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1.
A remarkable feature of the flagellar‐specific type III secretion system (T3SS) is the selective recognition of a few substrate proteins among the many thousand cytoplasmic proteins. Secretion substrates are divided into two specificity classes: early substrates secreted for hook‐basal body (HBB) construction and late substrates secreted after HBB completion. Secretion was reported to require a disordered N‐terminal secretion signal, mRNA secretion signals within the 5′‐untranslated region (5′‐UTR) and for late substrates, piloting proteins known as the T3S chaperones. Here, we utilized translational β‐lactamase fusions to probe the secretion efficacy of the N‐terminal secretion signal of fourteen secreted flagellar substrates in Salmonella enterica. We observed a surprising variety in secretion capability between flagellar proteins of the same secretory class. The peptide secretion signals of the early‐type substrates FlgD, FlgF, FlgE and the late‐type substrate FlgL were analysed in detail. Analysing the role of the 5′‐UTR in secretion of flgB and flgE revealed that the native 5′‐UTR substantially enhanced protein translation and secretion. Based on our data, we propose a multicomponent signal that drives secretion via the flagellar T3SS. Both mRNA and peptide signals are recognized by the export apparatus and together with substrate‐specific chaperones allowing for targeted secretion of flagellar substrates. 相似文献
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3.
The cytoplasmic C‐ring of the flagellum consists of FliG, FliM and FliN and acts as an affinity cup to localize secretion substrates for protein translocation via the flagellar‐specific type III secretion system. Random T‐POP transposon mutagenesis was employed to screen for insertion mutants that allowed flagellar type III secretion in the absence of the C‐ring using the flagellar type III secretion system‐specific hook–β‐lactamase reporter ( Lee and Hughes, 2006 ). Any condition resulting in at least a twofold increase in flhDC expression was sufficient to overcome the requirement for the C‐ring and the ATPase complex FliHIJ in flagellar type III secretion. Insertions in known and unknown flagellar regulatory loci were isolated as well as chromosomal duplications of the flhDC region. The twofold increased flhDC mRNA level coincided in a twofold increase in the number of hook‐basal bodies per cell as analysed by fluorescent microscopy. These results indicate that the C‐ring functions as a nonessential affinity cup‐like structure during flagellar type III secretion to enhance the specificity and efficiency of the secretion process. 相似文献
4.
The bacterial flagellum is assembled from over 20 structural components, and flagellar gene regulation is morphogenetically coupled to the assembly state by control of the anti-sigma factor FlgM. In the Gram-negative bacterium Salmonella enterica, FlgM inhibits late-class flagellar gene expression until the hook-basal body structural intermediate is completed and FlgM is inhibited by secretion from the cytoplasm. Here we demonstrate that FlgM is also secreted in the Gram-positive bacterium Bacillus subtilis and is degraded extracellularly by the proteases Epr and WprA. We further demonstrate that, like in S. enterica, the structural genes required for the flagellar hook-basal body are required for robust activation of σD-dependent gene expression and efficient secretion of FlgM. Finally, we determine that FlgM secretion is strongly enhanced by, but does not strictly require, hook-basal body completion and instead demands a minimal subset of flagellar proteins that includes the FliF/FliG basal body proteins, the flagellar type III export apparatus components FliO, FliP, FliQ, FliR, FlhA, and FlhB, and the substrate specificity switch regulator FliK. 相似文献
5.
Natural variation in the presence or the absence of STM0517-0529 genes allowing allantoin utilisation has been described in
field isolates of the multidrug resistant Salmonella enterica serovar Typhimurium belonging to the phage type DT104. Interestingly, S. enterica subspecies enterica serovar Typhimurium DT104 is quite frequent in pigs and cattle, but rarely present in egg-laying hens. Taking into account
the different mode of allantoin metabolism in birds and mammals, we were interested in whether the absence of STM0517-0529
genes may disable this clone in poultry colonisation. We have therefore constructed the allB (also designated as STM0523) mutants in S. enterica subspecies enterica serovar Typhimurium and S. enterica subspecies enterica serovar Enteritidis, and with these, we infected mice, newly hatched chickens and adult egg-laying hens to show that the
defect in allantoin utilisation does not influence S. enterica virulence for mice or adult hens, but slightly decreases virulence of S. enterica for chickens. The decrease in virulence of the allB mutant was relatively minor as it could be observed only after a mixed infection model, consistent with a lower prevalence,
but not a total absence of such clones in poultry flocks. 相似文献
6.
