This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.
In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.
Urinary tract infections (UTIs) are one of the most common bacterial infections. An estimated 40-50% of women are expected to experience at least one urinary tract infection (UTI) during their lifetime1. One of the main agents of UTI is uropathogenic E. coli (UPEC), which accounts for over 70% of uncomplicated UTIs2. Furthermore, approximately one quarter of those who have a UTI will have a recurrent infection, often caused by the same strain, despite appropriate antibiotic treatment3. The high incidence of UTI represents a substantial burden on healthcare systems, costing more than $2 billion a year in the US4. Furthermore, the use of antibiotics to treat UTIs also leads to rising antibiotic resistance rates, which is a major public health concern5.
Therefore, a large effort has been placed into understanding the mechanisms by which UPEC infects the urinary tract, as well as its ability to cause recurrent infections6,7,8. In particular, a mouse model of infection has been used to examine bacterial and host characteristics that contribute to UTI8. This mouse model has the benefit of being applicable to unmodified clinical strains isolated from human patients. This model has also led to the discovery of potentially druggable bacterial pathways important for establishment of UTI, such as the Type 1 pilus9 and iron acquisition systems10.
Compared to these successes in studying the early events in UTI, knowledge of the mechanisms underlying recurrent UTI is still lacking11. One hypothesis is that UPEC evades antibiotic therapy and causes recurrent infections in the bladder by forming intracellular bacterial communities (IBCs) within bladder epithelial cells. IBCs have been identified both in murine models of infection and in human UTI patients12,13. The presence of IBCs in urine samples from pediatric UTI patients has been associated with higher rates of recurrence14,15. However, isolating IBCs and studying the bacteria within them has proven to be technically challenging due to their rarity; it is estimated that an infected murine bladder typically only has 10-100 IBCs16. Furthermore, bladder epithelial cells are relatively large (50-120 µm)17, making it challenging to deploy fluorescence assisted cell sorting (FACS) given that typical FACS nozzles are designed with diameters of 70 µm or 100 µm. Thus, cells as large as bladder epithelial cells are often removed by filtration prior to FACS to avoid clogging the fluidics.
Our lab recently described a general and economical method to isolate rare infected cells from mixtures such as scraped epithelial cells of the bladder18. To effectively isolate IBCs, we used traditional mouth pipetting. Mouth micropipetting is a technique that has long been used for micromanipulation of single cells and embryos for downstream analysis19,20,21,22,23,24,25. Traditional mouth pipetting of large liquid volumes (in milliliters) has often been the cause of laboratory related accidents, and the technique has rightly been shunned by much of the research community outside of traditional embryology and single cell applications. Our protocol is inspired by the single cell versions of this technique19,20, which mitigate risk by providing a large buffer (>2 mL) of air between the researcher and sample compared to the volume of liquid transferred (<1 µL). This method also takes advantage of the fine control that mouth micropipetting provides, which translates to a low final volume of surrounding solution transferred and high purity of isolated cells. The technique uses inexpensive materials (<$50), and thus should be feasible to implement in all labs.
This visual protocol describes our IBC isolation technique, providing a reference to assist other researchers seeking to replicate this technique. The researcher will need access to a fluorescent dissecting microscope (or similar equipment) that can be used to visualize individual epithelial cells and the fluorescent bacteria during live imaging, with an open and accessible imaging stage for micropipetting (see the Table of Materials for the details of the microscope used, though other equivalent instrument models may also be used). While this protocol will focus on IBCs in a murine model of UTI, similar methods should be applicable to isolate infected cells from cell suspensions in other models of infection.
All methods described here regarding animal handling have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Genome Institute of Singapore and Biological Resource Center of the Agency for Science, Technology and Research, Singapore.
1. Mouse Infection
2. Bladder Epithelial Cell Harvesting to Obtain a Cell Suspension
3. Intracellular Bacterial Community (IBC) isolation: Mouth Pipetting of IBCs
NOTE: All methods described in this section have undergone an institutional risk assessment. Mouth pipetting carries the inherent risk of ingestion of the solution that is being transferred. This risk is largely mitigated by the nanoliter volumes that this protocol uses, and we recommend that all users of the protocol pay heed to the precautionary and practice notes listed here and in the discussion.
