scholarly journals Functional Characterization of the Type III Secretion Substrate Specificity Switch Protein HpaC from Xanthomonas campestris pv. vesicatoria

2011 ◽  
Vol 79 (8) ◽  
pp. 2998-3011 ◽  
Author(s):  
Steve Schulz ◽  
Daniela Büttner
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Type Iii Secretion ◽  
Protein Function ◽  
Xanthomonas Campestris ◽  
Functional Characterization ◽  
Effector Proteins ◽  
Content Type ◽  
Type Iii ◽  
In The Wild

ABSTRACTPathogenicity ofXanthomonas campestrispv.vesicatoriadepends on a type III secretion (T3S) system which translocates effector proteins into eukaryotic cells and is associated with an extracellular pilus and a translocon in the host plasma membrane. T3S substrate specificity is controlled by the cytoplasmic switch protein HpaC, which interacts with the C-terminal domain of the inner membrane protein HrcU (HrcUC). HpaC promotes the secretion of translocon and effector proteins but prevents the efficient secretion of the early T3S substrate HrpB2, which is required for pilus assembly. In this study, complementation assays with serial 10-amino-acid HpaC deletion derivatives revealed that the T3S substrate specificity switch depends on N- and C-terminal regions of HpaC, whereas amino acids 42 to 101 appear to be dispensable for the contribution of HpaC to the secretion of late substrates. However, deletions in the central region of HpaC affect the secretion of HrpB2, suggesting that the mechanisms underlying HpaC-dependent control of early and late substrates can be uncoupled. The results of interaction and expression studies with HpaC deletion derivatives showed that amino acids 112 to 212 of HpaC provide the binding site for HrcUCand severely reduce T3S when expressed ectopically in the wild-type strain. We identified a conserved phenylalanine residue at position 175 of HpaC that is required for both protein function and the binding of HpaC to HrcUC. Taking these findings together, we concluded that the interaction between HpaC and HrcUCis essential but not sufficient for T3S substrate specificity switching.

scholarly journals The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation

2016 ◽  
Vol 85 (2) ◽  
Author(s):  
Jens Hausner ◽  
Nadine Hartmann ◽  
Michael Jordan ◽  
Daniela Büttner
Keyword(s):  
Type Iii Secretion ◽  
Protein Function ◽  
Xanthomonas Campestris ◽  
Effector Proteins ◽  
Transport Pathway ◽  
Content Type ◽  
Bacterial Membranes ◽  
Type Iii ◽  
Lytic Transglycosylase

ABSTRACT The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which spans both bacterial membranes and translocates effector proteins into plant cells. The assembly of the T3S system presumably involves the predicted lytic transglycosylase (LT) HpaH, which is encoded adjacent to the T3S gene cluster. Bacterial LTs degrade peptidoglycan and often promote the formation of membrane-spanning macromolecular protein complexes. In the present study, we show that HpaH localizes to the bacterial periplasm and binds to peptidoglycan as well as to components of the T3S system, including the predicted periplasmic inner rod proteins HrpB1 and HrpB2 as well as the pilus protein HrpE. In vivo translocation assays revealed that HpaH promotes the translocation of various effector proteins and of early substrates of the T3S system, suggesting a general contribution of HpaH to type III-dependent protein export. Mutant studies and the analysis of reporter fusions showed that the N-terminal region of HpaH contributes to protein function and is proteolytically cleaved. The N-terminally truncated HpaH cleavage product is secreted into the extracellular milieu by a yet-unknown transport pathway, which is independent of the T3S system.

scholarly journals The Periplasmic HrpB1 Protein from Xanthomonas spp. Binds to Peptidoglycan and to Components of the Type III Secretion System

2013 ◽  
Vol 79 (20) ◽  
pp. 6312-6324 ◽  
Author(s):  
Jens Hausner ◽  
Nadine Hartmann ◽  
Christian Lorenz ◽  
Daniela Büttner
Keyword(s):  
Type Iii Secretion ◽  
Protein Function ◽  
Xanthomonas Campestris ◽  
Protein Complexes ◽  
Structural Similarity ◽  
Effector Proteins ◽  
Host Cells ◽  
Content Type ◽  
Type Iii ◽  
Inner Membrane Protein

