Polyphosphate Is an Extracellular Signal That Can Facilitate Bacterial Survival in Eukaryotic Cells

Polyphosphate is an extracellular signal that can facilitate bacterial survival in eukaryotic cells

Ramesh Rijala, Louis A. Cadenaa, Morgan R. Smitha, Joseph F. Carra, and Richard H. Gomera,1

aDepartment of Biology, Texas A&M University, College Station, TX 77843-3474

Edited by Julien Vaubourgeix, Imperial College London, London, United Kingdom, and accepted by Editorial Board Member Carl F. Nathan October 13, 2020 (received for review June 11, 2020) Polyphosphate is a linear chain of phosphate residues and is display decreased growth, reduced sensitivity to stress and star- present in organisms ranging from bacteria to humans. Pathogens vation, decreased survival, reduced invasiveness, defects in such as Mycobacterium tuberculosis accumulate polyphosphate, quorum sensing, and defects in other features associated with and reduced expression of the polyphosphate kinase that synthe- virulence (13, 14, 23, 24). However, the role of PPK in the sur- sizes polyphosphate decreases their survival. How polyphosphate vival of pathogenic bacteria is not clearly understood. potentiates pathogenicity is poorly understood. Escherichia coli K- The eukaryotic social amoeba D. discoideum uses phagocytosis 12 do not accumulate detectable levels of extracellular polyphos- to uptake nutrients such as bacteria (25). D. discoideum cells phate and have poor survival after phagocytosis by Dictyostelium accumulate extracellular polyphosphate, and, as the local cell discoideum or human macrophages. In contrast, Mycobacterium density increases, the extracellular polyphosphate concentration smegmatis and Mycobacterium tuberculosis accumulate detect- increases (26). To anticipate starvation when a colony of cells is able levels of extracellular polyphosphate, and have relatively bet- about to overgrow its food supply, the concomitant high extra- ter survival after phagocytosis by D. discoideum or macrophages. cellular polyphosphate concentration inhibits proliferation (26). Adding extracellular polyphosphate increased E. coli survival after The G-protein–coupled receptor glutamate receptor-like protein phagocytosis by D. discoideum and macrophages. Reducing ex- D (GrlD) binds, and helps cells sense, polyphosphate (26, 27). pression of polyphosphate kinase 1 in M. smegmatis reduced ex- Since not digesting nutrients might be a way to inhibit D. tracellular polyphosphate and reduced survival in D. discoideum discoideum proliferation, we examined whether polyphosphate and macrophages, and this was reversed by the addition of extra-

might inhibit phagosome maturation in D. discoideum and hu- CELL BIOLOGY cellular polyphosphate. Conversely, treatment of D. discoideum man macrophages. In this report, we show that both exogenous and macrophages with recombinant yeast exopolyphosphatase polyphosphate and polyphosphate released from bacteria inhibit reduced the survival of phagocytosed M. smegmatis or M. tuber- phagosome maturation in D. discoideum and that this effect is culosis. D. discoideum cells lacking the putative polyphosphate conserved in human macrophages. receptor GrlD had reduced sensitivity to polyphosphate and, compared to wild-type cells, showed increased killing of phagocytosed Results E. coli and M. smegmatis. Polyphosphate inhibited phagosome Polyphosphate Inhibits the Killing of Ingested Escherichia coli in D. acidification and lysosome activity in D. discoideum and macro- discoideum and Human Macrophages. We previously observed that phages and reduced early endosomal markers in macrophages. wild-type (WT) D. discoideum cells accumulate extracellular Together, these results suggest that bacterial polyphosphate potentiates pathogenicity by acting as an extracellular signal that inhibits phagosome maturation. Significance

polyphosphate | exopolyphosphatase | polyphosphate kinase | Macrophages use phagocytosis to engulf bacteria such as E. coli Mycobacterium tuberculosis | Burkholderia and kill them. Pathogenic bacteria such as Mycobacterium tuberculosis are similarly ingested, but not killed, by macro- n metazoans, cells such as macrophages use phagocytosis to phages. We find that M. smegmatis and M. tuberculosis Iobtain nutrients, remove cell debris, and engulf and kill path- bacteria accumulate high levels of extracellular polyphosphate ogens (1). Phagocytosis begins by recognition of particles by cell- compared to E. coli and survive better after phagocytosis in surface receptors and engulfment of the ingested particle to form Dictyostelium discoideum or macrophages. The addition of a phagosome. Ingested material in the phagolysosome is then polyphosphate causes some D. discoideum or macrophages to keep ingested nonpathogenic E. coli bacteria alive. Conversely, degraded by lysosomal acid, enzymes, and oxygen radicals (2, 3). exopolyphosphatase, an enzyme that degrades poly- Many successful pathogens, including Mycobacterium tuberculo- phosphate, causes D. discoideum and macrophages to kill more sis, Legionella pneumophila, Neisseria gonorrhoeae, and Strepto- of the ingested bacteria. Polyphosphate inhibits phagosome coccus pyogenes, have evolved countermeasures to evade phago- acidification and lysosome activity in D. discoideum and mac- lysosomal killing, allowing the pathogen to live in the arrested – rophages, suggesting that extracellular or intraphagosomal phagosome (4 8). One commonly used countermeasure is to polyphosphate activates a pathway to potentiate viability of inhibit phagosome acidification and fusion with lysosomes ingested bacteria. (9–11). How pathogens inhibit this process is poorly understood. Polyphosphate is a linear chain of phosphate residues present Author contributions: R.R. and R.H.G. designed research; R.R., L.A.C., M.R.S., and J.F.C. in all kingdoms of life (12). Polyphosphate metabolism is asso- performed research; R.R. and R.H.G. analyzed data; and R.R. and R.H.G. wrote the paper. ciated with the virulence of pathogens such as Burkholderia The authors declare no competing interest. mallei, M. tuberculosis, Salmonella enterica, Shigella flexneri, and This article is a PNAS Direct Submission. J.V. is a guest editor invited by the Pseudomonas aeruginosa (13–17). In a wide range of bacteria, Editorial Board. including pathogens, polyphosphate is synthesized from ATP by Published under the PNAS license. an essential enzyme polyphosphate kinase (PPK), and poly- 1To whom correspondence may be addressed. Email: [email protected]. phosphate levels are maintained by exopolyphosphatase (PPX), This article contains supporting information online at https://www.pnas.org/lookup/suppl/ an enzyme that degrades polyphosphate by removing terminal doi:10.1073/pnas.2012009117/-/DCSupplemental. phosphate residues (18–22). PPK mutants of many pathogens

www.pnas.org/cgi/doi/10.1073/pnas.2012009117 PNAS Latest Articles | 1of12 Downloaded by guest on October 4, 2021 polyphosphate, and, at high cell densities, corresponding to The uningested bacteria were removed, ingested bacteria were stationary phase (more than ∼2 × 107 cells per mL), the extra- cultured for 4 and 48 h, the D. discoideum cells were lysed, re- cellular polyphosphate concentration reaches ∼704 μg/mL, and leased bacteria were plated, and the colony-forming units (cfus) this concentration of polyphosphate inhibits proliferation and of viable ingested E. coli were counted. We noticed that low induces development (26). D. discoideum cells uptake nutrients concentrations of polyphosphate, which do not significantly af- from liquid medium by macropinocytosis (28). The endocytosed fect proliferation, pinocytosis, or exocytosis, affect bacterial di- material is transported to early and late endosomes and fuses gestion. At 4 h, compared to cells with no polyphosphate, D. with lysosomes before degradation. The undigested material in discoideum cells in the presence of low concentrations of poly- postlysosomes is then exocytosed (28). To test if polyphosphate phosphate showed no change in number of viable ingested E. coli inhibits D. discoideum macropinocytosis, we measured the en- (Fig. 1A). The addition of recombinant Saccharomyces cerevisiae docytosis and exocytosis of tetramethylrhodamine isothiocynate PPX, an enzyme that degrades polyphosphate (31), did not affect (TRITC)-labeled dextran (29). Endocytosis of TRITC–dextran the number of viable ingested E. coli at 4 h (Fig. 1A). At 48 h, in was decreased by 470 μg/mL and higher concentrations of ex- the absence of polyphosphate, there was no viable ingested tracellular polyphosphate (SI Appendix, Fig. S1 A and B). Simi- E. coli present in cells, whereas in the presence of low concen- larly, 470 μg/mL and higher polyphosphate concentrations trations of polyphosphate, there was viable ingested E. coli reduced exocytosis (SI Appendix, Fig. S1 C and D). Together, this (Fig. 1A). The effect of polyphosphate was reversed with the suggests that, as cells proliferate to high cell densities, the con- addition of PPX, indicating that the effect was due to poly- comitant high concentrations of extracellular polyphosphate in- phosphate, and not to a contaminant in the polyphosphate hibit proliferation by inhibiting nutrient uptake and subsequent (Fig. 1A). To determine the location of ingested bacteria, D. processing and exocytosis of waste products. discoideum cells were incubated with LysoTracker, which stains D. discoideum normally feed on bacteria (30). To determine if acidic compartments (32), and ptrc99A–enhanced yellow fluo- extracellular polyphosphate affects uptake and digestion of rescent protein (eYFP) E. coli (33) in the presence or absence of bacteria, D. discoideum cells were incubated with Escherichia coli polyphosphate. At 4 h, compared to D. discoideum cells with K-12 in the presence or absence of extracellular polyphosphate. no added polyphosphate, cells with 13 μg/mL polyphosphate

