Regulatory Role of PopN and Its Interacting Partners in Type III Secretion of Pseudomonas aeruginosa

ABSTRACT The type III secretion system (T3SS) of Pseudomonas aeruginosa plays a significant role in pathogenesis. We have previously identified type III secretion factor (TSF), which is required for effective secretion of the type III effector molecules, in addition to the low calcium signal. TSF includes many low-affinity high-capacity calcium binding proteins, such as serum albumin and casein. A search for the TSF binding targets on the bacterial outer membrane resulted in identification of PopN, a component of the T3SS that is readily detectable on the bacterial cell surface. PopN specifically interacts with Pcr1, and both popN and pcr1 mutants have a constitutive type III secretion phenotype, suggesting that the two proteins form a complex that functions as a T3SS repressor. Further analysis of the popN operon genes resulted in identification of protein-protein interactions between Pcr1 and Pcr4 and between Pcr4 and Pcr3, as well as between PopN and Pcr2 in the presence of PscB. Unlike popN and pcr1 mutants, pcr3 and pcr4 mutants are totally defective in type III secretion, while a pcr2 mutant exhibits reduced type III secretion. Interestingly, PopN, Pcr1, Pcr2, and Pcr4 are all secreted in a type III secretion machinery-dependent manner, while Pcr3 is not. These findings imply that these components have important regulatory roles in controlling type III secretion.

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

Infection and Immunity ◽

10.1128/iai.71.5.2404-2413.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2404-2413 ◽

Cited By ~ 166

Author(s):

Sachiko Miyata ◽

Monika Casey ◽

Dara W. Frank ◽

Frederick M. Ausubel ◽

Eliana Drenkard

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Galleria Mellonella ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Triple Mutant ◽

Type Iii ◽

Wax Moth

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

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Pseudomonas aeruginosa injects NDK into host cells through a type III secretion system

Microbiology ◽

10.1099/mic.0.078139-0 ◽

2014 ◽

Vol 160 (7) ◽

pp. 1417-1426 ◽

Cited By ~ 25

Author(s):

Dennis Neeld ◽

Yongxin Jin ◽

Candace Bichsel ◽

Jinghua Jia ◽

Jianhui Guo ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Cells ◽

Eukaryotic Cells ◽

Mammalian Host ◽

Type I ◽

Dependent Manner ◽

Type Iii

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen possessing a type III secretion system (T3SS) which injects toxic effector proteins into mammalian host cells. In previous studies, P. aeruginosa strains lacking all of the known type III effectors were shown to cause cytotoxicity upon prolonged infection time. In this study, we report the identification of a new cytotoxin, nucleoside diphosphate kinase (NDK), which is injected into eukaryotic cells in a T3SS-dependent manner. Injection of NDK is inhibited by the presence of previously known effectors of the T3SS, with an effectorless strain injecting the highest amount, suggesting active competition with the known T3SS effectors. NDK is shown to cause a cytotoxic response when expressed in eukaryotic cells, and P. aeruginosa strains harbouring NDK also show a greater toxicity than strains lacking it. Interestingly, the cytotoxic effect of intracellular NDK is independent of its kinase activity. In previous studies, NDK was shown to be secreted into culture supernatants via a type I secretion system and cause cytotoxicity in a kinase-dependent manner. Therefore, the current study highlights an alternative route of NDK secretion as well as two different cytotoxic mechanisms of NDK, depending on the extra- or intra-cellular location of the protein.

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MucA-Mediated Coordination of Type III Secretion and Alginate Synthesis in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.186.22.7575-7585.2004 ◽

2004 ◽

Vol 186 (22) ◽

pp. 7575-7585 ◽

Cited By ~ 71

Author(s):

Weihui Wu ◽

Hassan Badrane ◽

Shiwani Arora ◽

Henry V. Baker ◽

Shouguang Jin

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Regulatory Networks ◽

Protective Role ◽

Environmental Stresses ◽

Host Cells ◽

Calcium Signal ◽

Insertion Mutant ◽

Type Iii

ABSTRACT The type III secretion system (T3SS) of Pseudomonas aeruginosa is an important virulence factor. The T3SS of P. aeruginosa can be induced by a low calcium signal or upon direct contact with the host cells. The exact pathway of signal sensing and T3SS activation is not clear. By screening a transposon insertion mutant library of the PAK strain, mutation in the mucA gene was found to cause repression of T3SS expression under both type III-inducing and -noninducing conditions. Mutation in the mucA gene is known to cause alginate overproduction, resulting in a mucoid phenotype. Alginate production responds to various environmental stresses and plays a protective role for P. aeruginosa. Comparison of global gene expression of mucA mutant and wild-type PAK under T3SS-inducing conditions confirmed the down regulation of T3SS genes and up regulation of genes involved in alginate biosynthesis. Further analysis indicated that the repression of T3SS in the mucA mutant was AlgU and AlgR dependent, as double mutants mucA/algU and mucA/algR showed normal type III expression. An algR::Gm mutant showed a higher level of type III expression, while overexpression of the algR gene inhibited type III gene expression; thus, it seems that the AlgR-regulated product inhibits the expression of the T3SS genes. It is likely that P. aeruginosa has evolved tight regulatory networks to turn off the energy-expensive T3SS when striving for survival under environmental stresses.

