Role of EhRab7A in phagocytosis of type 1 fimbriated E. coli by Entamoeba histolytica

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1 Molecular Microbiology (2016) 102(6), doi: /mmi First published online 20 October 2016 Role of EhRab7A in phagocytosis of type 1 fimbriated E. coli by Entamoeba histolytica Kuldeep Verma, 1 Tomoyoshi Nozaki 2 and Sunando Datta 1 * 1 Department of Biological Science, Indian Institute of Science Education and Research Bhopal Bypass Road, Bhauri, Bhopal , Madhya Pradesh, India. 2 Department of Parasitology, National Institute of Infectious Diseases, Tokyo , Japan. Summary Entamoeba histolytica, the causative agent of amoebic colitis and liver abscess in human, ingests the intestinal bacteria and variety of host cells. Phagocytosis of bacteria by the amebic trophozoite has been reported to be important for the virulence of the parasite. Here, we set out to characterize different stages of phagocytosis of type 1 E. coli and investigated the role of a set of amoebic Rab GTPases in the process. The localizations of the Rab GTPases during different stages of the phagocytosis were investigated using laser scanning confocal microscopy and their functional relevance were determined using fluorescence activated cell sorter based assay as well as colony forming unit assay. Our results demonstrate that EhRab7A is localized on the phagosomes and involved in both early and late stages of type 1 E. coli phagocytosis. We further showed that the E. coli or RBC containing phagosomes are distinct from the large endocytic vacuoles in the parasite which are exclusively used to transport human holotransferrin and low density lipoprotein. Remarkably, type 1 E. coli uptake was found to be insensitive to cytochalasin D treatment, suggesting that the initial stage of E. coli phagocytosis is independent of the formation of actin filaments. Accepted 17 September, *For Correspondence: sunando@iiserb.ac.in; Tel ; Fax Introduction Entamoeba histolytica is an intestinal protozoan parasite that causes amoebic dysentery and liver abscess in human. As reported by WHO (1997), E. histolytica infection causes around 50,000 1,00,000 death each year. The highest prevalence of the disease is found in developing countries, particularly in Bangladesh, India, Mexico and South Africa, where it spreads by fecal-oral route mostly as cysts. The cyst form converts into the trophozoites, among which some live as a commensal in the large intestine feeding mainly on bacterial flora. The trophozoites can also invade the host intestinal epithelium and migrate to extra-intestinal sites. The enteric microbiota is a source of nutrient for the amoeba (Cleveland and Sanders, 1930). Moreover, an emerging body of work suggests that the intestinal microbiota could also be a crucial determinant of the outcome of amebiasis (Verma et al., 2012; Gilchrist et al., 2016). Although, it is not understood how the microbiota influences the progression of amebiasis. Phagocytosis is an evolutionary conserved process utilized by eukaryotes to ingest large particles, usually those over 0.5 microns in diameter. Phagocytosis is initiated by recognition and binding of the microorganism or debris to a specific membrane receptor which triggers a signal transduction event leading to the reorganization of the actin containing cytoskeleton and resulting in the formation of a nascent membrane bound compartment, the phagosome (Haas, 2007). The phagosome matures into the late phagosome which further fuses with lysosomes and leads to the formation of the phagolysosome, where the cargo is ultimately degraded by acidic proteases (Vieira et al., 2002; Kinchen and Ravichandran, 2008; Fairn and Grinstein, 2012). Thus, overall, phagocytosis represents a complex series of events involving multiple cellular machineries acting at different stages. In E. histoytica, several studies have been carried out to understand the molecular mechanism of the formation of the phagocytic cup which deciphered the early signalling events occurring during internalization of phagocytic cargo (Jain et al., 2008; Somlata et al., 2011; Mansuri et al., 2014; Babuta et al., 2015). Few studies have also been carried out which reported the VC 2016 John Wiley & Sons Ltd

2 1044 K. Verma, T. Nozaki, and S. Datta intracellular maturation of erythrophagosomes and identified the roles of some of the Rab family GTPases in this process (Rodriguez et al., 2000; Saito-Nakano et al., 2004; Welter and Temesvari, 2009). Rab proteins constitute the largest family of small GTPases that specifically controls various steps in the endosomal and the phagosomal transport process in eukaryotes (Gutierrez, 2013; Sherwood and Roy, 2013). The current knowledge about the roles of Rab GTPases in phagocytosis is majorly based on the studies in mammalian cells (Kinchen and Ravichandran, 2008; Stein et al., 2012). At the early stage of phagocytosis, the phagosome acquires Rab5, on its membrane to direct the fusion of phagosomes with early endosomes (Duclos et al., 2000; Koul et al., 2004). During maturation, the phagosomes loose Rab5 and acquire Rab7 (Vieira et al., 2003; Harrison et al., 2003; Becken et al., 2010). Rab7 is regarded as a marker of late phagosome (Rupper et al., 2001; Huynh et al., 2007). The genome of E. histolytica encodes more than 90 Rab proteins, a repertoire greater than that of mammals (Saito-Nakano et al., 2005). EhRab5 and EhRab7A are localized on large endocytic vacuoles (GEV) which transport human transferrin. Moreover, they are also essential for the formation of these compartments known as GEVs (Verma et al., 2015). EhRab5 and EhRab7A are also localized to the transient compartments known as prephagosomal vacuoles (PPV) which form transiently upon incubation of the trophozoites with red blood cells (RBCs) (Saito-Nakano et al., 2004). Isotypes of EhRab7 such as EhRab7A and EhRab7B are involved in lysosomal biogenesis and endosomal maturation (Saito- Nakano et al., 2007; Verma et al., 2015). EhRab8 (Juarez et al., 2001) and EhRab11B (Mitra et al., 2007) regulate the secretion pathway, whereas EhRab11A, the homologue of EhRab11B, is suggested to be involved in encystations (McGugan and Temesvari, 2003). Moreover, EhRabA and EhRab21 both are involved in the regulation of cytoskeleton dynamics through Gal/GalNAc mediated signalling (Welter et al., 2005; Emmanuel et al., 2015). Earlier studies using animal models have shown that the association of amoebic trophozoites with a mixture of bacterial species, including Escherichia, Klebsiella, Proteus etc enhances the parasite virulence (Luttermoser and Phillips, 1952; Phillips et al., 1958; Wittner and Rosenbaum, 1970; Bos and Hage, 1975). In vitro, phagocytosis of type 1 enteropathogenic E. coli or Shigella dysenteriae by the amoebic parasite increases its virulence and its capacity to damage epithelial layer in Gal/GalNAc dependent pathway (Galvan- Moroyoqui et al., 2008; Galvan-Moroyoqui et al., 2011). Altogether, these observations suggest that phagocytosis of certain enteric bacteria influences E. histolytica virulence during natural human infection. Interestingly, the interaction of certain gram-negative type 1 fimbriated bacteria with amoebic parasite has been well characterized by Mirelman and colleagues (Bracha et al., 1982; Mirelman and Bracha, 1982; Mirelman et al., 1983; Verdon et al., 1992; Pimenta et al., 2002; Mendoza-Macias et al., 2009). Further, it has been reported that a-methyl-mannose sugar (Bracha et al., 1982), transmembrane kinase 39 (Christy et al., 2012) and Gal/GalNAc lectin (Padilla-Vaca et al., 1999) regulate the adherence and ingestion of certain type 1 E. coli (E. coli BM079, E. coli 7343). Although the above reports have addressed some of the initial events in bacterial phagocytosis, the intracellular events of microbial phagocytosis by the parasite are largely unknown. Here we endeavored to dissect the trafficking route of type 1 uropathogenic E. coli (UPEC) in the parasite and investigated the role of the amoebic endocytic Rab GTPases in the process. Furthermore, we also investigated whether the endocytic cargos (low density lipoprotein and transferrin) segregate into distinct intracellular compartments from the phagocytic cargos (UPEC and human RBC) in the parasite. Results Type 1 uropathogenic E. coli phagosomes harbour EhRab5, EhRab7A and represent a distinct pool of compartments from giant endocytic vacuoles (GEVs) and other phagocytic cargos Earlier studies by various groups showed that E. histolytica phagocytose the certain type 1 fimbriated E. coli strains (Bracha and Mirelman, 1983; Galvan-Moroyoqui et al., 2008; Christy et al., 2012). E. histolytica trophozoites are very selective in their interaction with certain bacteria. This interaction is mediated by mannosebinding lectin present on the bacterial surface (Mirelman et al., 1980). Here we have developed a fluorescence microscopy based assay to study the intracellular trafficking of type 1 E. coli. We used UPEC and nonpathogenic E. coli MG1655 in this study. The presence of mannose-binding type 1 fimbriae of the transformed bacteria was confirmed by yeast agglutination assay (Supporting Information Fig. S1A). Both the strains were transformed with the bacterial expression vector harbouring egfp (see supplementary material for detail). Ingestion of the fluorescent bacteria was confirmed by confocal microscopy by probing the sub-cellular localization of the intracellular UPEC or E. coli MG1655 in the amoebic trophozoite (Supporting Information Fig. S1B). The ingested bacteria are transported via phagosomes and finally degraded in lysosomes. The uptake and