Fig. 1. Polyphosphate potentiates the survival of ingested E. coli in D. discoideum and human macrophages. (A) D. discoideum (Dicty) cells were incubated with E. coli, uningested E. coli were removed, and viable ingested E. coli per 106 D. discoideum cells, in the absence or in the presence of polyphosphate (PolyP) and/or S. cerevisiae PPX, were determined at 4 and 48 h. (B) D. discoideum cells with ingested HCB33 (RP437)/ptrc99A–eYFP E. coli (green), in the absence of polyphosphate (–) or in the presence of 15 μg/mL polyphosphate (+), were stained with LysoTracker (red) at 4 and 48 h. Arrows indicate E. coli puncta. (Bar: 10 μm.) (C) eYFP fluorescence intensity of at least 60 cells from three independent experiments from B were measured at 4 and 48 h. Total eYFP fluorescence − with no polyphosphate (−) at both time points was considered one. (D) Viable ingested E. coli per 106 WT and grlD D. discoideum cells, in the absence of polyphosphate (–) or in the presence of 15 μg/mL polyphosphate (+), were determined at 4 and 48 h. (E) Viable ingested E. coli in macrophages, as in A, were determined at 4 and 24 h. All values are mean ± SEM of five (A), three (C), five (D), and four (E) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant (t tests).

2of12 | www.pnas.org/cgi/doi/10.1073/pnas.2012009117 Rijal et al. Downloaded by guest on October 4, 2021 contained ptrc99A–eYFP E. coli that did not colocalize with donor blood with granulocyte-macrophage–colony-stimulating LysoTracker-stained acidic compartments (Fig. 1B). At 48 h, factor (GM-CSF) for 5 d. Macrophages in the presence or ab- enlarged regions of ptrc99A–eYFP E. coli were observed in the sence of extracellular polyphosphate were incubated with E. coli, presence of polyphosphate, and the relative eYFP fluorescence and the cfus of the ingested E. coli were determined at 4 and 24 per cell increased (Fig. 1 B and C). To determine if the h. At 4 h, polyphosphate did not affect the number of viable polyphosphate-induced increase in bacterial survival was due to ingested E. coli in macrophages (Fig. 1E). At 48 h, colony counts an increase in D. discoideum phagocytosis, D. discoideum cells for 0, 5, 8, 10, 13, and 15 μg/mL polyphosphate were 0 ± were incubated with pHrodo zymosan bioparticles in the pres- 0 (mean ± SEM n = 4 [two male and two female donors]), in- ence or absence of polyphosphate, and the number of bio- dicating that the macrophages killed all of the ingested E. coli in particles ingested by D. discoideum cells was measured. the presence of up to 15 μg/mL polyphosphate. However, at 24 h, Polyphosphate did not significantly affect the phagocytosis rate compared to macrophages with no added polyphosphate, mac- (SI Appendix, Fig. S1E). These data suggest that, while poly- rophages with 15 μg/mL additional extracellular polyphosphate phosphate inhibits lysis or clearance of most of the ingested showed an increase in the number of viable ingested E. coli for E. coli in D. discoideum cells, only some of the intracellular male and female donors (Fig. 1E and SI Appendix, Fig. S2). This E. coli actually survive and proliferate, giving rise to bacterial 15 μg/mL polyphosphate concentration is higher than the 5 ± counts at 48 h similar to those at 4 h. 1 μg/mL polyphosphate concentration in human plasma (36) and D. discoideum cells lacking a putative polyphosphate receptor, lower than the 470 μg/mL polyphosphate concentration that in- GrlD, are insensitive to polyphosphate-induced proliferation hibits human PBMC proliferation (37). inhibition and show strongly reduced binding of polyphosphate (27). To determine if polyphosphate inhibits the killing of bac- − Polyphosphate Inhibits the Killing of Ingested Mycobacterium teria in D. discoideum cells lacking GrlD (grlD cells), viable − smegmatis in D. discoideum and Human Macrophages. Reduced ingested E. coli were counted in WT and grlD cells. At 4 h expression of the polyphosphate-synthesizing enzyme PPK1 in without polyphosphate, compared to WT cells, there were ap- − M. tuberculosis decreases bacterial survival in human macro- proximately fivefold more viable ingested E. coli present in grlD cells (Fig. 1D). Adding polyphosphate did not affect the number phages (23). To determine if reduced expression of PPK1 in − of viable ingested E. coli in WT or grlD cells (Fig. 1D). At 48 h Mycobacterium smegmatis, a nonpathogenic model to study My- without polyphosphate, there were very few ingested E. coli in cobacterium infection, decreases bacterial survival in D. dis- − WT or grlD cells. Unlike in WT cells, polyphosphate did not coideum and human macrophages, the ppk1 gene in M.

− CELL BIOLOGY increase the number of viable ingested E. coli in grlD cells smegmatis was knocked down by using Clustered Regularly (Fig. 1D). This suggests that GrlD is required for D. discoideum Interspaced Short Palindromic Repeat (CRISPR) interference cells to sense polyphosphate to inhibit the killing of ingested (CRISPRi) to generate ppk1 knockdown cells (generated by E. coli. Thomas Snavely, Department of Biochemistry and Biophysics, Bacterial infection can be studied by using macrophages and Texas A&M University, College Station, TX), and reduced ex- D. discoideum cells as model systems (34). In metazoans with pression of ppk1 was confirmed by RT-PCR (SI Appendix, Fig. immune systems, macrophages protect their host by killing S3 A and B). E. coli did not accumulate detectable extracellular ingested microbes (35). To determine if extracellular poly- polyphosphate, while M. smegmatis accumulated detectable ex- phosphate inhibits the killing of ingested E. coli by human tracellular polyphosphate (Fig. 2A). Knockout of ppk in E. coli macrophages, human macrophages were generated by culturing did not affect the accumulation of extracellular polyphosphate peripheral blood mononuclear cells (PBMCs) isolated from (Fig. 2A and SI Appendix, Fig. S3C), whereas, compared to WT

Fig. 2. Polyphosphate potentiates the survival of M. smegmatis (M. smeg.) bacteria in D. discoideum and human macrophages. (A) Extracellular polyphosphate (PolyP) concentrations from WT and ppk knockout (Δppk) E. coli, or WT and ppk1 knockdown (ppk1 kd) M. smegmatis bacteria after 24 h of growth were determined by using DAPI. (B and C) Viable ingested WT and ppk1 knockdown M. smegmatis per 106 D. discoideum (Dicty) cells, in the absence − or presence of polyphosphate and/or PPX, were determined at 4 h (B) and 48 h (C). (D) Viable ingested M. smegmatis in WT and grlD D. discoideum cells were determined at 4 and 48 h, as in B.(E and F) Viable ingested WT and ppk1 knockdown M. smegmatis in human macrophages, in the absence of polyphosphate (–) or in the presence (+) of 470 μg/mL polyphosphate, were determined at 4 h (E) and 48 h (F). All values are mean ± SEM of three (A), at least five (B and C), at least three (D), and five (E and F) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (t tests).

