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

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.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-13-0019-r ◽

2013 ◽

Vol 26 (10) ◽

pp. 1176-1189 ◽

Cited By ~ 22

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.

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

Infection and Immunity ◽

10.1128/iai.00788-16 ◽

2016 ◽

Vol 85 (2) ◽

Cited By ~ 10

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.

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

Infection and Immunity ◽

10.1128/iai.00180-11 ◽

2011 ◽

Vol 79 (8) ◽

pp. 2998-3011 ◽

Cited By ~ 14

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.

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

Journal of Bacteriology ◽

10.1128/jb.00336-19 ◽

2019 ◽

Vol 201 (22) ◽

Cited By ~ 3

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.

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

mBio ◽

10.1128/mbio.02074-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 3

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.

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Dynamics of the Type III Secretion System Activity of Enteropathogenic Escherichia coli

mBio ◽

10.1128/mbio.00303-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 37

Author(s):

Erez Mills ◽

Kobi Baruch ◽

Gili Aviv ◽

Mor Nitzan ◽

Ilan Rosenshine

Keyword(s):

Escherichia Coli ◽

Type Iii Secretion ◽

Public Health Problem ◽

Effector Proteins ◽

Host Cells ◽

Global Public Health ◽

Secretion Systems ◽

Comprehensive Description ◽

Content Type ◽

Type Iii

ABSTRACT Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics. IMPORTANCE EPEC and the closely related enterohemorrhagic Escherichia coli (EHEC) represent a global public health problem. New strategies to combat EPEC and EHEC infections are needed, and development of such strategies requires better understanding of their virulence machinery. The TTSS is a critical virulence mechanism employed by these pathogens, and by others, including Salmonella. In this study, we aimed at elucidating new aspects of TTSS function. The results obtained provide a comprehensive description of the dynamics of TTSS activity of EPEC and new insights into the mechanisms that control these changes. This knowledge sets the stage for further analysis of the system and may accelerate the development of new ways to treat EPEC and EHEC infections. Further, the newly described microscopy-based assay can be readily adapted to study the dynamics of TTSS activity in other pathogens.

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Cytosporone B, an Inhibitor of the Type III Secretion System of Salmonella enterica Serovar Typhimurium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02421-12 ◽

2013 ◽

Vol 57 (5) ◽

pp. 2191-2198 ◽

Cited By ~ 40

Author(s):

Jianfang Li ◽

Chao Lv ◽

Weiyang Sun ◽

Zhenyu Li ◽

Xiaowei Han ◽

...

Keyword(s):

Type Iii Secretion ◽

Salmonella Enterica ◽

Bacterial Resistance ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Content Type ◽

Type Iii ◽

Serovar Typhimurium

ABSTRACTBacterial virulence factors have been increasingly regarded as attractive targets for development of novel antibacterial agents. Virulence inhibitors are less likely to generate bacterial resistance, which makes them superior to traditional antibiotics that target bacterial viability.Salmonella entericaserovar Typhimurium, an important food-borne human pathogen, has type III secretion system (T3SS) as its major virulence factor. T3SS secretes effector proteins to facilitate invasion into host cells. In this study, we identified several analogs of cytosporone B (Csn-B) that strongly block the secretion ofSalmonellapathogenicity island 1 (SPI-1)-associated effector proteins, without affecting the secretion of flagellar protein FliCin vitro. Csn-B and two other derivatives exhibited a strong inhibitory effect on SPI-1-mediated invasion to HeLa cells, while no significant toxicity to bacteria was observed. Nucleoid proteins Hha and H-NS bind to the promoters of SPI-1 regulator geneshilD,hilC, andrtsAto repress their expression and consequently regulate the expression of SPI-1 apparatus and effector genes. We found that Csn-B upregulated the transcription ofhhaandhns, implying that Csn-B probably affected the secretion of effectors through the Hha–H-NS regulatory pathway. In summary, this study presented an effective SPI-1 inhibitor, Csn-B, which may have potential in drug development against antibiotic-resistantSalmonella.

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Type III Secretion System Translocon Component EseB Forms Filaments on and Mediates Autoaggregation of and Biofilm Formation by Edwardsiella tarda

Applied and Environmental Microbiology ◽

10.1128/aem.01254-15 ◽

2015 ◽

Vol 81 (17) ◽

pp. 6078-6087 ◽

Cited By ~ 23

Author(s):

Zhi Peng Gao ◽

Pin Nie ◽

Jin Fang Lu ◽

Lu Yi Liu ◽

Tiao Yi Xiao ◽

...

Keyword(s):

Type Iii Secretion ◽

Biofilm Formation ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Edwardsiella Tarda ◽

Dependent Manner ◽

Content Type ◽

Type Iii

ABSTRACTThe type III secretion system (T3SS) ofEdwardsiella tardaplays an important role in infection by translocating effector proteins into host cells. EseB, a component required for effector translocation, is reported to mediate autoaggregation ofE. tarda. In this study, we demonstrate that EseB forms filamentous appendages on the surface ofE. tardaand is required for biofilm formation byE. tardain Dulbecco's modified Eagle's medium (DMEM). Biofilm formation byE. tardain DMEM does not require FlhB, an essential component for assembling flagella. Dynamic analysis of EseB filament formation, autoaggregation, and biofilm formation shows that the formation of EseB filaments occurs prior to autoaggregation and biofilm formation. The addition of an EseB antibody toE. tardacultures before bacterial autoaggregation prevents autoaggregation and biofilm formation in a dose-dependent manner, whereas the addition of the EseB antibody toE. tardacultures in which biofilm is already formed does not destroy the biofilm. Therefore, EseB filament-mediated bacterial cell-cell interaction is a prerequisite for autoaggregation and biofilm formation.

