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Type III Secretion Systems in Human/Animal Pathogenic Bacteria

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A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Molecular Microbiology and Immunology".

Deadline for manuscript submissions: closed (31 October 2020) | Viewed by 28146

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Prof. Dr. Francisco Ramos-Morales

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Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain
Interests: Salmonella; host–pathogen interaction; type III secretion; signal transduction; gene regulation
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Dr. Joaquín Bernal-Bayard

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Genetics Department, University of Seville, Sevilla, Spain
Interests: Salmonella; host–pathogen interaction; type III secretion; biofilms; zebrafish
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Dear Colleagues,

Type III secretion systems (T3SS) were discovered in the 1990s and initially described in the animal pathogen Yersinia, but they are present in many Gram-negative bacterial pathogens and symbionts of animal and plants. These systems are important virulence factors for many animal pathogenic bacteria and consist of a needle-like structure that spans inner and outer bacterial membranes and the host cell membrane to deliver effector proteins into the host cytosol. These effectors interfere with cellular functions to manipulate the host cells processes to benefit the pathogen.

Bacterial pathogens that use T3SS to communicate with host cells represent a major threat to human health and cause a broad spectrum of diseases and clinical manifestations. They include species of Shigella, Salmonella, Yersinia, Bordetella, Chlamydia, and Burkholderia, as well as enteropathogenic and enterohemorrhagic E. coli, and the opportunistic pathogen Pseudomonas aeruginosa. Some of them, together with species of Aeromonas, Citrobacter, Edwarsiella, Photorhabdus, or Vibrio, among others, can also cause infections in livestock and fisheries, leading to huge economic losses and, in some cases, increasing the risk of human infections.

In spite of the variety of lifestyles and diseases caused by these pathogens, there are also common themes arising from the fact that they share the use of T3SSs as key virulence mechanisms, and that effectors from different bacteria may have similar designs, biochemical activities, and/or functions. Therefore, researchers working in a particular T3SS can benefit from the studies carried out in other bacteria. Finally, these systems provide an opportunity to develop novel therapeutic approaches to respond to the growing number of antimicrobial-resistant pathogens, one of the ten threats to global health identified by the World Health Organization for this year.

This Special Issue focuses on the study of type III secretion systems in animal pathogenic bacteria and will publish high-quality research articles and reviews concerning this subject. We aim to address all aspects related to this topic, including: regulation of the expression of type III secretion systems and their effectors; mechanisms of type III protein secretion; conditions for secretion and translocation of effectors; host–pathogen specificity; structural studies; enzymatic activities, substrates, and interacting partners of the effectors; functional effects on the host; and biotechnological and biomedical applications.

Prof. Dr. Francisco Ramos-Morales
Dr. Joaquín Bernal-Bayard
Guest Editors

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Keywords

type III secretion
effectors
virulence
host-pathogen interactions
Aeromonas
Bordetella
Burkholderia
Chlamydia
Citrobacter
Edwardsiella
EPEC/EHEC
Photorhabdus
Pseudomonas
Salmonella
Shigella
Vibrio
Yersinia

Related Special Issue

Type III Secretion Systems in Human/Animal Pathogenic Bacteria 2.0 in Microorganisms (4 articles)

Published Papers (10 papers)

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Editorial

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3 pages, 219 KiB

Open AccessEditorial

Special Issue: Type III Secretion Systems in Human/Animal Pathogenic Bacteria

by Joaquín Bernal-Bayard and Francisco Ramos-Morales

Microorganisms 2022, 10(7), 1461; https://doi.org/10.3390/microorganisms10071461 - 20 Jul 2022

Viewed by 1390

Abstract

Type III secretion systems (T3SSs) are molecular devices that are essential for the communication of many Gram-negative bacteria with their eukaryotic hosts [...] Full article

(This article belongs to the Special Issue Type III Secretion Systems in Human/Animal Pathogenic Bacteria)

Research

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11 pages, 1623 KiB

Open AccessArticle

Novel Template Plasmids pCyaA’-Kan and pCyaA’-Cam for Generation of Unmarked Chromosomal cyaA’ Translational Fusion to T3SS Effectors in Salmonella

by Paulina A. Fernández, Marcela Zabner, Jaime Ortega, Constanza Morgado, Fernando Amaya, Gabriel Vera, Carolina Rubilar, Beatriz Salas, Víctor Cuevas, Camila Valenzuela, Fernando Baisón-Olmo, Sergio A. Álvarez and Carlos A. Santiviago

Microorganisms 2021, 9(3), 475; https://doi.org/10.3390/microorganisms9030475 - 25 Feb 2021

