scholarly journals Bcr4 is a Chaperone for the Inner Rod Protein in the Bordetella Type III Secretion System

2021 ◽  
Author(s):  
Masataka Goto ◽  
Akio Abe ◽  
Tomoko Hanawa ◽  
Asaomi Kuwae
Keyword(s):  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Sequence Similarity ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Virulence Proteins ◽  
Type Iii ◽  
Bacterial Proteins ◽  
Small Proteins

Bordetella bronchiseptica injects virulence proteins called effectors into host cells via a type III secretion system (T3SS) conserved among many Gram-negative bacteria. Small proteins called chaperones are required for stabilizing some T3SS components or localizing them to the T3SS machinery. In a previous study, we identified a chaperone-like protein named Bcr4 that regulates T3SS activity in B. bronchiseptica. Bcr4 does not show strong sequence similarity to well-studied T3SS proteins of other bacteria, and its function remains to be elucidated. Here, we investigated the mechanism by which Bcr4 controls T3SS activity. A pull-down assay revealed that Bcr4 interacts with BscI, based on its homology to other bacterial proteins, to be an inner rod protein of the T3SS machinery. A pull-down assay using truncated Bcr4 derivatives and secretion profiles of B. bronchiseptica producing truncated Bcr4 derivatives showed that the Bcr4 C-terminal region is necessary to interact with BscI and to activate the T3SS. Moreover, the deletion of BscI abolished the secretion of type III secreted proteins from B. bronchiseptica and the translocation of a cytotoxic effector into cultured mammalian cells. Finally, we showed that BscI is unstable in the absence of Bcr4. These results suggest that Bcr4 supports the construction of the T3SS machinery by stabilizing BscI. This is the first demonstration of a chaperone for the T3SS inner rod protein among the virulence bacteria possessing the T3SS.

scholarly journals Transcriptional Induction of the Pseudomonas aeruginosa Type III Secretion System by Low Ca2+ and Host Cell Contact Proceeds through Two Distinct Signaling Pathways

2006 ◽  
Vol 74 (6) ◽  
pp. 3334-3341 ◽  
Author(s):  
Nandini Dasgupta ◽  
Alix Ashare ◽  
Gary W. Hunninghake ◽  
Timothy L. Yahr
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Host Cell ◽  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Type Iii Secretion System ◽  
Growth Conditions ◽  
Secretion System ◽  
Host Cells ◽  
Dependent Manner ◽  
Type Iii

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to intoxicate eukaryotic host cells. Transcription of the T3SS is induced under calcium-limited growth conditions or following intimate contact of P. aeruginosa with host cells. In the present study, we demonstrate that expression of the T3SS is controlled by two distinct regulatory mechanisms and that these mechanisms are differentially activated in a host cell-dependent manner. The first mechanism is dependent upon ExsC, a regulatory protein that couples transcription of the T3SS to the activity of the type III secretion machinery. ExsC is essential for induction of the T3SS under low-calcium-growth conditions and for T3SS-dependent cytotoxicity towards social amoebae, insect cells, and erythrocytes. The second regulatory mechanism functions independently of ExsC and is sufficient to elicit T3SS-dependent cytotoxicity towards certain types of mammalian cells. Although this second pathway (ExsC independent) is sufficient, an exsC mutant demonstrates a lag in the induction of cytotoxicity towards Chinese hamster ovary cells and is attenuated for virulence in a mouse pneumonia model. We propose that the ExsC-dependent pathway is required for full cytotoxicity towards all host cell types tested whereas the ExsC-independent pathway may represent an adaptation that allows P. aeruginosa to increase expression of the T3SS in response to specific types of mammalian cells.

scholarly journals Transcriptional Downregulation of a Type III Secretion System under Reducing Conditions in Bordetella pertussis

2020 ◽  
Author(s):  
Masataka Goto ◽  
Tomoko Hanawa ◽  
Akio Abe ◽  
Asaomi Kuwae
Keyword(s):  
Bordetella Pertussis ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Culture Conditions ◽  
Secretion System ◽  
Host Cells ◽  
Secreted Proteins ◽  
Virulence Proteins ◽  
Type Iii ◽  
Reducing Conditions