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

Infection and Immunity ◽

10.1128/iai.00090-06 ◽

2006 ◽

Vol 74 (6) ◽

pp. 3334-3341 ◽

Cited By ~ 50

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.

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InvB Is a Type III Secretion Chaperone Specific for SspA

Journal of Bacteriology ◽

10.1128/jb.182.23.6638-6644.2000 ◽

2000 ◽

Vol 182 (23) ◽

pp. 6638-6644 ◽

Cited By ~ 58

Author(s):

Philip A. Bronstein ◽

Edward A. Miao ◽

Samuel I. Miller

Keyword(s):

Protein Interactions ◽

Type Iii Secretion ◽

Effector Proteins ◽

Amino Terminus ◽

Protein Protein Interactions ◽

Type Iii ◽

Protein Levels ◽

Serovar Typhimurium ◽

Secretion Chaperone ◽

Bacterial Cytoplasm

ABSTRACT A wide variety of gram-negative bacteria utilize a specialized apparatus called the type III secretion system (TTSS) to translocate virulence factors directly into the cytoplasm of eukaryotic cells. These translocated effectors contribute to the pathogen's ability to infect and replicate within plant and animal hosts. The amino terminus of effector proteins contains sequences that are necessary and sufficient for both secretion and translocation by TTSS. Portions of these sequences contain binding sites for type III chaperones, which facilitate efficient secretion and translocation of specific effectors through TTSS. In this study, we have utilized the yeast two-hybrid assay to identify protein-protein interactions between effector and chaperone proteins encoded within Salmonella pathogenicity island 1 (SPI-1). Several interactions were identified including a novel interaction between the effector protein, SspA (SipA), and a putative chaperone, InvB. InvB was demonstrated to bind to the amino terminus of SspA in the bacterial cytoplasm. Furthermore, InvB acts as a type III chaperone for the efficient secretion and translocation of SspA by SPI-1. InvB also permitted translocation of SspA through the SPI-2 TTSS, indicating that it is an important regulator in the recognition of SspA as a target of TTSS. Finally, it was determined that InvB does not alter the transcription of sspA but that its absence results in reduced SspA protein levels in Salmonella enterica serovar Typhimurium.

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Role of the Salmonella Pathogenicity Island 1 (SPI-1) Protein InvB in Type III Secretion of SopE and SopE2, Two Salmonella Effector Proteins Encoded Outside of SPI-1

Journal of Bacteriology ◽

10.1128/jb.185.23.6950-6967.2003 ◽

2003 ◽

Vol 185 (23) ◽

pp. 6950-6967 ◽

Cited By ~ 62

Author(s):

Kristin Ehrbar ◽

Andrea Friebel ◽

Samuel I. Miller ◽

Wolf-Dietrich Hardt

Keyword(s):

Type Iii Secretion ◽

Inflammatory Responses ◽

Pathogenicity Island ◽

Effector Proteins ◽

Host Cells ◽

Effector Protein ◽

Dependent Manner ◽

Type Iii ◽

Serovar Typhimurium

ABSTRACT Salmonella enterica subspecies 1 serovar Typhimurium encodes a type III secretion system (TTSS) within Salmonella pathogenicity island 1 (SPI-1). This TTSS injects effector proteins into host cells to trigger invasion and inflammatory responses. Effector proteins are recognized by the TTSS via signals encoded in their N termini. Specific chaperones can be involved in this process. The chaperones InvB, SicA, and SicP are encoded in SPI-1 and are required for transport of SPI-1-encoded effectors. Several key effector proteins, like SopE and SopE2, are located outside of SPI-1 but are secreted in an SPI-1-dependent manner. It has not been clear how these effector proteins are recognized by the SPI-1 TTSS. Using pull-down and coimmunoprecipitation assays, we found that SopE is copurified with InvB, the known chaperone for the SPI-1-encoded effector protein Sip/SspA. We also found that InvB is required for secretion and translocation of SopE and SopE2 and for stabilization of SopE2 in the bacterial cytosol. Our data demonstrate that effector proteins encoded within and outside of SPI-1 use the same chaperone for secretion via the SPI-1 TTSS.