ABSTRACTThe plant-pathogenic bacteriumXanthomonas campestrispv. vesicatoria employs a type III secretion (T3S) system to translocate bacterial effector proteins into eukaryotic host cells. The membrane-spanning secretion apparatus consists of 11 core components and several associated proteins with yet unknown functions. In this study, we analyzed the role of HrpB1, which was previously shown to be essential for T3S and the formation of the extracellular T3S pilus. We provide experimental evidence that HrpB1 localizes to the bacterial periplasm and binds to peptidoglycan, which is in agreement with its predicted structural similarity to the putative peptidoglycan-binding domain of the lytic transglycosylase Slt70 fromEscherichia coli. Interaction studies revealed that HrpB1 forms protein complexes and binds to T3S system components, including the inner membrane protein HrcD, the secretin HrcC, the pilus protein HrpE, and the putative inner rod protein HrpB2. The analysis of deletion and point mutant derivatives of HrpB1 led to the identification of amino acid residues that contribute to the interaction of HrpB1 with itself and HrcD and/or to protein function. The finding that HrpB1 and HrpB2 colocalize to the periplasm and both interact with HrcD suggests that they are part of a periplasmic substructure of the T3S system.

scholarly journals The Type III Secretion Chaperone HpaB Controls the Translocation of Effector and Noneffector Proteins From Xanthomonas campestris pv. vesicatoria

2018 ◽  
Vol 31 (1) ◽  
pp. 61-74 ◽  
Author(s):  
Felix Scheibner ◽  
Nadine Hartmann ◽  
Jens Hausner ◽  
Christian Lorenz ◽  
Anne-Katrin Hoffmeister ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Type Strain ◽  
Xanthomonas Campestris ◽  
Molecular Mechanisms ◽  
Wild Type Strain ◽  
Effector Proteins ◽  
Wild Type ◽  
Type Iii ◽  
In The Wild ◽  
Secretion Chaperone

Pathogenicity of the gram-negative bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which translocates effector proteins into plant cells. Effector proteins contain N-terminal T3S and translocation signals and interact with the T3S chaperone HpaB, which presumably escorts effectors to the secretion apparatus. The molecular mechanisms underlying the recognition of effectors by the T3S system are not yet understood. In the present study, we analyzed T3S and translocation signals in the type III effectors XopE2 and XopJ from X. campestris pv. vesicatoria. Both effectors contain minimal translocation signals, which are only recognized in the absence of HpaB. Additional N-terminal signals promote translocation of XopE2 and XopJ in the wild-type strain. The results of translocation and interaction studies revealed that the interaction of XopE2 and XopJ with HpaB and a predicted cytoplasmic substrate docking site of the T3S system is not sufficient for translocation. In agreement with this finding, we show that the presence of an artificial HpaB-binding site does not promote translocation of the noneffector XopA in the wild-type strain. Our data, therefore, suggest that the T3S chaperone HpaB not only acts as an escort protein but also controls the recognition of translocation signals.

scholarly journals The Inner Membrane Protein HrcV from Xanthomonas spp. Is Involved in Substrate Docking During Type III Secretion

2013 ◽  
Vol 26 (10) ◽  
pp. 1176-1189 ◽  
Author(s):  
Nadine Hartmann ◽  
Daniela Büttner
Keyword(s):  
Type Iii Secretion ◽  
Protein Function ◽  
Xanthomonas Campestris ◽  
Transmembrane Helix ◽  
Effector Proteins ◽  
Host Cells ◽  
System Component ◽  
Inner Membrane ◽  
Type Iii ◽  
Inner Membrane Protein

Pathogenicity of the gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a membrane-spanning type III secretion (T3S) system, which translocates effector proteins into eukaryotic host cells. In this study, we characterized the T3S system component HrcV, which is a member of the YscV/FlhA family of inner membrane proteins. HrcV consists of eight transmembrane helices and a cytoplasmic region (HrcVC). Mutant and protein–protein interaction studies showed that HrcVC is essential for protein function and binds to T3S substrates, including the early substrate HrpB2, the pilus protein HrpE, and effector proteins. Furthermore, HrcVC interacts with itself and with components and control proteins of the T3S apparatus. The interaction of HrcVC with HrpB2, HrpE, and T3S system components depends on amino acid residues in a conserved motif, designated flagella/hypersensitive response/invasion proteins export pore (FHIPEP), which is located in a cytoplasmic loop between transmembrane helix four and five of HrcV. Mutations in the FHIPEP motif abolish HrcV function but do not affect the interaction of HrcVC with effector proteins.

The YscU/FlhB homologue HrcU from Xanthomonas controls type III secretion and translocation of early and late substrates

Microbiology ◽  
2014 ◽  
Vol 160 (3) ◽  
pp. 576-588 ◽  
Author(s):  
Jens Hausner ◽  
Daniela Büttner
Keyword(s):  
Substrate Specificity ◽  
Type Iii Secretion ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Effector Proteins ◽  
Effector Protein ◽  
Amino Acid Residues ◽  
Type Iii ◽  
Cytoplasmic Region ◽  
Essential Components

