The PopN gate-keeper complex acts on the ATPase PscN to regulate the T3SS secretion switch from early to middle substrates in Pseudomonas aeruginosa

AbstractPseudomonas aeruginosa is an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction of PscN with a wide range of exported T3SS proteins including the needle, translocator, gate-keeper and effector. These interactions were further confirmed by Microscale Thermophoresis that also indicated a preferential interaction of PscN with secreted proteins or protein-chaperone complex rather than with chaperones alone, in line with the release of the chaperones in the bacterial cytoplasm after the dissociation from their exported proteins. Moreover, we suggest a new role of the gate-keeper complex and the ATPase in the regulation of early substrates recognition by the T3SS. This finding sheds a new light on the mechanism of secretion switching from early to middle substrates in P. aeruginosa.HighlightsT3SS substrates are secreted sequentially but information on the switches are missingInteraction of the T3SS ATPase with secreted proteins were investigated by different approachesMicroscale Thermophoresis revealed a lower affinity for chaperones alone compared to complexesThe Gate-keeper complex binds to the ATPase and increases its affinity for the needle complexA new role of the Gate-keeper complex is proposed, directly acting on the T3SS ATPase

Download Full-text

332 The diagnostic role of shunt series radiographs (SSR) in children presenting to the children’s emergency department (CED) with suspected ventriculoperitoneal (VP) shunt failure

Emergency Medicine Journal ◽

10.1136/emj-2020-rcemabstracts.56 ◽

2020 ◽

Vol 37 (12) ◽

pp. 852.3-853

Author(s):

Angharad Griffiths ◽

Ikechukwu Okafor ◽

Thomas Beattie

Keyword(s):

Clinical Outcome ◽

Sensitivity And Specificity ◽

Clinical Information ◽

Flow Diagram ◽

List Type ◽

Shunt Failure ◽

Vp Shunt ◽

Wide Range ◽

Diagnostic Role

Aims/Objectives/BackgroundVP shunts are used to drain CSF from the cranial vault because of a wide range of pathologies and, like any piece of hardware, can fail. Traditionally investigations include SSR and CT. This project examines the role of SSR in evaluating children with suspected VP shunt failure.Primary outcome: Sensitivity and specificity of SSR in children presenting to the CED with concern for shunt failure.Methods/DesignConducted in a single centre, tertiary CED of the national Irish Neurosurgical(NS) referral centre (ED attendance:>50,000 patients/year). 100 sequential SSR requested by the CED were reviewed. Clinical information was extracted from electronic requests. Shunt failure was defined by the need for NS intervention(Revision).Abstract 332 Figure 1Abstract 332 Figure 2Results/ConclusionsSensitivity and specificity is presented in figure 1 (two by two table).100 radiographs performed in 84 children.22% shunts revised (see flow diagram).7 SSR’s were abnormal.85% (n=6) shunts revised. [5 following abnormal CT].Of the normal SSR’s; 16 had abnormal CT and revised.85/100 received CT.64 of 85 CT’s (75%) were normal.□6 of the 64 had focal shunt concern.SSR’s shouldn’t be used in isolation. NPV&PPV, Sensitivity&Specificity is low.SSR’s are beneficial where there’s concern over focal shunt problems (injury/pain/swelling) or following abnormal CT.VP shunt failure is not well investigated with SSR alone.SSR’s could be omitted where there is no focal shunt concern/after normal CT (without impacting clinical outcome) reducing radiation exposure and reduce impact on CED’s.59 SSR’s could have been avoided without adverse clinical outcome.

Download Full-text

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.