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Factors triggering type III secretion in Pseudomonas aeruginosa

Microbiology ◽

10.1099/mic.0.28277-0 ◽

2005 ◽

Vol 151 (11) ◽

pp. 3575-3587 ◽

Cited By ~ 35

Author(s):

Jaewha Kim ◽

Kyungseop Ahn ◽

Sungran Min ◽

Jinghua Jia ◽

Unhwan Ha ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Calcium Binding ◽

Binding Proteins ◽

Calcium Binding Proteins ◽

Effective Control ◽

Type Iii Effector ◽

Effector Molecules ◽

Type Iii ◽

Low Calcium

The type III secretion system of Pseudomonas aeruginosa is tightly regulated by various environmental signals, such as low calcium and contact with the host cell. However, the exact signals triggering type III secretion are unknown. The present study describes the finding that secretion of P. aeruginosa type III effector molecules requires protein factors from serum and L broth, designated type III secretion factors (TSFs), in addition to the low-calcium environment. In the absence of TSF or calcium chelator EGTA, basal levels of type III effector molecules are accumulated intracellularly. Addition of TSF and EGTA together effectively triggers the secretion of pre-existing effector molecules in a short time, even before the active expression of type III genes; thus, active type III gene expression does not seem to be a prerequisite for type III secretion. A search for TSF molecules in serum and L broth resulted in the identification of albumin and casein as the functional TSF molecules. Although there is no clear sequence similarity between albumin and casein, both proteins are known to have a low-affinity, high-capacity calcium-binding property. Tests of well-studied calcium-binding proteins seemed to indicate that low-affinity calcium-binding proteins have TSF activity, although the requirement of low-affinity calcium-binding ability for the TSF activity is not clear. P. aeruginosa seems to have evolved a sensing mechanism to detect target cells for type III injection through host-derived proteins in combination with a low-calcium signal. Disruption of the bacterial ability to sense low calcium or TSF might be a valid avenue to the effective control of this bacterial pathogen.

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Alveolar Response to Pseudomonas aeruginosa: Role of the Type III Secretion System

Infection and Immunity ◽

10.1128/iai.73.7.4263-4271.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4263-4271 ◽

Cited By ~ 35

Author(s):

F. Ader ◽

R. Le Berre ◽

K. Faure ◽

P. Gosset ◽

O. Epaulard ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Lung Injury ◽

Type Iii Secretion ◽

A549 Cells ◽

Weight Ratio ◽

Type Iii Secretion System ◽

Secretion System ◽

Polymorphonuclear Neutrophils ◽

Type Iii

ABSTRACT The type III secretion system (TTSS) is a specialized cytotoxin-translocating apparatus of gram-negative bacteria which is involved in lung injury, septic shock, and a poor patient outcome. Recent studies have attributed these effects mainly to the ExoU effector protein. However, few studies have focused on the ExoU-independent pathogenicity of the TTSS. For the present study, we compared the pathogenicities of two strains of Pseudomonas aeruginosa in a murine model of acute lung injury. We compared the CHA strain, which has a functional TTSS producing ExoS and ExoT but not ExoU, to an isogenic mutant with an inactivated exsA gene, CHA-D1, which does not express the TTSS at all. Rats challenged with CHA had significantly increased lung injury, as assessed by the wet/dry weight ratio for the lungs and the protein level in bronchoalveolar lavage fluid (BALF) at 12 h, compared to those challenged with CHA-D1. Consistent with these findings, the CHA strain was associated with increased in vitro cytotoxicity on A549 cells, as assessed by the release of lactate dehydrogenase. CHA was also associated at 12 h with a major decrease in polymorphonuclear neutrophils in BALF, with a proinflammatory response, as assessed by the amounts of tumor necrosis factor alpha and interleukin-1β, and with decreased bacterial clearance from the lungs, ultimately leading to an increased mortality rate. These results demonstrate that the TTSS has a major role in P. aeruginosa pathogenicity independent of the role of ExoU. This report underscores the crucial roles of ExoS and ExoT or other TTSS-related virulence factors in addition to ExoU.

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Keeping in Touch with Type-III Secretion System Effectors: Mass Spectrometry-Based Proteomics to Study Effector–Host Protein–Protein Interactions

International Journal of Molecular Sciences ◽

10.3390/ijms21186891 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6891

Author(s):

Margaux De Meyer ◽

Joren De Ryck ◽

Sofie Goormachtig ◽

Petra Van Damme

Keyword(s):

Mass Spectrometry ◽

Protein Interactions ◽

Type Iii Secretion ◽

Large Scale ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Protein ◽

Protein Protein Interactions ◽

Type Iii ◽

Recent Advances

Manipulation of host cellular processes by translocated bacterial effectors is key to the success of bacterial pathogens and some symbionts. Therefore, a comprehensive understanding of effectors is of critical importance to understand infection biology. It has become increasingly clear that the identification of host protein targets contributes invaluable knowledge to the characterization of effector function during pathogenesis. Recent advances in mapping protein–protein interaction networks by means of mass spectrometry-based interactomics have enabled the identification of host targets at large-scale. In this review, we highlight mass spectrometry-driven proteomics strategies and recent advances to elucidate type-III secretion system effector–host protein–protein interactions. Furthermore, we highlight approaches for defining spatial and temporal effector–host interactions, and discuss possible avenues for studying natively delivered effectors in the context of infection. Overall, the knowledge gained when unravelling effector complexation with host factors will provide novel opportunities to control infectious disease outcomes.

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