ABSTRACTBordetella pertussis uses a type III secretion system (T3SS) to inject virulence proteins into host cells. Although the B. pertussis T3SS was presumed to be involved in host colonization, the efficient secretion of type III secreted proteins from B. pertussis has not been observed. To investigate the roles of type III secreted proteins during infection, we attempted to optimize culture conditions for the production and secretion of a type III secreted protein, BteA, in B. pertussis. We observed that B. pertussis efficiently secretes BteA in ascorbic acid-depleted (AsA−) medium. When L2 cells, a rat lung epithelial cell line, were infected with B. pertussis cultured in the AsA− medium, BteA-dependent cytotoxicity was observed. We also performed an immunofluorescence assay of L2 cells infected with B. pertussis. The clear fluorescence signals of Bsp22, a needle structure of T3SS, were detected on the bacterial surface of B. pertussis cultured in the AsA− medium. Since ascorbic acid is known as a reducing agent, we cultured B. pertussis in liquid medium containing other reducing agents such as 2-mercaptoethanol and dithioerythritol. Under these reducing conditions, the production of type III secreted proteins was repressed. These results suggest that in B. pertussis, the production and secretion of type III secreted proteins are downregulated under reducing conditions.IMPORTANCEThe type III secretion system (T3SS) of Bordetella pertussis forms a needle-like structure that protrudes from the bacterial cell surface. B. pertussis uses T3SS to translocate virulence proteins called effectors into host cells. The culture conditions for effector production in B. pertussis have not been investigated. We attempted to optimize culture medium compositions for producing and secreting type III secreted proteins. We found that B. pertussis secretes type III secreted proteins in reducing agent-deprived liquid medium, and that BteA-secreting B. pertussis provokes cytotoxicity against cultured mammalian cells. These results suggest that redox signaling is involved in the regulation of B. pertussis T3SS.

scholarly journals Burkholderia thailandensis as a Model System for the Study of the Virulence-Associated Type III Secretion System of Burkholderia pseudomallei

2008 ◽  
Vol 76 (11) ◽  
pp. 5402-5411 ◽  
Author(s):  
Andrea Haraga ◽  
T. Eoin West ◽  
Mitchell J. Brittnacher ◽  
Shawn J. Skerrett ◽  
Samuel I. Miller
Keyword(s):  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Burkholderia Pseudomallei ◽  
Clinical Symptoms ◽  
Type Iii Secretion System ◽  
Model System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Type Iii

ABSTRACT Burkholderia pseudomallei is a bacterial pathogen that causes a broad spectrum of clinical symptoms collectively known as melioidosis. Since it can be acquired by inhalation and is difficult to eradicate due to its resistance to a wide group of antibiotics and capacity for latency, work with B. pseudomallei requires a biosafety level 3 (BSL-3) containment facility. The bsa (Burkholderia secretion apparatus)-encoded type III secretion system (TTSS) has been shown to be required for its full virulence in a number of animal models. TTSSs are export devices found in a variety of gram-negative bacteria that translocate bacterial effector proteins across host cell membranes into the cytoplasm of host cells. Although the Bsa TTSS has been shown to play an important role in the ability of B. pseudomallei to survive and replicate in mammalian cells, escape from the endocytic vacuole, and spread from cell to cell, little is known about its effectors mediating these functions. Using bioinformatics, we identified homologs of several known TTSS effectors from other bacteria in the B. pseudomallei genome. In addition, we show that orthologs of these putative effectors exist in the genome of B. thailandensis, a closely related bacterium that is rarely pathogenic to humans. By generating a Bsa TTSS mutant B. thailandensis strain, we also demonstrated that the Bsa TTSS has similar functions in the two species. Therefore, we propose B. thailandensis as a useful BSL-1 model system to study the role of the Bsa TTSS during Burkholderia infection of mammalian cells and animals.

scholarly journals The Herbal Compound Thymol Targets Multiple Salmonella Typhimurium Virulence Factors for Lon Protease Degradation

2021 ◽  
Vol 12 ◽  
Author(s):  
Yong Zhang ◽  
Yan Liu ◽  
Jingjing Luo ◽  
Jing Jie ◽  
Xuming Deng ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Conformational Changes ◽  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Lon Protease ◽  
Type Iii ◽  
Pathogenic Factors

Many important bacterial pathogens are using the type III secretion system to deliver effectors into host cells. Salmonella Typhimurium (S. Typhimurium) is a pathogenic Gram-negative bacterium with the type III secretion system as its major virulence factor. Our previous studies demonstrated that thymol, a monoterpene phenol derivative of cymene, inhibited S. Typhimurium invasion into mammalian cells and protected mice from infection. However, the antibacterial mechanism of thymol is not clear. In this study, we revealed that thymol interferes with the abundance of about 100 bacterial proteins through proteomic analysis. Among the 42 proteins whose abundance was reduced, 11 were important virulence factors associated with T3SS-1. Further analyses with SipA revealed that thymol directly interacts with this protein to induce conformational changes, which makes it susceptible to the Lon protease. In agreement with this observation, thymol effectively blocks cell invasion by S. Typhimurium. Thus, thymol represents a class of anti-virulence compounds that function by targeting pathogenic factors for degradation.