Rijal et al. PNAS Latest Articles | 3of12 Downloaded by guest on October 4, 2021 M. smegmatis, ppk1 knockdown M. smegmatis had lower accumulation of extracellular polyphosphate (Fig. 2A). At 4 h, compared to M. smegmatis, ppk1 knockdown M. smegmatis showed unchanged viability in D. discoideum cells (Fig. 2B). Extracellular polyphosphate, PPX with no polyphosphate, or PPX with 15 μg/mL polyphosphate did not affect the viability of ingested WT or ppk1 knockdown M. smegmatis in D. discoideum cells at 4 h (Fig. 2B). However, at 48 h, compared to M. smegmatis, ppk1 knockdown M. smegmatis showed reduced viability in D. discoideum cells (Fig. 2C). Extracellular polyphosphate increased the number of viable WT and ppk1 knockdown M. smegmatis, and the effect of extracellular polyphosphate on both WT and ppk1 knockdown M. smegmatis viability was reversed by the addition of PPX (Fig. 2C). These data suggest that polyphosphate secreted by M. smegmatis in the phagosome may affect the host cells from within the phagosome. Since M. smegmatis appear to make their own extracellular polyphosphate to promote their survival in D. discoideum cells, − we used grlD cells to determine if this effect is mediated by GrlD. At 4 h, similar numbers of viable ingested M. smegmatis − were present in WT and grlD cells (Fig. 2D). At 48 h, the − number of viable ingested M. smegmatis were decreased in grlD cells compared to WT D. discoideum cells (Fig. 2D). This indi- cates that GrlD promotes the ability of polyphosphate to inhibit the killing of ingested M. smegmatis. Together, these data suggest that reduced expression of ppk1 in M. smegmatis decreases the accumulation of extracellular polyphosphate and increases their killing in D. discoideum cells and that GrlD potentiates the ability of M. smegmatis to survive in D. discoideum cells. As- suming that, like other G-protein–coupled receptors (38), GrlD is on the plasma membrane, one possibility is that some GrlD Fig. 3. PPX increases viability of human macrophages when infected with receptors are endocytosed along with bacteria and can sense M. tuberculosis (M. tb) and reduces ingested M. tb staining. (A) Differential polyphosphate released by bacteria in the phagosome (38). interference contrast (DIC) and fluorescence images of human macrophages Compared to D. discoideum cells, macrophages required rel- containing ingested M. tb (green) in the absence (control) or in the presence of 5 μg/mL PPX are shown. Representative images from at least three in- atively high concentrations of extracellular polyphosphate to μ inhibit the killing of E. coli (Fig. 1E). In cocultures with human dependent experiments are shown. (Bar: 20 m.) (B and C)At4and48hof incubation of macrophages with M. tb, macrophage numbers were counted macrophages, at 4 h, reduced expression of ppk1 did not affect (B), and total M. tb staining intensity was measured (C). The average mac- the viability of ingested M. smegmatis, and extracellular poly- rophage count in the absence of PPX (control) at 4 h, for each independent phosphate at relatively high concentrations compared to those experiment, was set to 100 in B. All values are mean ± SEM of nine (B and C) used in D. discoideum did not affect the viability of ingested WT independent experiments. *P < 0.05; ***P < 0.001 (t tests). and ppk1 knockdown M. smegmatis in macrophages (Fig. 2E). However, at 48 h, and compared to WT M. smegmatis, reduced expression of ppk1 in M. smegmatis decreased bacterial viability compared to male macrophages at 4 h, there was no significant in macrophages (Fig. 2F). At 48 h, extracellular polyphosphate difference between the response of macrophages from male and did not affect the viability of ingested WT M. smegmatis, but female donors (SI Appendix, Fig. S4 B and C). These results increased the viability of ppk1 knockdown M. smegmatis in suggest that polyphosphate secreted from M. tuberculosis may macrophages (Fig. 2F). These data suggest that M. smegmatis potentiate their survival in human macrophages and decrease the PPK1 potentiates M. smegmatis viability in macrophages, possi- viability of the ingesting macrophages. bly by secreting polyphosphate in the phagosome, and that WT M. smegmatis may produce sufficient polyphosphate to saturate Polyphosphate Inhibits Phagosome Acidification in D. discoideum and the polyphosphate response in human macrophages and survive Human Macrophages. Many successful pathogens have evolved in macrophages longer (48 h) than E. coli. ways to evade phagosome–lysosome killing, often by inhibiting phagosome acidification (9–11). To determine if polyphosphate PPX Potentiates the Killing of Ingested M. tuberculosis in Human inhibits the killing of ingested bacteria by inhibiting phagosome Macrophages. Similar to M. smegmatis, M. tuberculosis bacteria acidification, D. discoideum cells were allowed to ingest pHrodo accumulate extracellular polyphosphate as cell density increases zymosan bioparticles. When these bioparticles are acidified, their (SI Appendix, Fig. S4 A and B). To determine if polyphosphate fluorescence intensity increases. Cells were also stained with potentiates the survival of M. tuberculosis in macrophages, human macrophages were incubated with M. tuberculosis bacteria LysoTracker, which stains acidic compartments (32). In control with and without PPX and stained with antibodies to detect M. cells, the bioparticles tended to colocalize with LysoTracker tuberculosis. At 4 h, compared to control, PPX did not change staining (Fig. 4A). Extracellular polyphosphate decreased both the number of macrophages or the M. tuberculosis staining the bioparticle fluorescence intensity and LysoTracker staining (Fig. 3 B and C). At 48 h, the macrophage count in the cultures (Fig. 4 A, C, and E). These effects of polyphosphate were with M. tuberculosis was decreased compared to 4 h, and the abolished in cells lacking GrlD (Fig. 4 B, D, and F). These results addition of PPX partially reversed this (Fig. 3B). At 48 h, the suggest that polyphosphate may inhibit the killing of ingested total M. tuberculosis staining intensity increased compared to 4 h, bacteria by inhibiting phagosome acidification and that GrlD and the addition of PPX partially reversed this (Fig. 3 A and C). is necessary for polyphosphate inhibition of phagosome Aside from decreased M. tuberculosis staining intensity in female acidification.

4of12 | www.pnas.org/cgi/doi/10.1073/pnas.2012009117 Rijal et al. Downloaded by guest on October 4, 2021 CELL BIOLOGY

− Fig. 4. Polyphosphate inhibits phagosome acidification in D. discoideum cells. (A and B)WT(A) and grlD (B) D. discoideum cells were incubated with pHrodo zymosan bioparticles (red) in the presence of polyphosphate (PolyP) (μg/mL) for 1 h and stained with LysoTracker (green). (Bars: 10 μm.) (C–F) Fluorescence − intensities of pHrodo zymosan bioparticles (C and D) and LysoTracker (E and F) in WT and grlD D. discoideum cells were measured. The fluorescence intensity of pHrodo zymosan bioparticles with no polyphosphate (0) was set to 100. All values are mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (t tests).

To determine if polyphosphate also inhibits phagosome acid- alter levels of clathrin or the late endosomal marker Rab7, but ification in human macrophages, monocyte-derived macro- reduced levels of the early endosomal markers EEA1 and Rab 5 phages were allowed to ingest pHrodo zymosan bioparticles and at 4 and 48 h after M. smegmatis infection and reduced levels of stained with LysoTracker. In control macrophages, acidification the early endosomal marker VPS34 levels at 48 h, but not 4 h of the bioparticles occurred within 5 min of ingestion, and the (Fig. 5 B and C and SI Appendix, Fig. S5). bioparticles colocalized with LysoTracker staining (Movie S1). To investigate if extracellular polyphosphate alters phagosome Polyphosphate inhibited acidification of ingested bioparticles maturation and, thus, alters the distribution of endosome and colocalization of bioparticles with LysoTracker staining markers on phagosomes, macrophages were infected with (Movie S2 and Fig. 5A). These data suggest that polyphosphate ptrc99A–eYFP E. coli for 48 h and were stained for endosome inhibits phagosome acidification to prevent the killing of inges- markers. With no extracellular polyphosphate, very little ted bacteria in human macrophages. ptrc99A–eYFP E. coli was detected, indicating the digestion of ingested ptrc99A–eYFP E. coli (SI Appendix, Fig. S6A). In the Polyphosphate Reduces Early Endosome Markers and Causes Coronin presence of polyphosphate, the fluorescence of ptrc99A–eYFP to Be Retained on the Phagosomes in Human Macrophages. During E. coli was observed, indicating that there was less digestion of phagocytosis, cells extend their plasma membrane around par- the bacteria (SI Appendix, Fig. S6B). Although the presence of ticles to form a phagosome (39). Phagosome maturation is sep- extracellular polyphosphate reduced the levels of early endo- arated into early, late, and lysosomal fusion stages (1). Clathrin is some markers such as EEA1, VPS34, and Rab5, ptrc99A–eYFP a self-assembling protein that mediates micropinocytosis, but is E. coli partially colocalized with EEA1 and Rab5, as indicated by also associated with early phagosomes (40). The newly formed an approximately twofold increase in the Pearson’s correlation phagosome fuses with small vesicles to acquire the small GTPase coefficient (PCC) compared to that of clathrin, VPS34, and Rab5, which recruits class III phosphoinositide 3-kinase vacuolar Rab7 (SI Appendix, Fig. S6 B and C). PCC values close to zero protein-sorting 34 (Vps34) and early endosomal antigen 1 indicated no colocalization (51). These data indicate that poly- (EEA1) that are necessary for phagosome acidification via ac- phosphate reduced early endosome markers in M. smegmatis- quisition of V-ATPase and maturation to a late phagosome infected macrophages and caused the retention of early endo- (41–44). During maturation of the phagosome, Rab7 replaces some markers in phagosomes containing ptrc99A–eYFP E. coli. Rab5, and this mediates fusion of the phagosome with a lyso- These data also suggest that extracellular polyphosphate inhibits some to form a phagolysosome (45, 46). To determine if poly- phagosome maturation, possibly by retaining the early endosome phosphate affects levels of proteins involved in phagosome markers on phagosomes and, thus, preventing the recruitment of maturation, Western blots of human macrophages were stained the late endosome marker Rab7, which is necessary for the fu- for known endosomal markers (47–50). Polyphosphate did not sion of phagosomes with lysosomes (30).