3 Phagocytosis of type 1 E. coli in Entamoeba histolytica 1045 Fig. 1. Localization of EhRab GTPases during the UPEC phagocytosis in E. histolytica. A. Immunofluorescence microscopy showing sub-cellular localization of HA-EhRab5, Myc-EhRab7A, HAEhRab7B, HA-EhRab11B and HA-EhRab21 with UPEC containing phagosomes. Amoebic transformants were grown in media containing 20 lg/ml G418 for 24 h before incubating them with GFP-UPEC for 15 min. Cells were then fixed and processed for immunofluorescence using anti-ha or anti-myc antibodies. The square box indicates association of respective Rabs with UPEC positive compartments. The object under the square box has been represented in the zoomed panel. Arrow indicates an association between Rab and UPEC. Scale bar, 10 lm. B. Quantification of UPEC association with respective Rab compartments. Fraction of UPEC-E. coli associated with a specific Rab compartment was counted in HA- EhRab5, Myc-EhRab7A, HA-EhRab7B, HA-EhRab11B and HA-EhRab21 expressing trophozoite (45 cells/time intervals) and plotted. The total numbers of internalized UPEC were associated with a given Rab specific compartments considered as percentage association. Data points shown in bar graph represent percentage mean 6 SE.

4 1046 K. Verma, T. Nozaki, and S. Datta clearance of UPEC by amoebic trophozoites were quantified using colony forming unit (CFU) assay and confocal microscopy. The trophozoites were incubated with UPEC for 10 minutes (min) on ice followed by incubation for 20 min at 378C to allow internalization of the bacteria. The un-internalized E. coli was removed by several washes and the fate of intracellular bacteria was chased for different time intervals [2, 4, 8 and 18 h]. Supporting Information Figure S1C shows E. coli uptake and subsequent degradation by amoebic trophozoites