Identification of translocation inhibitors targeting the type III secretion system of enteropathogenic Escherichia coli

2021 ◽  
Author(s):  
Sabrina Mühlen ◽  
Viktor A. Zapol'skii ◽  
Ursula Bilitewski ◽  
Petra Dersch
Keyword(s):  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Immune Recognition ◽  
Type Iii ◽  
Compound Libraries ◽  
High Throughput Screening Assay

Infections with enteropathogenic E. coli (EPEC) cause severe diarrhea in children. The non-invasive bacteria adhere to enterocytes of the small intestine and use a type III secretion system (T3SS) to inject effector proteins into host cells to modify and exploit cellular processes in favor of bacterial survival and replication. Several studies have shown that the T3SSs of bacterial pathogens are essential for virulence. Furthermore, the loss of T3SS-mediated effector translocation results in increased immune recognition and clearance of the bacteria. The T3SS is, therefore, considered a promising target for antivirulence strategies and novel therapeutics development. Here, we report the results of a high-throughput screening assay based on the translocation of the EPEC effector protein Tir. Using this assay, we screened more than 13,000 small molecular compounds of six different compound libraries and identified three substances which showed a significant dose-dependent effect on translocation without adverse effects on bacterial or eukaryotic cell viability. Additionally, these substances reduced bacterial binding to host cells, effector-dependent cell detachment and abolished A/E lesion formation without affecting the expression of components of the T3SS or associated effector proteins. Moreover, no effects of the inhibitors on bacterial motility or Shiga-toxin expression were observed. In summary, we have identified three new compounds that strongly inhibit T3SS-mediated translocation of effectors into mammalian cells, which could be valuable as lead substances for treating EPEC and EHEC infections.

scholarly journals An NF-κB-Based High-Throughput Screen Identifies Piericidins as Inhibitors of the Yersinia pseudotuberculosis Type III Secretion System

2013 ◽  
Vol 58 (2) ◽  
pp. 1118-1126 ◽  
Author(s):  
Miles C. Duncan ◽  
Weng Ruh Wong ◽  
Allison J. Dupzyk ◽  
Walter M. Bray ◽  
Roger G. Linington ◽  
...  
Keyword(s):  
Natural Products ◽  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Block Type ◽  
Content Type ◽  
Type Iii

ABSTRACTThe type III secretion system (T3SS) is a bacterial appendage used by dozens of Gram-negative pathogens to subvert host defenses and cause disease, making it an ideal target for pathogen-specific antimicrobials. Here, we report the discovery and initial characterization of two related natural products with T3SS-inhibitory activity that were derived from a marine actinobacterium. Bacterial extracts containing piericidin A1 and the piericidin derivative Mer-A 2026B inhibitedYersinia pseudotuberculosisfrom triggering T3SS-dependent activation of the host transcription factor NF-κB in HEK293T cells but were not toxic to mammalian cells. As theYersiniaT3SS must be functional in order to trigger NF-κB activation, these data indicate that piericidin A1 and Mer-A 2026B block T3SS function. Consistent with this, purified piericidin A1 and Mer-A 2026B dose-dependently inhibited translocation of theY. pseudotuberculosisT3SS effector protein YopM inside CHO cells. In contrast, neither compound perturbed bacterial growthin vitro, indicating that piericidin A1 and Mer-A 2026B do not function as general antibiotics inYersinia. In addition, whenYersiniawas incubated under T3SS-inducing culture conditions in the absence of host cells, Mer-A 2026B and piericidin A1 inhibited secretion of T3SS cargo as effectively as or better than several previously described T3SS inhibitors, such as MBX-1641 and aurodox. This suggests that Mer-A 2026B and piericidin A1 do not block type III secretion by blocking the bacterium-host cell interaction, but rather inhibit an earlier stage, such as T3SS needle assembly. In summary, the marine-derived natural products Mer-A 2026B and piericidin A1 possess previously uncharacterized activity against the bacterial T3SS.

scholarly journals Design of small molecule inhibitors of type III secretion system ATPase EscN from enteropathogenic Escherichia coli.

1970 ◽  
Vol 64 (1) ◽  
Author(s):  
Lukasz Bzdzion ◽  
Hanna Krezel ◽  
Karol Wrzeszcz ◽  
Irmina Grzegorek ◽  
Katarzyna Nowinska ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Small Molecule ◽  
Active Site ◽  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Small Molecule Inhibitors ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Type Iii