Cited by 2 | Viewed by 2292

Abstract

The type III secretion systems (T3SS) encoded in pathogenicity islands SPI-1 and SPI-2 are key virulence factors of Salmonella. These systems translocate proteins known as effectors into eukaryotic cells during infection. To characterize the functionality of T3SS effectors, gene fusions to the CyaA’ reporter of Bordetella pertussis are often used. CyaA’ is a calmodulin-dependent adenylate cyclase that is only active within eukaryotic cells. Thus, the translocation of an effector fused to CyaA’ can be evaluated by measuring cAMP levels in infected cells. Here, we report the construction of plasmids pCyaA’-Kan and pCyaA’-Cam, which contain the ORF encoding CyaA’ adjacent to a cassette that confers resistance to kanamycin or chloramphenicol, respectively, flanked by Flp recombinase target (FRT) sites. A PCR product from pCyaA’-Kan or pCyaA’-Cam containing these genetic elements can be introduced into the bacterial chromosome to generate gene fusions by homologous recombination using the Red recombination system from bacteriophage λ. Subsequently, the resistance cassette can be removed by recombination between the FRT sites using the Flp recombinase. As a proof of concept, the plasmids pCyaA’-Kan and pCyaA’-Cam were used to generate unmarked chromosomal fusions of 10 T3SS effectors to CyaA’ in S. Typhimurium. Each fusion protein was detected by Western blot using an anti-CyaA’ monoclonal antibody when the corresponding mutant strain was grown under conditions that induce the expression of the native gene. In addition, T3SS-1-dependent secretion of fusion protein SipA-CyaA’ during in vitro growth was verified by Western blot analysis of culture supernatants. Finally, efficient translocation of SipA-CyaA’ into HeLa cells was evidenced by increased intracellular cAMP levels at different times of infection. Therefore, the plasmids pCyaA’-Kan and pCyaA’-Cam can be used to generate unmarked chromosomal cyaA’ translational fusion to study regulated expression, secretion and translocation of Salmonella T3SS effectors into eukaryotic cells. Full article

(This article belongs to the Special Issue Type III Secretion Systems in Human/Animal Pathogenic Bacteria)

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9 pages, 3190 KiB

Open AccessCommunication

Single Cell Analysis of Bistable Expression of Pathogenicity Island 1 and the Flagellar Regulon in Salmonella enterica

by María Antonia Sánchez-Romero and Josep Casadesús

Microorganisms 2021, 9(2), 210; https://doi.org/10.3390/microorganisms9020210 - 20 Jan 2021

Cited by 10 | Viewed by 2015

Abstract

Bistable expression of the Salmonella enterica pathogenicity island 1 (SPI-1) and the flagellar network (Flag) has been described previously. In this study, simultaneous monitoring of OFF and ON states in SPI-1 and in the flagellar regulon reveals independent switching, with concomitant formation of four subpopulations: SPI-1^OFF Flag^OFF, SPI-1^OFF Flag^ON, SPI-1^ON Flag^OFF, and SPI-1^ON Flag^ON. Invasion assays upon cell sorting show that none of the four subpopulations is highly invasive, thus raising the possibility that Flag^OFF cells might contribute to optimal invasion as previously proposed for SPI-1^OFF cells. Time lapse microscopy observation indicates that expression of the flagellar regulon contributes to the growth impairment previously described in SPI-1^ON cells. As a consequence, growth resumption in SPI-1^ON Flag^ON cells requires switching to both SPI-1^OFF and Flag^OFF states. Full article

(This article belongs to the Special Issue Type III Secretion Systems in Human/Animal Pathogenic Bacteria)

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10 pages, 1314 KiB

Open AccessArticle

Systematic Analysis of the Stress-Induced Genomic Instability of Type Three Secretion System in Aeromonas salmonicida subsp. salmonicida

by Pierre-Étienne Marcoux, Antony T. Vincent, Marie-Ange Massicotte, Valérie E. Paquet, Émilie J. Doucet, Nava Hosseini, Mélanie V. Trudel, Gabriel Byatt, Mathilde Laurent, Michel Frenette and Steve J. Charette

Microorganisms 2021, 9(1), 85; https://doi.org/10.3390/microorganisms9010085 - 31 Dec 2020

