scholarly journals A Proline-Rich Element in the Type III Secretion Protein FlhB Contributes to Flagellar Biogenesis in the Beta- and Gamma-Proteobacteria

2020 ◽  
Vol 11 ◽  
Author(s):  
John C. Hook ◽  
Vitan Blagotinsek ◽  
Jan Pané-Farré ◽  
Devid Mrusek ◽  
Florian Altegoer ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Transmembrane Protein ◽  
Rich Region ◽  
Type Iii ◽  
Gamma Proteobacteria ◽  
C Terminus ◽  
Complex Motor ◽  
Flagellar Biogenesis ◽  
Proline Rich Region

Flagella are bacterial organelles of locomotion. Their biogenesis is highly coordinated in time and space and relies on a specialized flagellar type III secretion system (fT3SS) required for the assembly of the extracellular hook, rod, and filament parts of this complex motor device. The fT3SS protein FlhB switches secretion substrate specificity once the growing hook reaches its determined length. Here we present the crystal structure of the cytoplasmic domain of the transmembrane protein FlhB. The structure visualizes a so-far unseen proline-rich region (PRR) at the very C-terminus of the protein. Strains lacking the PRR show a decrease in flagellation as determined by hook- and filament staining, indicating a role of the PRR during assembly of the hook and filament structures. Phylogenetic analysis shows that the PRR is a primary feature of FlhB proteins of flagellated beta- and gamma-proteobacteria. Taken together, our study adds another layer of complexity and organismic diversity to the process of flagella biogenesis.

scholarly journals Use of the Galleria mellonella Caterpillar as a Model Host To Study the Role of the Type III Secretion System in Pseudomonas aeruginosa Pathogenesis

2003 ◽  
Vol 71 (5) ◽  
pp. 2404-2413 ◽  
Author(s):  
Sachiko Miyata ◽  
Monika Casey ◽  
Dara W. Frank ◽  
Frederick M. Ausubel ◽  
Eliana Drenkard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Galleria Mellonella ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Triple Mutant ◽  
Type Iii ◽  
Wax Moth

ABSTRACT Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using ΔexoU ΔexoY, ΔexoT ΔexoY, and ΔexoT ΔexoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a ΔexoT ΔexoU ΔexoY triple mutant and a ΔpscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.

scholarly journals Mutations in Flk, FlgG, FlhA, and FlhE That Affect the Flagellar Type III Secretion Specificity Switch in Salmonella enterica

2009 ◽  
Vol 191 (12) ◽  
pp. 3938-3949 ◽  
Author(s):  
Takanori Hirano ◽  
Shino Mizuno ◽  
Shin-Ichi Aizawa ◽  
Kelly T. Hughes
Keyword(s):  
Type Iii Secretion ◽  
Secretion System ◽  
Gene Gain ◽  
Hook Length ◽  
Type Iii ◽  
C Terminus ◽  
Mutant Strains ◽  
Rod Structures ◽  
Control Protein ◽  
Filament Type

ABSTRACT Upon completion of the flagellar hook-basal body (HBB) structure, the flagellar type III secretion system switches from secreting rod/hook-type to filament-type substrates. The secretion specificity switch has been reported to occur prematurely (prior to HBB completion) in flk-null mutants (P. Aldridge, J. E. Karlinsey, E. Becker, F. F. Chevance, and K. T. Hughes, Mol. Microbiol. 60:630-643, 2006) and in distal rod gene gain-of-function mutants (flgG* mutants) that produce filamentous rod structures (F. F. Chevance, N. Takahashi, J. E. Karlinsey, J. Gnerer, T. Hirano, R. Samudrala, S. Aizawa, and K. T. Hughes, Genes Dev. 21:2326-2335, 2007). A fusion of β-lactamase (Bla) to the C terminus of the filament-type secretion substrate FlgM was used to select for mutants that would secrete FlgM-Bla into the periplasmic space and show ampicillin resistance (Apr). Apr resulted from null mutations in the flhE gene, C-terminal truncation mutations in the flhA gene, null and dominant mutations in the flk gene, and flgG* mutations. All mutant classes required the hook length control protein (FliK) and the rod cap protein (FlgJ) for the secretion specificity switch to occur. However, neither the hook (FlgE) nor the hook cap (FlgD) protein was required for premature FlgM-Bla secretion in the flgG* and flk mutant strains, but it was in the flhE mutants. Unexpectedly, when deletions of either flgE or flgD were introduced into flgG* mutant strains, filaments were able to grow directly on the filamentous rod structures.

