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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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Related in: MedlinePlus

L. pneumophila with macrophages under complement-opsonized or non-opsonized conditions (A) and antibody-opsonized conditions (B).
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Related In: Results  -  Collection


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ppat-1000501-g010: L. pneumophila with macrophages under complement-opsonized or non-opsonized conditions (A) and antibody-opsonized conditions (B).

Mentions: The strong requirement of phagocytosis for L. pneumophila effector translocation is remarkable. Other bacterial pathogens inject bacterial effectors without the requirement of phagocytosis and some pathogens like Yersinia enterocolitica and enteropathogenic E. coli even utilize type III-dependent effector translocation to inhibit phagocytosis [63]–[65]. We found that when macrophages are given non-opsonized Legionella, addition of actin depolymerizing agents that block phagocytosis severely decreased effector translocation. However, when the same cells were given antibody opsonized-Legionella, the actin depolymerizing agents did not inhibit translocation. We can imagine two different explanations for these results. One explanation is that in the presence of antibodies, binding of the bacteria to the macrophages is so efficient that, as seen by Kirby et al. [37] even in the presence of actin depolymerizing agents, there is a low level of phagocytosis that corresponds to the level of uptake observed in the absence of antibody and inhibitor. An alternative explanation is that the increased level of Legionella binding to the macrophages in the presence of antibody permits translocation even when the bacteria remain outside the cell in the presence of the inhibitors. We favor the second explanation because we found that opsonized Legionella were able to translocate effectors to CHO cells expressing mutant forms of the FcγRIIA receptor that are defective for signaling and cannot promote phagocytosis. Thus, the signaling cascade induced as a result of antibody-FcR binding is not required to trigger Icm/Dot-mediated effector translocation. Under these conditions, it is highly unlikely that the bacteria are being internalized. This supports the idea that artificially-induced intimate contact can trigger translocation in absence of phagocytosis and that phagocytosis is not required per se for translocation. These results provide a resolution for the apparent discrepancy between published work from our lab done with professional phagocytes in the absence of opsonization [25] and work from the Roy lab using CHO(FcγRIIΑ) cells and opsonized Legionella [66]. Real-time analysis of effector translocation showed that phagocytosis-dependent translocation is much slower than the phagocytosis-independent translocation triggered by antibody-FcR intimate binding. Presumably phagocytosis-dependent translocation is slower because the kinetics of phagocytosis adds time to the kinetics of effector translocation. The absence of evidence for protrusion of Icm/Dot system suggest that intimate binding may be needed to overcome the physical barrier established by the extracellular structures present on the surface of the bacterial or cellular membranes. Pulmonary macrophage infection by L. pneumophila generally occurs in the absence of pre-immune antibodies. Under in vivo conditions it is then likely that L. pneumophila relies on host cell signaling to stimulate uptake and achieve intimate contact required for effector translocation. Our data support a model in which L. pneumophila relies on the phagocytosis process to generate the intimate contact required for the translocation of pre-synthesized effector molecules (Figure 10). The phagocytosis requirement for effector translocation by L. pneumophila has consequences for its environmental lifestyle. Indeed, it is believed that amoebae are natural hosts for L. pneumophila providing a critical environment for its replication and survival [67]. L. pneumophila may only translocate its effectors when residing in a phagocytic membrane-bound compartment or once phagocytosis has been initiated. The translocated effectors can thereafter alter this compartment so as to create a vacuolar environment permissive for L. pneumophila replication. In addition, effector translocation will only be triggered by a potential host cell energetically competent to perform phagocytosis. Therefore relying phagocytosis to trigger translocation ensures that it is occurring only under conditions that could result in a successful infection, and thus, avoid delivery of effector to a host cell that cannot support replication. Interestingly, translocation of a Vibrio cholerae type VI secretion effector also requires internalization by host cells [68]. This is also somehow reminiscent of activation of the SPI-2 type III secretion system of Salmonella following internalization by macrophages [69]. Regardless of the secretion system used for translocating effector proteins, bacterial pathogens that interact with phagocytes may have evolved strategies to ensure that translocation occurs only in the phagosomal/endosomal microenvironment.


