Phosphoinositide3-kinase regulates actin polymerization during delayed phagocytosis of Helicobacter pylori

Abstract

We have shown previously that ulcerogenic (type I) strains of Helicobacter pylori (Hp) retard their entry into macrophages. However, the signaling pathways that regulate Hp phagocytosis are largely undefined. We show here that Hp strongly activated class IA phosphoinositide3-kinases (PI3Ks) in macrophages, coincident with phagocytosis, and endogenous p85 and active protein kinase Balpha accumulated on forming phagosomes. PI3K inhibitors, wortmannin and LY294002, inhibited phagocytosis of Hp in a dose-dependent manner, and blockade of engulfment correlated directly with loss of 3'-phosphoinositides in the membrane subjacent to attached bacteria. During uptake of large immunoglobulin G (IgG)-coated particles, PI3Ks regulate pseudopod extension and phagosome closure. In marked contrast, we show here that 3'-phosphoinositides regulated actin polymerization at sites of Hp uptake. Moreover, Hp and IgG beads activated distinct PI3K isoforms. Phagosomes containing IgG-coated particles accumulated 3'-phosphatase and tensin homologue deleted on chromosome 10 and Src homology 2 domain-containing inositol 5'-phosphatase, yet Hp phagosomes did not. Finally, rapid uptake of IgG-opsonized Hp or a less-virulent type II Hp was PI3K-independent. We conclude that Hp and IgG beads are ingested by distinct mechanisms and that PI3Ks regulate the actin cytoskeleton during slow phagocytosis of ulcerogenic Hp.

Fig. 1

Class IA PI3K is activated at sites of Hp uptake. (A and B) BMMs were infected with Hp 11637 for 0 –15 min, and postnuclear supernatants were fractionated into membranes and cytosol. (A) p85 immunoblots of membrane (M) and cytosol (S) fractions from resting (Control) and Hp-infected BMMs. Arrowheads indicate mobility shift of membrane p85 in Hp-infected cells. (B) PI3K activity at the membrane, as judged by in vitro kinase assays of p85 immune complexes. PI(3)P synthesized is shown as average counts per min (cpm) ±SD (n = 3). Pretreatment with 100 nM WTM abrogated PI3K activity (7 min infection). (C) Confocal sections of PMφ infected with Hp 11637 for 3–15 min show Hp (red) and p85 or PKBα (green). Arrows indicate enrichment of p85 and PKBα on forming phagosomes. Pretreatment with 100 nM WTM prevented translocation of PKBα to the membrane beneath Hp (far right, arrowheads). (D) Confocal sections of BMMs infected with Hp 11916 for 7 min show active P-Ser473 PKB (green) and Hp (red). Arrows indicate phagosomes. (E) BMMs were infected with Hp 11637 for 0 –20 min. Immunoblots of cell lysates show total PKB and PKB phosphorylated on Thre308 (PKB-P).

Fig. 2

Class IA PI3Ks are essential for Hp phagocytosis. (A) BMMs pretreated with 0 –100 nM WTM were infected with Hp 11637 for 30 min, and Hp internalization was quantified by differential staining. Data are the average ± SD of three determinations performed in triplicate. *, P = 0.032, and **, P < 0.010, versus no drug control. (B) BMMs pretreated with 0 –100 μM LY294002 were infected with Hp, and phagocytosis was quantified as in A. Data are the average ± SD of three experiments performed in triplicate. (C, upper panel) Immunoblot, demonstrating the effect of sense or antisense p85 oligos on BMM p85 content. Data shown are representative of three independent experiments. U, Untreated BMMs; AS, antisense oligo-treated; S, sense oligo-treated. (Lower) Internalization of Hp 11637 by control (Uninf.) or antisense (p85-AS)- or sense (p85-S)-treated BMMs. Data shown are the average ± SD of three independent experiments. *, P = 0.008, versus untreated or p85-S controls.

Fig. 3

Effect of WTM on PI3K activity, phagocytosis, and local accumulation of PKBα. (A and B) Macrophages were treated with 0 –100 nM WTM and then infected with IgG-Z (A) or Hp 11637 (B). Thereafter, cells were lysed, and total class IA PI3K activity was quantified by in vitro kinase assays of p85 immunoprecipitates (PI3K act.;●). Particle engulfment (Phago.;○;) was assessed by differential staining, and recruitment of PKBα; to forming phagosomes (PKB+; ▼) was scored using confocal microscopy. PI3K activity is normalized to cells infected in the absence of WTM. Phagocytosis and PKBα recruitment are shown as a percentage of cell-associated particles. In all cases, the data shown are the average ± SD of three to four independent experiments. *, P < 0.010, for Hp phagocytosis and PKBα recruitment versus PI3K activity at 25 nM WTM. (C and D) Effect of WTM on PKBα association with IgG-Z (C) and Hp (D) phagosomes. PMφ were treated with 0 –100 nM WTM and then infected with IgG-Z (C) or Hp (D). Arrowheads in each confocal section indicate areas of PKBα accumulation.

Fig. 4

PI3KC2, PTEN, and SHIP do not accumulate on Hp phagosomes. (A) Forming IgG-Z phagosomes recruit PI3KC2 (arrowhead, left panel), but Hp phagosomes do not (right panel, Hp, red; PI3KC2, green). (B) Confocal sections of PMφ show PTEN or SHIP (green) and Hp (red). Arrowheads, Rare SHIP-positive Hp phagosomes. (C) Time course of PTEN and SHIP accumulation on IgG-Z phagosomes (arrowheads). (D) Differential recruitment of PI3KC2, PTEN, and SHIP to IgG-Z and Hp phagosomes. Data are the average ± SD of three independent experiments performed in triplicate. *, P ≤ 0.003, for Hp versus IgG-Z. p’somes, Phagosomes.

Fig. 5

PI3K regulates actin polymerization at sites of Hp uptake. Control Mφ or cells treated with 100 nM WTM were infected with IgG-Z or Hp. F-actin was detected in fixed and permeabilized cells using rhodamine-phalloidin and confocal microscopy. (A) Unlike IgG-Z, forming Hp phagosomes accumulate F-actin in control macrophages but not in cells pretreated with WTM. Hp are green, and F-actin is red or gray. Arrows and arrowheads indicate actin-positive and actin-negative particles, respectively. (B) Differential effect of WTM on actin polymerization triggered by Hp and IgG-Z. Data shown are the average ± SD of three independent experiments performed in triplicate. *, P = 0.014, versus IgG-Z. Con, Control.

Fig. 6

PI3Kδ translocates in response to type I Hp but not IgG beads. (A) Membrane translocation of class IA PI3K isoforms. BMMs were left uninfected (U) or incubated with type I Hp strain DT61A (Hp), IgG-opsonized DT61A (IgG-Hp), or IgG beads for the indicated times. Immunoblots of isolated membranes were probed with pAb to p110α, p110β, or p110δ as indicated. Data shown are from one experiment that is representative of four independent determinations. Note the faster-migrating band reactive with p110α pAb in Hp-infected cells (arrow). (B) Unlike type I Hp (strain 11637), type II Hp (strain Tx30a) does not trigger membrane translocation of p110β or p110δ. U, Uninfected BMM. Immunoblots are from one experiment representative of three. (C) Relative effect of type II Hp (Tx30a), type I Hp 11637 (Hp), and IgG-opsonized Hp 11637 (IgG-Hp) on class IA PI3K activity in BMM. Data are normalized to uninfected BMMs (516 ± 94 cpm) and are the average ± SD (n = 3– 4). *, P < 0.010, versus uninfected BMM. Con, Control.