scholarly journals A revised model for the role of GacS/GacA in regulating type III secretion by Pseudomonas syringae pv. tomato DC3000

2019 ◽  
Vol 21 (1) ◽  
pp. 139-144 ◽  
Author(s):  
Megan R. O’Malley ◽  
Ching‐Fang Chien ◽  
Scott C. Peck ◽  
Nai‐Chun Lin ◽  
Jeffrey C. Anderson
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Tomato Dc3000 ◽  
Type Iii

scholarly journals Interaction of protein tyrosine phosphatase (PTP) 1B with its substrates is influenced by two distinct binding domains

2002 ◽  
Vol 364 (2) ◽  
pp. 377-383 ◽  
Author(s):  
Shrikrishna DADKE ◽  
Jonathan CHERNOFF
Keyword(s):  
Insulin Receptor ◽  
Protein Tyrosine Phosphatase ◽  
Insulin Signalling ◽  
Tyrosine Phosphatase ◽  
Rich Region ◽  
Protein Tyrosine ◽  
Binding Domains ◽  
C Terminus ◽  
Ptp 1B ◽  
Proline Rich Region

We have shown previously that protein tyrosine phosphatase (PTP) 1B interacts with insulin receptor and negatively regulates insulin signalling by an N-terminal binding domain [Dadke, Kusari and Chernoff (2000) J. Biol. Chem. 275, 23642–23647] and it also negatively regulates integrin signalling through a proline-rich region present in the C-terminus [Liu, Hill and Chernoff (1996) J. Biol. Chem. 271, 31290–31295; Liu, Sells and Chernoff (1998) Curr. Biol. 8, 173–176]. Here we show that PTP1B mutants that are defective in Src homology 3 domain binding fully retain the ability to inhibit insulin signalling, whereas mutants defective in insulin-receptor binding fully retain the ability to inhibit integrin signalling. In contrast, both the C-terminal proline-rich region and the tandem tyrosine residues present in the N-terminal region are required for the activation of Src family kinases. These data show that PTP1B can independently regulate insulin and integrin signals, and that Src might represent a convergence point for regulating signal transduction by this phosphatase.

scholarly journals Pseudomonas syringae HrpP Is a Type III Secretion Substrate Specificity Switch Domain Protein That Is Translocated into Plant Cells but Functions Atypically for a Substrate-Switching Protein

2009 ◽  
Vol 191 (9) ◽  
pp. 3120-3131 ◽  
Author(s):  
Joanne E. Morello ◽  
Alan Collmer
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Effector Proteins ◽  
Type Iii ◽  
Native Promoter ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Pseudomonas syringae delivers virulence effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). P. syringae pv. tomato DC3000 HrpP has a C-terminal, putative T3SS substrate specificity switch domain, like Yersinia YscP. A ΔhrpP DC3000 mutant could not cause disease in tomato or elicit a hypersensitive response (HR) in tobacco, but the HR could be restored by expression of HrpP in trans. Though HrpP is a relatively divergent protein in the T3SS of different P. syringae pathovars, hrpP from P. syringae pv. syringae 61 and P. syringae pv. phaseolicola 1448A restored HR elicitation and pathogenicity to DC3000 ΔhrpP. HrpP was translocated into Nicotiana benthamiana cells via the DC3000 T3SS when expressed from its native promoter, but it was not secreted in culture. N- and C-terminal truncations of HrpP were tested for their ability to be translocated and to restore HR elicitation activity to the ΔhrpP mutant. No N-terminal truncation completely abolished translocation, implying that HrpP has an atypical T3SS translocation signal. Deleting more than 20 amino acids from the C terminus abolished the ability to restore HR elicitation. HrpP fused to green fluorescent protein was no longer translocated but could restore HR elicitation activity to the ΔhrpP mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 ΔhrpP except the pilin subunit HrpA, which unexpectedly was secreted poorly. HrpP may function somewhat differently than YscP because the P. syringae T3SS pilus likely varies in length due to differing plant cell walls.

scholarly journals A C-Terminal Domain Targets the Pseudomonas aeruginosa Cytotoxin ExoU to the Plasma Membrane of Host Cells

2006 ◽  
Vol 74 (5) ◽  
pp. 2552-2561 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Jeffrey L. Veesenmeyer ◽  
Kathryn T. Bieging ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Type Iii ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.

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