Chemical genetics reveals bacterial and host cell functions critical for type IV effector translocation by Legionella pneumophila - PubMed (original) (raw)

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

Xavier Charpentier et al. PLoS Pathog. 2009 Jul.

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Use of the β-lactamase reporter system to study L. pneumophila effector translocation in macrophages.

A. Detection of translocation of the previously identified Legionella effector LepA. TEM-LepA or TEM-FabI fusions were produced in L. pneumophila and the bacteria were used to infect J774 cells for one hour at 37°C (MOI = 50). Cells were then incubated with the β-lactamase substrate CCF4/AM for two hours at room temperature. Fluorescence images were captured at 460 and 530 nm up 405 nm excitation and merged. B. Measurement of LepA translocation in J774 cells as function of multiplicity of infection. C. Measurement of RalF, LepA and LegAU13 translocation in J774 cells (MOI = 20) as function of TEM fusion protein expression. Bacteria were grown in the presence of varying amount of IPTG to obtain a range of increased TEM fusion protein expression.

Figure 2

Figure 2. Effect of the three libraries compounds on translocation of the LepA effector.

Mean percent inhibition (of two replicates) of translocation for each compound is plotted (blue circles, Biomol compounds; Orange circles, NINDS compounds; Pink circles, Prestwick compounds), along with untreated negative control wells (green circles) and positive control wells lacking L. pneumophila cells (red circles). The horizontal bar indicates 50% inhibition of signal and separates active compounds from inactive compounds and negative controls.

Figure 3

Figure 3

A. Categories or targets of inhibitors identified in initial chemical genetic screen. Number of inhibitors in each class is indicated. B. Dose-response curve of a selected inhibitors on LepA effector translocation. Data from three independent experiments (colored circles) were fitted (colored line) using the four parameter logistic model to determine the IC50.

Figure 4

Figure 4. The protonophore CCCP inhibits Icm/Dot-dependent activities.

A. Effect of translation inhibitors (kanamycin, erythromycin) and protonophore (CCCP) on translocation of the LepA effector in J774 cells. B. Icm/Dot-dependent red blood cell (RBC) lysis by L. pneumophila and effect of translation inhibitors (kanamycin, erythromycin) and respiratory chain poison sodium azide. C. Time course effect of the CCCP protonophore or the DMSO carrier on Icm/Dot-dependent RBC lysis by L. pneumophila. D. Effect of the CCCP protonophore on the bacterial ATP levels as a function of exposure time. In all panels error bars represent standard deviation from three independent experiments.

Figure 5

Figure 5. Identified translocation inhibitors inhibit RBC lysis or phagocytosis.

A. Effect of the identified translocation inhibitors on Icm/Dot-dependent RBC lysis by L. pneumophila. B. Effect of the identified translocation inhibitors on uptake of heat-killed E. coli by J774 cells. In both panels the data are reported as the percentage of untreated sample. Error bars represent standard deviation from three independent experiments.

Figure 6

Figure 6. Antibody opsonization of L. pneumophila restores effector translocation in the absence of phagocytosis.

Effect of antibody-mediated opsonization of L. pneumophila on uptake of L. pneumophila in J774 macrophages (A and B) and on effector translocation (C). A. Fluorescence microscopy of GFP-expressing L. pneumophila (JR32) infecting J774 cells in the absence or presence of anti-L. pneumophila antibodies. Extracellular bacteria were labeled with a rhodamine conjugated antibody. B. Uptake of fluorescein-labeled JR32 by J774 cells in the absence or presence of anti-L. pneumophila antibodies. Fluorescence signal of extracellular bacteria was quenched with trypan blue. Uptake is reported as the percentage of fluorescence signal that can not be quenched by trypan blue (i.e. the % of intracellular fluorescence signal). C. Effect of phagocytosis inhibitors or the DMSO carrier on L. pneumophila uptake by J774 cells in the absence or presence of anti-L. pneumophila antibodies. Uptake is reported as the percentage of untreated sample. D and E. Effect of phagocytosis inhibitors or the DMSO carrier on TEM-LepA (D) and TEM-RalF (D) effector translocation in J774 cells in the absence or presence of anti-L. pneumophila antibodies. Translocation is reported as the percentage of untreated sample. In all panels error bars represent standard deviation from three independent experiments.

Figure 7

Figure 7. CD45/CD148-deficient bone marrow-derived macrophages are defective for L. pneumophila phagocytosis.

A. Binding of L. pneumophila to WT and CD45/CD148-deficient bone-marrow derived macrophages (BMM). Errors bars represent standard deviation from six replicates of two independent experiments. B. Phagocytosis of E. coli and L. pneumophila by WT and CD45/CD148-deficient BMM. Error bars represent standard deviation from three experiments. C. Translocation of the TEM-LepA and TEM-RalF effectors in WT and CD45/CD148-deficient BMM (MOI = 10). Errors bars represent standard deviation from six replicates of two experiments.

Figure 8

Figure 8. 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.

Figure 9

Figure 9. Real time analysis of effector translocation by L. pneumophila.

A. Real time detection of CCF4 hydrolysis mediated by TEM-LepA translocation in J774 cells from L. pneumophila at various multiplicity of infection (MOI). B. Effect of MOI on the apparent maximal rate of product accumulation mediated by translocated TEM-LepA effector. The blue dotted line represents the ideal correlation between MOI and the apparent maximal rate of product accumulation (correlation coefficient of 1). C. Effect of antibody opsonization of the translocation efficiency of TEM-RalF, TEM-LepA and TEM-LegA3. D. Rate of product accumulation mediated by translocated TEM-RalF, TEM-LepA or TEM-LegA3 effectors as function of time in the presence or absence of antibodies. Legend of panel C applies also to panel D. Rates of product accumulation are expressed as arbitrary unit per unit of time (min.). The presented data are from a representative experiment.

Figure 10

Figure 10. L. pneumophila with macrophages under complement-opsonized or non-opsonized conditions (A) and antibody-opsonized conditions (B).

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