A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.
Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.
Nontyphoidal Salmonella are important causes of enteric diseases in all vertebrates. Salmonellosis in humans is among the top bacterial food-borne diseases1. Characterization of the molecular mechanisms that underpin the interactions of Salmonella with their animal hosts is mainly achieved through the study of Salmonella enterica serotype Typhimurium (STM) in tissue culture and animal models of infection. Gaining insights in STM-host interactions will help us understand how Salmonella survive and grow inside host cells. The first challenge in studying these interactions is to identify as many participating factors as possible from both host and pathogen, but these endeavors are largely obstructed by the significant difficulties of dealing with two independent complex biological systems simultaneously, i.e., host and Salmonella, under physiological conditions. Additionally, the large repertoire of Salmonella and host genes potentially encoding factors involved in host interactions require high-throughput biological platform to tackle this challenge.
A modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity was developed to examine a large set of genes likely engaging in Salmonella-host interactions. The Gentamicin protection assay was developed in 19732, but was first thoroughly described by Elsinghorst in 19943,4. It has now become a standard tool for studying many intracellular bacterial pathogens ex vivo, including Salmonella5,6. Internalized bacteria avoid being killed by some antibiotics, like Gentamicin, that cannot penetrate eukaryotic cells3. By taking advantage of this phenomenon, the Gentamicin protection assay measures the survival and growth of intracellular bacterial pathogens. Three events during the infection, i.e., association with eukaryotic cells, invasion and replication, can be evaluated for intracellular bacterial pathogens based on the time interval between infection, Gentamicin treatment, and further incubation (Figure 1). Eukaryotic cell lines provide a physiological environment that is less complex than relevant animal models for host-pathogen interaction studies.
The Gentamicin protection assay is an appropriate platform to study STM-host interactions, but the standard assay in a 24-well culture dish has low-throughput capacity. Computational analysis of in vivo datasets identified 149 Salmonella gene products that are predicted to interact with approximately 300 host gene products (unpublished data). The standard Gentamicin protection assay does not have the capacity to phenotype this number of mutants efficiently.
In addition, the Gentamicin protection assay can theoretically detect the invasion of even a single bacterium. Because of this inherent sensitivity, the raw data are susceptible to technical variances when repeated at different times. The internal controls and relative data presentation after normalization are essential for meaningful interpretation of the results. Given these considerations, a modified, standardized Gentamicin protection assay was developed to enhance testing capacity and increase precision.
The following protocol is detailed and illustrated to perform the modified Gentamicin protection assay using 96-well culture dishes and the murine macrophage RAW264.7 cell line. Compared to the standard protocol in 24-well culture dishes, the modified protocol has the following advantages: 1) Using 96-well culture dishes allows up to 10 different mutant strains to be phenotyped including internal positive and negative controls with sufficient statistical power; 2) The variance of results is significantly reduced, because the mutant strains are tested in the same culture dish and at the same time; 3) The use of multichannel pipettes increases throughput while reducing operator fatigue. Lastly, comparing to 24-well culture dishes, concerns of less host cells per well in 96-well culture dish were addressed through protocol optimization and standardization.
In summary, the modified, standardized assay to in vitro phenotype Salmonella or other bacterial association, invasion and replication in phagocytic cells increases precision and achieves high-throughput capacity while reducing operator fatigue.
1. Murine Macrophage RAW264.7 Cell Culture
2. Preparation of Salmonella Wild-type and Mutants
3. Invasion Assay in 96-well Culture Plate
4. Data Analysis
See representative results (Figure 2) after the data are plotted based on the modified phagocytic cell invasion assay. The data include five different strains, WT, ΔinvA, ΔphoP, mutant A, and mutant B. ΔinvA, known to be defective for invasion, and ΔphoP known to be defective for replication8, are used as positive controls to assess the experimental validity. Indeed, in the modified invasion assay, a ΔinvA mutant is internalized poorly by RAW264.7 cells and a ΔphoP mutant had reduced replication after 24 hr. Mutants A and B are representative strains assayed using this protocol. Mutant A shows significant reduction of replication (Figure 2), similar to a ΔphoP mutant; interestingly, mutant B shows a significant increase in replication (Figure 2). The data clearly indicate both of those mutants have altered phenotypes for invasion and survival in macrophages.