Download Full-text

Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals

Microbiology ◽

10.1099/mic.0.28368-0 ◽

2006 ◽

Vol 152 (1) ◽

pp. 143-152 ◽

Cited By ~ 12

Author(s):

Ciara M. Shaver ◽

Alan R. Hauser

Keyword(s):

Pseudomonas Aeruginosa ◽

Disease Severity ◽

Type Iii Secretion ◽

Disease Process ◽

Effector Proteins ◽

Host Cells ◽

Secreted Proteins ◽

Secretion Systems ◽

Type Iii

The effector proteins of the type III secretion systems of many bacterial pathogens act in a coordinated manner to subvert host cells and facilitate the development and progression of disease. It is unclear whether interactions between the type-III-secreted proteins of Pseudomonas aeruginosa result in similar effects on the disease process. We have previously characterized the contributions to pathogenesis of the type-III-secreted proteins ExoS, ExoT and ExoU when secreted individually. In this study, we extend our prior work to determine whether these proteins have greater than expected effects on virulence when secreted in combination. In vitro cytotoxicity and anti-internalization activities were not enhanced when effector proteins were secreted in combinations rather than alone. Likewise in a mouse model of pneumonia, bacterial burden in the lungs, dissemination and mortality attributable to ExoS, ExoT and ExoU were not synergistically increased when combinations of these effector proteins were secreted. Because of the absence of an appreciable synergistic increase in virulence when multiple effector proteins were secreted in combination, we conclude that any cooperation between ExoS, ExoT and ExoU does not translate into a synergistically significant enhancement of disease severity as measured by these assays.

Download Full-text

Novel Type III Effectors in Pseudomonas aeruginosa

mBio ◽

10.1128/mbio.00161-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 18

Author(s):

David Burstein ◽

Shirley Satanower ◽

Michal Simovitch ◽

Yana Belnik ◽

Meital Zehavi ◽

...

Keyword(s):

Machine Learning ◽

Pseudomonas Aeruginosa ◽

Host Cell ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Content Type ◽

Machine Learning Classification ◽

Type Iii ◽

Wide Range

ABSTRACT Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes chronic and acute infections in immunocompromised patients. Most P. aeruginosa strains encode an active type III secretion system (T3SS), utilized by the bacteria to deliver effector proteins from the bacterial cell directly into the cytoplasm of the host cell. Four T3SS effectors have been discovered and extensively studied in P. aeruginosa: ExoT, ExoS, ExoU, and ExoY. This is especially intriguing in light of P. aeruginosa's ability to infect a wide range of hosts. We therefore hypothesized that additional T3SS effectors that have not yet been discovered are encoded in the genome of P. aeruginosa. Here, we applied a machine learning classification algorithm to identify novel P. aeruginosa effectors. In this approach, various types of data are integrated to differentiate effectors from the rest of the open reading frames of the bacterial genome. Due to the lack of a sufficient learning set of positive effectors, our machine learning algorithm integrated genomic information from another Pseudomonas species and utilized dozens of features accounting for various aspects of the effector coding genes and their products. Twelve top-ranking predictions were experimentally tested for T3SS-specific translocation, leading to the discovery of two novel T3SS effectors. We demonstrate that these effectors are not part of the injection structural complex and report initial efforts toward their characterization. IMPORTANCE Pseudomonas aeruginosa uses a type III secretion system (T3SS) to secrete toxic proteins, termed effectors, directly into the cytoplasm of the host cell. The activation of this secretion system is correlated with disease severity and patient death. Compared with many other T3SS-utilizing pathogenic bacteria, P. aeruginosa has a fairly limited arsenal of effectors that have been identified. This is in sharp contrast with the wide range of hosts that this bacterium can infect. The discovery of two novel effectors described here is an important step toward better understanding of the virulence and host evasion mechanisms adopted by this versatile pathogen and may provide novel approaches to treat P. aeruginosa infections.

Download Full-text

Salmonella pathogenicity island 1 (SPI-1) type III secretion of SopD involves N- and C-terminal signals and direct binding to the InvC ATPase

Microbiology ◽

10.1099/mic.0.038117-0 ◽

2010 ◽

Vol 156 (6) ◽

pp. 1805-1814 ◽

Cited By ~ 12

Author(s):

R. Boonyom ◽

M. H. Karavolos ◽

D. M. Bulmer ◽

C. M. A. Khan

Keyword(s):

