The following antibodies were utilized for western blot and immunofluorescence studies. Mouse anti-PI3,4P2 mAb and mouse anti-PI3,4,5P3 mAb conjugated with FITC from Echelon Bioscience (Salt Lake City, UT). Rabbit anti-EEA1 mAb from Cell Signaling Technology (Danvers, MA). Goat anti-rabbit Ig-HRP conjugate, goat anti-mouse Ig-HRP conjugate, and goat anti-mouse pAb conjugated with Alexa Fluro 488 from Invitrogen (Carlsbad, CA). Rabbit anti-LAMP1 pAb, and rabbit anti-Rab5 pAb from Abcam (Cambridge, England). Mouse anti-Rab7 mAb from Sigma Aldrich (St. Louis, MO). For phagocytosis and phagosome maturation assays, pHrodo™ Red E. coli BioParticles™ Conjugate and DQ™-BSA Red (used in 96 well plate assay) and DQ™-BSA -Green (used for live cell assay) was purchased from Invitrogen.

The human acute monocytic leukemia cell line, THP-1, was obtained from ATCC (Manassas, VA); cells was maintained in RPMI1640-containing 10% FBS, 1 mM sodium pyruvate, 20μM 2-mercaptoethanol and 2% penicillin-streptomycin at 37°C with 5% CO₂ in a humidified incubator. THP-1 cells were differentiated into macrophages on 35mm glass bottom dishes (MatTek; Ashland, MA), 12 wells cell culture plate (Thermo Scientific; Waltham MA), or 96 wells cell culture plate (Thermo Scientific; Waltham MA) by incubating cells in the presence of 50ng/ml PMA for 48 hours at which time the cells were washed and incubated an additional 24 h in medium prior to use (Shenker et al., 2014).

Construction and expression of the plasmid containing the cdt genes for the holotoxin (pUCAacdtABChis) have previously been reported (Shenker et al., 2004). The plasmid was constructed so that cdt genes were under control of the lac promoter and transformed into E. coli DH5α. Cultures of transformed E. coli were grown in 1L LB broth and induced with 0.1 mM of isopropyl β-D-1-thiogalactopyranoside for 2 h; bacterial cells were harvested, washed, and resuspended in 50 mM of Tris (pH 8.0). The cells were frozen overnight, thawed, and sonicated. The histidine-tagged peptide holotoxin was isolated by nickel affinity chromatography as previously described (Shenker et al., 2000).

Cells (1×10⁶ cells/well) were incubated with Cdts or media for 240 min. Replicate cultures (0.5–1×10⁷cells) were pooled and harvested. Lipids were extracted as described (Shenker et al., 2014).. The cell pellet was treated with cold 0.5 TCA for 5 min, centrifuged and washed twice with 5% TCA containing 1 mM EDTA. Neutral lipids were extracted twice with methanol:chloroform (2:1) at room temperature. Acidic lipids were extracted with methanol:chloroform:12M HCl (80:40:1) for 15 min at room temperature; the samples were centrifuged for 5 min and the supernatant recovered. The supernatant was then treated with 0.75 ml chloroform and 0.1 M HCl and centrifuged to separate organic and aqueous phases; the organic phase was collected and dried. The dried lipids were resuspended in 120μl 50 mM Hepes buffer (pH 7.4) containing 150 mM NaCl and 1.5% sodium cholate and left overnight at 4°C. PI(4,5)P3, PI(3,4)P3, and PI(3,4,5)P3 levels were then determined using commercially available competitive ELISA according to the manufacturer’s directions (Echelon).

Cells were treated with Cdts and solubilized in 20mM Tris-HCl buffer (pH7.5) containing 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, and protease inhibitor cocktail (ThermoFisher Scientific; Waltham, MA). Samples (15μg) were separated on 12% SDS-PAGE and then transferred to PVDF membranes. The membrane was blocked with BLOTTO and then incubated with primary antibodies overnight (18hrs) at 4^°C (Boesze-Battaglia et al., 2017). Membranes were washed and incubated with secondary antibodies conjugated to horseradish peroxidase (Boesze-Battaglia et al., 2017). Western blots were developed using chemiluminescence and analyzed by digital densitometry (LiCor Biosciences; Lincoln, NE) (Shenker et al., 2020). Each protein was normalized to either actin or GAPDH.

