A Genome-Wide siRNA Screen Implicates Spire1/2 in SipA-Driven Salmonella Typhimurium Host Cell Invasion

Abstract

Salmonella Typhimurium (S. Tm) is a leading cause of diarrhea. The disease is triggered by pathogen invasion into the gut epithelium. Invasion is attributed to the SPI-1 type 3 secretion system (T1). T1 injects effector proteins into epithelial cells and thereby elicits rearrangements of the host cellular actin cytoskeleton and pathogen invasion. The T1 effector proteins SopE, SopB, SopE2 and SipA are contributing to this. However, the host cell factors contributing to invasion are still not completely understood. To address this question comprehensively, we used Hela tissue culture cells, a genome-wide siRNA library, a modified gentamicin protection assay and S. TmSipA, a sopBsopE2sopE mutant which strongly relies on the T1 effector protein SipA to invade host cells. We found that S. TmSipA invasion does not elicit membrane ruffles, nor promote the entry of non-invasive bacteria "in trans". However, SipA-mediated infection involved the SPIRE family of actin nucleators, besides well-established host cell factors (WRC, ARP2/3, RhoGTPases, COPI). Stage-specific follow-up assays and knockout fibroblasts indicated that SPIRE1 and SPIRE2 are involved in different steps of the S. Tm infection process. Whereas SPIRE1 interferes with bacterial binding, SPIRE2 influences intracellular replication of S. Tm. Hence, these two proteins might fulfill non-redundant functions in the pathogen-host interaction. The lack of co-localization hints to a short, direct interaction between S. Tm and SPIRE proteins or to an indirect effect.

Fig 1

(A and B) Scanning electron micrographs of wild type (A) and S. Tm^SipA strains (B) infecting HeLa cells. (C) Representation of Salmonella Typhimurium infection steps (adapted from [31]). (D) Representative image of the genome-wide RNAi screen, (E) spot- and nuclei-detection of the infectX pipeline (right) at MOI150. (F) HeLa Kyoto cells were infected at increasing MOI using different SPI-1 effector mutant strains (Table 1) and the infection efficiency infection was monitored using the modified gentamicin protection assay. (G) Helper assay using different T1 effector mutants in HeLa cells. (H) Representation of helper activity of different T1 effector mutants.

Fig 2

(A) Quality control 1 of the genome wide screen. Cell numbers of chosen controls throughout the whole genome wide screen. (B) Quality control 2 of the genome wide screen. Infection index of chosen controls throughout the whole genome wide screen. Red bars = median of all data points. (C-G) Results of the genome-wide screen are functionally grouped. Color of data points indicates the type of experiment and type of RNAi used. QU = Qiagen unpooled siRNA in HeLa Kyoto cells (blue; original screen). Additional data for knockdown of the presented genes are indicated in green or black: AU = Ambion unpooled siRNA in HeLa CCL-2 cells (green). SE = Sigma esiRNA in HeLa CCL-2 cells (black). Bars represent median of all data points. Asterisks after gene names indicate hit for genome wide QU screen. Panels show proteins indicated in ARP2/3 complex and regulation (C), WAVE, WASH, WASP and interacting proteins (D), RhoGTPases (E), additional actin interacting proteins and regulators (F) and formins (G). (H) KEGG pathway mapping of decreasing hits from the Qiagen unpooled S. Tm^SipA siRNA screen. Hits reducing the fraction of infected cells below -0.5 median z-score infection index were mapped on all KEGG pathways. KEGG pathway for regulation of actin cytoskeleton is shown. Color code: Strength of the observed phenotype (median z-score). White fields: no hits or no data available from the genome-wide screen. Grey fields were not included in the library or had to be excluded due to low cell number. Adapted from Kreibich et al., 2015.

Fig 3

(A) SPIRE1 and SPIRE2 expression as measured by RT-qPCR upon RNAi treatment. Columns represent mean and error bars show standard deviation. (B) Small scale validation screen using S. Tm^SipA and the modified gentamicin protection assay to confirm phenotype of SPIRE2. (C) Modified gentamicin protection assay using Spire1^gt/gt and Spire2^-/- iMEFs infected with S. Tm^SipA normalized to the wild type iMEF control cell line. A shows data from 2 independent experiments with 2 replicates each in HeLa Kyoto cells. B and C show data from 3 independent experiments with 3 replicates each. Asteriscs indicate significant differences. *: p<0.05.

Fig 4. Infection step assays using iMEF cell lines.

(A) Binding assay using Spire1^gt/gt and Spire2^-/- iMEFs normalized to the data from the wild type iMEF control cell line. Spire1^gt/gt show reduced Salmonella binding. (B) Effector injection assay using Spire1^gt/gt and Spire2^-/- iMEFs normalized to the wild type iMEF control cell line. Spire1^gt/gt and Spire2^-/- cells show attenuation and increase in effector injection, respectively. (C) Modified gentamicin protection assay using Spire1^gt/gt and Spire2^-/- iMEFs normalized to the wild type iMEF control cell line. Spire1^gt/gt and Spire2^-/- cell lines show decrease in invasion after 4h. Invasion is not dependent on Salmonella effectors or invasion type. (D) Intracellular replication in Spire1^gt/gt and Spire2^-/- iMEFs normalized to the wild type iMEF control cell line measure by plating assay. Spire2^-/- cell line shows decrease of intracellular bacterial replication. (E) Absolute number of CFUs corresponding to D. A-C show data from 3 independent experiments with 2 replicates each in HeLa Kyoto cells. D and E show data from 2 independent experiments with 3 replicates each. Asteriscs indicate significant differences. *: p<0.05.

Fig 5. SPIRE1 and SPIRE2 do not co-localize with Salmonella Typhimurium after invasion.

(A and B) SPIRE2 overexpressing HeLa cells infected with Salmonella Typhimurium. SPIRE2 does not co-localize with bacteria as shown in merge. (C and D) Quantification of immunofluorescence images as shown in A and B. Same phenotype was observed for SPIRE1 in two different cell lines and two different Salmonella Typhimurium strains. No co-localization observed after 15min (data not shown).