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The type III secretion effector NleE inhibits NF-kappaB activation.

Nadler C, Baruch K, Kobi S, Mills E, Haviv G, Farago M, Alkalay I, Bartfeld S, Meyer TF, Ben-Neriah Y, Rosenshine I - PLoS Pathog. (2010)

Bottom Line: Importantly, we show that NleE inhibits NF-kappaB activation by preventing activation of IKKbeta and consequently the degradation of the NF-kappaB inhibitor, IkappaB.This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB.In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-kappaB signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, The Hebrew University, Jerusalem, Israel.

ABSTRACT
The complex host-pathogen interplay involves the recognition of the pathogen by the host's innate immune system and countermeasures taken by the pathogen. Detection of invading bacteria by the host leads to rapid activation of the transcription factor NF-kappaB, followed by inflammation and eradication of the intruders. In response, some pathogens, including enteropathogenic Escherichia coli (EPEC), acquired means of blocking NF-kappaB activation. We show that inhibition of NF-kappaB activation by EPEC involves the injection of NleE into the host cell. Importantly, we show that NleE inhibits NF-kappaB activation by preventing activation of IKKbeta and consequently the degradation of the NF-kappaB inhibitor, IkappaB. This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB. In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-kappaB signaling pathways.

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NleE of IE2 is deficient in translocation and inhibition of IκB degradation.(A) HeLa cells were either uninfected (N/I) or infected with a strain deleted of nleEIE6 (SC3680) (see Fig. 2A), complemented or not complemented with plasmids expressing NleEIE2 or NleEIE6 (indicated as E2 and E6, respectively). After 3h, cells were washed, treated with TNFα for 30 min and extracted. The extracts were analyzed by western blot with anti-IκB and anti-tubulin antibodies. (B and C) HeLa cells were infected with wild-type EPEC harboring plasmids expressing nleEIE2 or nleEIE6 fused to the blaM reporter gene (indicated as pnleE2 and pnleE6, respectively). As a negative control, cells were infected with EPEC harboring the parental vector pCX341 (Vector). The β-lactamase activity in the infecting bacteria, reflecting the expression levels of the fusion proteins (B), and the β-lactamase activity in the infected HeLa cells, reflecting the levels of translocation of the fusion proteins into the HeLa cells (C) were determined. The experiment was done twice in triplicates and typical results are shown. Standard errors are indicated by bars.
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ppat-1000743-g003: NleE of IE2 is deficient in translocation and inhibition of IκB degradation.(A) HeLa cells were either uninfected (N/I) or infected with a strain deleted of nleEIE6 (SC3680) (see Fig. 2A), complemented or not complemented with plasmids expressing NleEIE2 or NleEIE6 (indicated as E2 and E6, respectively). After 3h, cells were washed, treated with TNFα for 30 min and extracted. The extracts were analyzed by western blot with anti-IκB and anti-tubulin antibodies. (B and C) HeLa cells were infected with wild-type EPEC harboring plasmids expressing nleEIE2 or nleEIE6 fused to the blaM reporter gene (indicated as pnleE2 and pnleE6, respectively). As a negative control, cells were infected with EPEC harboring the parental vector pCX341 (Vector). The β-lactamase activity in the infecting bacteria, reflecting the expression levels of the fusion proteins (B), and the β-lactamase activity in the infected HeLa cells, reflecting the levels of translocation of the fusion proteins into the HeLa cells (C) were determined. The experiment was done twice in triplicates and typical results are shown. Standard errors are indicated by bars.

Mentions: EPEC encode two very similar nleE alleles. One allele, identified in our screen, is located in the IE6 region and the other is in the IE2 region [2]. We initially found that deletion of the IE6 region, but not of the IE2 region, caused deficiency in inhibition of IκB degradation (data not shown). However, the two proteins, NleEIE2 and NleEIE6, are identical, apart from an internal deletion of 56 residues in NleEIE2 (Fig. S1), and this similarity between the two proteins urged us to determine the activity of each of the two proteins. We first tested their ability to complement IκB destabilization in a strain deleted of nleEIE6. To this end, we expressed each of them on a plasmid carrying an identical promoter and ribosomal binding site. Results showed that only NleEIE6, but not NleEIE2, was able to attenuate IκB degradation (Fig. 3A). These results indicate that NleEIE2 is either not active in the host cell or is not translocated into the host cell. To differentiate between these two possibilities, we used the above mentioned plasmid where both proteins were fused to the β-lactamase translocation reporter protein, BlaM. The plasmids were introduced into EPEC and the ability to translocate them into infected cells was tested. We found that both NleEIE2-BlaM and NleEIE6-BlaM were expressed at similar levels in the bacteria (Fig. 3B). Importantly, however, only NleEIE6 was translocated into the host cell (Fig. 3C), suggesting that NleEIE2 is a cryptic effector. Cumulatively, these results define NleEIE6, but not NleEIE2, as the effector needed for inhibition of IκB degradation. Therefore, in this report the term “NleE” specifically refers to “NleEIE6”.


