerae deletion mutants lacking hlyA together with one additional gene encoding a known or putative toxin do not significantly alter the occurrence of macrophage death. Bars represent the mean values, and error bars denote the standard deviation. The changes in macrophage death for the different bacterial strains in comparison to ΔhlyA are not statistically significant. Statistical significance was calculated using one-way ANOVA (n = 9–25 independent biological replicates). Error bars denote the standard deviation.

Figure 6

V. cholerae cells break the human intestinal epithelial barrier and subsequently form biofilms on macrophages underneath (A) Differentiated human enteroid monolayers (donor #1) were grown on a permeable membrane insert (3 μm pore size) and placed above THP-1 macrophages as illustrated in the schematic diagram of the experimental setup. After adding V. cholerae to the apical side of the epithelium, bacteria grew and accumulated. Cell numbers at the start of infection: 3.2 × 10⁶ bacteria, 9.1 × 10⁴ epithelial cells, and 2.5 × 10⁴ THP-1 macrophages. Different MOIs are shown in Figure S5B, results for primary macrophages are shown in Figure S5A, and results for enteroid monolayers from a different donor are shown in Figure S6. Over time, V. cholerae broke through the monolayer, reached the basal side, and formed biofilms on the macrophage surface. Representative confocal fluorescence images of n = 3 independent biological replicates show the xz side view (maximum projection) of enteroid monolayers, macrophages, and bacteria at the start of the co-culture and at the time of peak biofilm formation on macrophages (magenta: epithelial cells, red: macrophages, cyan: V. cholerae). For the same time points, xy images show the macrophages and V. cholerae biofilms underneath the epithelial cells. (B) Visualization of MSHA pili, TC pili, and TcpF using fluorescently conjugated antibodies (shown in yellow) inside V. cholerae biofilms formed on macrophages during co-culture of enteroid monolayers, macrophages, and bacteria. Images are representative of n = 3 independent biological replicates. (C–F) Co-culture of enteroid monolayers and macrophages infected with different V. cholerae mutants (mutations are indicated above each panel). Representative fluorescence images of n = 3 independent biological replicates show xz side views (maximum projection) and xy images in the same format as for (A). Images show the bacteria interaction with epithelial cells and macrophages at the start of co-culture and peak time of biofilm formation on macrophages (for C and D) or a time at which WT bacteria would have normally formed biofilms (for E and F). Results for additional mutants are shown in Figures S5C–S5F. Cell numbers at the start of infection: 3.2 × 10⁶ bacteria, 9.1 × 10⁴ epithelial cells, and 2.5 × 10⁴ THP-1 macrophages.

Figure S5

Infection of the enteroid-macrophage co-culture model with V. cholerae for different MOIs, different bacterial mutants, and different macrophages, refers to Figure 6 (A) A confluent intestinal epithelial monolayer comprising 9.1 × 10⁴ cells was grown from human enteroids (donor #1) on a permeable membrane insert (3 μm pore size) and placed above 8 × 10⁴ macrophages that were differentiated from primary monocytes obtained from human blood. After adding 3.2 × 10⁶V. cholerae WT cells, the co-culture dynamics resembled the ones observed for THP-1 macrophages shown in Figure 6A. Representative confocal fluorescence microscopy images of n = 3 independent biological replicates show the xz side view (maximum projection) at the start of the co-culture and at the time of peak biofilm formation on primary macrophages (magenta: epithelial cells, red: macrophages, cyan: V. cholerae). For the same time points, xy images show macrophages and V. cholerae biofilms underneath the epithelial cells. (B) Co-culture of intestinal epithelial monolayers grown from enteroids (donor #1) on a membrane insert with 3 μm pore size and THP-1 macrophages, which was inoculated with varying numbers of V. cholerae WT cells (for 9.1 × 10⁴ epithelial cells, 2.5 × 10⁴ THP-1 macrophages). Higher bacterial cell numbers at the start of infection resulted in an earlier crossing of the epithelial barrier and biofilm formation on macrophages. Representative fluorescence images of n = 3 independent biological replicates show the xz side view (maximum projection) of enteroid monolayers, macrophages, and bacteria at the start of the co-culture and at the time of peak biofilm formation on macrophages. For the same time points, xy images show macrophages and V. cholerae biofilms underneath the epithelial cells. (C–F) Co-culture of confluent intestinal epithelial monolayers grown from enteroids (donor #1) on a membrane insert with 3 μm pore size with THP-1-derived macrophages and different V. cholerae deletion mutants (mutations are indicated above each panel). Cell numbers at the start of infection: 3.2 × 10⁶ bacteria, 9.1 × 10⁴ epithelial cells, and 2.5 × 10⁴ THP-1 macrophages. Representative confocal fluorescence microscopy images of n = 3 independent biological replicates for each mutant show xz side views (maximum projection) and xy images in the same format as for (A). (C)–(E) show that V. cholerae impaired in the production of the known biofilm matrix components Bap1, RbmA, RbmC, and VPS as well as cells lacking cholera toxin (ΔctxBA) or hemolysin (ΔhlyA) accumulated on the apical side of monolayers, crossed the epithelial barrier, and formed biofilms on the surface of the underlying macrophages, similar to the WT. Microscopy images show the bacterial interaction with enteroid monolayers and macrophages at the start of co-culture and the time of peak biofilm formation on macrophages. (F) shows V. cholerae deficient in the production of MSHA pili, TC pili, and hemolysin (ΔmshA ΔtcpA ΔhlyA) were unable to form biofilms on the surface of macrophages after crossing the epithelial barrier. Microscopy images show the start of the experiment and a time point when the WT would have formed biofilms.

Figure S6

Co-culture results of different V. cholerae strains with THP-1 macrophages and human enteroid monolayers from donor #2, refers to Figure 6 These results are analogous to those shown in Figures 6A and 6C–6F but using a confluent intestinal epithelial monolayer that was generated from enteroids of donor #2. The human enteroid monolayer was grown on a permeable membrane insert (3 μm pore size) and placed above THP-1 macrophages as illustrated in the schematic diagram of the experimental setup (magenta: epithelial cells, red: macrophages, cyan: V. cholerae). Representative confocal fluorescence microscopy images of n = 3 independent biological replicates are shown for each bacterial strain. Cell numbers at the start of infection: 3.2 × 10⁶ bacteria, 31 × 10⁴ epithelial cells, and 2.5 × 10⁴ THP-1 macrophages. (A–F) V. cholerae WT cells and bacteria lacking MSHA pili (ΔmshA) or TC pili (ΔtcpA) formed biofilms on the surface of macrophages after crossing the epithelial barrier. Similarly, biofilm formation on macrophages was observed for bacteria that cannot produce the biofilm matrix components RbmA, RbmC, Bap1, and VPS (Δbap1 ΔrbmA ΔrbmC ΔvpsL) and bacteria that cannot produce the cholera toxin (ΔctxBA) or hemolysin (ΔhlyA). Images show the xy plane at the start of the co-culture and at peak time of biofilm formation on macrophages. (G–H) Bacterial strains that cannot produce MSHA pili and TC pili (ΔmshA ΔtcpA or ΔmshA ΔtcpA ΔhlyA) were unable to form biofilms on macrophages after breaching the epithelial barrier. Images show the xy plane at the start of the co-culture and a time point when WT bacteria would have formed biofilms on macrophages. (I) Non-motile bacteria (ΔflaA) could not break through the epithelial barrier, and ΔflaA mutants were consequently not observed at the basal side of enteroid monolayers. Microscopy images show the start of the experiment and a time point when WT bacteria would have formed biofilms on macrophages.