In summary, numerous Salmonella deletion mutants were individually phenotyped for involvement in the bacterial association, invasion, and replication in RAW264.7 macrophages using the modified invasion assay. Twenty of 38 tested mutants so far displayed variable but significant defects in bacterial infection of macrophages (Table 1), thus the modified assay to in vitro phenotype Salmonella or other bacterial association, invasion and replication in phagocytic cells increases precision and high-throughput capacity while reducing operator fatigue.
Figure 1. Working scheme of high-throughput assay. Gentamicin protection assay is modified, standardized to phenotype numerous Salmonella mutants simultaneously in 96-well plates. The key steps are performed as above to examine association, invasion and replication of Salmonella in RAW264.7 macrophage cells.
Figure 2. Representative results. The high-throughput assays are performed for Salmonella strains — WT, ΔinvA, ΔphoP, mutant A, and mutant B — in RAW264.7 macrophage cells. For each strain, the geometric means of CFU per macrophage are obtained from three independent experiments for cell association, invasion and replication, respectively, which are further normalized to the WT and graphic representation is shown. Error bars denote standard error, and statistical significance of each strain referenced to WT was determined by Student’s t-test. *p<0.05. MOI=20.
Table 1. Phenotypes of candidate genes. Candidate Salmonella mutants are phenotyped by the modified, standardized assay for association, invasion and replication in RAW264.7 macrophage cells. 20 mutants have significant defects in association, invasion and/or replication.
The Gentamicin protection assay is widely used to study the invasion and replication of intracellular bacterial pathogens inside host cell, and it is especially an important biological tool for studying pathogens, like Salmonella, whose invasion is the prerequisite step for establishing infection1. The standard Gentamicin protection assay in Salmonella research community is implemented in 24-well culture dish5. Though the use of 48 or even 96-well plates were discussed before for high-throughput capacity9, no actual protocol has been demonstrated in detail and successfully employed to test a collection of bacterial mutants. Here, a modified and standardized protocol was developed to use 96-well culture dishes to phenotype Salmonella mutants. Notably, this protocol was only tested for Salmonella and phagocytic cells, thus modifications and optimization would obviously be necessary with other bacteria or host cells.
There are several advantages to the modified Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells. First, the 96-well culture dish format allows high-throughput testing and analysis, up to 10 different strains can be tested simultaneously with sufficient repetition statistical analysis and internal controls. Second, because of the high sensitivity of the assay, the raw data of the Gentamicin protection assay may be susceptible to technical variances when repeated at different times. Under this modified protocol, several strains are phenotyped under the same conditions throughout the process, reducing variance. Lastly, the use of multichannel pipettes increases efficiency and reduces operator fatigue. The new protocol facilitated the rapid phenotyping numerous Salmonella mutants while generating large amounts of quantitative, reproducible data.