Protein Interactions ◽

Type Iii Secretion ◽

Pathogenicity Island ◽

Effector Proteins ◽

Amino Acid Residues ◽

Type Iii ◽

Protein Protein Interaction ◽

Secretion Signal ◽

Serovar Typhimurium ◽

Bacterial Cytoplasm

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important pathogen and a causative agent of gastroenteritis. During infection, S. Typhimurium assembles molecular-needle complexes termed type III secretion (T3S) systems to translocate effector proteins from the bacterial cytoplasm directly into the host cell. The T3S signals that direct the secretion of effectors still remain enigmatic. SopD is a key T3S effector contributing to the systemic virulence of S. Typhimurium and the development of gastroenteritis. We have scrutinized the distribution of the SopD T3S signals using in silico analysis and a targeted deletion approach. We show that amino acid residues 6–10 act as the N-terminal secretion signal for Salmonella pathogenicity island 1 (SPI-1) T3S. Furthermore, we show that two putative C-terminal helical regions of SopD are essential for its secretion and also help prevent erroneous secretion through the flagellar T3S machinery. In addition, using protein–protein interaction assays, we have identified an association between SopD and the SPI-1 T3S system ATPase, InvC. These findings demonstrate that T3S of SopD involves multiple signals and protein interactions, providing important mechanistic insights into effector protein secretion.

Download Full-text

Direct and indirect interactions promote complexes of the lipoprotein LbcA, the CtpA protease and its substrates, and other cell wall proteins in Pseudomonas aeruginosa

10.1101/2021.07.29.454410 ◽

2021 ◽

Author(s):

Dolonchapa Chakraborty ◽

Andrew J Darwin

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Wall ◽

Type Iii Secretion ◽

Protein Complexes ◽

Peptidoglycan Hydrolases ◽

Carboxyl Terminal ◽

Processing Protease ◽

Enzyme Complexes

The Pseudomonas aeruginosa lipoprotein LbcA was discovered because it copurified with and promoted the activity of CtpA, a carboxyl-terminal processing protease (CTP) required for type III secretion system function, and for virulence in a mouse model of acute pneumonia. In this study we explored the role of LbcA by determining its effect on the proteome and its participation in protein complexes. lbcA and ctpA null mutations had strikingly similar effects on the proteome, suggesting that facilitating CtpA might be the most impactful role of LbcA in the bacterial cell. Independent complexes containing LbcA and CtpA, or LbcA and substrate, were isolated from P. aeruginosa cells, indicating that LbcA facilitates proteolysis by recruiting the protease and its substrates independently. An unbiased examination of proteins that copurified with LbcA revealed an enrichment for proteins associated with the cell wall. One of these copurification partners was found to be a new CtpA substrate, and the first substrate that is not a peptidoglycan hydrolase. Many of the other LbcA copurification partners are known or predicted peptidoglycan hydrolases. However, some of these LbcA copurification partners were not cleaved by CtpA, and an in vitro assay revealed that while CtpA and all of its substrates bound to LbcA directly, these non-substrates did not. Subsequent experiments suggested that the non substrates might co-purify with LbcA by participating in multi-enzyme complexes containing LbcA-binding CtpA substrates.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.01680-06 ◽

2007 ◽

Vol 189 (7) ◽

pp. 2599-2609 ◽

Cited By ~ 29

Author(s):

Hongjing Yang ◽

Zhiying Shan ◽

Jaewha Kim ◽

Weihui Wu ◽

Wei Lian ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Protein Interactions ◽

Type Iii Secretion ◽

Calcium Binding ◽

High Capacity ◽

Calcium Signal ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Type Iii

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.

Download Full-text

Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.12.137 ◽

2016 ◽

Vol 12 ◽

pp. 1428-1433 ◽

Cited By ~ 8

Author(s):

Bernardas Morkunas ◽

Balint Gal ◽

Warren R J D Galloway ◽

James T Hodgkinson ◽

Brett M Ibbeson ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Cell Signalling ◽

Opportunistic Pathogen ◽

Wild Type ◽

Wide Range ◽

Potential Applications ◽

Therapeutic Context ◽

Pyocyanin Production

Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H)-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable.

Download Full-text

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.

Download Full-text