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)

L. pneumophila with macrophages under complement-opsonized or non-opsonized conditions (A) and antibody-opsonized conditions (B).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000501-g010: L. pneumophila with macrophages under complement-opsonized or non-opsonized conditions (A) and antibody-opsonized conditions (B).
Mentions: The strong requirement of phagocytosis for L. pneumophila effector translocation is remarkable. Other bacterial pathogens inject bacterial effectors without the requirement of phagocytosis and some pathogens like Yersinia enterocolitica and enteropathogenic E. coli even utilize type III-dependent effector translocation to inhibit phagocytosis [63]–[65]. We found that when macrophages are given non-opsonized Legionella, addition of actin depolymerizing agents that block phagocytosis severely decreased effector translocation. However, when the same cells were given antibody opsonized-Legionella, the actin depolymerizing agents did not inhibit translocation. We can imagine two different explanations for these results. One explanation is that in the presence of antibodies, binding of the bacteria to the macrophages is so efficient that, as seen by Kirby et al. [37] even in the presence of actin depolymerizing agents, there is a low level of phagocytosis that corresponds to the level of uptake observed in the absence of antibody and inhibitor. An alternative explanation is that the increased level of Legionella binding to the macrophages in the presence of antibody permits translocation even when the bacteria remain outside the cell in the presence of the inhibitors. We favor the second explanation because we found that opsonized Legionella were able to translocate effectors to CHO cells expressing mutant forms of the FcγRIIA receptor that are defective for signaling and cannot promote phagocytosis. Thus, the signaling cascade induced as a result of antibody-FcR binding is not required to trigger Icm/Dot-mediated effector translocation. Under these conditions, it is highly unlikely that the bacteria are being internalized. This supports the idea that artificially-induced intimate contact can trigger translocation in absence of phagocytosis and that phagocytosis is not required per se for translocation. These results provide a resolution for the apparent discrepancy between published work from our lab done with professional phagocytes in the absence of opsonization [25] and work from the Roy lab using CHO(FcγRIIΑ) cells and opsonized Legionella [66]. Real-time analysis of effector translocation showed that phagocytosis-dependent translocation is much slower than the phagocytosis-independent translocation triggered by antibody-FcR intimate binding. Presumably phagocytosis-dependent translocation is slower because the kinetics of phagocytosis adds time to the kinetics of effector translocation. The absence of evidence for protrusion of Icm/Dot system suggest that intimate binding may be needed to overcome the physical barrier established by the extracellular structures present on the surface of the bacterial or cellular membranes. Pulmonary macrophage infection by L. pneumophila generally occurs in the absence of pre-immune antibodies. Under in vivo conditions it is then likely that L. pneumophila relies on host cell signaling to stimulate uptake and achieve intimate contact required for effector translocation. Our data support a model in which L. pneumophila relies on the phagocytosis process to generate the intimate contact required for the translocation of pre-synthesized effector molecules (Figure 10). The phagocytosis requirement for effector translocation by L. pneumophila has consequences for its environmental lifestyle. Indeed, it is believed that amoebae are natural hosts for L. pneumophila providing a critical environment for its replication and survival [67]. L. pneumophila may only translocate its effectors when residing in a phagocytic membrane-bound compartment or once phagocytosis has been initiated. The translocated effectors can thereafter alter this compartment so as to create a vacuolar environment permissive for L. pneumophila replication. In addition, effector translocation will only be triggered by a potential host cell energetically competent to perform phagocytosis. Therefore relying phagocytosis to trigger translocation ensures that it is occurring only under conditions that could result in a successful infection, and thus, avoid delivery of effector to a host cell that cannot support replication. Interestingly, translocation of a Vibrio cholerae type VI secretion effector also requires internalization by host cells [68]. This is also somehow reminiscent of activation of the SPI-2 type III secretion system of Salmonella following internalization by macrophages [69]. Regardless of the secretion system used for translocating effector proteins, bacterial pathogens that interact with phagocytes may have evolved strategies to ensure that translocation occurs only in the phagosomal/endosomal microenvironment.

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