Rijal et al. PNAS Latest Articles | 5of12 Downloaded by guest on October 4, 2021 phagosomes containing pHrodo zymosan bioparticles and increased coronin staining on those phagosomes, suggesting that extracellular polyphosphate causes coronin to be retained on the phagosomes in human macrophages (Fig. 5 D and E). Human coronin shares ∼30% homology with D. discoideum coronin A (54). Although D. discoideum coronin is involved in phagocytosis, loss of coronin A does not alter the survival of pathogens such as Mycobacterium marinum and Legionella pneumonia in D. discoideum (55–57). To determine if polyphosphate alters coronin localization on D. discoideum phagosomes, coronin-null D. discoideum cells expressing Coronin–GFP were incubated with pH-independent Alexa Fluor 594-conjugated zymosan A bioparticles for 1 h, and cells were imaged. Unlike with human coronin, polyphosphate did not appear to alter the localization of coronin A in phagosomes containing bioparticles within D. discoideum cells (SI Appendix, Fig. S6D). Taken together, these data suggest that polyphosphate inhibits phagosome acidification, possibly due to retention of coronin on the phagosomes in human macrophages, but does not alter coronin localization on D. discoideum phagosomes, indicating that other factors in D. discoideum contribute to polyphosphate-mediated inhibition of phagosome acidification.

Polyphosphate Reduces Lysosome Activity in D. discoideum and Human Macrophages, and Reduced Expression of ppk1 in M. smegmatis Causes Increased Lysosome Activity. Polyphosphate reduced LysoTracker staining of acidic compartments in D. discoideum and human macrophages (Fig. 4 and Movie S2). To determine if polyphosphate alters lysosome activity, cells were incubated with the Biovision Lysosomal Intracellular Activity Assay Kit (Cell-Based) lysosome substrate, which becomes fluorescent after being cleaved in the lysosome. Compared to control, polyphosphate or the presence of WT M. smegmatis reduced the lysosome substrate fluorescence in D. discoideum cells, and, for M. smegmatis, this effect was reversed by knock- Fig. 5. Polyphosphate inhibits phagosome acidification, reduces early down of ppk1 (Fig. 6 A and C). In macrophages, polyphosphate endosome markers, and retains coronin in human macrophages. (A) Fluo- also reduced the lysosome substrate fluorescence, and, compared rescence intensities of pHrodo zymosan bioparticles in human macrophages, to WT M. smegmatis, knockdown of M. smegmatis ppk1 increased in the absence (−) or presence (+) of 470 μg/mL polyphosphate (PolyP), were the lysosome substrate fluorescence in macrophages (Fig. 6 B measured. Fluorescence intensity of control with no polyphosphate (−) was and D). As a control, cytochalasin D, an inhibitor of endocytosis set to 100. (B) Human macrophages containing ingested M. smegmatis in the (58), reduced the lysosome substrate fluorescence in both D. − μ absence ( ) or presence (+) of 470 g/mL polyphosphate were lysed, the discoideum cells and macrophages (Fig. 6). Taken together, lysates was resolved by SDS/PAGE, and Western blots of lysates were stained polyphosphate appears to reduce lysosome activity in D. dis- for the indicated endosome markers. Representative blots from four independent experiments are shown. Coomassie staining of samples as loading coideum and macrophages, and this might prevent lysosomal controls is shown in SI Appendix, Fig. S5.(C) Band intensities of endosome degradation of ingested bacteria. markers from B were quantified by densitometric analysis. The intensity of the control was set to one. (D) Human macrophages containing ingested Polyphosphate Causes D. discoideum to Carry Bacteria during pHrodo zymosan bioparticles (red) in the absence (−) or presence (+) of 470 Development. The soil-dwelling amoeba D. discoideum feed on μg/mL polyphosphate were fixed and stained for coronin (green). Repre- prey bacteria during the vegetative stage of their life cycle, but sentative images from three independent experiments are shown. (Bar: 10 amoebas start aggregating as soon as the food bacteria become μm.) (E) Fluorescence intensity of coronin relative to the fluorescence in- scarce (59). Developing D. discoideum cells form a fruiting body tensity of pHrodo zymosan bioparticles from 30 cells was measured in each − consisting of a column of stalk cells holding a mass of spores off experiment. Fluorescent intensity of control ( ) was set to 100. All values are the ground (60). Burkholderia bacteria or lectins secreted by D. mean ± SEM of three (A and E) and four (C) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (t tests). discoideum cells induce carriage of food bacteria by D. discoideum cells (61, 62). To determine if polyphosphate causes a retention of internalized bacteria during development, cells were – Coronin 1 (henceforth simplified as coronin) is a cytoskeleton- grown on a lawn of ptrc99A eYFP E. coli in the presence or absence of polyphosphate. After 4 d, spore masses were picked associated protein that connects actin filaments to the plasma with a pipette tip and spotted on Luria–Bertani (LB) agar plates membrane and transiently localizes on a phagosome membrane to check for bacterial growth. Polyphosphate increased the per- (52). Coronin is retained on mycobacteria-containing phag- centage of spore masses that contained at least one viable bac- osomes, and this retention may be responsible for blocking terium (Fig. 7A). These data suggest that, similar to secreted D. phagosome maturation (53). To investigate if extracellular pol- discoideum discoidin I (62), polyphosphate can cause some, but yphosphate alters coronin localization on phagosomes, human not all, D. discoideum cells to retain unkilled bacteria during macrophages were incubated with pHrodo zymosan bioparticles development. for 1 h, fixed, and stained for coronin. Compared to macro- As a social amoeba, D. discoideum can form a stable symbiotic phages with no extracellular polyphosphate, macrophages with relationship with Burkholderia species (63). Specific Burkholderia extracellular polyphosphate showed reduced fluorescence of species called “farming strains” colonize D. discoideum during

6of12 | www.pnas.org/cgi/doi/10.1073/pnas.2012009117 Rijal et al. Downloaded by guest on October 4, 2021 CELL BIOLOGY

Fig. 6. Polyphosphate inhibits lysosome activity in D. discoideum and human macrophages. (A and B) Fluorescence and differential interference contrast (DIC) images of D. discoideum cells (A) or human macrophages (B) with lysosome substrate (green) in the presence of polyphosphate or WT or ppk1 knockdown (ppk1 kd) M. smegmatis are shown. Cytochalasin D (Cyt. D), an inhibitor of endocytosis, was used as a positive control. Images are representative of three independent experiments. (Bars: 10 μm.) (C and D) The fluorescence intensity of the lysosome substrate in D. discoideum (C) and human macrophages (D) was measured from A and B. The fluorescence intensity of control (0) was set to 100. All values are mean ± SEM of four independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (t tests).