5 Phagocytosis of type 1 E. coli in Entamoeba histolytica 1047 Fig. 2. Phagocytic and endocytic cargo are transported via morphologically distinct vesicular compartments. A C. Amoebic trophozoites stably expressing (A) HA-EhRab5 (B,C) Myc-EhRab7A transformants were incubated with (1:100) UPEC and (A, B) 100 lg/ ml Alexa-647 transferrin or (C) 100 lg/ml Dil LDL for 5 min at 378C. Trophozoites were fixed and subsequently subjected to immunofluorescence using (A) anti-ha and (B,C) anti-myc antibodies. Arrowheads indicate (A, B) transferrin or (C) LDL in (A) HA-EhRab5 and (B,C) Myc-EhRab7A positive GEVs, the arrow represents UPEC containing phagosome which are distinct from GEVs. D. Myc-EhRab7A expressing stable cells incubated with 100 lg/ml Alexa-647 transferrin and 100 lg/ml Dil-LDL for 5 min at 378C. Trophozoites were fixed and subsequently subjected to immunofluorescence using anti- Myc antibody. Arrows indicate LDL or transferrin associated with Myc- EhRab7A GEVs. E-F. Amoebic trophozoites stably expressing (E) HA-EhRab5 or (F) Myc-EhRab7A transformants incubated with both (1:100) RBC labeled with CellTracker Orange and 100 lg/ml Alexa-488 transferrin for 5 min at 378C. Trophozoites were fixed and subsequently subjected to immunofluorescence using (E) anti-ha and (F) anti-myc antibodies. Arrowheads indicate transferrin positive for (E) HA-EhRab5 and (F) Myc-EhRab7A GEVs, arrows represent RBC containing phagosome. Scale bars, 10 lm. using CFU assay. 30 min post-incubation, the intracellular bacteria were found to be localized in lysotracker positive compartments (r , n cells) as evident from our confocal micrographs (Supporting Information Fig. S1D). We further investigated the intracellular trafficking of type 1 UPEC in the amoebic trophozoites. Rab GTPases along with the adaptors, SNARES and effectors play a major role in intracellular vesicular trafficking of various cargo molecules. Here, we studied the roles of some of the amoebic Rab proteins in intracellular transport of type 1 UPEC. Earlier studies have demonstrated the involvement of EhRab5 and EhRab7A in RBC phagocytosis (Saito-Nakano et al., 2004). Moreover, the association of these Rab proteins with phagosome have also been demonstrated using proteomics based studies (Okada et al., 2005; Okada and Nozaki, 2006; Okada et al., 2006). We also included EhRab21, a member of Rab5 subfamily, and EhRab11B, a bona fide member of secretory/recycling Rab GTPases in our study. We first asked whether any of the above amoebic Rab proteins is associated with the E. coli phagosomes. Amoebic trophozoites were stably transfected with the expression plasmid for a given Rab GTPase fused to HA or Myc tag. Stably-transfected trophozoites were incubated with UPEC for 15 min at 378C followed by paraformaldehyde fixation and immunostaining with appropriate epitope specific antibodies. As evident from Fig. 1A, EhRab5, EhRab7A are colocalized with the UPEC phagosomes with a Pearson s correlation coefficient of r and r , respectively (n 5 30 cells). We also observed a few UPEC within the EhRab7B positive compartments (Fig. 1A). EhRab11B and EhRab21 did not colocalize with UPEC phagosomes. It is evident from the microscopic images (Fig. 1A) that UPEC are localized inside or within the Rab compartments resulting in low Pearson s correlation coefficients. Therefore, to support our co-localization data, we also quantified the association of the UPEC with the Rab GTPases. A fraction of the internalized UPEC which were associated with particular Rab positive compartments is expressed as the percentage association for the given Rab. We found that EhRab7A showed the highest percentage association ( , n 5 45 cells) with UPEC phagosomes (Fig. 1B). The association of the Rab GTPases with the type 1 uropathogenic E. coli containing phagosomes was further validated by biochemical studies. HA-EhRab5 or Myc-EhRab7A expressing transgenic trophozoites were incubated with GFP-UPEC for 15 min to allow internalization of the bacteria following purification of phagosomes as described previously with some modification (Okada et al., 2005; Okada et al., 2006). Immunoblot and immunofluorescence analysis of the purified phagosomes showed that the HA-EhRab5 or Myc-EhRab7A were associated with the GFP-UPEC containing compartments (Supporting Information Fig. S2). In a recent study, we have shown that EhRab5 and EhRab7A are colocalized on large endocytic vacuoles (diameter lm, n 5 30 cells) which transport human transferrin in the trophozoites (Supporting Information Fig. S3). These vacuoles are known as GEVs (Verma et al., 2015). We also demonstrated that both EhRab5 and EhRab7A are important for biogenesis of these endocytic compartments. As it is evident from Fig. 1A, the UPEC containing phagosomes, with more tubular morphology are distinct from the GEVs. The distinct morphological features prompted us to ask whether the E. coli containing phagosomes maintain a separate pool of compartments from the other endocytic cargos during transport via the endo-membrane system. To address the above question, we set up an uptake assay with multiple cargo molecules at a time. We used phagocytic cargo such as, UPEC or RBC in combination with endocytic cargo transferrin, dextran or low density lipoprotein (LDL). These studies were conducted on stable amoebic cell lines expressing HA-EhRab5 or Myc-EhRab7A. EhRab5 or EhRab7A expressing trophozoites were incubated with the transferrin and UPEC for 5 min at 378C and subsequently fixed, and further processed for immunofluorescence. The intracellular localization of the cargos and the EhRab GTPases were determined using confocal microscopy. Figures 2A and B shows that UPEC containing phagosomes are positive for EhRab5 (diameter mm, n 5 30 cells), EhRab7A (diameter mm, n 5 30 cells), and are distinct

6 1048 K. Verma, T. Nozaki, and S. Datta from transferrin containing GEVs. Similar experiments were performed in the EhRab7A transgenic trophozoites with LDL and UPEC. As shown in Fig. 2C, while LDL is localized to EhRab7A positive GEVs (diameter mm, n 5 30 cells), UPEC phagosomes maintains a separate pool distinct from GEVs. We also observed that LDL is colocalized with EhRab5 or EhRab7A (EhRab5 r and EhRab7A r , n 5 30 cells) and trafficked via enlarge vacuoles (Supporting Information Fig. S4A, B). As shown in Figure 2D, LDL and transferrin both progress through EhRab7A positive GEVs. We further found that RBC progressed through EhRab5 (diameter mm, n 5 30 cells) or EhRab7A (diameter mm,

