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As a common food-borne pathogen, Salmonella can infect the host by contaminating food or water sources.
After human infection, it usually manifests as typhoid fever or gastroenteritis.
In severe cases, it can be life-threatening.
Salmonella can secrete effector proteins to host cells through the three-type secretion system during the pathogenic process, helping them to invade host cells and survive, multiply and spread in host cells.
The SopD family is a class of very conservative effector proteins unique to Salmonella.
Studies have shown that SopD plays an important role in the invasion, colonization and later systemic infections of Salmonella [1,2], making it a potential antibacterial drug.
However, the lack of specific molecular mechanism severely limits the progress of related research.
On February 19, 2021, Gao Xiang's research group from the State Key Laboratory of Microbial Technology of Shandong University and Professor Jorge Galán from Yale University published an article The Salmonella effector protein SopD targets Rab8 to positively and negatively modulate the inflammatory response in Nature Microbiology.
Progress has been made in the pathogenic molecular mechanism of the Salmonella effector protein SopD.
In this work, the author first screened the substrates of SopD protein through a series of biochemical methods, and finally found that Rab8 is a specific substrate of SopD from 25 kinds of Rab small G protein family involved in cell membrane vesicle transport.
And it further proved that SopD can exert GAP (GTPase activating protein, GTPase activating protein) function on Rab8 through its Arg(R) at position 312, promote the GTPase activity of Rab8, and make Rab8 change from the "activated state" that binds to GTP to that that binds GDP.
"Inactive".Studies have shown that Rab8 can negatively regulate the inflammatory response mediated by TLRs (Toll-like receptors) [3].
The authors also further proved that SopD can inhibit the function of Rab8 through its GAP activity, thereby promoting the inflammatory response.
In order to understand the molecular interaction mode of SopD and Rab8 in detail, the author analyzed the structure of SopD-Rab8 binding complex with a resolution of about 2.
3 Å through protein crystallography.
By analyzing the structure, the authors unexpectedly discovered that they may have obtained SopD's other action modes for Rab8 than GAP; and through site-directed mutagenesis, it was proved that Glu(E) at position 293 in SopD plays an important role in this combination.
Further studies have shown that although the SopDE293A mutant can disrupt the stable binding of SopD and Rab8, it does not affect the GAP activity of SopD, while SopDR312A loses GAP activity but can still form a stable complex with Rab8.
Functional experiments also show that SopDE293A will further reduce (inactivate) GTP-binding Rab8 compared to SopD; and SopDR312A will further increase (activate) GTP-binding Rab8 compared to SopD, suggesting that SopD itself has the opposite activity to GAP.
Function.
Studies have shown that there are four main regulatory proteins involved in the work cycle of Rab family proteins.
GAP and GDI (GDP dissociation inhibitor) proteins have functions similar to "inactivating" Rab activity, GEF (guanine nucleotide exchange factor) and GDF (GDI) Displacement factors) The protein functions similar to "activating" Rab activity.
Among them, the GDI protein has a high affinity with the Rab protein that binds to GDP, making it unable to open its downstream signaling pathway; the GDF protein can help the Rab protein to dissociate from GDI; the GEF protein can make the Rab from the inactive state of binding GDP to Combined with the activation state of GTP, promote its activity [4].
The author first proved that SopD does not have GEF function.
Through further structural analysis, it is found that the binding interface of SopD and Rab8 and the interaction interface of GDI and Rab protein have obvious steric hindrance, and the competitive binding experiment in vitro and in vivo proves that SopD and SopDR312A can compete with Rab8 from GDI protein.
, While SopDE293A cannot, indicating that SopD also has a function similar to GDF activity.
Finally, the authors further infected cells and mice with Salmonella containing different SopD mutants, and the results showed that SopDR312A significantly reduced inflammation (promoting the activity of Rab8) compared to SopD, and SopDE293A significantly promoted compared to SopD Inflammation (inhibition of the activity of Rab8).
The above results indicate that SopD is a bifunctional effector protein with inhibition and promotion functions for Rab8.
This study, through a combination of microbial genetics, biochemistry, structural biology, and cell biology, revealed for the first time the mechanism of action of the Salmonella effector protein SopD at the molecular level, and unexpectedly found that SopD is a rare and both.
"Yin-Yang" bidirectional effector protein.
On the one hand, SopD inhibits the anti-inflammatory pathway mediated by Rab8 through its GAP activity, enhances the inflammatory response, and promotes the invasion and proliferation of Salmonella.