Apart from confirmation (Figure 3D) of the presence of a single isolated IBC in the collection tube via the dissecting microscope, the purity of the isolated IBC can also be confirmed by confocal microscopy. As shown in Figure 4A, the isolated cells should stain for both E. coli and uroplakin, and are the expected size for IBCs (50-120 µm)17. Furthermore, E. coli staining is not present in the surrounding liquid. Based on our data, more than 90% of cells isolated with this technique are IBCs18. After isolation, the presence and viability of the bacterial cells in the individual IBC can be confirmed through colony forming unit (CFU) enumeration (Figure 4B) or quantitative polymerase chain reaction (qPCR) for genomic equivalents (Figure 4C). Figure 4C also demonstrates that uninfected epithelial cells isolated with the same protocol do not have quantifiable amounts of bacteria. Based on these data, we estimate that the range of CFUs in a single IBC is 102-103 in the murine model of urinary tract infection. One of the main goals of single IBC isolation is to perform downstream analyses such as RNA sequencing. To verify that our isolation method is able to obtain RNA from bacteria in IBCs for analysis, we performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) quantification of three genes (16S, cyoB, and frdA) for a range of individually isolated and pooled IBCs (Figure 4D). All the data shown in Figure 4 has been adapted with permission from Duraiswamy et al.18. An overview schematic of our IBC isolation protocol can be seen in Figure 5, which is reproduced from Duraiswamy et al.18.
Figure 1: Hand-pulled capillaries retain narrow openings. (A) Samples of hand-pulled capillary tubes are displayed on a black background for contrast. From bottom to top, an unpulled capillary, a capillary that was not pulled to a sufficient extent, a capillary that can be used for single bladder epithelial cell harvesting, and a capillary that was pulled too thin (and thus separated into two pieces) are shown. A 15 cm ruler is placed at the bottom of the image for scale. The estimated point for snapping off the usable capillary is indicated on the figure by the red arrow. (B) Image taken with a dissecting microscope confirming the hollow internal diameter of a pulled capillary (bottom). An unpulled capillary is positioned above to demonstrate the relative size difference of the two capillaries. Scale bar = 4.0 mm. Please click here to view a larger version of this figure.
Figure 2: Dissection of mouse to harvest bladder epithelial cells. (A) An image of a mouse with white lines added to indicate the estimated location and angle of incisions to expose the murine peritoneal cavity and bladder. (B) An image of the exposed mouse peritoneal cavity post-incision. (C) An image of the exposed bladder (red arrow) protruding from between the fat pads. (D) An image of the murine bladder with the tip of the forceps inserted into the lumen, with arrows to indicate the direction of motion needed to invert the bladder. The bladder is first pulled slightly outwards, then around and off the first shaft of the forceps. The directions of motion for both actions are as indicated by white arrows numbered 1 and 2. (E) An image showing the final position of the inverted bladder inserted onto the second shaft of the forceps. The shafts of the forceps are labeled in both panels D and E with red arrows and text. Please click here to view a larger version of this figure.
Figure 3: IBC harvesting from bladder cells. (A) An infected and inverted bladder in cold PBS solution before cell scraping. (B) An image showing scraped bladder cells as seen under a microscope. IBCs can be identified as large green fluorescent aggregates in both images (see red arrows). (C) An image of the completed mouth micropipetting apparatus. The aspirating pipette, pipette tip, aspirator tube, and pulled capillary tube are identified with numbered arrows as indicated on the right. (D) An image of a single isolated IBC within a 1.5 mL collection tube (outlined in red). Scale bars (as indicated) are represented by white lines in panels A, B, and D. Please click here to view a larger version of this figure.
