scholarly journals Characterization of the binding motif for the T3SS master regulator LcrF in Yersinia pseudotuberculosis

2021 ◽  
Author(s):  
Luyi Liu ◽  
Shaojia Huang ◽  
Keke Fei ◽  
Wei Zhou ◽  
Shiyun Chen ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Bioinformatics Analysis ◽  
Promoter Sequence ◽  
Binding Motif ◽  
Binding Region ◽  
Master Regulator ◽  
Or Genes ◽  
Target Promoters

Abstract LcrF is the master regulator that positively regulates the Ysc type III secretion system (T3SS) in Yersinia and shares high similarity to the DNA binding domain of the T3SS master regulator ExsA in Pseudomonas aeruginosa. Based on these features, bioinformatics analysis has predicted putative LcrF binding site in its target promoters. Here we experimentally characterized its binding motif. An A-rich LcrF binding region in the lcrG promoter sequence, a typical regulatory target of LcrF, was first confirmed. To obtain detailed information, this binding region was cloned into a synthetized promoter and mutations in this region were further constructed. We demonstrated that the 5’-AAAAA-n5-GnCT-3’ sequence is required for LcrF regulation and this motif is strictly located 4-bp upstream of a noncanonical promoter, in which the -35 and -10 elements are separated by a 21-bp spacer. Consistently, the putative binding motif was found in promoters of nine T3SS related operons or genes positively regulated by LcrF. Transcriptome analysis further confirmed that LcrF specifically activates T3SS genes in Yersinia. Collectively, our data suggest that LcrF is evolved to be a specific T3SS activator with stringent sequence requirement for transcriptional regulation.

scholarly journals Phenotypic characterization of a virulence-associated protein, VagH, of Yersinia pseudotuberculosis reveals a tight link between VagH and the type III secretion system

Microbiology ◽  
2007 ◽  
Vol 153 (5) ◽  
pp. 1464-1473 ◽  
Author(s):  
Sara Garbom ◽  
Martina Olofsson ◽  
Ann-Catrin Björnfot ◽  
Manoj Kumar Srivastava ◽  
Victoria L Robinson ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Phenotypic Characterization ◽  
Type Iii

scholarly journals Characterization of Pyrin Dephosphorylation and Inflammasome Activation in Macrophages as Triggered by the Yersinia Effectors YopE and YopT

2019 ◽  
Vol 87 (3) ◽  
Author(s):  
Natasha P. Medici ◽  
Maheen Rashid ◽  
James B. Bliska
Keyword(s):  
Immune Responses ◽  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Effector Proteins ◽  
Host Cells ◽  
Cell Physiology ◽  
Inflammasome Activation ◽  
Wild Type ◽  
Content Type

ABSTRACT Pathogenic Yersinia species deliver Yop effector proteins through a type III secretion system into host cells. Among these effectors, YopE and YopT are Rho-modifying toxins, which function to modulate host cell physiology and evade immune responses. YopE is a GTPase-activating protein (GAP) while YopT is a protease, and they inhibit RhoA by different modes of action. Modifications to RhoA are sensed by pyrin, which, once activated, assembles a caspase-1 inflammasome, which generates cytokines such as interleukin-1β (IL-1β) and cell death by pyroptosis. In Yersinia-infected macrophages, YopE or YopT triggers inflammasome assembly only in the absence of another effector, YopM, which counteracts pyrin by keeping it inactive. The glucosyltransferase TcdB from Clostridium difficile, a well-studied RhoA-inactivating toxin, triggers activation of murine pyrin by dephosphorylation of Ser205 and Ser241. To determine if YopE or YopT triggers pyrin dephosphorylation, we infected lipopolysaccharide (LPS)-primed murine macrophages with ΔyopM Yersinia pseudotuberculosis strains expressing wild-type (wt) or YopE mutant variants or YopT. By immunoblotting pyrin after infection, we observed that wt YopE triggered dephosphorylation of Ser205 and inflammasome activation. Pyrin dephosphorylation was reduced if a YopE variant had a defect in stability or RhoA specificity but not membrane localization. We also observed that wt YopT triggered pyrin dephosphorylation but more slowly than YopE, suggesting that YopE is dominant in this process. Our findings provide evidence that RhoA-modifying toxins trigger activation of pyrin by a conserved dephosphorylation mechanism. In addition, by characterization of YopE and YopT, we show that different features of effectors, such as RhoA specificity, affect the efficiency of pyrin dephosphorylation.

scholarly journals Bioinformatics analysis and characterization of a secretory cystatin from Thelohanellus kitauei