development, and these bacteria are stably carried through polyphosphate than “nonfarming” strains B. xenovorans, B. fun- multiple D. discoideum generations (61). We observed that the gorum,andB. phymatum (Fig. 7 B and C). These data suggest that farming strains B. agricolaris, B. hayleyella, and B. bonniea, which polyphosphate may potentiate the ability of farming Burkholderia can stably colonize D. discoideum, accumulated more extracellular species to colonize D. discoideum cells. To determine if exogenous

Fig. 7. Polyphosphate potentiates carriage of E. coli during D. discoideum development, and farming Burkholderia accumulate more polyphosphate than nonfarming Burkholderia bacteria. (A)WTD. discoideum cells were grown in the presence of E. coli in the absence (−) or presence (+) of 15 μg/mL polyphosphate and allowed to starve and develop. Spore masses from fruiting bodies were spotted onto bacterial growth plates, and the percent of fruiting bodies with spore masses containing live E. coli bacteria were counted. (B) Farming Burkholderia (B. agricolaris, B. hayleyella, and B. bonniea) and nonfarming Burkholderia (B. xenovorans, B. fungorum, and B. phymatum) bacteria were grown in SM/5 for 72 h, and the polyphosphate level in the conditioned medium was measured. (C) Polyphosphate from the conditioned medium (B) was resolved in 10% acrylamide gels. We used 14-mer (14 p), 60-mer (60 p), and 130-mer (130 p) polyphosphate standards as markers. Image is representative of four independent experiments. All values are mean ± SEM of four (A and B) independent experiments. *P < 0.05 (t tests for A and one-way ANOVA with Fisher’s least-significant difference test for B).

Rijal et al. PNAS Latest Articles | 7of12 Downloaded by guest on October 4, 2021 polyphosphate can increase the percentage of viable nonfarming extracellular polyphosphate, ∼1 in 1,000 D. discoideum cells start Burkholderia in D. discoideum spore masses, D. discoideum cells storing rather than digesting bacteria, and, at high cell densities, were grown on a lawn of a mixture of farming or nonfarming all of the cells respond to the concomitant high concentration of Burkholderia and ptrc99A–eYFP E. coli in the presence or ab- polyphosphate (∼704 μg/mL) by inhibiting nutrient uptake and sence of polyphosphate. After 4 d, D. discoideum spore masses initiating development. Whereas digesting rather than storing were picked with a pipette tip and spotted on SM/5 agar plates to nutrients is advantageous if there will be more nutrients avail- check for bacterial growth. When D. discoideum cells were grown able, storing rather than digesting nutrients might be advanta- on farming Burkholderia,100± 0% of spore masses contained geous if the cell does not encounter more bacteria to eat. viable bacteria (n = 4 independent experiments). Lower percent- Together, this suggests the possibility of a stochastic or deter- ages of spore masses contained viable bacteria when D. dis- ministic bet-hedging strategy driving some cells to store, and coideum cells were grown on nonfarming Burkholderia (SI other cells to digest, phagocytosed bacteria. Appendix,Fig.S7). The addition of polyphosphate to the agar In humans, in a limited subset of antigen-presenting cells plates did not significantly increase the percentage of D. dis- (APCs) such as macrophages, dendritic cells, and B cells, the coideum spore masses containing viable nonfarming Burkholderia major histocompatibility complex class II molecules bind and (SI Appendix,Fig.S7). These data suggest that extracellular pol- display small peptides at the cell surface, such that T lympho- yphosphate in the substrate material is not sufficient to increase cytes recognize the peptides and induce antigenic response the retention of nonfarming Burkholderia in the spore masses that against foreign peptides (66). APCs are less proteolytically active are held up off the substrate. and show reduced degradative capacity to maximize antigen presentation (67–69). We observed that polyphosphate causes Discussion ∼1 in 100 macrophages to store rather than digest bacteria, Proliferating D. discoideum cells accumulate extracellular poly- suggesting that a stochastic or deterministic process causes most phosphate as their local cell density increases, and the cells sense of the macrophages to kill ingested bacteria and some macro- extracellular polyphosphate to help them sense the local cell phages to slow the killing of ingested bacteria, possibly to allow density and anticipate when the cells will overgrow their food antigen presentation (66). For the macrophages, the extracellu- source and starve (26, 27). We found that exogenous poly- lar polyphosphate that triggers this may come from platelet de- phosphate, at concentrations that do not induce development, granulation during blood clotting and wound healing (70). These inhibits the killing of ingested E. coli and M. smegmatis bacteria results suggest an evolutionarily conserved process in D. dis- in D. discoideum. A possible explanation for this is that the re- coideum and human macrophages, where exogenous poly- sponse to extracellular polyphosphate would then allow some of phosphate or polyphosphate secreted by bacteria induce only a the D. discoideum cells to start carrying ingested bacteria as a small subset of cells to not immediately kill and digest ingested future food source when the D. discoideum cell density starts bacteria. getting high and the cells are about to starve. Remarkably, hu- D. discoideum can uptake nutrients by macropinocytosis (28). man macrophages similarly respond to polyphosphate by inhib- Polyphosphate at a concentration of ≥470 μg/mL inhibits D. iting digestion of phagocytosed bacteria, but why they do this discoideum proliferation (26), and we found that this poly- is unclear. phosphate concentration partially inhibits macropinocytosis, in- Pathogens such as M. tuberculosis inhibit phagosome matura- dicating that polyphosphate may inhibit D. discoideum tion and, thus, phagolysosomal killing to survive in host cells (7), proliferation in part by inhibiting macropinocytosis. Poly- and reduced expression of PPK in these pathogens diminishes phosphate at a concentration between 5 and 47 μg/mL inhibits their survival in macrophages (20). In agreement, we found that, the killing of ingested bacteria, but does not significantly inhibit in comparison to parental M. smegmatis cells, ppk1 knockdown macropinocytosis or proliferation (26). This, then, suggests that M. smegmatis cells survive poorly in D. discoideum and human as the cell density and extracellular polyphosphate concentration macrophages. Exogenous polyphosphate rescued the diminished in a colony of D. discoideum cells increase, some of the cells start survival of the ppk1 knockdown M. smegmatis cells in both D. storing bacteria, and, then, at higher cell densities and higher discoideum and human macrophages, suggesting that poly- extracellular polyphosphate concentrations, closer to the point phosphate is a signal released from M. smegmatis cells to po- where cells will overgrow their food supply and starve, a larger tentiate their survival in both D. discoideum and human percentage of the cells decrease macropinocytosis for unknown macrophages. This, in turn, suggests a similarity in the poly- reasons and inhibit cytokinesis to increase the percentage of phosphate response that has been conserved from D. discoideum large cells. to humans (10). During the exponential growth phase of a culture of M. After infecting human macrophages, M. tuberculosis can es- smegmatis bacteria, with N0 the initial cell density and t time, cell cape the host cells by inducing macrophage cell death (64). density N(t) will be When M. tuberculosis bacilli number per macrophage is less than kt approximately five, the bacterial proliferation is slow, and the N(t) = N0e . [1] bacteria have less cytotoxic effect in the host macrophages (65). However, when M. tuberculosis bacilli number exceeds ∼18 per Assuming no breakdown of polyphosphate, and cells secreting m macrophage, M. tuberculosis induces macrophage cell death (65). molecules of polyphosphate per cell per second, the change in We observed that ∼10 to 20 M. tuberculosis bacilli per macro- extracellular polyphosphate concentration [pP] at time t will be phage induced some macrophage cell death. The addition of PPX partially reversed both the increase in M. tuberculosis and d[pP] = N(t) m. the M. tuberculosis-induced macrophage death. Together, this dt suggests that extracellular polyphosphate from M. tuberculosis potentiates M. tuberculosis survival in macrophages. How M. tu- The extracellular polyphosphate concentration will then be berculosis and other bacteria secrete polyphosphate is unknown. At very low cell densities, and, thus, low extracellular poly- N m [pP] = ∫ t N(t) m dt = N m∫ t ekt dt = 0 ekt + C. [2] phosphate concentrations, D. discoideum cells digest all bacteria 0 0 0 k (unless the bacteria are secreting polyphosphate). As the cell density increases (and the competition for, and depletion of, From the growth curves in SI Appendix, Fig. S3D, for the interval nutrients increases), as indicated to cells by ∼10 μg/mL of 12 to 24 h, for each experiment, we calculated k, and from the

