Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration.
AllmondL. R.,
KaracaT. J.,
NguyenV. N.,
NguyenT.,
Wiener-KronishJ. P.,
SawaT.2003; Protein binding between PcrG-PcrV and PcrH-PopB/PopD encoded by the pcrGVH-popBD operon of the Pseudomonas aeruginosa type III secretion system. Infect Immun 71:2230–2233
BarberD. F.,
BartoloméA.,
HernandezC.,
FloresJ. M.,
RedondoC.,
Fernandez-AriasC.,
CampsM.,
RückleT.,
SchwarzM. K.other authors2005; PI3K γ inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus. Nat Med 11:933–935
CollinsS. J.,
RuscettiF. W.,
GallagherR. E.,
GalloR. C.1978; Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci U S A 75:2458–2462
CornelisG. R.,
BiotT.,
Lambert de RouvroitC.,
MichielsT.,
MulderB.,
SluitersC.,
SoryM.-P.,
Van BouchauteM.,
VanooteghemJ.-C.1989; The Yersinia Yop regulon. Mol Microbiol 3:1455–1459
DacheuxD.,
GoureJ.,
ChabertJ.,
UssonY.,
AttreeI.2001; Pore-forming activity of type III system-secreted proteins leads to oncosis of Pseudomonas aeruginosa-infected macrophages. Mol Microbiol 40:76–85
DaviesS. P.,
ReddyH.,
CaivanoM.,
CohenP.2000; Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351:95–105
FaudryE.,
VernierG.,
NeumannE.,
ForgeV.,
AttreeI.2006; Synergistic pore formation by type III toxin translocators of Pseudomonas aeruginosa. Biochemistry 45:8117–8123
FraylickJ. E.,
La RocqueJ. R.,
VincentT. S.,
OlsonJ. C.2001; Independent and coordinate effects of ADP-ribosyltransferase and GTPase-activating activities of exoenzyme S on HT-29 epithelial cell function. Infect Immun 69:5318–5328
FraylickJ. E.,
RieseM. J.,
VincentT. S.,
BarbieriJ. T.,
OlsonJ. C.2002; ADP-ribosylation and functional effects of Pseudomonas exoenzyme S on cellular RalA. Biochemistry 41:9680–9687
GaoY.,
DickersonJ. B.,
GuoF.,
ZhengJ.,
ZhengY.2004; Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci U S A 101:7618–7623
GoureJ.,
PastorA.,
FaudryE.,
ChabertJ.,
DessenA.,
AttreeA.2004; The V antigen of Pseudomonas aeruginosa is required for assembly of the functional PopB/PopD translocation pore in host cell membranes. Infect Immun 72:4741–4750
HartM. J.,
CallowM. G.,
SouzaB.,
PolakisP.1996; IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs. EMBO J 15:2997–3005
HauertA. B.,
MartinelliS.,
MaroneC.,
NiggliV.2002; Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis. Int J Biochem Cell Biol 34:838–854
HaywardR. D.,
CainR. J.,
McGhieE. J.,
PhillipsN.,
GarnerM. J.,
KoronakisV.2005; Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells. Mol Microbiol 56:590–603
HotzeE. M.,
Wilson-KubalekE. M.,
RossjohnJ.,
ParkerM. W.,
JohnsonA. E.,
TwetenR. K.2001; Arresting pore formation of a cholesterol-dependent cytolysin by disulfide trapping synchronizes the insertion of the transmembrane-sheet from a prepore intermediate. J Biol Chem 276:8261–8268
HuetC.,
Sahuquillo-MerinoC.,
CoudrierE.,
LuvardD.1987; Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation. J Cell Biol 105:345–357
JainM.,
RamirezD.,
SeshadriR.,
CullinaJ. F.,
PowersC. A.,
SchulertG. S.,
Bar-MeirM.,
SullivanC. L.,
McColleyS. A.other authors2004; Type III secretion phenotypes of Pseudomonas aeruginosa strains change during infection of individuals with cystic fibrosis. J Clin Microbiol 42:5229–5237
KlinghofferR. A.,
SachsenmaierC.,
CooperJ. A.,
SorianoP.1999; Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J 18:2459–2471
McGuffieE. M.,
FraylickJ. E.,
VincentT. S.,
OlsonJ. C.1999; Differential sensitivity of human epithelial cells to Pseudomonas aeruginosa exoenzyme S. Infect Immun 67:3494–3503
MuellerC. A.,
BrozP.,
MullerS. A.,
RinglerP.,
Erne-BrandF.,
SorgI.,
KuhnM.,
EngelA.,
CornelisG. R.2005; The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles. Science 310:674–676
NobesC. D.,
HallA.1995; Rho, Rac and Cdc42 regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia and filopodia. Cell 81:53–62
Ohno-IwashitaY.,
ShimadaY.,
WaheedA. A.,
HayashiM.,
InomataM.,
NakamuraM.,
MaruyaM.,