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Fengli Zhang ◽  
Yalin Yang ◽  
Chenchen Gao ◽  
Yuanyuan Yao ◽  
Rui Xia ◽  
...  
Keyword(s):  
Bioinformatics Analysis

scholarly journals A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology inChlamydomonas reinhardtii

2019 ◽  
Vol 116 (37) ◽  
pp. 18445-18454 ◽  
Author(s):  
Alan K. Itakura ◽  
Kher Xing Chan ◽  
Nicky Atkinson ◽  
Leif Pallesen ◽  
Lianyong Wang ◽  
...  
Keyword(s):  
Surface Area ◽  
Structure And Function ◽  
Binding Motif ◽  
Starch Granules ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
Biophysical Mechanism ◽  
Mechanism Function ◽  
And Function

A phase-separated, liquid-like organelle called the pyrenoid mediates CO2fixation in the chloroplasts of nearly all eukaryotic algae. While most algae have 1 pyrenoid per chloroplast, here we describe a mutant in the model algaChlamydomonasthat has on average 10 pyrenoids per chloroplast. Characterization of the mutant leads us to propose a model where multiple pyrenoids are favored by an increase in the surface area of the starch sheath that surrounds and binds to the liquid-like pyrenoid matrix. We find that the mutant’s phenotypes are due to disruption of a gene, which we call StArch Granules Abnormal 1 (SAGA1) because starch sheath granules, or plates, in mutants lacking SAGA1 are more elongated and thinner than those of wild type. SAGA1 contains a starch binding motif, suggesting that it may directly regulate starch sheath morphology. SAGA1 localizes to multiple puncta and streaks in the pyrenoid and physically interacts with the small and large subunits of the carbon-fixing enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), a major component of the liquid-like pyrenoid matrix. Our findings suggest a biophysical mechanism by which starch sheath morphology affects pyrenoid number and CO2-concentrating mechanism function, advancing our understanding of the structure and function of this biogeochemically important organelle. More broadly, we propose that the number of phase-separated organelles can be regulated by imposing constraints on their surface area.

Characterization of the human nebulette gene: a polymorphism in an actin-binding motif is associated with nonfamilial idiopathic dilated cardiomyopathy

2000 ◽  
Vol 107 (5) ◽  
pp. 440-451 ◽  
Author(s):  
Takuro Arimura ◽  
Takeyuki Nakamura ◽  
Shitoshi Hiroi ◽  
Manatsu Satoh ◽  
Megumi Takahashi ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Idiopathic Dilated Cardiomyopathy ◽  
Actin Binding ◽  
Binding Motif

scholarly journals Characterization of a γ-adaptin ear-binding motif in enthoprotin

FEBS Letters ◽  
2003 ◽  
Vol 555 (3) ◽  
pp. 437-442 ◽  
Author(s):  
Sylwia Wasiak ◽  
Alexei Yu. Denisov ◽  
Zhaozhong Han ◽  
Peter A. Leventis ◽  
Elaine de Heuvel ◽  
...  
Keyword(s):  
Binding Motif

scholarly journals Diminished LcrV Secretion Attenuates Yersinia pseudotuberculosis Virulence

2007 ◽  
Vol 189 (23) ◽  
pp. 8417-8429 ◽  
Author(s):  
Jeanette E. Bröms ◽  
Matthew S. Francis ◽  
Åke Forsberg
Keyword(s):  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Signal Sequence ◽  
Infection Model ◽  
Target Cells ◽  
Cell Infection ◽  
Host Cell Membrane ◽  
Type Iii ◽  
A Cell

ABSTRACT Many gram-negative bacterial pathogenicity factors that function beyond the outer membrane are secreted via a contact-dependent type III secretion system. Two types of substrates are predestined for this mode of secretion, namely, antihost effectors that are translocated directly into target cells and the translocators required for targeting of the effectors across the host cell membrane. N-terminal secretion signals are important for recognition of the protein cargo by the type III secretion machinery. Even though such signals are known for several effectors, a consensus signal sequence is not obvious. One of the translocators, LcrV, has been attributed other functions in addition to its role in translocation. These functions include regulation, presumably via interaction with LcrG inside bacteria, and immunomodulation via interaction with Toll-like receptor 2. Here we wanted to address the significance of the specific targeting of LcrV to the exterior for its function in regulation, effector targeting, and virulence. The results, highlighting key N-terminal amino acids important for LcrV secretion, allowed us to dissect the role of LcrV in regulation from that in effector targeting/virulence. While only low levels of exported LcrV were required for in vitro effector translocation, as deduced by a cell infection assay, fully functional export of LcrV was found to be a prerequisite for its role in virulence in the systemic murine infection model.

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