8of12 | www.pnas.org/cgi/doi/10.1073/pnas.2012009117 Rijal et al. Downloaded by guest on October 4, 2021 extracellular polyphosphate concentration measurements in SI provided by Ludwig Eichinger, University of Cologne, Cologne, Germany Appendix, Fig. S3E, we calculated the change in [pP] between (74). Cells were grown at 21 °C in shaking culture in synthetic medium 12 and 24 h, Δ[Pp]. With N being the cell density at 12 h and t (SIH-defined minimal medium) (Formedium) and on SM/5 agar (2 g of glu- 12 · being 12 h (43,200 s), cose, 2 g of bactopeptone, 0.2 g of yeast extract, 0.2 g of MgCl2 7H2O, 1.9 g of KH2PO4,1gofK2HPO4, and 15 g of agar per liter) (http://www.dictybase. org)onlawnsofE. coli (DBS0350636). A quantity of 100 μg/mL dihydros- = Δ[] ()()()kt − m Pp k / N12 e 1 , treptomycin and 100 μg/mL ampicillin was used to kill E. coli in D. discoideum − cultures obtained from SM/5 agar (75). grlD cells were grown under se- − we find that for WT M. smegmatis, m is 53 ± 3 × 10 12 μgof lection with 5 μg/mL blasticidin. Bacterial survival assays were performed in polyphosphate per cell per second, and for the ppk1 knockdown type 353047 24-well plates (Corning) with 106 D. discoideum cells per well in − strain, m is 3 ± 1 × 10 12 μg of polyphosphate per cell per second 1 mL of SIH, and phagocytosis and lysosome activity assays were performed ± = in type 353219, 96-well, black/clear, tissue-culture-treated, glass-bottom (means SEMs, n 6). 5 μ From figure 1 of ref. 71, at 15 min after ingesting a 3.23-μm- plates (Corning) with 10 cells per well in 100 L. diameter yeast, the inner diameter of the phagosome is 3.64 μm. Human peripheral blood was collected from healthy volunteers who gave written consent, with specific approval from the Texas A&M University hu- Assuming that both the yeast and the phagosome are spherical, man subjects institutional review board. PBMCs were isolated as described the annular space volume (difference in volumes of the two (76). The PBMCs were cultured in Roswell Park Memorial Institute Medium −12 spheres) is 7.6 × 10 mL. From the above calculation for the (RPMI) (Lonza) containing 10% bovine calf serum (VWR Life Science Ser- polyphosphate secretion rate from a M. smegmatis bacterium, adigm), 2 mM L-glutamine (Lonza), and 50 ng/mL human GM-CSF (Biol- −8 there will be 5 × 10 μg of polyphosphate secreted in 15 min, so egend) at 37 °C in a humidified chamber with 5% CO2 in type 353047, the concentration in the space between the bacterium and the 24-well plates with 106 cells per well in 1 mL or type 353219, 96-well, black/ 5 inner wall of the yeast-sized phagosome will be ∼6,100 μg/mL. clear, tissue-culture-treated, glass-bottom plates with 10 cells per well in μ For the ppk1 knockdown, the concentration will be ∼360 μg/mL. 100 L. After 5 d, loosely adhered cells were removed by gentle pipetting. Since the bacteria are smaller than a yeast, the volume between a Bacterial survival assays were performed in 24-well plates, and phagocytosis and lysosome activity assays were performed in 96-well plates. bacterium and the inner wall of the phagosome will be smaller, making these concentrations even higher. If the initial ingestion Bacterial Culture. E. coli K-12 (BW25113) (CGSC#7636) (77, 78), E. coli JW2486- event involves the plasma membrane touching the bacterium, so 2[Δppk-749::Kan] from the E. coli Genetic Stock Center (77, 78), and HCB33 that both the annular space and the polyphosphate concentra- (RP437)/ptrc99A–eYFP E. coli (a gift from Pushkar Lele, Texas A&M Univer- tion in this space are initially both 0, and assuming that the fluid sity, College Station, TX) (33) were grown at 37 °C in LB broth (BD). A leakage through the phagosome membrane is roughly linear with quantity of 100 μg/mL ampicillin was used to select for HCB33 (RP437)/ time, causing the annular volume to increase linearly with time, ptrc99A–eYFP E. coli. M. smegmatis (mc21555 strain) (79), and attenuated CELL BIOLOGY and assuming the polyphosphate secretion rate is also linear with (mc-ΔleuDΔpanCD) Biosafety Level-2 strain of M. tuberculosis (derivatives of H37Rv strain) (80) (gifts from Jim Sacchettini, BioBio, Texas A&M University, time, then the polyphosphate concentration in this annular space – will be roughly constant, and, thus, for the WT M. smegmatis, the College Station, TX) were grown as described (81 83) in Middlebrook 7H9 ∼ μ broth (BD) at 37 °C (M. smegmatis) or at 37 °C in a rotator in a humidified polyphosphate concentration will be 6,100 g/mL or more incubator (M. tuberculosis) or 7H10 agar (Sigma) containing 0.5% glycerol within a time much shorter than 15 min. (VWR Life Science Seradigm), 0.05% Tween 80 (MP Biomedicals), and Mid- We observed that the concentration of phosphate monomers dlebrook albumin dextrose catalase (ADC) Enrichment (M. smegmatis)orthe in polyphosphate needed to keep some bacteria from being digested Middlebrook Oleic ADC Enrichment (M. tuberculosis)(BD).M. tuberculosis is ∼10 μg/mL, well below the concentrations in a phagosome with ΔleuDΔpanCD liquid cultures were additionally supplemented with 50 μg/mL WT and ppk1 knockdown M. smegmatis calculated above. Assuming leucine (VWR Life Science Seradigm) and 50 μg/mL pantothenate (Beantown that a few polyphosphate receptors will be internalized with the Chemical). CRISPRi-mediated knockdown of ppk1 (Gene ID: 4535776) in ’ region of the plasma membrane used to form the phagosome, this M. smegmatis was performed by Thomas Snavely (James Sacchettini s laboratory, concentration of polyphosphate in the phagosome might be suffi- Texas A&M University, College Station, TX) as described (81, 82). The M. smegmatis ppk1 DNA sequence 5′-AGTGCCGGGCGCACCGAGTC-3′ wasclonedintothe cient to prevent digestion of the M. smegmatis bacterium by cells > μ PLJR962 vector (Addgene plasmid no. 115162) (81) to generate the guide RNA that have not accumulated 10 g/mL extracellular polyphosphate. that target the template DNA strand within the open reading frame of ppk1 to How the addition of extracellular polyphosphatase affects the ability generate ppk1 knockdown M. smegmatis. Anhydrotetracycline (ATc) hydrochlo- of phagocytosed Mycobacteria to inhibit their digestion by D. dis- ride (Sigma), an inducer of CRISPRi knockdown, was used at a concentration of coideum or macrophages is unknown. One possibility is that the 200 ng/mL for ppk1 knockdown M. smegmatis. Burkholderia strains (B. agricolaris polyphosphatase hydrolyzes polyphosphate weakly bound to the QS159, B. hayleyella QS11, B. bonniea QS859, B. xenovorans LB400, B. fungorum bacterium, so that for a few critical seconds after phagocytosis, there [ATCC no. BAA-463], and B. phymatum STM-815) (provided by Debra A. Brock, is not enough polyphosphate to activate from within the phagosome Strassmann/Queller Laboratory, Washington University in St. Louis, St. Louis, MO) were grown at 21 °C on SM/5 agar plates as described (63, 84). the pathway that inhibits lysosome fusion. Another possibility is that some polyphosphatase, either free or weakly binding to the bacte- RNA Extraction and PCR. To examine the CRISPRi-mediated knockdown of rium, is brought into the phagosome. ppk1 in M. smegmatis and the deletion of ppk in E. coli K-12, M. smegmatis