Yukio Furukawa Yumi Inoue Aya Sakaguchi Yoko Mori Takuma Fukumura Tomoko Miyata Keiichi Namba Tohru Minamino 《Molecular microbiology》2016,102(3):405-416
FliS chaperone binds to flagellin FliC in the cytoplasm and transfers FliC to a sorting platform of the flagellar type III export apparatus through the interaction between FliS and FlhA for rapid and efficient protein export during flagellar filament assembly. FliS also suppresses the secretion of an anti‐σ factor, FlgM. Loss of FliS results in a short filament phenotype although the expression levels of FliC are increased considerably due to an increase in the secretion level of FlgM. Here to clarify the rate limiting step of FliC export in the absence of FliS, we isolated bypass mutants from a Salmonella ΔfliS mutant. All the bypass mutations were identified in FliC. These bypass mutations increased the export rate of FliC by ca. twofold, allowing the bypass mutant cells to produce longer filaments than the parental ΔfliS cells. Both far‐UV CD measurements and limited proteolysis revealed that the bypass mutations significantly destabilize the folded structure of FliC monomer. These results suggest that an unfolding step of FliC limits the export rate of FliC in the ΔfliS mutant, thereby producing short filaments. We propose that FliS promotes FliC docking at the FlhA platform to facilitate subsequent unfolding of FliC. 相似文献
7.
Lynne S. Cairns Victoria L. Marlow Taryn B. Kiley Christopher Birchall Adam Ostrowski Phillip D. Aldridge Nicola R. Stanley-Wall 《Journal of bacteriology》2014,196(12):2216-2226
The assembly of the bacterial flagellum is exquisitely controlled. Flagellar biosynthesis is underpinned by a specialized type III secretion system that allows export of proteins from the cytoplasm to the nascent structure. Bacillus subtilis regulates flagellar assembly using both conserved and species-specific mechanisms. Here, we show that YvyG is essential for flagellar filament assembly. We define YvyG as an orthologue of the Salmonella enterica serovar Typhimurium type III secretion system chaperone, FlgN, which is required for the export of the hook-filament junction proteins, FlgK and FlgL. Deletion of flgN (yvyG) results in a nonmotile phenotype that is attributable to a decrease in hag translation and a complete lack of filament polymerization. Analyses indicate that a flgK-flgL double mutant strain phenocopies deletion of flgN and that overexpression of flgK-flgL cannot complement the motility defect of a ΔflgN strain. Furthermore, in contrast to previous work suggesting that phosphorylation of FlgN alters its subcellular localization, we show that mutation of the identified tyrosine and arginine FlgN phosphorylation sites has no effect on motility. These data emphasize that flagellar biosynthesis is differentially regulated in B. subtilis from classically studied Gram-negative flagellar systems and questions the biological relevance of some posttranslational modifications identified by global proteomic approaches. 相似文献
8.
Claudia Hoffmann Marlies Galle Sabrina Dilling Rina K?ppeli Andreas J. Müller Pascal Songhet Rudi Beyaert Wolf-Dietrich Hardt 《PloS one》2010,5(8)
The innate immune system is of vital importance for protection against infectious pathogens. Inflammasome mediated caspase-1 activation and subsequent release of pro-inflammatory cytokines like IL-1β and IL-18 is an important arm of the innate immune system. Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium, SL1344) is an enteropathogenic bacterium causing diarrheal diseases. Different reports have shown that in macrophages, S. Typhimurium may activate caspase-1 by at least three different types of stimuli: flagellin, the type III secretion system 1 (T1) and the T1 effector protein SopE. However, the relative importance and interdependence of the different factors in caspase-1 activation is still a matter of debate. Here, we have analyzed their relative contributions to caspase-1 activation in LPS-pretreated RAW264.7 macrophages. Using flagellar mutants (fliGHI, flgK) and centrifugation to mediate pathogen-host cell contact, we show that flagellins account for a small part of the caspase-1 activation in RAW264.7 cells. In addition, functional flagella are of key importance for motility and host cell attachment which is a prerequisite for mediating caspase-1 activation via these three stimuli. Using site directed mutants lacking several T1 effector proteins and flagellin expression, we found that SopE elicits caspase-1 activation even when flagellins are absent. In contrast, disruption of essential genes of the T1 protein injection system (invG, sipB) completely abolished caspase-1 activation. However, a robust level of caspase-1 activation is retained by the T1 system (or unidentified T1 effectors) in the absence of flagellin and SopE. T1-mediated inflammasome activation is in line with recent work by others and suggests that the T1 system itself may represent the basic caspase-1 activating stimulus in RAW264.7 macrophages which is further enhanced independently by SopE and/or flagellin. 相似文献
9.