A.actinomycetemcomitans strains, D7S-SA (wild type Aa) and D7S-SA CHE001 (Cdt deficient Aa mutant), were obtained as described by Nalbant, A., et al (Nalbant et al., 2003). Each strain was plated on AAGM agar which consisted of 20g of BBL trypticase soy agar (BD; Sparks, MD); 3g of yeast extract (ThermoFisher) supplemented with 0.4% sodium bicarbonate and 0.8% dextrose (Sreenivasan et al., 1993). After bacteria were grown on plates for 24 or 48 hours in the incubator with 10% CO₂ at 37°C, they were inoculated in 10ml of AAGM broth until OD₆₀₀ close to 0.2. Bacteria were plated from different dilutions at various time points on AAGM agar plates and incubated for 24 hours in the incubator with 10% CO2 at 37°C (Nalbant et al., 2003). OD₆₀₀ was measured using DU 650 Spectrophotometer (Beckman Coulter, Indianapolis IN) for each time point.

THP-1 macrophages (10⁶cells/well) were differentiated on 12 well plates and incubated in the presence of medium only or with Cdt for 240 min. THP-1 macrophages were washed three times with cell media without penicillin/streptomycin prior to inoculation. Aa strains were cultured until log phase (OD₆₀₀ of 0.5 to 1) and used to inoculate with Multiplicity of Infection (MOI) (eukaryotic cells: bacteria) of 1:10 for 12 hours at 37°C with 5% CO₂ in media without penicillin/streptomycin. Cells were then washed with PBS three times and treated with gentamicin (50μg/ml) to wash away and kill extracellular bacteria (Ando-Suguimoto et al., 2014; Sharma and Puhar, 2019). Soy broth with 1% saponin (500μl/well) was added and the mixture incubated at 37°C with 10% CO₂ for 15 min to lyse macrophages (Perez-Stuardo et al., 2019). Lysates were plated (100μl/plate) on AAGM agar plates and incubated for 1 to 2 days at 37°C with 10% CO₂. The number of survived bacteria was determined by the number of colonies forming unit (CFU). Same method was applied for 0 hour to measure initial CFU of phagocyted Aa population in THP-1 macrophages. Percent of Aa surviving after 12 hours was calculated by using equation below:

Cdt treated macrophages exhibit perturbations in intracellular PI pools consistent with the CdtB subunit’s function as a PIP3 phosphatase (Shenker et al., 2014). Cells were exposed to either Cdt holotoxin containing the wildtype CdtB submit (Cdt^WT) or Cdt holotoxin containing the phosphatase deficient CdtB subunit (Cdt^R117A). Macrophages treated with Cdt^WT exhibited a two-fold increase in PI(3,4)P2 levels with a corresponding decrease in PI(3,4,5)P3 levels ( Figure 1A ). As expected due to CdtB’s enzymatic 5’-phosphatase specificity, no change in PI(4,5)P2 was observed ( Figure 1A ). Furthermore, no change in PI(3,4)P2 or PI(3,4,5)P3 levels was observed in cells treated with Cdt^R117A ( Figure 1A ). The increase in PI(3,4)P2 observed with Cdt^WT appeared to be associated with intracellular and plasma membrane regions (indicated by white arrows, Figure 1B ); semi-quantitative analyses indicated a 50% increase in PI(3,4)P2 association within these regions. No increase in PI(3,4)P2 levels were observed in cells treated with CdtB^R117A. Figure 1C , showed distinct PI(3,4)P2 domains (white arrows) with CdtB^WT concentrations of 125ng/ml and higher. There was a ~35% increase in PI(3,4)P2 with 125ng/ml and an almost 80% increase when cells were treated with 500ng/ml Cdt^WT (Shenker et al., 2016).