D. discoideum cells appear to use GrlD to sense both extra- and E. coli K-12 were grown to optical density (OD600) of 0.5 to 1.0. M. cellular polyphosphate and the polyphosphate released by bac- smegmatis was grown in the presence of 200 ng/mL ATc. RNA was extracted teria in a phagosome. Assuming that macrophages also use a from M. smegmatis and E. coli as described (85). Complementary DNA receptor to sense polyphosphate to inhibit phagosome maturation, (cDNA) was synthesized from 2 μg of RNA by using the Maxima H Minus First this implies that there is a polyphosphate signal-transduction Strand cDNA Synthesis kit (Thermo Scientific). PCR was performed to verify pathway in macrophages exploited by pathogens such as M. tu- the reduction or loss of cDNA associated with the gene knockdown or de- ′ berculosis to promote their own survival. An intriguing possibility letion using M. smegmatis ppk1 gene-specific primers (forward primer, 5 - TGACGATCCCGAACTGCTGC-3′, and reverse primer, 5′-GGGCGCGTTTCTTGT is that blocking this pathway would cause macrophages to digest CGTTG-3′) and E. coli K-12 ppk gene-specific primers (forward primer, 5′- all, rather than most, internalized pathogens such as M. tubercu- GGGTCAGGAAAAGCTATACATCG-3′, and reverse primer, 5′-GTCATAAAT losis, suggesting a therapeutic approach to promote clearance of CGCCAACTGCGCCC-3′). PCR products were separated by agarose gel elec- these pathogens in patients. trophoresis. Band intensities on gels were quantified by using Image Lab (Bio-Rad). Materials and Methods − D. discoideum and Human Cell Culture. WT AX2 (DBS0237699) and grlD Recombinant Polyphosphatase and Polyphosphate Assays. Sodium poly- (DBS0350227) D. discoideum strains were obtained from the Dictyostelium phosphate (Spectrum; average chain length 45 monomers) was dissolved in Stock Center (72, 73). D. discoideum (corA-/[act15]:corA:GFP) cells were water to a concentration of 15 mg/mL, and the pH was verified to be pH ∼ 6.9.

Rijal et al. PNAS Latest Articles | 9of12 Downloaded by guest on October 4, 2021 This stock diluted 1,000 × in cultures was defined as 15 μg/mL poly- images was done by using the Richardson–Lucy algorithm (81) in NIS- phosphate. Higher dilutions were used to make lower polyphosphate con- Elements AR software. Macrophage count and fluorescence intensity of M. centrations. The sodium polyphosphate was also dissolved in water to a tuberculosis were analyzed by ImageJ. Figures were prepared by using concentration of 70.4 mg/mL, the pH was checked, and this stock was diluted CorelDRAW X8. 100 × in cultures to make 704 μg/mL polyphosphate; higher dilutions were Viable bacteria in D. discoideum spore masses was quantified as described used to make culture concentrations down to 15 μg/mL. The plasmid for (61) with the following modifications. Stationary-phase HCB33 (RP437)/ purifying S. cerevisiae PPX1 was a gift from Michael Gray, University of ptrc99A–eYFP E. coli grown in LB agar or Burkholderia grown on SM/5 agar Alabama at Birmingham, Birmingham, AL (86). Recombinant PPX1 was pu- was collected in SM/5 (SM/5 without agar). Log-phase D. discoideum cells rified as described in a protein-purification protocol (87). A quantity of 5 μg/ grown in SIH and bacteria were washed two times in SM/5 by centrifugation 5 mL PPX with 5 mM MgCl2 was used for treatment of D. discoideum and at 500 × g for 3 min. A quantity of 2 × 10 D. discoideum cells in 50 μLofSM/ human macrophages in the assays. To check PPX enzymatic activity, poly- 5 was added to 300 μL of SM/5 containing HCB33 (RP437)/ptrc99A–eYFP μ phosphate (Spectrum) was treated with 5 g/mL PPX with 5 mM MgCl2 for E. coli (OD600 5) or 300 μL of SM/5 containing 90% HCB33 (RP437)/ 1 h at 37 °C. Extracellular polyphosphate secreted by E. coli or M. smegmatis, ptrc99A–eYFP E. coli (OD600 5) and 10% Burkholderia (OD600 0.001). The or polyphosphate from the above assay, was assessed by adding 25 μg/mL mixture was plated on SM/5 agar plates in the presence or absence of pol- DAPI (Biolegend), and measuring fluorescence at 415/550 nm (excitation/ yphosphate and incubated at 21 °C for 3 to 4 d. The D. discoideum cells emission), as described (88). Culture supernatants were clarified by centri- overgrew the bacteria, starved, and formed fruiting bodies. Individual D. fugation at 12,000 × g for 2 min. Extracellular polyphosphate secreted by discoideum spore masses were picked with a sterile pipette tip, spotted on Burkholderia was assayed by growing cells on SM/5 agar for 48 h, and the SM/5 agar plate, and incubated at 21 °C for 24 to 48 h, and bacterial colonies agar was crushed and resuspended in 10 mL of sodium phosphate buffer were counted.

(10 mM NaPO4, pH 7.4), incubated for 5 min, and clarified by centrifugation at 12,000 × g for 2 min, and polyphosphate in the suspension was measured. Endocytosis and Exocytosis Assays. Endocytosis and exocytosis of TRITC– Phagosome volume was calculated from ref. 71. dextran (average molecular weight 65,000 to 85,000) (Sigma) were performed as described (89). For control endocytosis, the time point 180 min Bacterial Survival Assay. D. discoideum cells or human macrophages were was assigned 100% relative fluorescence. For control exocytosis, the time seeded in a 24-well plate. After 30 min, either polyphosphate or PPX, or point 0 was assigned 100% relative fluorescence. Rate constant K (1/min) of polyphosphate and PPX was added to the cells and mixed by gentle pipet- endocytosis and exocytosis, expressed in reciprocal of the x-axis time units ting. E. coli K-12 or M. smegmatis bacteria were washed two times in Sor- (minutes), was generated by fitting the curve to one phase-association

