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Chemical genetics reveals bacterial and host cell functions critical for type IV effector translocation by Legionella pneumophila.

Charpentier X, Gabay JE, Reyes M, Zhu JW, Weiss A, Shuman HA - PLoS Pathog. (2009)

Bottom Line: We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin.Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148.We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Columbia University Medical Center, New York, NY, USA.

ABSTRACT
Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host-pathogen interactions.

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Effector translocation in the presence of antibody does not require immunoreceptor signaling.A. Antibody-stimulated translocation of the LepA and RalF effector in CHO cells expressing the wild-type or signaling-deficient (Y2F/Y3F) FcγRIIA. B. Effect of phagocytosis inhibitors or the DMSO carrier on antibody-stimulated LepA and RalF translocation in CHO cells expressing the wild-type or signaling-deficient (Y2F/Y3F) FcγRIIA. Error bars represent standard deviation from three independent experiments.
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ppat-1000501-g008: Effector translocation in the presence of antibody does not require immunoreceptor signaling.A. Antibody-stimulated translocation of the LepA and RalF effector in CHO cells expressing the wild-type or signaling-deficient (Y2F/Y3F) FcγRIIA. B. Effect of phagocytosis inhibitors or the DMSO carrier on antibody-stimulated LepA and RalF translocation in CHO cells expressing the wild-type or signaling-deficient (Y2F/Y3F) FcγRIIA. Error bars represent standard deviation from three independent experiments.

Mentions: The fact that phagocytosis is dispensable for translocation when bacteria contact macrophages through the antibody-FcR interaction suggests that either the tight binding of the bacteria to the cells or signaling events due to clustering of the high affinity Fc receptors, are promoting effector translocation. Binding of antibody-coated particles to FcR induces clustering of the receptors and initiates a signal transduction cascade leading to phagocytosis [51],[52]. Clustering is then followed by phosphorylation of two specific tyrosines on the FcR ITAM domain (Immunoreceptor Tyrosine-based Activation Motifs) by enzymes of the Src tyrosine-kinase family [53]. The phosphorylated ITAMs then recruit Syk kinase which is required for efficient phosphorylation of phosphatidylinositol 3-kinase and is critical for signal transduction and phagocytosis mediated by FcR [54]. As initial evidence that FcR signaling is not required for antibody-stimulated effector translocation, we found that the Syk kinase inhibitor R406 [55] does not inhibit LepA translocation in J774 cells (data not shown). To further determine if binding of antibody-opsonized bacteria to the FcR is sufficient to trigger effector translocation, we used CHO cells expressing the human FcγRIIA receptor or a signaling deficient mutant of FcγRIIA, Y2F/Y3F in which the two tyrosines of the ITAM domain have been substituted by phenylalanine [56]. Opsonization of Legionella with antibody strongly stimulated translocation of LepA and RalF in cells expressing the wild-type or signaling deficient FcγRIIA receptors (Figure 8A). Whereas the wild-type FcγRIIA is fully functional and capable of promoting phagocytosis, the Y2F/Y3F mutant can bind antibody but does not support phagocytosis [56]. Consistently, translocation of LepA and RalF in CHO cells expressing either the wild type or the signaling-defective receptors is insensitive to phagocytosis inhibitors (Figure 8B). We conclude that the intimate binding of antibody-opsonized L. pneumophila to FcR on host cells can substitute for naturally occurring phagocytosis to trigger effector translocation.


Chemical genetics reveals bacterial and host cell functions critical for type IV effector translocation by Legionella pneumophila.

Charpentier X, Gabay JE, Reyes M, Zhu JW, Weiss A, Shuman HA - PLoS Pathog. (2009)

Effector translocation in the presence of antibody does not require immunoreceptor signaling.A. Antibody-stimulated translocation of the LepA and RalF effector in CHO cells expressing the wild-type or signaling-deficient (Y2F/Y3F) FcγRIIA. B. Effect of phagocytosis inhibitors or the DMSO carrier on antibody-stimulated LepA and RalF translocation in CHO cells expressing the wild-type or signaling-deficient (Y2F/Y3F) FcγRIIA. Error bars represent standard deviation from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2698123&req=5

ppat-1000501-g008: Effector translocation in the presence of antibody does not require immunoreceptor signaling.A. Antibody-stimulated translocation of the LepA and RalF effector in CHO cells expressing the wild-type or signaling-deficient (Y2F/Y3F) FcγRIIA. B. Effect of phagocytosis inhibitors or the DMSO carrier on antibody-stimulated LepA and RalF translocation in CHO cells expressing the wild-type or signaling-deficient (Y2F/Y3F) FcγRIIA. Error bars represent standard deviation from three independent experiments.
Mentions: The fact that phagocytosis is dispensable for translocation when bacteria contact macrophages through the antibody-FcR interaction suggests that either the tight binding of the bacteria to the cells or signaling events due to clustering of the high affinity Fc receptors, are promoting effector translocation. Binding of antibody-coated particles to FcR induces clustering of the receptors and initiates a signal transduction cascade leading to phagocytosis [51],[52]. Clustering is then followed by phosphorylation of two specific tyrosines on the FcR ITAM domain (Immunoreceptor Tyrosine-based Activation Motifs) by enzymes of the Src tyrosine-kinase family [53]. The phosphorylated ITAMs then recruit Syk kinase which is required for efficient phosphorylation of phosphatidylinositol 3-kinase and is critical for signal transduction and phagocytosis mediated by FcR [54]. As initial evidence that FcR signaling is not required for antibody-stimulated effector translocation, we found that the Syk kinase inhibitor R406 [55] does not inhibit LepA translocation in J774 cells (data not shown). To further determine if binding of antibody-opsonized bacteria to the FcR is sufficient to trigger effector translocation, we used CHO cells expressing the human FcγRIIA receptor or a signaling deficient mutant of FcγRIIA, Y2F/Y3F in which the two tyrosines of the ITAM domain have been substituted by phenylalanine [56]. Opsonization of Legionella with antibody strongly stimulated translocation of LepA and RalF in cells expressing the wild-type or signaling deficient FcγRIIA receptors (Figure 8A). Whereas the wild-type FcγRIIA is fully functional and capable of promoting phagocytosis, the Y2F/Y3F mutant can bind antibody but does not support phagocytosis [56]. Consistently, translocation of LepA and RalF in CHO cells expressing either the wild type or the signaling-defective receptors is insensitive to phagocytosis inhibitors (Figure 8B). We conclude that the intimate binding of antibody-opsonized L. pneumophila to FcR on host cells can substitute for naturally occurring phagocytosis to trigger effector translocation.

Bottom Line: We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin.Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148.We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Columbia University Medical Center, New York, NY, USA.

ABSTRACT
Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host-pathogen interactions.

Show MeSH
Related in: MedlinePlus