TLR4-mediated expulsion of bacteria from infected bladder epithelial cells Jeongmin Songa,1, Brian L. Bishopa,1, Guojie Lib, Richard Gradyc, Ann Stapletond,e, and Soman N. Abrahama,b,f,g,2 Departments of aMolecular Genetics and Microbiology, bPathology, and fImmunology, Duke University Medical Center, Durham, NC 27710; gProgram in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, Singapore 169547; and Departments of cUrology and dMedicine, and eDivision of Allergy and Infectious Diseases, University of Washington, Seattle, WA 98195 Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved July 16, 2009 (received for review January 18, 2009) Uropathogenic Escherichia coli invade bladder epithelial cells bladder expansion. That E. coli are harbored in fusiform compart- (BECs) by direct entry into specialized cAMP regulated exocytic ments of infected BECs was confirmed by an immune microscopy, compartments. Remarkably, a significant number of these intra- which revealed bacteria encased in membranes closely associated cellular bacteria are subsequently expelled in a nonlytic and piece- with Rab27b, a marker of fusiform vesicles (9). meal fashion by infected BECs. Here, we report that expulsion of Surprisingly, a significant number of intracellular E. coli, har- intracellular E. coli by infected BECs is initiated by the pattern bored within exocytic compartments of cultured BECs, were sub- recognition receptor, Toll-like receptor (TLR)4, after activation by sequently returned into the extracellular medium in piecemeal LPS. Also, we reveal that caveolin-1, Rab27b, PKA, and MyRIP are fashion (9). Seemingly, BECs have the innate capacity to sense and components of the exocytic compartment, and that they form a expel infecting bacteria. Here, we sought to examine the specificity complex involved in the exocytosis of bacteria. This capacity of of this host cell response and elucidate the underlying mechanism. TLR4 to mediate the expulsion of intracellular bacteria from in- fected cells represents a previously unrecognized function for this Results innate immune receptor. Expulsion of E. coli from Infected Human BECs. Our earlier studies have suggested that infected BECs have the capacity to spontane- caveolin ͉ exocytosis ͉ signaling ͉ urinary tract infection ͉ ously exocytose UPEC (9). We sought to examine whether this uropathogenic Escherichia coli exocytic response of BECs was specific to E. coli, and if so, to identify the bacterial component(s) responsible. We infected the uring their lifetimes, 10–20% of American females will receive 5637 human BEC line with E. coli CI5, a previously described UTI Dmedical attention for a urinary tract infection (UTI), and Ϸ3% isolate (12), as well as ORN103(pSH2), a K12 strain of E. coli will experience more than one infection per year (1, 2). UTIs expressing type 1 fimbriae, the critical bacterial component respon- represent the second leading cause of physician visits in the U.S., sible for E. coli entry into BECs (10). To examine the specificity of costing the health care system over $2 billion per year (1, 2). the BEC response, we also used two other bacterial strains Salmo- Interestingly, when compared with other mucosal surfaces, the UT nella typhimurium SL1344 and Staphyloccus aureus 54 in this assay. is difficult to colonize. Much of the resistance of the UT is After1hofinfection with each of the 4 strains of bacteria, a attributable to the flushing actions of urine and to the imperme- standard gentamicin protection assay was undertaken, and after ability of the epithelial lining. Because of their specialized role in 24 h, intracellular bacterial numbers assessed again. Whereas the storing urine, the apical surface of superficial bladder epithelial numbers of intracellular S. typhimuium SL1344 and S. aureus 54 had cells (BECs) is lined by scalloped-shaped plaques comprising a markedly increased implying intracellular growth, the same could not be said of E. coli CI5 or E. coli ORN103(pSH2) (Fig. 1A). We tightly interlaced latticework of proteins called uroplakins (3). Ϸ These proteins are closely associated with a collection of lipids, found a 60% drop in intracellular E. coli numbers (Fig. 1A), E. coli sphingolipids, and cholesterol, often referred to as lipid rafts that suggesting active exocytosis by infected BECs, although we cannot rule out the possibility of intracellular bacterial killing. In cumulatively constitute a surface that is highly impregnable to any case, our findings suggest that this drop in intracellular numbers urine, solutes, and potential pathogens (3). of E. coli is a unique phenomenon, because significant increases in Uropathogenic Escherichia coli (UPEC) are uniquely successful intracellular S. typhimurium and S. aureus numbers were observed in overcoming the bladder defenses, accounting for Ͼ85% of all under the same incubation conditions (Fig. 1A). bladder infections. The singular success