Isabel Martinez-Argudo Andreas K. J. Veenendaal Xia Liu A. Dorothea Roehrich Maria C. Ronessen Giulia Franzoni Katerine N. van Rietschoten Yusuke V. Morimoto Yumiko Saijo-Hamano Matthew B. Avison David J. Studholme Keiichi Namba Tohru Minamino Ariel J. Blocker 《PloS one》2013,8(1)
Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB
(P28T) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors'' molecular target(s) using motility assays in the ΔfliH-fliI/flhB
(P28T) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the FOF1-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18''s phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification. 相似文献
10.
Compromised outer membrane integrity in Vibrio cholerae Type II secretion mutants 总被引:1,自引:0,他引:1
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The type II secretion (T2S) system of Vibrio cholerae is a multiprotein complex that spans the cell envelope and secretes proteins important for pathogenesis as well as survival in different environments. Here we report that, in addition to the loss of extracellular secretion, removal or inhibition of expression of the T2S genes, epsC-N, results in growth defects and a broad range of alterations in the outer membrane that interfere with its barrier function. Specifically, the sensitivity to membrane-perturbing agents such as bile salts and the antimicrobial peptide polymyxin B is increased, and periplasmic constituents leak out into the culture medium. As a consequence, the σE stress response is induced. Furthermore, due to the defects caused by inactivation of the T2S system, the Δeps deletion mutant of V. cholerae strain N16961 is incapable of surviving the passage through the infant mouse gastrointestinal tract. The growth defect and leaky outer membrane phenotypes are suppressed when the culture medium is supplemented with 5% glucose or sucrose, although the eps mutants remain sensitive to membrane-damaging agents. This suggests that the sugars do not restore the integrity of the outer membrane in the eps mutant strains per se but may provide osmoprotective functions. 相似文献
11.
Kane PM 《Journal of bioenergetics and biomembranes》2007,39(5-6):415-421
V-ATPases are structurally conserved and functionally versatile proton pumps found in all eukaryotes. The yeast V-ATPase has
emerged as a major model system, in part because yeast mutants lacking V-ATPase subunits (vma mutants) are viable and exhibit a distinctive Vma- phenotype. Yeast vma mutants are present in ordered collections of all non-essential yeast deletion mutants, and a number of additional phenotypes
of these mutants have emerged in recent years from genomic screens. This review summarizes the many phenotypes that have been
associated with vma mutants through genomic screening. The results suggest that V-ATPase activity is important for an unexpectedly wide range
of cellular processes. For example, vma mutants are hypersensitive to multiple forms of oxidative stress, suggesting an antioxidant role for the V-ATPase. Consistent
with such a role, vma mutants display oxidative protein damage and elevated levels of reactive oxygen species, even in the absence of an exogenous
oxidant. This endogenous oxidative stress does not originate at the electron transport chain, and may be extra-mitochondrial,
perhaps linked to defective metal ion homeostasis in the absence of a functional V-ATPase. Taken together, genomic data indicate
that the physiological reach of the V-ATPase is much longer than anticipated. Further biochemical and genetic dissection is
necessary to distinguish those physiological effects arising directly from the enzyme’s core functions in proton pumping and
organelle acidification from those that reflect broader requirements for cellular pH homeostasis or alternative functions
of V-ATPase subunits. 相似文献
12.
PAUL A. LEFEBVRE SARA-ELLEN BARSEL DANIEL E. WEXLER 《The Journal of eukaryotic microbiology》1988,35(4):559-564
Amputating the flagella of Chlamydomonas reinhardtii stimulates increased synthesis of many flagellar proteins within 30 min. We have isolated a series of mutants which are defective in this stimulation, taking advantage of the fact that cells which cannot stimulate flagellar protein synthesis cannot regenerate flagella. More than a dozen mutants which have flagella, but cannot regenerate them after amputation, were isolated and studied by in vivo labeling to identify those non-regenerator mutants which were specifically defective in the induction of flagellar protein synthesis. Ten such mutants have been identified, and in each of them flagellar amputation does not stimulate the synthesis of any of the major flagellar proteins. At least four of the mutants display an interesting conditional phenotype. The synthesis of flagellar proteins after deflagellation is defective only in gametic cells; vegetative cells of these mutants are capable of flagellar protein synthesis after flagellar amputation. 相似文献
13.
