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Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System.

Gawthorne JA, Audry L, McQuitty C, Dean P, Christie JM, Enninga J, Roe AJ - Appl. Environ. Microbiol. (2016)

Bottom Line: Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors.We found the Escherichia coli O157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference.Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of the Shigella flexneri effector IpaB.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Quantification of bacterial uptake in S. flexneri. Bacterial invasiveness for the shown strains was quantified scoring Shigella-induced actin rearrangements within host cells at the bacterial entry site using phalloidin-rhodamine. Expression of IpaB-phiLOV or phiLOV alone did not affect the internalization of Shigella after 45 min of host cell challenge. For each condition, 100 cells were scored (n = 3). Error bars indicate the standard deviations.
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Figure 6: Quantification of bacterial uptake in S. flexneri. Bacterial invasiveness for the shown strains was quantified scoring Shigella-induced actin rearrangements within host cells at the bacterial entry site using phalloidin-rhodamine. Expression of IpaB-phiLOV or phiLOV alone did not affect the internalization of Shigella after 45 min of host cell challenge. For each condition, 100 cells were scored (n = 3). Error bars indicate the standard deviations.

Mentions: The simplicity of the phiLOV reporter makes it an attractive tool that might be applied to study translocation events in a wide range of T3SS-expressing bacteria. We wanted to test this potential wider applicability using a completely different pathogen and effector combination. To this end, we tested Shigella T3SS effector translocation with an IpaB-phiLOV fusion. We chose IpaB because its secretion kinetics have been analyzed previously (7), it can be modified at its C terminus without significantly impeding its secretion, and its function has been analyzed in some detail (see the introduction). It is expressed at high levels compared to other effectors; therefore, it is a good candidate for the establishment and validation of a new effector labeling approach. The fusion protein was generated by cloning the sequences encoding ipaB and phiLOV into pBAD18, thereby creating pBADIpaBphiLOV, allowing inducible expression upon the addition of arabinose (see Materials and Methods). An advantage of Shigella is that interactions with host cells result in a rapid injection of T3SS effectors and effects on the host cell in comparison to EHEC. Shigella effector translocation occurs within minutes rather than hours after initial infection (7). When expressed in the WT S. flexneri strain M90T, IpaB-phiLOV could be readily visualized within the bacteria, yielding a typical polar localization that has been previously identified for a number of bacterial effectors using different fluorescent techniques (7, 26) (Fig. 5). Remarkably, after a time course between 15 and 45 min of contact with the epithelial host cell line HeLa, fluorescence was rapidly dissipated from the bacterial cytoplasm, a finding consistent with its translocation. On the other hand, we could detect IpaB-phiLOV during the investigated time course within the targeted HeLa cells in proximity to the invading bacteria. IpaB-phiLOV localization within host cells was similar to the localization of IpaB using other fluorescent techniques (7) highlighting the potential of the phiLOV labeling only minimally perturbing the localization of the tagged effectors. As an additional control, an isogenic ΔmxiD strain of S. flexneri was also used. In this T3SS-deficient mutant, IpaB-phiLOV was retained within the bacterial cell, and no attachment to host cells was observed (Fig. 5). Quantification of the level of fluorescence within the bacteria to that outside revealed the dramatic loss of the effector pool within the injecting bacteria. We also tested whether Shigella invasion of HeLa cells was perturbed through the expression of IpaB-phiLOV. Scoring the number of entry foci as a marker for ongoing bacterial uptake for M90T, M90T/IpaB-phiLOV, M90T/phiLOV, and the noninvasive mutant mxiD/IpaB-phiLOV, we could not detect a significant difference in the number of entry foci for the WT strains expressing the different fluorescently tagged proteins (Fig. 6). This showed that IpaB-phiLOV expression did not perturb the entry of the pathogen into the host cells.


Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System.

Gawthorne JA, Audry L, McQuitty C, Dean P, Christie JM, Enninga J, Roe AJ - Appl. Environ. Microbiol. (2016)

Quantification of bacterial uptake in S. flexneri. Bacterial invasiveness for the shown strains was quantified scoring Shigella-induced actin rearrangements within host cells at the bacterial entry site using phalloidin-rhodamine. Expression of IpaB-phiLOV or phiLOV alone did not affect the internalization of Shigella after 45 min of host cell challenge. For each condition, 100 cells were scored (n = 3). Error bars indicate the standard deviations.
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Figure 6: Quantification of bacterial uptake in S. flexneri. Bacterial invasiveness for the shown strains was quantified scoring Shigella-induced actin rearrangements within host cells at the bacterial entry site using phalloidin-rhodamine. Expression of IpaB-phiLOV or phiLOV alone did not affect the internalization of Shigella after 45 min of host cell challenge. For each condition, 100 cells were scored (n = 3). Error bars indicate the standard deviations.
Mentions: The simplicity of the phiLOV reporter makes it an attractive tool that might be applied to study translocation events in a wide range of T3SS-expressing bacteria. We wanted to test this potential wider applicability using a completely different pathogen and effector combination. To this end, we tested Shigella T3SS effector translocation with an IpaB-phiLOV fusion. We chose IpaB because its secretion kinetics have been analyzed previously (7), it can be modified at its C terminus without significantly impeding its secretion, and its function has been analyzed in some detail (see the introduction). It is expressed at high levels compared to other effectors; therefore, it is a good candidate for the establishment and validation of a new effector labeling approach. The fusion protein was generated by cloning the sequences encoding ipaB and phiLOV into pBAD18, thereby creating pBADIpaBphiLOV, allowing inducible expression upon the addition of arabinose (see Materials and Methods). An advantage of Shigella is that interactions with host cells result in a rapid injection of T3SS effectors and effects on the host cell in comparison to EHEC. Shigella effector translocation occurs within minutes rather than hours after initial infection (7). When expressed in the WT S. flexneri strain M90T, IpaB-phiLOV could be readily visualized within the bacteria, yielding a typical polar localization that has been previously identified for a number of bacterial effectors using different fluorescent techniques (7, 26) (Fig. 5). Remarkably, after a time course between 15 and 45 min of contact with the epithelial host cell line HeLa, fluorescence was rapidly dissipated from the bacterial cytoplasm, a finding consistent with its translocation. On the other hand, we could detect IpaB-phiLOV during the investigated time course within the targeted HeLa cells in proximity to the invading bacteria. IpaB-phiLOV localization within host cells was similar to the localization of IpaB using other fluorescent techniques (7) highlighting the potential of the phiLOV labeling only minimally perturbing the localization of the tagged effectors. As an additional control, an isogenic ΔmxiD strain of S. flexneri was also used. In this T3SS-deficient mutant, IpaB-phiLOV was retained within the bacterial cell, and no attachment to host cells was observed (Fig. 5). Quantification of the level of fluorescence within the bacteria to that outside revealed the dramatic loss of the effector pool within the injecting bacteria. We also tested whether Shigella invasion of HeLa cells was perturbed through the expression of IpaB-phiLOV. Scoring the number of entry foci as a marker for ongoing bacterial uptake for M90T, M90T/IpaB-phiLOV, M90T/phiLOV, and the noninvasive mutant mxiD/IpaB-phiLOV, we could not detect a significant difference in the number of entry foci for the WT strains expressing the different fluorescently tagged proteins (Fig. 6). This showed that IpaB-phiLOV expression did not perturb the entry of the pathogen into the host cells.

Bottom Line: Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors.We found the Escherichia coli O157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference.Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of the Shigella flexneri effector IpaB.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, United Kingdom.

No MeSH data available.


Related in: MedlinePlus