Figure 4: Harvested IBCs are pure and can be used for downstream analysis. This figure has been modified with permission from Duraiswamy et al18. (A) Images of two isolated GFP-positive cells that were stained with anti-uroplakin and anti-E.coli antibodies. The first cell (IBC 1) has images of individual channels (at low-magnification) on the left, and a high-magnification merged image is on the right. The second cell (IBC 2) is shown in high magnification in merged and individual channels. Scale bars are as indicated. DNA is stained with 4′,6-diamidino-2-phenylindole (DAPI) and represented in the blue channel. Anti-E. coli is stained with a secondary antibody conjugated to fluorescein isothiocyanate (FITC) and represented in the green channel. Anti-uroplakin is stained with a secondary antibody conjugated to tetramethylrhodamine isothiocyanate (TRITC) and represented in the red channel. (B) Bacterial CFUs from isolated IBCs. IBCs were processed immediately, or incubated in 0.1% Triton-X for 10 or 30 min. Pooled CFU counts of individual IBCs isolated from n = 3 separate experiments are shown. Limit of detection = 0.7 log10 CFUs/IBC. Red dots plotted at the limit of detection indicate samples for which no colonies were recovered. All IBC-containing samples are not significantly different (p > 0.05, Mann-Whitney test); the uninfected epithelial cells are significantly different from the IBC (10 min) data (p < 0.001, Mann-Whitney test). (C) qPCR quantification of bacteria on individual IBCs and uninfected epithelial cells after a 10 min incubation in 0.1% Triton-X (*, p < 0.0001, Mann-Whitney test, n = 4). Limit of detection = 1.18 log10 bacterial genome equivalents/IBC. Red dots indicate samples for which no colonies were recovered on titering in panel B. (D) Quantification of the 16S rRNA, cyoB, and frdA genes for varying numbers of individually isolated and pooled IBCs (n = 1 experiment; each point indicates the mean of 3 technical replicates). NC = no DNA negative control. Please click here to view a larger version of this figure.
Figure 5: A schematic and its associated photographs representing the isolation of IBCs via mouth micropipetting from infected mice bladders. This figure is reproduced from Duraiswamy et al.18. (A) A harvested whole bladder; (B) an inverted whole bladder exposing the GFP expressing IBCs; (C) a close-up of the edge of a scraped bladder showing individual IBCs in suspension in the adjacent buffer; (D) a single isolated IBC pipetted into a tube. Red arrows in panel B indicate examples of GFP-positive IBCs on the luminal surface of the bladder. The red dotted line in panel C indicates the right border of the inverted bladder (indicated as "BL"); red arrows in panel C indicate apparent individual GFP-positive epithelial cells that have been scraped off the bladder surface. White dotted line in panel D indicates a micropipetted sub-microliter droplet containing an isolated IBC, which is indicated by a white arrow. Scale bars = 2 mm. Please click here to view a larger version of this figure.
The protocol we have described allows for the isolation of single IBCs from a murine model of UTI. This protocol isolates IBCs containing viable intracellular bacteria, which can be verified by culturing for CFU. The protocol results in intracellular bacteria from IBCs with little contamination by extracellular bacteria, allowing for further characterization of both bacteria and host cell from an IBC (Figure 4C). We also show that the bacteria from a single IBC can be used in downstream applications such as qPCR (Figure 4C), suggesting that our technique can be used to process IBCs for other in vitro analyses. By pooling the bacteria harvested from as few as 5 IBCs, we further demonstrate our ability to perform qRT-PCR analysis on three bacterial genes, suggesting that good quality RNA can be harvested from the bacteria in our isolated IBCs (Figure 4D). Combined, the data we have shown indicate that performing genome-wide RNA analysis (such as RNA sequencing) on single IBCs may be possible using this isolation technique.
In this protocol, we have focused on the 6 h time point because that is when IBC numbers peak in the bladders of black 6 mice infected by UTI8927. Furthermore, we have also used a static bacterial culture system to enhance the level of type 1 pilus expression in UTI89. The expression of type 1 pilus is critical for E. coli to attach to and infect bladder epithelial cells28. However, this expression is tightly regulated29 and environmental cues are known to alter it30. In order to maintain a consistent infection phenotype and sufficient numbers of IBCs, we recommend using a 2 x 24 h static bacterial culture (slightly modified from Hung et al.8) and the 6 h infection time point when working with previously tested E. coli strains such as NU14 and UTI8928,29. However, it is possible that these variables will need to be adjusted in other UTI strains or in other mice strains to obtain the ideal number of IBCs from each infection.