Cited by 9 | Viewed by 1938

Abstract

The type three secretion system (TTSS) locus of Aeromonas salmonicida subsp. salmonicida, located on the plasmid pAsa5, is known to be lost when the bacterium is grown at temperatures of 25 °C. The loss of the locus is due to the recombination of the insertion sequences flanking the TTSS region. However, the mechanism involved in this recombination is still elusive. Here, we analyzed 22 A. salmonicida subsp. salmonicida strains that had already lost their TTSS locus, and we systematically explored another 47 strains for their susceptibility to lose the same locus when grown at 25 °C. It appeared that strains from Europe were more prone to lose their TTSS locus compared to Canadian strains. More specifically, it was not possible to induce TTSS loss in Canadian strains that have AsaGEI2a, a genomic island, and prophage 3, or in Canadian strains without a genomic island. A comparative genomic approach revealed an almost perfect correlation between the presence of a cluster of genes, not yet characterized, and the susceptibility of various groups of strains to lose their locus. This cluster of genes encodes putative proteins with DNA binding capacity and phage proteins. This discovery creates new opportunities in the study of pAsa5 thermosensitivity. Full article

(This article belongs to the Special Issue Type III Secretion Systems in Human/Animal Pathogenic Bacteria)

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13 pages, 2155 KiB

Open AccessCommunication

Fluorescence Detection of Type III Secretion Using a Glu-CyFur Reporter System in Citrobacter rodentium

by Heather A. Pendergrass, Adam L. Johnson, Julia A. Hotinger and Aaron E. May

Microorganisms 2020, 8(12), 1953; https://doi.org/10.3390/microorganisms8121953 - 09 Dec 2020

Cited by 3 | Viewed by 1875

Abstract

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea worldwide. EPEC and the closely related murine model of EPEC infection, Citrobacter rodentium, utilize a type III secretion system (T3SS) to propagate the infection. Since the T3SS is not essential for the bacteria to survive or propagate, inhibiting the virulence factor with a therapeutic would treat the infection without causing harm to commensal bacteria. Studying inhibitors of the T3SS usually requires a BSL-2 laboratory designation and eukaryotic host cells while not indicating the mechanism of inhibition. We have designed a BSL-1 assay using the murine model C. rodentium that does not require mammalian cell culture. This CPG2-reporter assay allows for more rapid analysis of secretion efficiency than Western blotting and is sensitive enough to differentiate between partial and total inhibition of the T3SS. Here we present our method and the results of a small collection of compounds we have screened, including known T3SS inhibitors EGCG, regacin, and aurodox and related quorum sensing inhibitors tannic acid and ellagic acid. We have further characterized EGCG as a T3SS inhibitor and established its IC₅₀ of 1.8 ± 0.4 μM. We also establish tannic acid as a potent inhibitor of the T3SS with an IC₅₀ of 0.65 ± 0.09 μM. Full article

(This article belongs to the Special Issue Type III Secretion Systems in Human/Animal Pathogenic Bacteria)

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14 pages, 1866 KiB

Open AccessArticle

A Reporter System for Fast Quantitative Monitoring of Type 3 Protein Secretion in Enteropathogenic E. coli

by Luit Barkalita, Athina G. Portaliou, Maria S. Loos, Biao Yuan, Spyridoula Karamanou and Anastassios Economou

Microorganisms 2020, 8(11), 1786; https://doi.org/10.3390/microorganisms8111786 - 14 Nov 2020

Cited by 2 | Viewed by 2121

Abstract

The type 3 secretion system is essential for pathogenesis of several human and animal Gram-negative bacterial pathogens. The T3SS comprises a transmembrane injectisome, providing a conduit from the bacterial cytoplasm to the host cell cytoplasm for the direct delivery of effectors (including toxins). Functional studies of T3SS commonly monitor the extracellular secretion of proteins by SDS-PAGE and western blot analysis, which are slow and semi-quantitative in nature. Here, we describe an enzymatic reporter-based quantitative and rapid in vivo assay for T3SS secretion studies in enteropathogenic E. coli (EPEC). The assay monitors the secretion of the fusion protein SctA-PhoA through the injectisome based on a colorimetric assay that quantifies the activity of alkaline phosphatase. We validated the usage of this reporter system by following the secretion in the absence of various injectisome components, including domains of the gatekeeper essential for T3SS function. This platform can now be used for the isolation of mutations, functional analysis and anti-virulence compound screening. Full article

(This article belongs to the Special Issue Type III Secretion Systems in Human/Animal Pathogenic Bacteria)

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11 pages, 2283 KiB

Open AccessArticle

Early Intracellular Trafficking and Subsequent Activity of Programmed Cell Death in Channel Catfish Macrophages Infected with Edwardsiella ictaluri

by Lidiya P. Dubytska and Ronald L. Thune

Microorganisms 2020, 8(11), 1649; https://doi.org/10.3390/microorganisms8111649 - 24 Oct 2020