Having established that Cdt treatment of THP1 macrophages increases intracellular pools of phosphoinositides involved in phagosome processing, we next assessed the ability of Cdt to modulate macrophage phagocytic function with an opsonized pHrodo™ red E. coli BioParticles™ conjugate using live cell imaging. In preliminary experiments we determined that 10μg/ml pHrodo™ was ideal for imaging studies, (data not shown). The red pH-sensitive fluorogenic dye contributing to pHrodo™ intensity (cyan pseudo colored for visualization) was followed for 180min. During this time, pHrodo™ intensity in control (untreated) cells started to dramatically increase at 90 min (4.5x10⁶ to 9.4x10⁶ relative intensity) reaching 2.9x10⁷ relative intensity units by 180 min. In contrast, when cells were pre-treated with Cdt^WT (500ng/ml) the magnitude of change in pHrodo fluorescence intensity was comparatively suppressed with minimum of 1.3x10⁶ relative intensity to maximum of 1.7x10⁷ relative intensity, at 180 min. The maximum intensity observed with Cdt^WT treatment was 40% less than of that observed in control at 180min ( Figure 2A ), suggesting decreased phagocytic function. In order to conserve materials in our analysis of phosphatase deficient CdtB subunit (Cdt^R117A), we turned to a 96 well plate format. Macrophages were pre-treated with Cdt^WT or Cdt^R117A, at concentrations of 125-500ng/ml, prior to the addition of pHrodo™. Macrophage phagocytic function, represented by pHrodo™ fluorescence intensity, decreased by over 40% in cells pretreated with 250ng/ml Cdt^WT and by over 50% with 500ng/ml Cdt^WT ( Figure 2B ). There was no change in pHrodo intensity compared to controls when cells were pretreated with Cdt^R117A at any concentration studied ( Figure 2B ). These studies suggest modulation macrophage phagocytic function was dependent on CdtB phosphatase activity.

The pHrodo™ studies suggested that Cdt^WT may modulate phagosome maturation, a process during which effector proteins associate with specific phosphoinositide pools. We sought to determine if there was a spatio-temporal relationship between phosphoinositide pools and phagocytic cargo, specifically ingested latex beads. We first wanted to determine if Cdt treatment altered the composition of phosphoinositides associated with phagosomes. Using opsonized red latex beads (1μm diameter) as the phagocytic cargo, we analyzed the region around the phagocytosed bead for phosphoinositide association using multi-fluor confocal imaging. To assess difference in latex bead-PI(3,4)P2 association in the presence of Cdt^WT, macrophages were pre-treated with 500ng/ml of Cdt^WT, Cdt^R117A, or no treatment (control) for 4 hours. Using a synchronized challenge protocol, opsonized latex beads were added at 4°C with a 30min incubation and phagocytosis intuited with an increase in temperature to 37°C). After 60 minutes of phagocytosis, we analyzed a region centered around the bead in the Z axis with fluorescence intensity measured within a 2μm x 2μm. A PI(3,4)P2 pool was observed associated with the phagocytosed bead in the presence of Cdt^WT ( Figure 3A ). There was no detectable PI(3,4)P2-latex bead association when macrophages were pretreated with Cdt^R117A or in untreated controls ( Figures 3B, C ). The observation that Cdt^WT enhanced PI3,4 P2 levels in phagosome membranes led us to consider if effector protein association with phagosomes was altered in the presence of toxin.