The majority of Gram-negative plant- and animal-pathogenic bacteria employ a type III secretion (T3S) system to deliver effector proteins to eukaryotic cells. Members of the YscU protein family are essential components of the T3S system and consist of a transmembrane and a cytoplasmic region that is autocatalytically cleaved at a conserved NPTH motif. YscU homologues interact with T3S substrate specificity switch (T3S4) proteins that alter the substrate specificity of the T3S system after assembly of the secretion apparatus. We previously showed that the YscU homologue HrcU from the plant pathogen Xanthomonas campestris pv. vesicatoria interacts with the T3S4 protein HpaC and is required for the secretion of translocon and effector proteins. In the present study, analysis of HrcU deletion, insertion and point mutant derivatives led to the identification of amino acid residues in the cytoplasmic region of HrcU (HrcUC) that control T3S and translocation of the predicted inner rod protein HrpB2, the translocon protein HrpF and the effector protein AvrBs3. Mutations in the vicinity of the NPTH motif interfered with HrcU cleavage and/or the interaction of HrcUC with HrpB2 and the T3S4 protein HpaC. However, HrcU function was not completely abolished, suggesting that HrcU cleavage is not crucial for pathogenicity and T3S. Given that mutations in HrcU differentially affected T3S and translocation of HrpB2 and effector proteins, we propose that HrcU controls the secretion of different T3S substrate classes by independent mechanisms.

scholarly journals Functional Characterization of the Type III Secretion ATPase HrcN from the Plant Pathogen Xanthomonas campestris pv. vesicatoria

2008 ◽  
Vol 191 (5) ◽  
pp. 1414-1428 ◽  
Author(s):  
Christian Lorenz ◽  
Daniela Büttner
Keyword(s):  
Type Iii Secretion ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Functional Characterization ◽  
Effector Proteins ◽  
Host Cells ◽  
Dependent Manner ◽  
Phosphate Binding ◽  
Type Iii

ABSTRACT Many gram-negative plant and animal pathogenic bacteria employ a type III secretion (T3S) system to inject effector proteins into the cytosol of eukaryotic host cells. The membrane-spanning T3S apparatus is associated with an ATPase that presumably provides the energy for the secretion process. Here, we describe the role of the predicted ATPase HrcN from the plant pathogenic bacterium Xanthomonas campestris pathovar vesicatoria. We show that HrcN hydrolyzes ATP in vitro and is essential for T3S and bacterial pathogenicity. Stability of HrcN in X. campestris pv. vesicatoria depends on the conserved HrcL protein, which interacts with HrcN in vitro and in vivo. Both HrcN and HrcL bind to the inner membrane protein HrcU and specifically localize to the bacterial membranes under T3S-permissive conditions. Protein-protein interaction studies revealed that HrcN also interacts with the T3S substrate specificity switch protein HpaC and the global T3S chaperone HpaB, which promotes secretion of multiple effector proteins. Using an in vitro chaperone release assay, we demonstrate that HrcN dissociates a complex between HpaB and the effector protein XopF1 in an ATP-dependent manner, suggesting that HrcN is involved in the release of HpaB-bound effectors. Effector release depends on a conserved glycine residue in the HrcN phosphate-binding loop, which is crucial for enzymatic activity and protein function during T3S. There is no experimental evidence that T3S can occur in the absence of the ATPase, in contrast to recent findings reported for animal pathogenic bacteria.

scholarly journals RNase E Promotes Expression of Type III Secretion System Genes in Pseudomonas aeruginosa

2019 ◽  
Vol 201 (22) ◽  
Author(s):  
Josh S. Sharp ◽  
Arne Rietsch ◽  
Simon L. Dove
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Rnase E ◽  
Content Type ◽  
Type Iii ◽  
Small Regulatory Rnas

ABSTRACT Pseudomonas aeruginosa is an important opportunistic pathogen that employs a type III secretion system (T3SS) to inject effector proteins into host cells. Using a protein depletion system, we show that the endoribonuclease RNase E positively regulates expression of the T3SS genes. We also present evidence that RNase E antagonizes the expression of genes of the type VI secretion system and limits biofilm production in P. aeruginosa. Thus, RNase E, which is thought to be the principal endoribonuclease involved in the initiation of RNA degradation in P. aeruginosa, plays a key role in controlling the production of factors involved in both acute and chronic stages of infection. Although the posttranscriptional regulator RsmA is also known to positively regulate expression of the T3SS genes, we find that RNase E does not appreciably influence the abundance of RsmA in P. aeruginosa. Moreover, we show that RNase E still exerts its effects on T3SS gene expression in cells lacking all four of the key small regulatory RNAs that function by sequestering RsmA. IMPORTANCE The type III secretion system (T3SS) is a protein complex produced by many Gram-negative pathogens. It is capable of injecting effector proteins into host cells that can manipulate cell metabolism and have toxic effects. Understanding how the T3SS is regulated is important in understanding the pathogenesis of bacteria with such systems. Here, we show that RNase E, which is typically thought of as a global regulator of RNA stability, plays a role in regulating the T3SS in Pseudomonas aeruginosa. Depleting RNase E results in the loss of T3SS gene expression as well as a concomitant increase in biofilm formation. These observations are reminiscent of the phenotypes associated with the loss of activity of the posttranscriptional regulator RsmA. However, RNase E-mediated regulation of these systems does not involve changes in the abundance of RsmA and is independent of the known small regulatory RNAs that modulate RsmA activity.