of UPEC in the UT is We next sought to establish that the drop in the number of ascribed primarily to its capacity to penetrate and harbor within the intracellular bacteria in E. coli infected BECs was attributable to superficial BECs (4). Several studies have reported that UPEC also expulsion of bacteria rather than loss of viability. For these and for assume distinct intracellular shapes and form ‘‘intracellular bacte- most of the subsequent studies, we used E. coli ORN103(pSH2), rial communities’’ (IBCs) in the cytoplasm of superficial epithelial because the genome of this strain is only a fraction of that found on cells of both rodent and human bladders (5–7). This ability of UPEC strains, making it infinitely easier to pinpoint the genetic UPEC to successfully breach the mucosal barrier and colonize these factor(s) responsible for reduced intracellular numbers. We in- cells represents a critical initiating event in the development of fected multiple cultures of BECs with E. coli ORN103(pSH2) for UTIs. 1 h followed by 30-min gentamicin treatment to eliminate extra- E. coli invasion of BECs has been reported to involve several components of lipid rafts such as caveolin-1, an integral membrane protein found in the inner leaflet of the lipid bilayer on many Author contributions: J.S., B.L.B., G.L., and S.N.A. designed research; J.S., B.L.B., and G.L. mammalian cells, and Rac1, a member of the Rho family of performed research; R.G. and A.S. contributed new reagents/analytic tools; J.S., B.L.B., G.L., GTPases (8). A remarkable aspect of E. coli entry into BECs came and S.N.A. analyzed data; and J.S., B.L.B., and S.N.A. wrote the paper. from the recent finding that, after entry, the bacteria were harbored The authors declare no conflict of interest. within specialized exocytic vesicles (9). These compartments (or This article is a PNAS Direct Submission. fusiform vesicles) in BECs perform an important physiologic 1J.S. and B.L.B. contributed equally to this work. function by providing the necessary membranes required for blad- 2To whom correspondence should be addressed. E-mail: [email protected]. der expansion. As urine volume increases, these vesicles fuse with This article contains supporting information online at www.pnas.org/cgi/content/full/ the apical bladder surface in a cAMP-dependent manner allowing 0900527106/DCSupplemental. 14966–14971 ͉ PNAS ͉ September 1, 2009 ͉ vol. 106 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900527106 Downloaded by guest on September 26, 2021 A B 450 *** 30000 400 0 hr 350 25000 24 hr Fig. 1. E. coli exocytosis from infected BECs. (A) The 300 5637 BECs were infected with various bacteria for 1 h, 20000 ** after which a standard gentamicin protection assay was 250 performed to quantify the number of intracellular bac- 15000 200 ** teria (0 h). A significant decrease in intracellular E. coli ORN103(pSH2) and E. coli CI5 was seen at 24 h after the 150 10000 addition of gentamicin (24 h). Unlike E. coli 100 ORN103(pSH2) and E. coli CI5, the numbers of intracel- 5000 lular S. typhimurium SL1344 and S. aureus 54 were 50 *** ** Viable bacteria (CFU/well) significantly increased at 24 h after initial infection and 0 Intracellular bacteria (% of 0 hour) 0 Intracellular Extracellular after gentamicin treatment. (B) Intracellular and extra- Escherichia coli Escherichia coli Salmonella Staphylococcus Intracellular ORN103(pSH2) CI5 typhimurium aureus E. coli 0 hr E. coli 4 hr E. coli 4 hr cellular E. coli ORN103(pSH2) numbers in BECs at 0 and 4 h after gentamicin treatment; 5637 BECs were in- fected with 1000 MOI E. coli ORN103(pSH2). The sum of C 150 D 3500 human primary BECs numbers of intracellular and extracellular E. coli at4h after gentamicin treatment was similar to intracellular 125 3000 bacteria numbers at 0 h, suggesting bacterial exocytosis from infected BECs. (C) Treatment of infected BECs with 2500 100 NH4Cl and Bafilomycin, which neutralize bactericidal activity within lysosomes, caused no change of numbers 2000 of intracellular bacteria, indicating the compartment 75 survival 1500 harboring E. coli did not possess bactericidal activity. (D) Quantitative E. coli exocytosis assays were per- 50 1000 formed by using human primary BECs and a clinical E. coli (% Control) isolate of UPEC strain E. coli CI5. Significant numbers of 25 500 intracellular E. coli CI5 were expelled from infected IMMUNOLOGY primary BECs at 4 h after the gentamicin treatment. Viable bacteria (CFU/well) 0 Bars represent the mean ϩ SEM. , P Ͻ 0.05; , P Ͻ 0.03; Control Bafilomycin NH Cl 0 * ** 4 Intracellular Intracellular Extracellular , P Ͻ 0.001, relative to Intracellular E. coli titers at 0 h (1μ M) (10mM) *** E. coli 0 hr E. coli 4 hr E. coli 4 hr in A, B, and D. cellular bacteria. The intracellular bacterial numbers at this time (0 protein), and cytokeratin, all of which are hallmarks of terminal h) were assessed in one of the cultures.