Enteropathogenic E. coli (EPEC) is a human pathogen using type III secretion system for delivery of proteins directly into the human host. The system contains a single ATPase, EscN, which is essential for uncoupling of proteins from their complexes with chaperones before the delivery. The structure of EscN ATPase (PDB code: 2obm) was used to screen computationally for small molecule inhibitors blocking its active site. Two lead candidates were examined but only one, Compound 54, was selected for further optimization. After extended QSAR optimization, two derivatives were found to be competitive inhibitors of EscN capable of blocking ATPase activity with a Ki below 50 µM. One candidate, WEN05-03, with a Ki=16±2 µM, was also minimally toxic to mammalian cells as determined by other assays. In the cell infection model of HeLa cells with EPEC, Compound WEN05-03 completely blocked actin cluster formation at 100 µM concentration, when analyzed by confocal microscopy. The second best inhibitor of EscN ATPase activity was WEN04-34 with a Ki=46±2 µM. However, the compound was highly toxic to the BALB/3T3 cell line. In summary, the work identifies a compound blocking bacterial ATPase in its active site without causing cellular toxicity to the host cells. It is the first report showing feasibility of using bacterial virulence system ATPase as a target for safe, non-toxic compounds and offering a proof-of-concept for non-antibiotic alternatives.

scholarly journals Cre Reporter System To Monitor the Translocation of Type III Secreted Proteins into Host Cells

2006 ◽  
Vol 74 (2) ◽  
pp. 1084-1090 ◽  
Author(s):  
Gabriel Briones ◽  
Dirk Hofreuter ◽  
Jorge E. Galán
Keyword(s):  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Type Iii Secretion System ◽  
Cre Recombinase ◽  
Secretion System ◽  
Host Cells ◽  
Secretion Systems ◽  
Bacteriophage P1 ◽  
Type Iii

ABSTRACT Central to the study of type III secretion systems is the availability of reporter systems to monitor bacterial protein translocation into host cells. We report here the development of a bacteriophage P1 Cre recombinase-based system to monitor the translocation of bacterial proteins into mammalian cells. Bacteriophage P1 Cre recombinase fused to the secretion and translocation signals of Salmonella enterica serovar Typhimurium of the type III secreted protein SopE was secreted in a type III secretion system-dependent fashion. More importantly, the SopE-Cre chimera was translocated into host cells via the type III secretion system and activated the expression of luciferase and green fluorescent protein reporters of Cre recombinase activity.

scholarly journals Chlamydial Lytic Exit from Host Cells Is Plasmid Regulated

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Chunfu Yang ◽  
Tregei Starr ◽  
Lihua Song ◽  
John H. Carlson ◽  
Gail L. Sturdevant ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Actin Polymerization ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Cytoskeletal Proteins ◽  
Host Cells ◽  
Genetic Mechanism ◽  
Developmental Cycle ◽  
Obligate Intracellular ◽  
Type Iii

ABSTRACTChlamydia trachomatisis an obligate intracellular bacterium that is a globally important human pathogen. The chlamydial plasmid is an attenuating virulence factor, but the molecular basis for attenuation is not understood. Chlamydiae replicate within a membrane-bound vacuole termed an inclusion, where they undergo a biphasic developmental growth cycle and differentiate from noninfectious into infectious organisms. Late in the developmental cycle, the fragile chlamydia-laden inclusion retains its integrity by surrounding itself with scaffolds of host cytoskeletal proteins. The ability of chlamydiae to developmentally free themselves from this cytoskeleton network is a fundamental virulence trait of the pathogen. Here, we show that plasmidless chlamydiae are incapable of disrupting their cytoskeletal entrapment and remain intracellular as stable mature inclusions that support high numbers of infectious organisms. By using deletion mutants of the eight plasmid-carried genes (Δpgp1to Δpgp8), we show that Pgp4, a transcriptional regulator of multiple chromosomal genes, is required for exit. Exit of chlamydiae is dependent on protein synthesis and is inhibited by the compound C1, an inhibitor of the type III secretion system (T3S). Exit of plasmid-free and Δpgp4organisms, which failed to lyse infected cells, was rescued by latrunculin B, an inhibitor of actin polymerization. Our findings describe a genetic mechanism of chlamydial exit from host cells that is dependent on an unknownpgp4-regulated chromosomal T3S effector gene.IMPORTANCEChlamydia's obligate intracellular life style requires both entry into and exit from host cells. Virulence factors that function in exiting are unknown. The chlamydial inclusion is stabilized late in the infection cycle by F-actin. A prerequisite of chlamydial exit is its ability to disassemble actin from the inclusion. We show that chlamydial plasmid-free organisms, and also a plasmid gene protein 4 (pgp4) null mutant, do not disassociate actin from the inclusion and fail to exit cells. We further provide evidence that Pgp4-regulated exit is dependent on the chlamydial type III secretion system. This study is the first to define a genetic mechanism that functions in chlamydial lytic exit from host cells. The findings also have practical implications for understanding why plasmid-free chlamydiae are highly attenuated and have the ability to elicit robust protective immune responses.

Sign in / Sign up

Export Citation Format

Share Document