While the protocol from Hung et al.8 uses only female mice, other established protocols for establishing urinary tract infection in male mice have been reported31. In this model, cystitis in male mice also followed the IBC pathway. As the bladders of male and female mice are similar in size, we anticipate that our IBC isolation protocol can be used on infected male mice as well.
The relatively simple technology utilized in this protocol also ensures that it can be deployed in most laboratories. One of the key steps involved in this protocol is the pulling of glass capillaries to create microcapillaries for selecting the cell type of interest. This step allows for flexibility in the diameters of microcapillaries created, and thus the method can be extended to multiple different target cell types. However, due to the inherent variation in creating these capillaries, care must be taken to ensure that the final diameter is in a usable range. If the capillaries are too narrow, they fail to pick up the cell of interest, but if they are made too wide, multiple cells could be selected in a single attempt. Furthermore, the use of an open flame during the process of capillary pulling carries an inherent risk of burns and fire, so the researcher attempting to create microcapillaries should take care to prevent such events from occurring. To reduce the variability as well as the open fire risk involved in making these capillaries, the researcher could make use of a traditional micropipette pulling machine, such as those used for electrophysiological experiments (e.g., PC-100, Narishige group). As these machines make use of either gravity or robotic platforms to pull the capillaries, they can be tailored to meet the needs of the infection model. However, the wide range of micropipette pulling machines available will mean that the individual researcher will need to go through some trial and error to determine the appropriate final capillary diameter for use with this protocol.
The presented protocol makes use of bacteria expressing a fluorescent tag to visually identify the IBC. Thus, this technique is limited by the researchers' ability to genetically modify the infectious organism. Specifically for UPEC, IBC-forming strains such as CFT073 and NU14 have been successfully transformed with GFP-expressing plasmids32,33,34; these should therefore be usable in the same protocol. Based on the area of the mouse bladder (70 mm2)35, the length of individual epithelial cells (50-120 µm)17, and the frequency of IBCs in a single bladder16, a conservative estimate for the incidence of IBCs is about 1 in 1,000 cells (or 0.1%). This estimate showcases the utility of our cell isolation protocol to target rare events. The precision of cell selection through our protocol and the wide range of capillary diameters that can be pulled suggest that this protocol can be used to isolate intracellular bacteria from other in vivo and in vitro models of infection. Indeed, we have successfully used this technique to isolate infected cultured bladder epithelial cells (data not shown).
One of the more technically challenging steps in the protocol is inverting the bladder to expose the epithelial cells for scraping. We have found that it is also possible to make an incision on the bladder to splay it out for scraping. However due care should be taken to reduce the damage to the epithelial cells of the bladder during the process of cutting it open; ideally a single cut should be utilized to splay the bladder open. Additionally, the cut should be made in cold PBS, to prevent accidental loss of cells or bladder tissue during the process.
Mouth pipetting of the IBCs in this protocol provides greater control over the cell selection process, as well as limiting the final volume of solution transferred together with the cell. The fine control and large separation of solution from the researchers' mouth also maximizes the safety of the researcher, as the volumes transferred are within the nanoliter to microliter range. In contrast, our experience with the modern micropipette is that it tends to transfer more surrounding liquid and cells with the IBC, potentially leading to contamination with extracellular luminal bacteria. Our finding that mouth pipetting provides a higher performance over other single cell isolation methods has also been reported by other labs22,23,24. Aside from single cell isolation, mouth pipetting has even been used in single-cell electroporation for neurons25, which further demonstrates the utility and the minute control that a trained researcher can attain with the technique. However, safety is of primary importance, and we suggest potential additional measures that can be taken depending on the pathogens being used: (i) the extension of the air buffer between the researcher and the biological material, for example by using an aspirating pipette with a larger volume (e.g., 5 mL), or (ii) adding a physical filter such as cotton wool into the aspirating pipette to act as an additional barrier.