ensen buffer (14.6 mM KH2PO4 and 2 mM Na2HPO4,pH6)forD. discoideum equation. and phosphate-buffered saline (PBS) (Lonza) for human macrophages by centrifuging at 12,000 × g for 2 min, and resuspended in Sorensen buffer for Fluorescence Microscopy. D. discoideum cells were seeded in a 96-well, black/ D. discoideum or PBS for human macrophages to a final OD600 of 0.1. E. coli clear, tissue-culture-treated plate. After 30 min, polyphosphate was added K-12 (50 μL), or M. smegmatis (10 μL) was added to D. discoideum cells or to the cells and mixed by gentle pipetting. pHrodo zymosan bioparticles human macrophages and incubated for 2 h. Bacteria outside the cells were (ThermoFisher) were resuspended in Sorensen buffer to a concentration of removed by gently washing D. discoideum cells with SIH or human macro- 0.5 mg/mL. Ten microliters of bioparticles and 100 nM LysoTracker (Ther- phages with RPMI. The remaining uningested bacteria were killed by adding moFisher) was added to the cells and mixed by gentle pipetting, and the 200 μg/mL gentamicin (Sigma). Cells were washed as above to remove plates were spun down at 300 × g for 2 min. After 1 h, images of D. dis- gentamicin. After 4, 24, or 48 h, D. discoideum cells or human macrophages coideum cells were taken with a 40× objective on a Nikon Eclipse Ti2 (Nikon), were washed as above and lysed by using 0.1% Triton X-100 (Alfa Aesar) in and we used the Richardson–Lucy algorithm (90) for deconvolution of im- PBS for 5 min at room temperature by gentle pipetting, and lysates were ages in NIS-Elements AR software. The number of bioparticles ingested per plated on LB agar for E. coli K-12 growth or 7H10 agar for M. smegmatis cell per hour was calculated as a mean number of ingested bioparticles per growth and incubated at 37 °C. Polyphosphate or PPX, when assayed, was phagocytosing D. discoideum cell multiplied by the percentage of D. dis- present in all incubation steps up to the Triton lysis step. E. coli K-12 colonies coideum cells engaged in phagocytosis. Fluorescence intensity was analyzed were visible after 24 h, whereas M. smegmatis colonies were visible after 72 by ImageJ software. Figures were prepared by using CorelDRAW X8. h. Bacterial colonies were counted, and viable bacteria in D. discoideum cells To investigate coronin localization, D. discoideum coronin A knockout as cfu/106 D. discoideum cells or viable bacteria in human macrophages as mutant with overexpressed coronin A-GFP (corA-/[act15]:corA:GFP) or hu- cfu/mL were calculated. man macrophages was seeded in 96-well tissue-culture plates. D. discoideum For imaging, D. discoideum cells or human macrophages were incubated cells were incubated with Alexa Fluor 594-conjugated zymosan bioparticles with HCB33 (RP437)/ptrc99A–eYFP E. coli for 4 and 48 h as described above, (ThermoFisher), and macrophages were incubated with pHrodo zymosan and cells were stained with 100 nM of LysoTracker (ThermoFisher) for 30 min bioparticles for 1 h; uningested extracellular bioparticles were removed by after 3.5 or 47.5 h of incubation. Cells were washed two times with SIH (D. washing D. discoideum with SIH or macrophages with RPMI, and cells were discoideum) or RPMI (macrophages), images were taken with a 40× objective fixed with 4% (wt/vol) paraformaldehyde in PBS for 10 min. Cells were on an FV1000 confocal microscope (Olympus), using Olympus Fluoview washed two times with PBS, and 100 μL of PBS was added to the well. Images Version 4.2a software, and figures were prepared by using CorelDRAW X8. of fixed D. discoideum cells were taken to analyze coronin A localization. Fluorescence intensity was measured, and colocalization was analyzed with Fixed macrophages were washed once with PBS, permeabilized, blocked the Coloc2 plugin in Fiji (ImageJ; NIH). with BSA, and stained with 1:2,000 anti-coronin antibody (catalog no. To study M. tuberculosis survival in human macrophages, a bacterial ab123574, Abcam) as described in Bacterial Survival Assay. Macrophages survival assay was performed as described above, except that there were were washed three times with PBST, and 200 μL of PBS was then added to there were ∼10 to 20 M. tuberculosis bacteria per macrophage. After 4 and the well. Images of D. discoideum and macrophages were taken with a 40× 48 h, macrophages with ingested M. tuberculosis were fixed with 4% objective on a Nikon Eclipse Ti2 (Nikon), and deconvolution of images was (weight [wt]/volume [vol]) paraformaldehyde (Electron Microscopy Sciences) done by using the Richardson–Lucy algorithm (90) in NIS-Elements AR soft- in PBS for 10 min. Cells were washed two times with PBS, and 1 mL of PBS ware. Fluorescence intensity of coronin and pHrodo zymosan bioparticle was was added to the well. Fixed macrophages were washed once with PBS and analyzed by ImageJ. Figures were prepared by using CorelDRAW X8. permeabilized with 0.1% Triton X-100 (Alfa Aesar) in PBS for 5 min. Mac- rophages were washed two times with PBS, blocked with 1 mg/mL type 0332 Live-Cell Microscopy. Human macrophages in 96-well, black/clear, bovine serum albumin (BSA) (VWR Life Science Seradigm) in PBS for 1 h, and tissue-culture-treated, glass-bottom plates were incubated with 100 nM washed once with PBS. A concentration of 1:2,000 rabbit anti-M. tubercu- LysoTracker (ThermoFisher) for 30 min, followed by incubation with 10 μLof losis antibody (catalog no. OBT0947, Bio-Rad) in PBS/0.1% Tween 20 (PBST; 0.5 mg/mL pHrodo zymosan bioparticles, and the plates were spun down at Fisher Scientific) was added to macrophages and incubated at 4 °C over- 300 × g for 2 min. Imaging of cells took place in a controlled chamber night. Macrophages were washed three times with PBST and incubated with maintaining 37 °C with 5% CO2. Confocal images were taken with a 60× 1:500 Alexa 488 anti-rabbit (Jackson Immunoresearch) and 10 μg/mL DAPI in glycerol-immersion objective at 15-s intervals for 45 min by using a Leica SP8 PBST for 1 h. Macrophages were washed three times with PBST, and 1 mL of confocal microscope (Leica Microsystems). PBS was then added to the well. Each washing step was done for 5 min, and all steps were performed at room temperature, if not indicated otherwise. Lysosome Activity Assay. D. discoideum cells or human macrophages were Images of macrophages were taken with a 4× dry or 100× oil-immersion seeded in 96-well plates. Cytochalasin D (BioVision) or polyphosphate was objective on a Nikon Eclipse Ti2 microscope (Nikon), and deconvolution of added to the cells and incubated for 30 min. Lysosome activity assays were

10 of 12 | www.pnas.org/cgi/doi/10.1073/pnas.2012009117 Rijal et al. Downloaded by guest on October 4, 2021 performed with a K448-50 lysosomal intracellular activity assay kit (Bio- chain lengths (14-, 60-, and 130-mer polyphosphate) were provided by Vision) following the manufacturer’s directions. Cells were imaged with a Toshikazu Shiba, RegeneTiss Inc. (Japan). The running buffer was 1× TAE 40× objective on a Nikon Eclipse Ti2 (Nikon) and processed with NIS- (4.84 g Tris, 1.14 mL glacial acetic acid, and 0.37 g of ethyl- Elements AR software. The fluorescence intensity of the lysosome sub- enediaminetetraacetic acid [EDTA]) per liter (all reagents were from VWR strate was analyzed by ImageJ software. Figures were prepared by using Life Science Seradigm), and the 6× sample buffer was 0.01% Orange G CorelDRAW X8. (Fisher Scientific); 30% glycerol; 10 mM Tris (VWR Life Science Seradigm), pH 7.4; and 1 mM EDTA (VWR Life Science Seradigm). PAGE was performed at Immunoblot Analysis. Macrophages were lysed in sample buffer (50 mM Tris/ 100 V for 2 to 3 h at room temperature until the Orange G had run through HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate [SDS] [J. T. Baker], 1% two-thirds of the gel. Gels were stained with 0.05% toluidine blue (Fisher β-mercaptoethanol [Sigma], and 0.02% Bromophenol blue [Matheson]) Scientific), 20% methanol (VWR Life Science Seradigm), and 2% glycerol for containing 1x Pierce protease inhibitor (ThermoFisher) and heated to 95 °C for 5 min. Samples were separated by SDS/polyacrylamide gel electropho- 1 h and destained 2 to 3 d with several changes of destaining solution resis (PAGE) using Tris–glycine 4 to 20% polyacrylamide gels (Lonza), trans- (staining solution without toluidine blue), and images were taken in white ferred to polyvinylidene fluoride blotting membrane (GE Healthcare), and light by using a Bio-Rad scanner (Bio-Rad). Images were prepared in immunoblotted following the manufacturers’ directions. Blots were blocked CorelDRAW X8. with 5% skim milk (BD) in PBS. The following primary antibodies were used: rabbit monoclonal anti-clathrin heavy chain (catalog no. 4796), rabbit Statistical Analysis. Statistical analyses were performed by using GraphPad monoclonal anti-EEA1 (catalog no. 3288), rabbit monoclonal anti-Rab5 Prism 8 (GraphPad) or Microsoft Excel. P < 0.05 was considered significant. (catalog no. 46449), rabbit monoclonal anti-Rab7 (catalog no. 9367) (all from Cell Signaling Technology; 1:2,000 dilution in PBST), and rabbit poly- Materials and Data Availability. Readers can access associated materials by clonal anti-VPS34 (catalog no. NB110-87320SS, Novus Biologicals; 1:2,000). contacting R.R. or R.H.G. All study data are included in the article and Bound antibodies were detected with an enhanced chemiluminescence SI Appendix. Western blotting kit (Thermo Fisher). Band intensities on blots were quantified by using Image J. For loading control, samples were separated by SDS/ ACKNOWLEDGMENTS. We thank Thomas Snavely for kindly generating the PAGE and stained with Coomassie Brilliant Blue R-250 (Bio-Rad), band in- CRISPRi ppk1 knockdown M. smegmatis strain; Dr. Pushkar Lele for kindly tensities were quantified, and adjusted equivalent amount of total cell ly- providing HCB33 (RP437)/ptrc99A–eYFP E. coli; Sara A. Kirolos for assistance sates were used for immunoblotting. with experiments; Dr. Ludwig Eichinger (University of Cologne, Germany) for providing the D. discoideum corA-/[act15]:corA:GFP strain; the volunteers Toluidine Blue Staining of Polyphosphate. Extracellular polyphosphate se- who donated blood; the phlebotomy staff at the Texas A&M Beutel Student 2 creted by Burkholderia was resolved by PAGE using a 16- × 16-cm 10% Health Center; and three anonymous referees for helpful suggestions. The polyacrylamide (Acryl/Bis 19:1 40% (wt/vol) solution; VWR Life Science Ser- use of the Microscopy and Imaging Center facility at Texas A&M University is adigm) gel as described (91, 92). Conditioned medium from Burkholderia acknowledged. The Olympus FV1000 and Leica SP8 confocal microscopes CELL BIOLOGY agar cultures was concentrated ∼20 times by using a 1-kDa spin filter (Pall) acquisition was supported by the Office of the Vice President for Research by centrifugation at 4,000 × g for 2 h. Polyphosphate standards of specific at Texas A&M University. This work was supported by NIH Grant GM118355.

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