7 Phagocytosis of type 1 E. coli in Entamoeba histolytica 1049 Fig. 3. EhRab7A is participating in the early stage of UPEC phagocytosis. A. FACS histogram showing the fluorescence intensity distribution for GFP-UPEC containing transgenic trophozoites. Amoebic trophozoites, stably transfected with pehex, HA-EhRab5, Myc-EhRab7A and HA-EhRab7B and their respective DN mutants, were incubated with (1:100) UPEC for 15 min at 378C. B. The flow cytometry quantification of ingested GFP-UPEC in amoebic transgenic trophozoites. Bar graph shows the mean 6SE of mean for fluorescence intensity for individual cell lines. The error bar is based on three independent flow cytometry-based experiments (50, 000 events/ each experiment). P-values from t-tests: *P < 0.05, **P < 0.01, ***P < (ns 5 non significant). C. HA-EhRab5 and Myc-EhRab7A expressing trophozoites were incubated with UPEC for 2 and 5 min at 378C. After each time point, cells were fixed and subsequently subjected to immunofluorescence using anti-ha and anti-myc antibodies. The square box indicates association of respective Rabs with UPEC positive compartments. Arrows indicate association between Rab and UPEC. The object under the square box has been represented in the zoomed panel. D. Untransfected amoebic trophozoites incubated with UPEC for 2 min and subsequently immunostained with anti-hgl antibody. The square box indicates association of HG with UPEC positive compartments. The object under the square box has been represented in the zoomed panel. Arrowheads (zoom panel) indicate the association between HGL and UPEC. E. HA-EhRab5 and Myc-EhRab7A expressing trophozoites were incubated with UPEC for 2 min at 378C. After, cells were fixed and subsequently subjected to immunofluorescence using anti-ha or anti-myc and anti-hgl antibodies. The square box indicates localization of respective Rabs with HGL and UPEC positive compartments. The object within the square box has been represented in the zoomed panel. Arrowhead indicates the association between EhRab5 and EhRab7A with HGL and UPEC. F. Myc-EhRab7A and Myc-DN EhRab7A expressing trophozoites were incubated for 2, 5 and 10 min with UPEC at 378C. After each time point, cells were fixed and subsequently subjected to immunofluorescence. Quantification of number of UPEC at different time intervals in respective Rab transformants. Total number of internalized UPEC counted in EhRab7A WT and DN expressing trophozoite (50 cells/time intervals) and plotted. Scale bars, 10 lm. n 5 30 cells) positive compartments which were distinct from the transferrin containing GEVs (Figs. 2E and F). We also observed association of GEVs with dextran, a fluid phase cargo (Supporting Information Fig. S5). Thus, our results suggest that the phagocytic and endocytic cargos are transported via separate membrane compartments. EhRab7A participates in early and late stages of type 1 E. coli phagocytosis Both biochemical and image based localization studies revealed that the type 1 UPEC containing phagosomes harbour some of the endocytic Rab GTPases like EhRab5 and EhRab7A. Earlier study by Nozaki et al. showed that EhRab5 play important role in erythrophagocytosis (Saito-Nakano et al., 2004). In an earlier study, EhRab7B was shown to be important in the biogenesis of lysosome like compartments (Saito-Nakano et al., 2007). In the current study, a minor population of UPEC phagosomes was found to be associated with EhRab7B. To find out whether the Rabs associated with the phagosomes; EhRab5, EhRab7A and EhRab7B have any role in phagocytosis of E. coli, we investigated the phagocytic efficiency of the transgenic trophozoites, expressing wild-type (WT) or dominant negative (DN) mutants of these Rab GTPases. Dominant negative Rab mutants represent the GDP bound inactivated form and have been frequently used as a useful tool in cell biology of Ras superfamily of small GTPases. Previous studies have demonstrated that the overexpression of these mutant proteins does not have any significant effect on the growth pattern of the amoebic trophozoites (Saito-Nakano et al., 2004). We quantified the percentage of trophozoites which could express the gene of our interest and their expression levels using immunofluorescence. Our quantification data suggest that both the percentage and the level of expression for the DN mutants were significantly lower than that of the WT trophozoites (Supporting Information Fig. S6). To study the steady-state effect of expression of the DN mutants on UPEC phagocytosis, stably transfected trophozoites were incubated with UPEC for 15 min at 378C. The intracellular UPEC population was then quantified using flow cytometry. While the overexpression of the WT Rab GTPases did not show any significant effect on UPEC phagocytosis (Fig. 3A, B), DN EhRab7A expressing trophozoites showed significant compromise in its ability to uptake the UPEC. In contrast, DN EhRab5 and DN EhRab7B did not show any inhibition of UPEC internalization (Fig. 3A, B). The steady state effect might be due to involvement of EhRab7A in early, late or even both phases during intracellular transport of E. coli phagosome. To study stage specific roles of the GTPases, we performed fixed-cell imaging in EhRab5 and EhRab7A expressing trophozoites (Fig. 3C). A continuous uptake experiment was carried out in which the EhRab5 and EhRab7A expressing trophozoites were incubated with UPEC at 378C for different time intervals. Trophozoites were fixed at 2 and 5 min post incubation and processed for immunofluorescence. As shown in the representative images (Fig. 3C), UPEC was internalized by the trophozoites and were found to be positive for EhRab5, EhRab7A compartment within 2 min of incubation. UPEC phagosomes were also found to be positive for HGL (heavy subunit of Gal/GalNac lectin) (Fig. 3D), a well known marker for early phagosomes (Boettner et al., 2008), localized with EhRab5 and

8 1050 K. Verma, T. Nozaki, and S. Datta Fig. 4. EhRab7A is involved in UPEC containing phagosomes maturation. A. HA-EhRab5, Myc-EhRab7A and HA-EhRab7B stable transgenic cells incubated with UPEC for 15 min at 378C. After removing the extracellular UPEC the intracellular E. coli was chased for 0 min, 4 h and 18 h. At each time point, cells, were fixed and prepared for immunofluorescence. The arrow indicates the localization of UPEC and Rab GTPase, arrowheads representing Rab compartments which did not show any close proximity with UPEC. The square box (with arrow) indicates association of respective Rabs with UPEC positive compartments. The object under the square box has been represented in the zoomed panel. Scale bars, 10 lm. B. Vector control, WT and DN mutant of Myc-EhRab7A and HA-EhRab7B transformants incubated with UPEC for 15 min at 378C. After removing uninternalized UPEC the intracellular UPEC was chased for 6 h, 8 h and 18 h. After each time interval, trophozoites were lysed and the lysate was uniformly spread on LB agar plate. After 16 h of incubation, the number of colonies was counted. The graph represents mean 6 SE of the mean from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-way analysis of variance (ANOVA) with Bonferroni post-tests compared with vector as a control (ns 5 non significant). EhRab7A (Fig. 3E). Further, we also investigated the uptake kinetics of UPEC by amoebic trophozoites expressing WT and DN mutant of EhRab7A using microscopy. The transgenic trophozoites were incubated with UPEC at 378C for different time intervals (2, 5 and 10 min) and subsequently processed for immunofluorescence microscopy. The numbers of ingested UPEC were counted using confocal images. As shown in Figure 3F, overexpression of DN EhRab7A leads to inhibition of UPEC phagocytosis by amoebic trophozoites. Altogether, these results suggest that the EhRab7A and EhRab5 are localized on the early UPEC phagosomes. However, only EhRab7A is found to be functionally associated with UPEC uptake. Next we sought to decipher the role of EhRab5, EhRab7A and EhRab7B in the late stage of phagocytosis. Transgenic trophozoites were incubated with UPEC for 15 min at 378C, washed extensively to remove the extracellular bacteria, and then chased for 4 h and 18 h in complete medium without the cargo. Confocal images