The type III secretion effector NleE inhibits NF-kappaB activation.

Nadler C, Baruch K, Kobi S, Mills E, Haviv G, Farago M, Alkalay I, Bartfeld S, Meyer TF, Ben-Neriah Y, Rosenshine I - PLoS Pathog. (2010)

NleE of IE2 is deficient in translocation and inhibition of IκB degradation.(A) HeLa cells were either uninfected (N/I) or infected with a strain deleted of nleEIE6 (SC3680) (see Fig. 2A), complemented or not complemented with plasmids expressing NleEIE2 or NleEIE6 (indicated as E2 and E6, respectively). After 3h, cells were washed, treated with TNFα for 30 min and extracted. The extracts were analyzed by western blot with anti-IκB and anti-tubulin antibodies. (B and C) HeLa cells were infected with wild-type EPEC harboring plasmids expressing nleEIE2 or nleEIE6 fused to the blaM reporter gene (indicated as pnleE2 and pnleE6, respectively). As a negative control, cells were infected with EPEC harboring the parental vector pCX341 (Vector). The β-lactamase activity in the infecting bacteria, reflecting the expression levels of the fusion proteins (B), and the β-lactamase activity in the infected HeLa cells, reflecting the levels of translocation of the fusion proteins into the HeLa cells (C) were determined. The experiment was done twice in triplicates and typical results are shown. Standard errors are indicated by bars.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2813277&req=5

ppat-1000743-g003: NleE of IE2 is deficient in translocation and inhibition of IκB degradation.(A) HeLa cells were either uninfected (N/I) or infected with a strain deleted of nleEIE6 (SC3680) (see Fig. 2A), complemented or not complemented with plasmids expressing NleEIE2 or NleEIE6 (indicated as E2 and E6, respectively). After 3h, cells were washed, treated with TNFα for 30 min and extracted. The extracts were analyzed by western blot with anti-IκB and anti-tubulin antibodies. (B and C) HeLa cells were infected with wild-type EPEC harboring plasmids expressing nleEIE2 or nleEIE6 fused to the blaM reporter gene (indicated as pnleE2 and pnleE6, respectively). As a negative control, cells were infected with EPEC harboring the parental vector pCX341 (Vector). The β-lactamase activity in the infecting bacteria, reflecting the expression levels of the fusion proteins (B), and the β-lactamase activity in the infected HeLa cells, reflecting the levels of translocation of the fusion proteins into the HeLa cells (C) were determined. The experiment was done twice in triplicates and typical results are shown. Standard errors are indicated by bars.
Mentions: EPEC encode two very similar nleE alleles. One allele, identified in our screen, is located in the IE6 region and the other is in the IE2 region [2]. We initially found that deletion of the IE6 region, but not of the IE2 region, caused deficiency in inhibition of IκB degradation (data not shown). However, the two proteins, NleEIE2 and NleEIE6, are identical, apart from an internal deletion of 56 residues in NleEIE2 (Fig. S1), and this similarity between the two proteins urged us to determine the activity of each of the two proteins. We first tested their ability to complement IκB destabilization in a strain deleted of nleEIE6. To this end, we expressed each of them on a plasmid carrying an identical promoter and ribosomal binding site. Results showed that only NleEIE6, but not NleEIE2, was able to attenuate IκB degradation (Fig. 3A). These results indicate that NleEIE2 is either not active in the host cell or is not translocated into the host cell. To differentiate between these two possibilities, we used the above mentioned plasmid where both proteins were fused to the β-lactamase translocation reporter protein, BlaM. The plasmids were introduced into EPEC and the ability to translocate them into infected cells was tested. We found that both NleEIE2-BlaM and NleEIE6-BlaM were expressed at similar levels in the bacteria (Fig. 3B). Importantly, however, only NleEIE6 was translocated into the host cell (Fig. 3C), suggesting that NleEIE2 is a cryptic effector. Cumulatively, these results define NleEIE6, but not NleEIE2, as the effector needed for inhibition of IκB degradation. Therefore, in this report the term “NleE” specifically refers to “NleEIE6”.

Bottom Line: Importantly, we show that NleE inhibits NF-kappaB activation by preventing activation of IKKbeta and consequently the degradation of the NF-kappaB inhibitor, IkappaB.This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB.In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-kappaB signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, The Hebrew University, Jerusalem, Israel.

ABSTRACT
The complex host-pathogen interplay involves the recognition of the pathogen by the host's innate immune system and countermeasures taken by the pathogen. Detection of invading bacteria by the host leads to rapid activation of the transcription factor NF-kappaB, followed by inflammation and eradication of the intruders. In response, some pathogens, including enteropathogenic Escherichia coli (EPEC), acquired means of blocking NF-kappaB activation. We show that inhibition of NF-kappaB activation by EPEC involves the injection of NleE into the host cell. Importantly, we show that NleE inhibits NF-kappaB activation by preventing activation of IKKbeta and consequently the degradation of the NF-kappaB inhibitor, IkappaB. This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB. In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-kappaB signaling pathways.

Show MeSH
Related in: MedlinePlus