On the other hand, SopD can also release Rab8 from its tight binding with GDI through its GDF-like function, and promote the reactivation of Rab8 protein; by enhancing the anti-inflammatory response mediated by Rab8, it helps host cells to restore homeostasis, which is beneficial to Salmonella Long-term colonization in host cells.
The above research results not only enrich the understanding of the pathogenic mechanism of Salmonella, but also show an outstanding product of the Salmonella effector protein in the evolution process, laying a theoretical foundation for the subsequent research on SopD protein and the development of antibacterial drugs targeting SopD .
The schematic diagram of SopD's regulation mechanism on Rab8 Lian Huan, Jiang Kun, Tong Ming are the co-first authors of the paper, and Gao Xiang and Professor Jorge Galán are the co-corresponding authors of the study.
The public technology platform for life environment research of Shandong University and the Shanghai synchrotron radiation source provided important support for this work.
Gao Xiang’s team mainly used intestinal Bacteroides and Salmonella as model bacteria to conduct research on the molecular mechanism of the interaction between intestinal bacteria-intestinal pathogens and hosts.
They are invited to have microbiology, cell biology, immunology, and structural biology Various young talents (fixed personnel, post-doctoral students, graduate students and research assistants) with related research backgrounds such as bioinformatics and other related researches will jointly carry out relevant research! For details, please refer to the website of the research group: https://gaolab.
qd.
sdu.
edu.
cn/http:// Original link: https:// dx.
doi.
org/10.
1038/s41564-021-00866-3 Platemaker: Eleven References [1] Bakowski, MA et al.
SopD acts cooperatively with SopB during Salmonella enterica serovar Typhimurium invasion.
Cellular microbiology .
2007,9, 2839 -2855.
[2] Giacomodonato, MN et al.
SipA, SopA, SopB, SopD and SopE2 effector proteins of Salmonella enterica serovar Typhimurium are synthesized at late stages of infection in mice.
Microbiology.
2007, 153, 1221-1228.
[3 ] Luo, L.
et al.
TLR Crosstalk Activates LRP1 to Recruit Rab8a and PI3Kγ for Suppression of Inflammatory Responses.
Cell Reports.
2018, 24, 3033-3044,[4] Olena Pylypenko et al.
After human infection, it usually manifests as typhoid fever or gastroenteritis.
In severe cases, it can be life-threatening.
Salmonella can secrete effector proteins to host cells through the three-type secretion system during the pathogenic process, helping them to invade host cells and survive, multiply and spread in host cells.
The SopD family is a class of very conservative effector proteins unique to Salmonella.
Studies have shown that SopD plays an important role in the invasion, colonization and later systemic infections of Salmonella [1,2], making it a potential antibacterial drug.
However, the lack of specific molecular mechanism severely limits the progress of related research.
On February 19, 2021, Gao Xiang's research group from the State Key Laboratory of Microbial Technology of Shandong University and Professor Jorge Galán from Yale University published an article The Salmonella effector protein SopD targets Rab8 to positively and negatively modulate the inflammatory response in Nature Microbiology.
Progress has been made in the pathogenic molecular mechanism of the Salmonella effector protein SopD.
In this work, the author first screened the substrates of SopD protein through a series of biochemical methods, and finally found that Rab8 is a specific substrate of SopD from 25 kinds of Rab small G protein family involved in cell membrane vesicle transport.
And it further proved that SopD can exert GAP (GTPase activating protein, GTPase activating protein) function on Rab8 through its Arg(R) at position 312, promote the GTPase activity of Rab8, and make Rab8 change from the "activated state" that binds to GTP to that that binds GDP.
"Inactive".Studies have shown that Rab8 can negatively regulate the inflammatory response mediated by TLRs (Toll-like receptors) [3].
The authors also further proved that SopD can inhibit the function of Rab8 through its GAP activity, thereby promoting the inflammatory response.
In order to understand the molecular interaction mode of SopD and Rab8 in detail, the author analyzed the structure of SopD-Rab8 binding complex with a resolution of about 2.
3 Å through protein crystallography.
By analyzing the structure, the authors unexpectedly discovered that they may have obtained SopD's other action modes for Rab8 than GAP; and through site-directed mutagenesis, it was proved that Glu(E) at position 293 in SopD plays an important role in this combination.
Further studies have shown that although the SopDE293A mutant can disrupt the stable binding of SopD and Rab8, it does not affect the GAP activity of SopD, while SopDR312A loses GAP activity but can still form a stable complex with Rab8.