9 Phagocytosis of type 1 E. coli in Entamoeba histolytica 1051 of the amoebic trophozoites were acquired and scored for UPEC localization with Rab compartments. We observed that while EhRab5, EhRab7A and EhRab7B are associated with the phagosomes at 0 min chase, EhRab7A remained associated even at later stages (4 h and 18 h) (Fig. 4A). Previous studies have shown that EhRab7B is involved in the late phase of endosomal as well as phagosomal maturation (Saito-Nakano et al., 2007; Verma et al., 2015). To examine whether these GTPases play any role in the maturation of UPEC containing phagosome, amoebic trophozoites were stably transfected with expression plasmids for WT and the DN form of each Rab proteins. Transfected cells were allowed to phagocytose UPEC for 15 min at 378C. The un-internalized UPEC was removed by lysozyme treatment. The internalized UPEC population was chased for different time intervals (6, 8 and 18 h). At different time points, the trophozoites were lysed and plated on Luria- Bertani (LB) agar plates. The number of live UPEC at different chase-time was determined by counting the bacterial colonies. As shown in Fig. 4B, the degradation kinetics of UPEC was significantly impaired in DN EhRab7A trophozoites. However, EhRab7B WT and DN mutants did not show any significant effect. Altogether, these observations suggest that EhRab7A is involved in the early phase of UPEC phagocytosis as well as in the late phase of phagosome maturation. Phagocytosis of type 1 E. coli by E. histolytica is insensitive to cytochalasin D treatment We further studied the role of microfilaments in the amoeba for engulfment of the prey. In bacterial phagocytosis, the remodeling of actin at the phagocytic cup is known to be conserved from higher mammalian cells to social amoeba (Coppolino et al., 2001; Clarke et al., 2006). Earlier studies had demonstrated that the RBC phagocytosis by the amoeba is as an actin dependent process (Bailey et al., 1985). Therefore, we tested whether the internalization of UPEC is an actin dependent process in E. histolytica. Trophozoites were incubated with UEPC for 10 min on ice followed by incubation for 5 min at 378C. Subsequently, the trophozoites were fixed and probed for actin using immunofluorescence microscopy (see experimental procedure in detail). We did not observe any proximity or colocalization between the actin and UPEC (Fig. 5A). Further, we checked the effect of actin inhibitor, cytochalasin D on UPEC phagocytosis. Cytochalasin D is a well-established inhibitor of actin polymerization (Schliwa, 1982; Goddette and Frieden, 1986) and has been widely used in the study of endocytosis (Meza and Clarke, 2004), phagocytosis of chinese hamster ovary (CHO) cells (Ravdin et al., 1980; Ralston et al., 2014) and other actin dependent processes in E. histolytica (Emmanuel et al., 2015). Earlier, it has been shown that up to a concentration of 100 mm, cytochalasin D does not show any inhibitory effect on the growth pattern of the amoebic trophozoites although it (de la Garza et al., 1989) significantly abrogated the phagocytosis of RBC by this treatment (Bailey et al., 1985). As reported in earlier studies, the drug did not show any effect on the viability of the amoeba (Supporting Information Fig S7). Amoebic trophozoites, pretreated with 20 mm of the inhibitor, were used for UPEC uptake for 15 min at 378C and analyzed by flow cytometer. Our results did not show any significant difference in UPEC uptake between the treated and control (untreated) trophozoites (Fig. 5B). As shown in the representative image (Fig. 5C), trophozoites treated with cytochalasin D were able to ingest the UPEC. We also carried out a similar study on a non-pathogenic E. coli strain, MG1655 which is often used as a control in the studies involving type 1 UPEC (Martinez et al., 2000; Eto et al., 2006; Dikshit et al., 2015) to show that the insensitivity towards cytochalasin D is not only limited to the UPEC (Fig. 5D). It is well-known that attachment of pathogenic bacteria can lead to activation of signal transduction events, sometimes inducing dramatic arrangement of cytoskeleton components of eukaryotic cells that can result in the uptake of adherent bacteria (Finlay and Falkow, 1997). Ingestion of UPEC or E. coli MG1655 did not cause any rearrangement of the actin filaments in the amoebic trophozoites (Fig. 5E,F). Further, we performed the kinetics of UPEC phagocytosis by amoebic trophozoites in the absence and presence of cytochalasin D for the different time interval (10, 15 and 20 min). Cytochalasin D treatment did not show any inhibitory effect on the uptake kinetics of UPEC (Fig. 5G). The above observations suggest that the initial stage of phagocytosis of type 1 E. coli likely to be independent of microfilaments in E. histolytica. In contrast, we observed that the other phagocytic cargo such as RBCs is associated with actin positive phagocytic cup and nascent phagosome (Supporting Information Fig. S8). Moreover, the uptake of RBC and CHO is significantly abrogated by cytochalasin D treatment (Fig. 5D). Discussion E. histolytica is an intestinal protozoan parasite, which ingests a variety of bacteria to fulfill its nutritional requirement (Cleveland and Sanders, 1930; Lesh, 1975; Mirelman, 1987). Dissecting the intracellular events during the transport of type 1 E. coli in the amoeba is therefore of significant importance. In this report, we have used flow cytometry, confocal microscopy and CFU based assays to study the intracellular transport of uropathogenic type 1 E. coli in the enteric parasite. Further investigation led us to