Type-III protein secretion systems are utilized by gram-negative pathogens to secrete building blocks of the bacterial flagellum, virulence effectors from the cytoplasm into host cells, and structural subunits of the needle complex. The flagellar type-III secretion apparatus utilizes both the energy of the proton motive force and ATP hydrolysis to energize substrate unfolding and translocation. We report formation of functional flagella in the absence of type-III ATPase activity by mutations that increased the proton motive force and flagellar substrate levels. We additionally show that increased proton motive force bypassed the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion. Our data support a role for type-III ATPases in enhancing secretion efficiency under limited secretion substrate concentrations and reveal the dispensability of ATPase activity in the type-III protein export process. 相似文献
14.
Daofeng Qu Mengxue Jiang Calder Duffin Kelly T. Hughes Fabienne F. V. Chevance 《PLoS genetics》2022,18(7)
The Salmonella flagellar secretion apparatus is a member of the type III secretion (T3S) family of export systems in bacteria. After completion of the flagellar motor structure, the hook-basal body (HBB), the flagellar T3S system undergoes a switch from early to late substrate secretion, which results in the expression and assembly of the external, filament propeller-like structure. In order to characterize early substrate secretion-signals in the flagellar T3S system, the FlgB, and FlgC components of the flagellar rod, which acts as the drive-shaft within the HBB, were subject to deletion mutagenesis to identify regions of these proteins that were important for secretion. The β-lactamase protein lacking its Sec-dependent secretion signal (Bla) was fused to the C-terminus of FlgB and FlgC and used as a reporter to select for and quantify the secretion of FlgB and FlgC into the periplasm. Secretion of Bla into the periplasm confers resistance to ampicillin. In-frame deletions of amino acids 9 through 18 and amino acids 39 through 58 of FlgB decreased FlgB secretion levels while deleting amino acid 6 through 14 diminished FlgC secretion levels. Further PCR-directed mutagenesis indicated that amino acid F45 of FlgB was critical for secretion. Single amino acid mutagenesis revealed that all amino acid substitutions at F45 of FlgB position impaired rod assembly, which was due to a defect of FlgB secretion. An equivalent F49 position in FlgC was essential for assembly but not for secretion. This study also revealed that a hydrophobic patch in the cleaved C-terminal domain of FlhB is critical for recognition of FlgB at F45. 相似文献
15.
Joyce E. Karlinsey Michael E. Maguire Lynne A. Becker Marie‐Laure V. Crouch Ferric C. Fang 《Molecular microbiology》2010,78(3):669-685
The phage shock protein (Psp) system is induced by extracytoplasmic stress and thought to be important for the maintenance of proton motive force. We investigated the contribution of PspA to Salmonella virulence. A pspA deletion mutation significantly attenuates the virulence of Salmonella enterica serovar Typhimurium following intraperitoneal inoculation of C3H/HeN (Ityr) mice. PspA was found to be specifically required for virulence in mice expressing the natural resistance‐associated macrophage protein 1 (Nramp1) (Slc11a1) divalent metal transporter, which restricts microbial growth by limiting the availability of essential divalent metals within the phagosome. Salmonella competes with Nramp1 by expressing multiple metal uptake systems including the Nramp‐homologue MntH, the ABC transporter SitABCD and the ZIP family transporter ZupT. PspA was found to facilitate Mn2+ transport by MntH and SitABCD, as well as Zn2+ and Mn2+ transport by ZupT. In vitro uptake of 54Mn2+ by MntH and ZupT was reduced in the absence of PspA. Transport‐deficient mutants exhibit reduced viability in the absence of PspA when grown under metal‐limited conditions. Moreover, the ZupT transporter is required for Salmonella enterica serovar Typhimurium virulence in Nramp1‐expressing mice. We propose that PspA promotes Salmonella virulence by maintaining proton motive force, which is required for the function of multiple transporters mediating bacterial divalent metal acquisition during infection. 相似文献
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Sebastian E. Winter Maria G. Winter Victor Poon A. Marijke Keestra Torsten Sterzenbach Franziska Faber Luciana F. Costa Fabiane Cassou Erica A. Costa Geraldo E. S. Alves Tatiane A. Paix?o Renato L. Santos Andreas J. B?umler 《PLoS pathogens》2014,10(7)
Delivery of microbial products into the mammalian cell cytosol by bacterial secretion systems is a strong stimulus for triggering pro-inflammatory host responses. Here we show that Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, tightly regulates expression of the invasion-associated type III secretion system (T3SS-1) and thus fails to activate these innate immune signaling pathways. The S. Typhi regulatory protein TviA rapidly repressed T3SS-1 expression, thereby preventing RAC1-dependent, RIP2-dependent activation of NF-κB in epithelial cells. Heterologous expression of TviA in S. enterica serovar Typhimurium (S. Typhimurium) suppressed T3SS-1-dependent inflammatory responses generated early after infection in animal models of gastroenteritis. These results suggest that S. Typhi reduces intestinal inflammation by limiting the induction of pathogen-induced processes through regulation of virulence gene expression. 相似文献
18.
Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition. Here we show that flagellar basal bodies fail to proceed to hook assembly at high frequency in the absence of the monotopic protein SwrB of Bacillus subtilis. Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP. Furthermore, mutants defective in the flagellar C-ring phenocopy the absence of SwrB for reduced hook frequency and C-ring defects may be bypassed either by SwrB overexpression or by a gain-of-function allele in the polymerization domain of FliG. We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize. 相似文献
19.
The flagellar-specific anti-sigma factor, FlgM, inhibits the expression of late flagellar genes until the hook–basal body structure is assembled and competent for export of the flagellins and hook-associated proteins (flagellar late proteins). FlgM monitors this assembly checkpoint by being a substrate for export via the hook–basal body structure, which includes a type III protein secretion complex. Amino acid sequence alignment of late-secreted flagellar proteins identified a region of homology present in the amino-terminus of FlgM and the other late flagellar proteins, but not in flagellar proteins secreted earlier during flagellar biosynthesis. Single amino acid substitutions at specific positions within this motif decreased the export of FlgM. Deletion of this region (S3-P11) resulted in lower intracellular FlgM levels, but did not prevent recognition and export by the flagellar-specific secretion system. Mutations were isolated in a second region of FlgM spanning residues K27 to A65 that exhibited increased anti-σ28 activity. These FlgM 'hyperinhibitor' mutants were secreted less than wild-type FlgM. Mutations that interfere with the secretion of FlgM without abolishing anti-σ28 activity have a negative effect upon the secretion of a His-tagged FlgM mutant that lacks anti-σ28 activity. Models are proposed to explain the dominant negative phenotype of the FlgM secretion mutants reported in this study. 相似文献
20.
Aurélien Michau Ghislaine Guillemain Alexandra Grosfeld Sandrine Vuillaumier-Barrot Teddy Grand Mathilde Keck Sébastien L'Hoste Danielle Chateau Patricia Serradas Jacques Teulon Pascale De Lonlay Rapha?l Scharfmann Edith Brot-Laroche Armelle Leturque Maude Le Gall 《The Journal of biological chemistry》2013,288(43):31080-31092
The structure-function relationships of sugar transporter-receptor hGLUT2 coded by SLC2A2 and their impact on insulin secretion and β cell differentiation were investigated through the detailed characterization of a panel of mutations along the protein. We studied naturally occurring SLC2A2 variants or mutants: two single-nucleotide polymorphisms and four proposed inactivating mutations associated to Fanconi-Bickel syndrome. We also engineered mutations based on sequence alignment and conserved amino acids in selected domains. The single-nucleotide polymorphisms P68L and T110I did not impact on sugar transport as assayed in Xenopus oocytes. All the Fanconi-Bickel syndrome-associated mutations invalidated glucose transport by hGLUT2 either through absence of protein at the plasma membrane (G20D and S242R) or through loss of transport capacity despite membrane targeting (P417L and W444R), pointing out crucial amino acids for hGLUT2 transport function. In contrast, engineered mutants were located at the plasma membrane and able to transport sugar, albeit with modified kinetic parameters. Notably, these mutations resulted in gain of function. G20S and L368P mutations increased insulin secretion in the absence of glucose. In addition, these mutants increased insulin-positive cell differentiation when expressed in cultured rat embryonic pancreas. F295Y mutation induced β cell differentiation even in the absence of glucose, suggesting that mutated GLUT2, as a sugar receptor, triggers a signaling pathway independently of glucose transport and metabolism. Our results describe the first gain of function mutations for hGLUT2, revealing the importance of its receptor versus transporter function in pancreatic β cell development and insulin secretion. 相似文献