We next sought to determine if Cdt modulated phagosome association with effector proteins, EEA1, and Rab5. Using opsonized latex beads as the phagocytic cargo, we followed the association of Rab5 and EEA1 with this cargo in the presence of Cdt^WT (500ng/ml) using multi-fluor confocal imaging. In these latex bead–protein association studies, we measured fluorescence intensity around the 1μm diameter latex bead. The area of analysis was confined to a region centered around the bead as in the latex bead-PI(3,4)P2 association studies above ( Figure 3 ) and described in Methods. In macrophages treated with Cdt^WT, we observed a decrease in Rab5 association with beads when a 2μm square area around the latex bead was analyzed for Rab5 recruitment. At 30 min, the association of Rab5 with beads decreased by 28% with a further decrease to 32% at 60 min, compared to untreated control cells ( Figure 4A ). Rab5-latex bead association was also unchanged in cells pretreated with Cdt^R117A ( Figure 4A ) implicating CdtB phosphatase activity as decreasing dissociation of phagosome-Rab5.

Both Rab5 and EEA1 are associated with phagosomes early in the maturation process. In contrast to Rab5 association, EEA1-latex bead association was unaltered in macrophages treated with Cdt^WT or Cdt^R117A at both the 30 and 60 min time points ( Figure 4B ). Moreover, no change in latex-bead association with either Rab7 or LAMP1 was observed in Cdt^WT or Cdt^R117A treated cells ( Supplementary Figure 1 ) compared to untreated controls. Neither Cdt^WT nor Cdt^R117A (500ng/ml) treatment of macrophages altered the levels of any of the intracellular trafficking proteins studied; EEA1, Rab5, Rab7 or LAMP1, compared to untreated control ( Figure 5 ). Collectively, these studies suggest that CdtB phosphatase activity dependent changes in phosphoinositide decreases Rab5 association with phagosomes thereby delaying phagosome maturation.

The pHrodo™ studies suggested decreased phagocytic capacity but they did not specifically focus on later steps in phagosome maturation. To determine if CdtB phosphatase activity modulates phagolysosome formation, we followed DQ™-BSA fluorescence in macrophages treated with Cdt^WT or Cdt^R117A. Cleavage of the self-quenched DQ™-BSA protease substrate in an acidic compartment generates a highly fluorescent product (Frost et al., 2017). DQ™-BSA intensity was followed for 180min, the control (untreated) cells reached maximum intensity (5.5x10⁸ intensity) by 120min while cells pre-treated with Cdt^WT (500ng/ml) reached maximum intensity (3.7x10⁸ intensity) at 150min. Also, when we employed the 96-well plate assay at 180min, macrophages pretreated with Cdt^WT (500ng/ml) exhibited a 40% decrease in DQ™-BSA fluorescence ( Figure 6A ) suggesting a reduction in phagolysosome formation. Cdt^R117A treatment did not alter DQ™-BSA fluorescence implicating phosphatase activity as the major contributor to decreased phagolysosome formation ( Figure 6B ). Lastly, neither Cdt^WT nor Cdt^R117A, altered lysosome integrity ( Supplementary Figure 2 )

The studies detailed above suggest that Cdt^WT decreases phagosome maturation by modulating PI pools and decreasing phago-lysosome fusion. In this final series of experiments, we assessed Aa survival to determine if Cdt^WT alters macrophage bactericidal function. When THP1 macrophages were inoculated with wild type Aa (D7S), or the mutant Aa lacking Cdt (D7SΔCdt) at a multiplicity of infection (MOI) of 1:10 for 12 hours, both bacterial strains exhibited virtually the same extent of survival when to compared 0 hour (time-zero) CFUs ( Figure 7A ). To determine if macrophage exposure to Cdt, as may occur in vivo alters Aa phagocytic processing, macrophages were pretreated with Cdt at a concentration shown to decrease phagosome maturation and phago-lysosome fusion ( Figure 6A ). When macrophages were pretreated with Cdt^WT (500ng/ml) and subsequently inoculated with either wild type Aa or Cdt deficient mutant Aa, there was a 17.2% increase in Aa (D7S), and an 18.8% increase in Aa (D7SΔCdt) survival. Thus, pretreatment of THP1 derived macrophages with Cdt^WT resulted in an increase in Aa survival and a decrease in bactericidal effect ( Figure 7A ). When we utilized an MOI of 1:100 for 12 hours, macrophage cell death was observed (data not shown).