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Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins

mBio ◽

10.1128/mbio.00769-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 9

Author(s):

Vera Göser ◽

Carina Kommnick ◽

Viktoria Liss ◽

Michael Hensel

Keyword(s):

Type Iii Secretion ◽

Effector Proteins ◽

Host Cells ◽

Secretion Systems ◽

Content Type ◽

Superresolution Microscopy ◽

Type Iii ◽

T3ss Effector ◽

Novel Approach ◽

Temporal And Spatial

ABSTRACTType III secretion systems (T3SS) are molecular machines in Gram-negative pathogens that translocate effector proteins with central roles in virulence. The analyses of the translocation, subcellular localization, and mode of action of T3SS effector proteins are of central importance for the understanding of host-pathogen interaction and pathogenesis of bacterial infections. The analysis of translocation requires dedicated techniques to address the temporal and spatial dynamics of translocation. Here we describe a novel approach to deploy self-labeling enzymes (SLE) as universal tags for localization and tracking of translocated effector proteins. Effector-SLE fusion proteins allow live-cell imaging of translocation by T3SS, superresolution microscopy, and single-molecule tracking of effector motility in living host cells. We describe the application of the approach to T3SS effector proteins for invasion and intracellular lifestyle ofSalmonella entericaserovar Typhimurium and to a T3SS effector ofYersinia enterocolitica. The novel approach enables analyses of the role of T3SS in host-pathogen interaction at the highest temporal and spatial resolution, toward understanding the molecular mechanisms of their effector proteins.IMPORTANCEType III secretion systems mediate translocation of effector proteins into mammalian cells. These proteins interfere with host cell functions, being main virulence factors of Gram-negative pathogens. Analyses of the process of translocation, the subcellular distribution, and the dynamics of effector proteins in host cells have been hampered by the lack of suitable tags and detection systems. Here we describe the use of self-labeling enzyme tags for generation of fusions with effector proteins that are translocated and functional in host cell manipulation. Self-labeling reactions with cell-permeable ligand dyes are possible prior to or after translocation. We applied the new approach to superresolution microscopy for effector protein translocation. For the first time, we show the dynamic properties of effector proteins in living host cells after translocation by intracellular bacteria. The new approach of self-labeling enzyme tags fusions will enable analyses of type III secretion system effector proteins with new dimensions of temporal and spatial resolution.

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The ADP-Ribosyltransferase Domain of the Effector Protein ExoS Inhibits Phagocytosis of Pseudomonas aeruginosa during Pneumonia

mBio ◽

10.1128/mbio.01080-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 22

Author(s):

Stephanie M. Rangel ◽

Latania K. Logan ◽

Alan R. Hauser

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Severe Disease ◽

Effector Proteins ◽

Host Cells ◽

Gtpase Activating Protein ◽

Protein Activity ◽

Content Type ◽

Type Iii ◽

Adp Ribosyltransferase

ABSTRACTPseudomonas aeruginosais a Gram-negative pathogen commonly associated with nosocomial infections such as hospital-acquired pneumonia. It uses a type III secretion system to deliver effector proteins directly into the cytosol of host cells. Type III secretion inP. aeruginosahas been linked to severe disease and worse clinical outcomes in animal and human studies. The majority ofP. aeruginosastrains secrete ExoS, a bifunctional toxin with GTPase-activating protein and ADP-ribosyltransferase activities. Numerousin vitrostudies have investigated the targets and cellular effects of ExoS, linking both its enzymatic activities with inhibition of bacterial internalization. However, little is known about how this toxin facilitates the progression of infectionin vivo. In this study, we used a mouse model to investigate the role of ExoS in inhibiting phagocytosis during pneumonia. We first confirmed previous findings that the ADP-ribosyltransferase activity of ExoS, but not the GTPase-activating protein activity, was responsible for bacterial persistence and decreased host survival in this model. We then used two distinct assays to demonstrate that ExoS inhibited phagocytosis during pneumonia. In contrast to the findings of severalin vitrostudies, thisin vivoinhibition was also dependent on the ADP-ribosyltransferase activity, but not the GTPase-activating protein activity, of ExoS. These results demonstrate for the first time the antiphagocytic function of ExoS in the context of an actual infection and indicate that blocking the ADP-ribosyltransferase activity of ExoS may have potential therapeutic benefit.IMPORTANCEPseudomonas aeruginosais a major cause of hospital-acquired infections. To cause severe disease, this bacterium uses a type III secretion system that delivers four effector proteins, ExoS, ExoT, ExoU, and ExoY, into host cells. The majority ofP. aeruginosastrains secrete ExoS, a bifunctional toxin with GTPase-activating protein and ADP-ribosyltransferase activities. In cell culture models, both enzymatic activities have been associated with decreased bacterial internalization. However, our study is the first to examine a role for ExoS in blocking phagocytosis in an animal model. We report that ExoS does inhibit phagocytosis during pneumonia. The ADP-ribosyltransferase activity, but not the GTPase-activating protein activity, of ExoS is necessary for this effect. Our findings highlight the ability ofP. aeruginosato manipulate the inflammatory response during pneumonia to facilitate bacterial survival.

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