Functional experiments also show that SopDE293A will further reduce (inactivate) GTP-binding Rab8 compared to SopD; and SopDR312A will further increase (activate) GTP-binding Rab8 compared to SopD, suggesting that SopD itself has the opposite activity to GAP.
Function.
Studies have shown that there are four main regulatory proteins involved in the work cycle of Rab family proteins.
GAP and GDI (GDP dissociation inhibitor) proteins have functions similar to "inactivating" Rab activity, GEF (guanine nucleotide exchange factor) and GDF (GDI) Displacement factors) The protein functions similar to "activating" Rab activity.
Among them, the GDI protein has a high affinity with the Rab protein that binds to GDP, making it unable to open its downstream signaling pathway; the GDF protein can help the Rab protein to dissociate from GDI; the GEF protein can make the Rab from the inactive state of binding GDP to Combined with the activation state of GTP, promote its activity [4].
The author first proved that SopD does not have GEF function.
Through further structural analysis, it is found that the binding interface of SopD and Rab8 and the interaction interface of GDI and Rab protein have obvious steric hindrance, and the competitive binding experiment in vitro and in vivo proves that SopD and SopDR312A can compete with Rab8 from GDI protein.
, While SopDE293A cannot, indicating that SopD also has a function similar to GDF activity.
Finally, the authors further infected cells and mice with Salmonella containing different SopD mutants, and the results showed that SopDR312A significantly reduced inflammation (promoting the activity of Rab8) compared to SopD, and SopDE293A significantly promoted compared to SopD Inflammation (inhibition of the activity of Rab8).
The above results indicate that SopD is a bifunctional effector protein with inhibition and promotion functions for Rab8.
This study, through a combination of microbial genetics, biochemistry, structural biology, and cell biology, revealed for the first time the mechanism of action of the Salmonella effector protein SopD at the molecular level, and unexpectedly found that SopD is a rare and both.
"Yin-Yang" bidirectional effector protein.
On the one hand, SopD inhibits the anti-inflammatory pathway mediated by Rab8 through its GAP activity, enhances the inflammatory response, and promotes the invasion and proliferation of Salmonella.
On the other hand, SopD can also release Rab8 from its tight binding with GDI through its GDF-like function, and promote the reactivation of Rab8 protein; by enhancing the anti-inflammatory response mediated by Rab8, it helps host cells to restore homeostasis, which is beneficial to Salmonella Long-term colonization in host cells.
The above research results not only enrich the understanding of the pathogenic mechanism of Salmonella, but also show an outstanding product of the Salmonella effector protein in the evolution process, laying a theoretical foundation for the subsequent research on SopD protein and the development of antibacterial drugs targeting SopD .
The schematic diagram of SopD's regulation mechanism on Rab8 Lian Huan, Jiang Kun, Tong Ming are the co-first authors of the paper, and Gao Xiang and Professor Jorge Galán are the co-corresponding authors of the study.
The public technology platform for life environment research of Shandong University and the Shanghai synchrotron radiation source provided important support for this work.
Gao Xiang’s team mainly used intestinal Bacteroides and Salmonella as model bacteria to conduct research on the molecular mechanism of the interaction between intestinal bacteria-intestinal pathogens and hosts.
They are invited to have microbiology, cell biology, immunology, and structural biology Various young talents (fixed personnel, post-doctoral students, graduate students and research assistants) with related research backgrounds such as bioinformatics and other related researches will jointly carry out relevant research! For details, please refer to the website of the research group: https://gaolab.
qd.
sdu.
edu.
cn/http:// Original link: https:// dx.
doi.
org/10.
1038/s41564-021-00866-3 Platemaker: Eleven References [1] Bakowski, MA et al.
SopD acts cooperatively with SopB during Salmonella enterica serovar Typhimurium invasion.
Cellular microbiology .
2007,9, 2839 -2855.
[2] Giacomodonato, MN et al.
SipA, SopA, SopB, SopD and SopE2 effector proteins of Salmonella enterica serovar Typhimurium are synthesized at late stages of infection in mice.
Microbiology.
2007, 153, 1221-1228.
[3 ] Luo, L.
et al.
TLR Crosstalk Activates LRP1 to Recruit Rab8a and PI3Kγ for Suppression of Inflammatory Responses.
Cell Reports.
2018, 24, 3033-3044,[4] Olena Pylypenko et al.