10 1052 K. Verma, T. Nozaki, and S. Datta identify EhRab7A as one of the important components of phagocytic machinery in the amoeba. Phagocytosis is a complex process consisting of a series of molecular events tightly regulated by multiple cellular machineries. It begins with the attachment of the phagocytic cargo with the cell surface receptors. Previous reports suggest that the cell surface lectin, Gal/Gal- NAc (Katz et al., 2002; Petri et al., 2002; Padilla-Vaca et al., 1999), transmembrane kinase 39 (Christy et al., 2012) and adhesin (Rodriguez et al., 1994) function like receptor during phagocytosis. Earlier studies on erythrophagocytosis in E. histolytica provided a wealth of information related to internalization processes. The involvements of the actin filaments in the formation of phagocytic cups (Bailey et al., 1985; Mansuri et al., 2014) has been of particular interest. Through a series of studies, (Jain et al., 2008; Somlata et al., 2011, 2012; Mansuri et al., 2014) it has been demonstrated how binding of RBC to the amoebic trophozoite, triggers sequential recruitment of EhC2PK, Eh alpha kinase 1 and actin molecules to initiate cup formation. In the current study, we observed that RBC internalization is significantly inhibited by cytochalasin D suggesting a direct involvement of actin filaments. This is consistent with an earlier report, which showed that the cytochalasin D treatment, notably decreases the RBC internalization via partial disassembly of actin filaments (Bailey et al., 1985). In the current study, we demonstrated that the uptake of UPEC and E. coli MG1655 is unaffected by cytochalasin D treatment, suggesting a distinct mechanism for internalization. It has also been reported that the inhibitory effect of cytochalasin D depends on the size of the phagocytic cargo indicating the existence of an actin independent internalization process (Koval et al., 1998; May and Machesky, 2001; Tse et al., 2003; Tollis et al., 2010). Moreover, the involvement of a-tubulin has also been reported in phagocytosis and pseudopod extension in mammalian cells (Harrison and Grinstein, 2002; Khandani et al., 2007). Therefore, the involvement of other cytoskeletal components cannot be ruled out in type 1 E. coli phagocytosis by the amoeba. Rab GTPases being master regulators of vesicular trafficking, play crucial role in the intracellular transport of phagosomes. Most of our current knowledge about the roles of the Rab GTPases in phagocytosis has come from the studies carried out in mammalian cells. Multiple Rab proteins are recruited to the phagocytic compartments progressively during the phagosomal maturation in mammalian cells (Stein et al., 2012; Gutierrez, 2013). Rab5 and Rab7 are well characterized Rab protein in bacterial phagocytosis and functionally important in the early and late stage of phagocytosis respectively (Harrison et al., 2003; Kitano et al., 2008; Fairn and Grinstein, 2012). Till date, the mechanism of phagosomal maturation in the amoeba has been addressed in a single study where the host RBCs were used as the phagocytic cargo and its intracellular trafficking was studied (Saito-Nakano et al., 2004). This study reported that the RBC phagocytosis involved a transient compartment positive for EhRab5/EhRab7A called, PPV. Further, EhRab5 dissociates from theses PPVs and EhRab7A harbouring PPVs fuse with phagosomes. Over expression of DN mutant of EhRab5 impaired RBC phagocytosis by the amoeba. Thus EhRab5 decorated PPV formation is essential for erythrophagocytosis. In the current study, we observed that EhRab5 is associated with UPEC containing phagosome but it neither affects the uptake nor alters the clearance of the bacteria. Thus EhRab5 plays a selective role in erythrophagocytosis (Saito-Nakano et al., 2004). However, the existence of such transient compartment cannot be ruled out during E. coli phagocytosis which could only be detected by real time live cell imaging of the phagocytic cargo in the amoebic trophozoites. Rab7 is a well-established marker for the late endosome and implicated in the lysosome biogenesis and phagosomal maturation in mammalian cells (Rupper et al., 2001; Vieira et al., 2003; Kinchen and Ravichandran, 2008). Interestingly, we have recently shown that the amoebic EhRab7A participates in the early events in transferrin endocytosis where it plays an essential role in the formation of large endocytic vacuoles (GEVs). Overexpression of DN Rab7 inhibits the uptake of latex beads) in Dictyostelium discoideum cells (Buczynski et al., 1997). It is also evident in Xenopus laevis and Dictyostelium discoideum, that Rab7 regulates both early and late stages of endocytosis and phagocytosis (Mukhopadhyay et al., 1997; Rupper et al., 2001). Interestingly, our results show that the activity of EhRab7A is important in the early stage of phagocytosis (Fig. 3B, F) in the amoeba. Accordingly, DN EhRab7A significantly impaired the uptake of UPEC (Fig. 3B, F). In an earlier report, it has demonstrated that EhRab7A resides on PPV along with EhRab5. The PPVs fuse with RBC containing compartments to form erythrophagosomes which eventually lose EhRab5. EhRab7A continues to localize on erythrophaogosomes which fuse with amoebic lysosome like compartments to form phagolysosomes. In contrast to EhRab7A, EhRab7B did not show any significant effect on the UPEC clearance by the amoebic trophozoites. A recent study from our group has shown that this particular Rab7 isotype regulates transferrin maturation (Verma et al., 2015). We hypothesize that the function of EhRab7B might be redundant in this parasite due to the presence of various isotypes of EhRab7 (Saito-Nakano et al., 2005).

11 Phagocytosis of type 1 E. coli in Entamoeba histolytica 1053 Fig. 5. (Continued) While phagocytic and endocytic pathways mainly differ in terms of the size and nature of the cargo they transport (Herant et al., 2006; Tollis et al., 2010) they still share common molecular machinery for endolysosomal transport of the respective cargo in higher eukaryotes (Kinchen and Ravichandran, 2008). These two processes majorly differ in the initiation phase when the cargo molecules bind to their respective cell surface receptors. The binding process activates a series of downstream signalling events including triggering of the deformation in a membrane to engulf the receptor-cargo complex. The internalized vesicles fuse with early endosomes, which then matures into late endosomes and eventually fuses with the lysosome. Even though numerous studies have been carried out to decipher the various steps in both the processes, our knowledge about

12 1054 K. Verma, T. Nozaki, and S. Datta Fig. 5. (Continued) co-transportation of endocytic and phagocytic cargo is very shallow in mammalian cells (Booth et al., 2002; Harrison et al., 2003). Recently, we observed that EhRab5 and EhRab7A are localized on GEVs and regulate its biogenesis (Verma et al., 2015). In this study, we investigated whether the GEVs are also involved in transporting the phagocytic cargo. Our results suggest (Fig. 2 and Supporting Information Fig. S4) that the internalized transferrin and LDL are transported via GEVs which are distinct from UPEC or RBC containing