scholarly journals Control of Type III Secretion System Effector/Chaperone Ratio Fosters Pathogen Adaptation to Host-Adherent Lifestyle

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Netanel Elbaz ◽  
Yaakov Socol ◽  
Naama Katsowich ◽  
Ilan Rosenshine
Keyword(s):  
Gene Expression ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Host Colonization ◽  
Content Type ◽  
Type Iii ◽  
Attached State

ABSTRACT The transition from a planktonic lifestyle to a host-attached state is often critical for bacterial virulence. Upon attachment to host cells, enteropathogenic Escherichia coli (EPEC) employs a type III secretion system (T3SS) to inject into the host cells ∼20 effector proteins, including Tir. CesT, which is encoded from the same operon downstream of tir, is a Tir-bound chaperone that facilitates Tir translocation. Upon Tir translocation, the liberated CesT remains in the bacterial cytoplasm and antagonizes the posttranscriptional regulator CsrA, thus eliciting global regulation in the infecting pathogen. Importantly, tight control of the Tir/CesT ratio is vital, since an uncontrolled surge in free CesT levels may repress CsrA in an untimely manner, thus abrogating colonization. We investigated how fluctuations in Tir translation affect the regulation of this ratio. By creating mutations that cause the premature termination of Tir translation, we revealed that the untranslated tir mRNA becomes highly unstable, resulting in a rapid drop in cesT mRNA levels and, thus, CesT levels. This mechanism couples Tir and CesT levels to ensure a stable Tir/CesT ratio. Our results expose an additional level of regulation that enhances the efficacy of the initial interaction of EPEC with the host cell, providing a better understanding of the bacterial switch from the planktonic to the cell-adherent lifestyle. IMPORTANCE Host colonization by extracellular pathogens often entails the transition from a planktonic lifestyle to a host-attached state. Enteropathogenic E. coli (EPEC), a Gram-negative pathogen, attaches to the intestinal epithelium of the host and employs a type III secretion system (T3SS) to inject effector proteins into the cytoplasm of infected cells. The most abundant effector protein injected is Tir, whose translocation is dependent on the Tir-bound chaperon CesT. Upon Tir injection, the liberated CesT binds to and inhibits the posttranscriptional regulator CsrA, resulting in reprogramming of gene expression in the host-attached bacteria. Thus, adaptation to the host-attached state involves dynamic remodeling of EPEC gene expression, which is mediated by the relative levels of Tir and CesT. Fluctuating from the optimal Tir/CesT ratio results in a decrease in EPEC virulence. Here we elucidate a posttranscriptional circuit that prevents sharp variations from this ratio, thus improving host colonization.

scholarly journals Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions

2011 ◽  
Vol 79 (8) ◽  
pp. 3036-3045 ◽  
Author(s):  
B. Chellas-Géry ◽  
K. Wolf ◽  
J. Tisoncik ◽  
T. Hackstadt ◽  
K. A. Fields
Keyword(s):  
Type Iii Secretion ◽  
De Novo ◽  
Ectopic Expression ◽  
Effector Proteins ◽  
Developmental Cycle ◽  
Inclusion Membrane ◽  
Content Type ◽  
Type Iii ◽  
Intracellular Parasites ◽  
The Many

ABSTRACTChlamydiaspp. are among the many pathogenic Gram-negative bacteria that employ a type III secretion system (T3SS) to overcome host defenses and exploit available resources. Significant progress has been made in elucidating contributions of T3S to the pathogenesis of these medically important, obligate intracellular parasites, yet important questions remain. Chief among these is how secreted effector proteins traverse eukaryotic membranes to gain access to the host cytosol. Due to a complex developmental cycle, it is possible that chlamydiae utilize a different complement of proteins to accomplish translocation at different stages of development. We investigated this possibility by extending the characterization ofC. trachomatisCopB and CopB2. CopB is detected early during infection but is depleted and not detected again until about 20 h postinfection. In contrast, CopB2 was detectible throughout development. CopB is associated with the inclusion membrane. Biochemical and ectopic expression analyses were consistent with peripheral association of CopB2 with inclusion membranes. This interaction correlated with development and required both chlamydialde novoprotein synthesis and T3SS activity. Collectively, our data indicate that it is unlikely that CopB serves as the sole chlamydial translocation pore and that CopB2 is capable of association with the inclusion membrane.

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.

Sign in / Sign up

Export Citation Format

Share Document