IwashitaS.2004; Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts. Anaerobe 10:125–134
OlsonJ. C.,
McGuffieE. M.,
FrankD. W.1997; Effects of differential expression of the 49-kilodalton exoenzyme S by Pseudomonas aeruginosa on cultured eukaryotic cells. Infect Immun 65:248–256
PedersonK. J.,
KrallR.,
RieseM. J.,
BarbieriJ. T.2002; Intracellular localization modulates targeting of ExoS, a type III cytotoxin, to eukaryotic signalling proteins. Mol Microbiol 46:1381–1390
PfeltzR. F.,
SchmidtJ. L.,
WilkinsonB. J.2001; A microdilution plating method for population analysis of antibiotic-resistant staphylococci. Microb Drug Resist 7:289–295
PlotkowskiM. C.,
de BentzmannS.,
PereiraS. H.,
ZahmJ. M.,
Bajolet-LaudinatO.,
RogerP.,
PuchelleE.1999; Pseudomonas aeruginosa internalization by human and epithelial respiratory cells depends on cell differentiation, polarity, and junctional complex integrity. Am J Respir Cell Mol Biol 20:880–890
RidleyA. J.,
SchwartzM. A.,
BurridgeK.,
FirtelR. A.,
GinsbergM. H.,
BorisyG.,
ParsonsJ. T.,
HorwitzA. R.2003; Cell migration: integrating signals from front to back. Science 302:1704–1709
RiffJ. D.,
CallahanJ. W.,
ShermanP. M.2005; Cholesterol-enriched membrane microdomains are required for inducing host cell cytoskeleton rearrangements in response to attaching–effacing Escherichia coli. Infect Immun 73:7113–7125
RoseJ. J.,
FoleyJ. F.,
YiL.,
HerrenG.,
VenkatesanS.2008; Cholesterol is obligatory for polarization and chemotaxis but not for endocytosis and associated signaling from chemoattractant receptors in human neutrophils. J Biomed Sci 15:441–461
RucksE. A.,
FraylickJ. E.,
BrandtL. M.,
VincentT. S.,
OlsonJ. C.2003; Cell line differences in bacterially translocated ExoS ADP-ribosyltransferase substrate specificity. Microbiology 149:319–331
SawaT.,
YahrT. L.,
OharaM.,
KurahashiK.,
GropperM. A.,
Wiener-KronishJ. P.,
FrankD. W.1999; Active and passive immunization with the Pseudomonas V antigen protects against type III intoxication and lung injury. Nat Med 5:392–398
SchoehnG.,
Di GuilmiA. M.,
LemaireD.,
AttreeI.,
WeissenhornW.,
DessenA.2003; Oligomerization of type III secretion proteins PopB and PopD precedes pore formation. EMBO J 22:4957–4967
ServantG.,
WeinerO. D.,
HerzmarkP.,
BallaT.,
DedatJ. W.,
BourneH. R.2000; Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science 287:1037–1040
ShepardL. A.,
HeuckA. P.,
HammanB. D.,
RossjohnJ.,
ParkerM. W.,
RyanK. R.,
JohnsonA. E.,
TwetenR. K.1998; Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an α-helical to β-sheet transition identified by fluorescence spectroscopy. Biochemistry 37:14563–14574
ShimadaY.,
MaruyaM.,
IwashitaS.,
Ohno-IwashitaY.2002; The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains. Eur J Biochem 269:6195–6203
SlosbergE. D.,
YaoY.,
XingF.,
IkuiA.,
JirousekM. R.,
WeinsteinI. B.2000; The protein kinase C β-specific inhibitor LY379196 blocks TPA-induced monocytic differentiation of HL60 cells. Mol Carcinog 27:166–176
TonettiD. A.,
Henning-ChubbC.,
YamanishiD. T.,
HubermanE.1994; Protein kinase C- β is required for macrophage differentiation of human HL-60 leukemia cells. J Biol Chem 269:23230–23235
van der GootF. G.,
Tran van NhieuG.,
AllaouiA.,
SansonettiP.,
LafontF.2004; Rafts can trigger contact-mediated secretion of bacterial effectors via a lipid-based mechanism. J Biol Chem 279:47792–47798
WaheedA. A.,
ShimadaY.,
HeijnenH. F.,
NakamuraM.,
InomataM.,
HayashiM.,
IwashitaS.,
SlotJ. W.,
Ohno-IwashitaY.2001; Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts). Proc Natl Acad Sci U S A 98:4926–4931
WolfA. A.,
FujinagaY.,
LencerW. I.2002; Uncoupling of the cholera toxin-G(M1) ganglioside receptor complex from endocytosis, retrograde Golgi trafficking, and downstream signal transduction by depletion of membrane cholesterol. J Biol Chem 277:16249–16256
XuB.,
PelishH.,
KirchhausenT.,
HammondG. B.2006; Large scale synthesis of the Cdc42 inhibitor secramine A and its inhibition of cell spreading. Org Biomol Chem 4:4149–4157