Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation

Abstract

Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.

Figure 1. Yeast Growth Inhibition Screen

The entire yeast haploid deletion strain collection was transformed four separate times, twice with a plasmid that conditionally expresses GFP and twice with a plasmid that conditionally expresses GFP-OspF. Transformants were spotted in quadruplicate onto solid media trays such that up to four biological replicas were examined in each screen. Each screen was conducted in duplicate. Images shown reflect growth after 48 h.

Figure 2. Ontologies Enriched among the 83 Genes That Are Essential in Yeast Expressing OspF

A black cell indicates membership of its column's gene in its row's ontology. Two independent types of hierarchy are represented in the matrix. Columns and rows have been hierarchically clustered and the dendrogram on the left shows the results of row clustering (column dendrogram not shown). Gene ontologies are hierarchical classifications, and the color bands indicate hierarchically related groups of process ontologies (in bold type). Ontologies in italics are molecular functions; the remaining five are components.

Figure 3. OspF Inhibits the Yeast CWI Pathway by Inhibition of MAPK Phosphorylation

(A) Summary of the activity of an RLM1-regulated β-galactosidase reporter in response to heat shock in the presence or absence of OspF. (B) Outline of the CWI pathway. (C) Yeast containing empty vector or a plasmid that conditionally expresses OspF were subjected to the designated stress 2 h after the induction of expression of OspF (see Materials and Methods). Representative immunoblots used to assay for activation of each of four MAPK signaling pathways: SLT2 (CWI pathway), FUS3 (mating pathway), KSS1 (invasive growth pathway), and HOG1 (high-osmolarity glycerol pathway) are shown. The circled P denotes phosphorylated versions of the proteins. The blots were probed with the anti-PSTAIRE antibody that recognizes CDC28 and PHO85 as a loading control. Each experiment was conducted at least in triplicate with similar results.

Figure 4. OspF Inhibits ERK and p38 Phosphorylation

(A) Immunoblots of extracts of HeLa cells infected with wild-type, ΔospF, or ΔospF/pOspF Shigella for 1 h. (B) pOspF indicates the OspF complementing plasmid. Cell lysates were probed with the designated antibodies. Immunoblots of extracts of uninfected HeLa cells or HeLa infected with wild-type or ΔospF Shigella for 1 h and then exposed to 50 ng/ml EGF or 0.4 M sorbitol to activate ERK and p38 signaling, respectively. (C) Immunoblots of extracts of HeLa cells infected with wild-type Shigella (MOI 10:1), ΔospF Shigella (MOI 10:1), or mix of wild-type Shigella (MOI 5:1) plus ΔospF Shigella (MOI 5:1) for 1 h. In all cases, each experiment was conducted at least in triplicate with similar results.

Figure 5. OspF Stimulates MAPKK Activation and Inhibits MAPK Activation

Immunoblots of extracts of HeLa cells infected with wild-type, ΔospF, or ΔospF/pOspF Shigella or exposed to anisomycin or EGF, to activate p38 and ERK signaling, respectively. The latter two reagents were added as positive controls for detection of activation of phosphorylated MAPK and MAPKK. Cell lysates were probed with the designated antibodies. Each experiment was conducted at least in triplicate with similar results.

Figure 6. OspF Is Associated with Attenuation of the Host Innate Immune Response

The images shown are of hematoxylin- and eosin-stained sections of lungs of mice 24 h after infection with wild-type or ΔospF Shigella or injection with equivalent volume of phosphate buffered saline.