One concern in doing invasion assays in 96-well culture dishes is that the wells contain fewer eukaryotic cells, and because of this quantitation could be compromised. To address this concern, a large series of assays were performed to optimize reproducibility. First, the RAW264.7 macrophages used in this protocol had fewer than 20 passages, and the cells were seeded into 96-well culture dish overnight before the assay. Second, the MOI was reduced to 20:1 from the regularly used 50:1 or 100:1. A MOI 20:1 ensures that 99% of macrophages will be exposed to infection by 10 to 30 bacteria calculated by Poisson distribution according to the stochastic point. It is thought that, not restricted to biological factors, cellular damage could be caused from overwhelming bacteria physical contacts10. Additionally, Salmonella invasion could induce apoptosis of macrophages, which is positively associated with high MOI11. Using a MOI of 20:1, cellular damage was found to be minimal, presumably due to limited bacteria-macrophage physical contact. Third, the incubation time interval was optimized for the three time points: association for 30 min with no Gentamicin; invasion for additional 1 hr (total 1.5 hr post-infection) with 100 μg/ml Gentamicin; replication for additional 22.5 hr (total 24 hr after infection) with 10 μg/ml Gentamicin. In our test, 5 μg/ml Gentamicin is sufficient to kill all the Salmonella in cell-free DMEM with 10% fetal bovine serum (unpublished data), thus, 100 μg/ml Gentamicin would be expected to ensure the rapid killing and 10 g/ml Gentamicin would maintain the clearance of extracellular bacteria in the cell culture medium for overnight incubation. In vivo, it takes about 15 min to detect tissue associated Salmonella with calf intestine epithelial cells12,13; in vitro, 30 min incubation is reported to be adequate for the wild-type to invade14. For the standard invasion assay, the work load of the first two time points is quite intense due to washing, serial dilution and plating. The precise time interval for each time point was achieved by utilizing the multichannel pipettes, because the device significantly increases the operator’s efficiency and precision. Furthermore, the sample plating technique was altered after serial dilution, instead of plating 100 μl and manually spreading, 5 separate drops of 10 μl were plated for each repeat well on plates which greatly reduces the plating time and increases the accuracy, because the final count per well is scored from 5 plated samples. Collectively, these optimization and standardization procedures increase the reproducibility of the assay, different operators can repeatedly obtain consistent results at different times.
This protocol employs phagocytic cells instead of epithelial cells for this assay in a smaller 96-well format. In studying the pathogenesis of salmonellosis, the most widely used epithelial cell line is Caco-2, derived from heterogeneous human epithelial colorectal adenocarcinoma. Using the regular 24-well culture dish invasion test with Caco-2 cells, the actual number of recovered bacteria at each time point, particularly the 22.5 hr time point for phenotyping replication, is much lower than when phagocytic cells, such as J774 or RAW264.7 macrophages, are used (unpublished data). Thus, this makes it more difficult to perform phenotyping using a 96-well culture dish when each well has fewer host cells. It is known that macrophages cells could actively engulf bacteria, which could lead to more bacteria being internalized, and it also cannot be ruled out that Salmonella apparently replicate more slowly in Caco-2 cells. The ability of Salmonella to survive and replicate inside macrophage cells is related to key roles in the pathogenesis of salmonellosis, i.e., triggering massive host inflammation responses and facilitating systemic infection1. We mainly employed the modified and standardized Gentamicin protection assay to phenotype a large set of computation pipeline selected genes for their potential engagements in host interactions. It turned out nearly 50% of the Salmonella mutants were phenotyped as genes involved in intracellular replication in phagocytic cells.
The authors have nothing to disclose.
This project was supported partly by a grant for National Institutes of Health NIAID (for A.J.B. and L.G.A., R01 AI076246). The Salmonella mutant collection was partly supported by National Institutes of Health grants (for M.M., U01 A152237-05, R01 AI07397-01, R01 AI039557-11 and R01 AI075093-01), partly by National Institutes of Health grants (for H.A.P, R21 AI083964-01, 1R0 1AI083646-01, 1R56AI077645, R01 AI075093). We thank Steffen Prowollik for replica plating and confirming the mutants in the collection.
Dulbecco's Modified Eagle Medium (DMEM) | Life Technologies | 11965 | |
Fetal bovine serum | HyClone | SH30910.03 | |
T-75 cell culture flask vented filter cap | Nest Biotechnology | 708003 | |
100X Non-Essential Amino Acids | Life Technologies | 11140 | |
Cell scraper | BD Falcon | 353086 | |
96-well cell culture plate | Corning Incorporated | 3595 | |
Luria-Bertani (LB) broth | MP Biomedicals | 3002-075 | |
14 mL Polypropylene Round-Bottom Tube | BD Falcon | 352059 | |
PBS pH7.4 (1x) | Life Technologies | 10010 | |
Triton X-100 | Sigma | T-8787 | |
Kanamycin solution | Sigma | K0254 | |
Gentamicin solution | Sigma | G1272 | |
0.25% Trypsin-EDTA | Life Technologies | 25200 | |
Trypan blue | Sigma | T8154 |