13 Phagocytosis of type 1 E. coli in Entamoeba histolytica 1055 Fig. 5. Cytochalasin D insensitive phagocytosis of type 1 fimbriated E. coli in E. histolytica. A. Entamoeba trophozoites were incubated with UPEC (1:100) for 10 min on ice. The trophozoites were then transferred onto glass slides and further incubated for 5 min at 378C, and probed for actin using alexa-568 labeled phalloidin. Scale bar, 10 lm. B. Amoebic trophozoites were pretreated with cytochalasin D (20 lm) for 60 min and further incubated with UPEC for 15 min at 378C. Cells were then washed with ice chilled 1X PBS buffer for three times, and finally suspend in 1X PBS and immediately scanned in flow cytometer. A pseudocolored dot plot is shown for SSC and fluorescence channel to gate amoebic population only. In each panel dot plot, the y-axis reflects the intracellular GFP-UPEC fluorescence and the x-axis shows SSC (20, 000 events). Value is represented as the fluorescence intensity distribution of trophozoites ingested UPEC. C. Representative micrograph shows the uptake of UPEC in cytochalasin D (20 lm) treated amoebic trophozoites. Scale bar, 10 lm. D. Amoebic trophozoites were incubated with dextran or UPEC or E. coli MG1655 or RBC or CHO cells for 15 min at 378C. Relative cellular uptake is the percentage of the trophozoites which are positive for a given cargo. Data from three independent flow cytometry based experiments ( events/each experiment) are presented as mean 6 SE. P-values from t-tests: *P < 0.05, **P < 0.01, ***P < (ns 5 non significant). E F. Trophozoites were incubated without (E) and with (F) UEPC or E. coli MG1655 for 10 min on ice, followed by incubation for 5 min at 378C, and probed for actin using alexa-568 labeled phalloidin. Scale bar, 10 lm. G. Entamoeba trophozoites were pretreated with cytochalasin D (20 lm) and incubated with E. coli (1:100) for 10, 15 and 20 min at 378C. Graph represents the mean 6SE of the mean from three independent flow cytometry based experiments (20,000 events/each experiment). phagosome. Particularly, they differ significantly in their sizes. However, our experimental observation does not rule out the possibility of transient intermixing between the phagocytic and endocytic cargoes. Further experimental investigation using live cell imaging would be necessary to explore these events. It is also evident from earlier studies that transferrin (Lopez-Soto et al., 2009) and LDL (Christy et al., 2012) internalized by receptor mediated endocytosis in E. histolyitca. In mammalian cells, transferrin and LDL are internalized through clathrin mediated endocytosis (Lakadamyali et al., 2006) and believed to enter a common pool of early endosomes (Dickson et al., 1983; Dunn et al., 1989; Ghosh et al., 1994). Early endosomes fuse with sorting endosomes from which, transferrin is recycled back to the cell surface; LDL follows the degradative route to lysosome via late endosome. While GEVs transport LDL and transferrin in the amoeba, PPVs have been shown to be essential for RBC phagocytosis (Saito-Nakano et al., 2004). Interestingly, these compartments transiently form at a very early stage of erythrophagocytosis and fuse with the early RBC phagosomes. We believe that amoebic trophozoites employ the GEVs and PPVs to provide membrane to newly internalized cargo molecules. These compartments probably maintain the intracellular ph and also supplying membrane during endosome and phagosome maturation process. While the GEVs are always observed in the amoebic trophozoites and the PPVs are transiently formed in response to the incubation of the trophozoites with RBC. It could also be possible that PPVs are generated from the GEVs in response to attachment of RBC with the trophozoites. Our results also does not rule out very early and rapid sorting of endocytic cargos from the phagocytic ones. In conclusion, the current study presents the first intracellular description of UPEC phagocytosis (Fig. 6). It further demonstrates that the amoeba maintains a morphologically distinct pool of compartments to transport various endocytic and phagocytic cargo. Experimental procedures Entamoeba histolytica culture E. histolytica isolate HM-1: IMSS was cultured axenically in BI-S-33 medium with 15% (v/v) heat-inactivated Adult Bovine Serum (Cat. B-9433, Sigma-Aldrich, St. Louis, MO) at 35.58C as described previously (Diamond et al., 1978; Gillin and Diamond, 1980). Plasmids and antibodies Construction of EhRab5, EhRab7A, EhRab7B, EhRab11B and EhRab21 and their dominant negative mutants in amoebic expression vector and rabbit polyclonal anti- EhRab7A were reported previously (Saito-Nakano et al., 2004; Saito-Nakano et al., 2007; Nakada-Tsukui et al., 2005; Emmanuel et al., 2015; Verma et al., 2015). Cytochalasin D treatment Trophozoites harvested during logarithmic growth were plated on BI-S-33 medium with 20 lm Cytochalasin D and incubated for an additional hour at 378C. After the Cytochalasin D treatment, trophozoites remained viable and impermeable to propidium iodide (50 lg/ml) and trypan blue (0.4%) during the experiments. Non-treated (negative) or 70% ethanol treated (positive) trophozoites were used as viability controls. Transfection Transfection of E. histolytica was performed by electroporation as described previously (Hamann et al., 1995). The trophozoites were collected from the log phase cultures and washed with phosphate buffer saline (PBS) followed by incomplete cytomix buffer (10 mm K 2 HPO 4 /KH 2 PO 4 (ph 7.6), 120 mm KCl, 0.15 mm CaCl 2, 25 mm HEPES (ph 7.4), 2 mm EGTA, 5 mm MgCl 2 ). The washed cells were then re-suspended in 0.4 ml of complete cytomix buffer (incomplete cytomix containing 4 mm adenosine triphosphate, 10 mm reduced glutathione) containing 100 mg of plasmid DNA and subjected to consecutive pulses of 500 V voltage and 500 lf capacitance (Bio-Rad Gene M Pulser

14 1056 K. Verma, T. Nozaki, and S. Datta Fig. 6. Intracellular trafficking of UPEC in E. histolytica. The process begins at the plasma membrane, where UPEC are internalized in an actin independent pathway. EhRab7A activity is important for uptake and maturation of UPEC containing phogosomes. Endocytosis of transferrin and LDL progress via EhRab5 and EhRab7A positive GEVs. PPVs (EhRab5 and EhRab7A positive) are transient compartments which formed during RBC phagocytosis. These PPV loose EhRab5 and EhRab7A positive PPVs fused with RBC phagosome (Saito Nakano et al., 2004). In contrast, UPEC was observed in EhRab5 and EhRab7A positive compartments which are morphologically distinct from the transferrin and LDL containing GEVs. In this study, we also observed EhRab5 positive, EhRab7A positive as well as EhRab5 and EhRab7A positive RBC phagosomes (Supporting Information Fig. S9). Xcell, Hercules, CA). The transfectants were initially allowed to grow without any selection. Screening of stable transgenic amoeba was performed as described earlier (Verma et al., 2015). All the experiments were performed on the stable transformants maintained at 20 lg/ml G418 (Cat. 1720, Sigma-Aldrich). Colony forming unit assay Uropathogenic E. coli type 1 phagocytosis by Entamoeba was examined. Briefly, the day before commencing the experiment, UPEC carrying the GFP gene on the plasmid was grown from a glycerol stock in LB broth containing 100 mg/ml ampicillin for 16 h at 378C without shaking. Bacteria were then washed in BI-S-33 medium and resuspended at the in BI-S-33 to a concentration of colony forming unit (CFU)/mL without serum or antibiotics. Before the uptake experiments, trophozoites were washed in BI-S-33 and then mixed with UPEC in BI-S-33 media without serum or antibiotics, at 378C for 15 min. After uptake, trophozoites were incubated with UPEC lysis buffer (lysozyme solution (10 mg/ml) in 10 mm Tris- HCl, ph 8.0.) and washed several times with cold 1X PBS in order to stop additional bacterial uptake or destruction of bacteria in the phagosome. The ingested bacteria by amoebic parasite were further chased different time (2, 4, 6, 8, and 18 h) for the study of phagosome maturation. After the indicated time interval, trophozoites were centrifuged at 900g for 3 min and the pellet was lysed in LB broth containing 0.2% Triton X-100, and an aliquot of the cell lysate was plated on the LB agar plates to determine the number of viable bacteria present in amoeba as colony-forming units. Bacteria were incubated at 1008C for 30 min. None of the colony was observed with heat killed UPEC bacteria spared on LB agar plate. CHO cell culture and labeling CHO cells were cultured in F12 medium (Cat , Life Technologies) supplemented with 15% fetal bovine serum (Cat , Life Technologies). CHO cells were briefly washed with 1X PBS and immediately incubated with 5 mm of CellTracker orange (F12 medium) (Cat. C2927, Life Technologies) for 30 min at 378C. After CHO cells were washed once with PBS. For use in in vitro assays, CHO cells were released from the monolayer by 5 min incubation in trypsin. CHO cells labeling confirmed under the fluorescence microscopy and subsequently suspended in the BI-S-33 medium for experiments. Flow cytometry Around trophozoites were incubated in 6 well plates for 15 min. After the medium was replaced with 2 3