In cases where a risk assessment leads to the conclusion that mouth pipetting is still too risky, commercially available robotic setups (such as those used for nanoinjections) can be combined with the other sections of our technique to provide a safer method for isolating infected cells from mixed populations. It should be noted that our experience with using a robotic micromanipulator has demonstrated a decreased rate of IBC isolation compared to mouth pipetting, as the large intra-experimental variation in bladder epithelial cell size makes it challenging for the user of a robotic arm to determine the force required to pick up single IBCs. Nevertheless, it remains a viable, though costlier, option for those working with highly infectious agents of disease.
The authors have nothing to disclose.
This research was supported by the National Research Foundation, Prime Minister's Office, Singapore, under its NRF Research Fellowship Scheme (NRF award no. NRF-RF2010-10); the Singapore Ministry of Health's National Medical Research Council (NMRC/CIRG/1358/2013); and the Genome Institute of Singapore (GIS)/Agency for Science, Technology, and Research (A*STAR).
1.5ml eppendorf tube | For static bacterial culture and OD measurement | ||
100% ethanol | For Alcohol Burner | ||
15 ml conical tube | For static bacterial culture and OD measurement | ||
1ml Tuberculin Syringe | BD Biosciences | 302100 | |
3% Bacterial Agar | For static bacterial culture and OD measurement | ||
70% ethanol | For static bacterial culture and OD measurement | ||
Aesculap anatomic forceps | Braun/Kruuse | BD222R | For initial dissection of mouse (skin, fascia) |
Alcohol Burner | Wheaton | 237070 | |
Aspirating pipette | BD Biosciences | 357558 | |
Aspirator tube | Sigma-Aldrich | A5177 | |
Bacterial loops | For static bacterial culture and OD measurement | ||
Benchtop centrifuge | Eppendorf | 5424 | Any centrifuge for 1.5ml eppendorf tubes |
Conical flasks | For static bacterial culture and OD measurement | ||
Digital camera for microscope | Olympus | DP71 | For image capture and harvesting of IBCs. Any other fluorescent microscope with a GFP channel will suffice |
Glass Capillaries | Kimax | 6148K07 | |
Iris Scissors STR SS 110MM | Braun | BC110R | |
Isoflurane (Isothesia) | Henry Schein Animal Health | 29405 | |
Kanamycin Sulfate | Calbiochem | 420311 | For static bacterial culture and OD measurement |
LB broth (Miller) | Thermo/Gibco | 10855021 | For static bacterial culture and OD measurement |
Light source unit for microscope | Olympus | LG-PS2 | For image capture and harvesting of IBCs. Any other fluorescent microscope with a GFP channel will suffice |
Lubricant | KY | Any similar commercial medical lubricant will suffice | |
Macro fluorescence microscope | Olympus | MVX10 | For image capture and harvesting of IBCs. Any other fluorescent microscope with a GFP channel will suffice |
Micropipette + micropipette tips | For static bacterial culture and OD measurement | ||
PBS 1x | For static bacterial culture and OD measurement | ||
Pipette controller + Pipettes | For static bacterial culture and OD measurement | ||
Polyethylene Tubing | BD Intramedic | 427401 | |
Precision Glide needle 30G | BD Biosciences | 305107 | Possibly under new catalogue number (305106) |
Splinter forceps curved | Braun | BD312R | |
Spray bottle (for ethanol) | For static bacterial culture and OD measurement | ||
Square cuvettes | Elkay | 127-1010-400 | For static bacterial culture and OD measurement |
Sterilgard III Advance Safety Cabinet | Baker | SG403 | Any biosafety cabinet with a UV irridiator |
Sterilin 90mm Standard Petri Dish | Thermo | 101VR20 | Any sterile petri dish |
Stevens, vascular and tendon scissors, curved, delicate, 110 mm | Braun | OK366R | Recommended for harvesting of bladder |
Surgical Scissors STR S/B 105MM | Braun | BC320R | |
Tabletop Centrifuge | Eppendorf | 5810R | Any refridgerated centrifuge for 15ml conicals |
WPA C08000 cell density meter | Biowave (Biochrom) | 80-3000-45 | For static bacterial culture and OD measurement |