15 Phagocytosis of type 1 E. coli in Entamoeba histolytica UPEC or E. coli MG1655 or Texas Red dextran (Cat. D-1864, Life Technologies) or RBC or CHO cells [labeled with CellTracker Orange (Cat. C2927, Life Technologies)] and incubate for 15 min at 378C. After indicating incubation time, replace the medium by adding 10 ml of ice cold 1X PBS and centrifuged at g for 3 min and washed twice with 1X PBS. The pellet was resuspended in 0.5 ml of 1X PBS and intracellular fluorescence analyzed by flow cytometry using the BD-FACS Aria III (Becton, Dickinson, East Rutherford, NJ, USA). The ingestion of CHO cells by E. histolytica was performed as described previously (Nakada-Tsukui et al., 2009). The results represent three independent experiments. All the flow cytometry based quantification were performed using live trophozoites by suspending in ice cold PBS. Uptake of transferrin, acetylated low density lipoprotein, dextran, UPEC and RBCs Trophozoites were incubated in BI-S-33 media for 20 min at 378C before initiation of uptake assays. To follow the Alexa- 647 transferrin (Cat. T-23366, Life Technologies) or acetylated Dil-LDL (Cat. L-3482, Life Technologies) or Texas Red dextran or UPEC or RBCs in trophozoites. Transgenic trophozoites were incubated with above cargo in prewarmed BI-S-33 media for 5 or 10 min at 378C. After each time point, cells were fixed and images were acquired. Immunofluorescence confocal microscopy E. histolytica grown in glass culture tubes were chilled on ice for 10 min. After inverting to detach the trophozoites from the glass wall, cells were transferred to a falcon tube and centrifuged at g for 5 min. The pellet was washed with BI-S-33 medium. Harvested trophozoites suspended in the BI-S-33 medium and transferred to 8-mm round wells on glass slides and incubated for 20 min at 378C in the water bath to let trophozoites attach to the glass surface. After a final wash, the cells were fixed in 4.0% paraformaldehyde for 15 min. After washing with 1X PBS, the trophozoites were permeabilized using 0.1% Triton X-100 in 1X PBS for 10 min and blocked with 5% fetal calf serum in PBS for 30 min at room temperature. Trophozoites were incubated for 1 h in primary antibodies at room temperature. Rabbit polyclonal anti-ehrab7a antibody was provided by Prof. Nozaki. Mouse monoclonal anti-ha (Cat. sc-7392), rabbit polyclonal anti-ha (Cat. sc-789) and mouse monoclonal (Cat. sc-40) anti-myc antibodies were purchased from Santa Cruz, Texas]. After three washes in blocking solution, trophozoites were co-incubated with Alexa-conjugated (Life Technologies) secondary antibodies (1:500 dilutions), Alexa-568 phalloidin (1:50 dilutions) and 4 0, 6-diamidino-2-phenylindole (DAPI) for 1hr at room temperature. After three washes with blocking solution, coverslips were mounted on the glass slide using Mowiol. Slides were examined using a LSM-780 laser scanning confocal microscope (Carl Zeiss, GmbH, Jena, Germany) with a 633/1.4 NA oil immersion objective lens. Immunofluorescence signals were captured in individual en face (x-y axes) planes throughout the cellular z-axis at 0.35 mm intervals. Colocalization analysis The degree of colocalization of UPEC, transferrin, LDL with or the EhRab constructs was quantified. To quantify colocalization, a randomly chosen field of cells (30 cells/experiment) was selected as a region of interest, and analysis was carried using ImageJ-Fiji Colocalization Threshold (developed by Tony Collins (Collins, 2007), McMaster Biophotonics Facility, McMaster University, Hamilton, ON, Canada; available at confirmed by eye. Pearson s correlation coefficient VR was calculated in z-stacks between the two fluorescent signals. Values represent the mean 6 standard deviations (SD) from each image. Counting of GFP-UPEC phagosomes associated with amoebic Rab GTPase Association of amoebic Rab GTPases (HA-EhRab5, Myc- EhRab7A, HA-EhRab7B, HA-EhRab11B and HA-EhRab21) was analyzed by confocal microscopy. The fraction of internalized bacteria within the amoebic Rab specific compartment were counted and normalized with respect to the total number of ingested bacteria. Statistical analysis Statistical significance was determined by the differences between treatments using unpaired two-tailed Student s t- test and two-way ANOVA using the program GraphPad Prism version 4.0 (Graph-Pad Software, San Diego, CA). Acknowledgements The authors thank Dr. Atul Aher (Peoples Hospital, Bhopal, India) and Dr. Vikash Jain (Indian Institute of Science Education and Research Bhopal, India) for their generous gift of uropathogenic type 1 E. coli and E. coli MG1655 strain, respectively. They are thankful to Professor David Mirelman (Weizmann Institute of Science, Rehovot, Israel) for his discussion and encouragement in initiating this project. They are deeply grateful to Dr. William A. Petri Jr (University of Virginia, USA) for kindly provide monoclonal antibodies against Gal/ GalNac lectin. We are grateful to Dr. Christopher D. Huston (University of Vermont College of Medicine, USA) and Dr. Lesly A. Temesvari (Clemson University, USA) for sharing the protocols. They also thank Dr. Amulya Priya, Prateek Raj and Chitra Asati for helping in ultracentrifugation, western blot and flow cytometry respectively. Kuldeep Verma acknowledges the Department of Biotechnology, New Delhi, India for DBT- IISc Postdoctoral Fellowship. This work was supported by Max Planck Gesellschaft, Germany and Department of Science and Technology, India partner group funding. Author contributions K.V. and S.D. conceived and designed the experiments; K.V. performed the experiments; K.V. and S.D. analyzed