Cited by 6 | Viewed by 1769

Abstract

The development of Edwardsiella-containing-vacuoles (ECV) and the ability of Edwardsiella ictaluri to survive and replicate within macrophages suggests a unique process relative to normal phagosomal/lysosomal maturation and programed cell death. Developing ECV showed that endosomal membrane markers Rab5, EEA1, and Rab7 were all detected in both the wild type (WT) and an E. ictaluri type-3 secretion system (T3SS) mutant, 65ST. Co-localization with Lamp1, however, was significantly lower in the WT. The host cell endoplasmic reticulum marker, calnexin, co-localized to 65ST ECV significantly more than WT ECV, while Golgi vesicle marker, giantin, was recruited to WT ECV significantly more than 65ST. The autophagosomal marker LC3 was significantly lower in WT than in 65ST and Western blotting demonstrated significantly greater induction of the membrane localized, lipidated form, LC3-II, in 65ST ECV than in WT ECV. Activity of the apoptosis initiator caspase-8 increased post-infection in 65ST and was significantly lower in WT-infected cells. Executioner caspase-3/7 activity also increased significantly in 65ST-infected cells compared to WT-infected cells. Repression of apoptosis was further demonstrated with flow cytometry using Alexa Fluor 647-labeled Annexin V and propidium iodide. Results indicate that WT ECV fused with early and late endosomes but that phagosomal/lysosomal fusion did not occur. Additionally, WT-infected cells recruited Golgi vesicles for vacuolar size increase and bacterial growth material, and both autophagy and apoptosis were repressed in the WT. This activity was all based on the function of the E. ictaluri T3SS. Full article

(This article belongs to the Special Issue Type III Secretion Systems in Human/Animal Pathogenic Bacteria)

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Review

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26 pages, 1291 KiB

Open AccessReview

The Shigella Type III Secretion System: An Overview from Top to Bottom

by Meenakumari Muthuramalingam, Sean K. Whittier, Wendy L. Picking and William D. Picking

Microorganisms 2021, 9(2), 451; https://doi.org/10.3390/microorganisms9020451 - 22 Feb 2021

Cited by 26 | Viewed by 4244

Abstract

Shigella comprises four species of human-restricted pathogens causing bacillary dysentery. While Shigella possesses multiple genetic loci contributing to virulence, a type III secretion system (T3SS) is its primary virulence factor. The Shigella T3SS nanomachine consists of four major assemblies: the cytoplasmic sorting platform; the envelope-spanning core/basal body; an exposed needle; and a needle-associated tip complex with associated translocon that is inserted into host cell membranes. The initial subversion of host cell activities is carried out by the effector functions of the invasion plasmid antigen (Ipa) translocator proteins, with the cell ultimately being controlled by dedicated effector proteins that are injected into the host cytoplasm though the translocon. Much of the information now available on the T3SS injectisome has been accumulated through collective studies on the T3SS from three systems, those of Shigella flexneri, Salmonella typhimurium and Yersinia enterocolitica/Yersinia pestis. In this review, we will touch upon the important features of the T3SS injectisome that have come to light because of research in the Shigella and closely related systems. We will also briefly highlight some of the strategies being considered to target the Shigella T3SS for disease prevention. Full article

(This article belongs to the Special Issue Type III Secretion Systems in Human/Animal Pathogenic Bacteria)

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27 pages, 4640 KiB

Open AccessReview

The T3SS of Shigella: Expression, Structure, Function, and Role in Vacuole Escape

by Waad Bajunaid, Nathaline Haidar-Ahmad, Anwer Hasil Kottarampatel, France Ourida Manigat, Navoun Silué, Caetanie F. Tchagang, Kyle Tomaro and François-Xavier Campbell-Valois

Microorganisms 2020, 8(12), 1933; https://doi.org/10.3390/microorganisms8121933 - 05 Dec 2020

Cited by 27 | Viewed by 5555

Abstract

Shigella spp. are one of the leading causes of infectious diarrheal diseases. They are Escherichia coli pathovars that are characterized by the harboring of a large plasmid that encodes most virulence genes, including a type III secretion system (T3SS). The archetypal element of the T3SS is the injectisome, a syringe-like nanomachine composed of approximately 20 proteins, spanning both bacterial membranes and the cell wall, and topped with a needle. Upon contact of the tip of the needle with the plasma membrane, the injectisome secretes its protein substrates into host cells. Some of these substrates act as translocators or effectors whose functions are key to the invasion of the cytosol and the cell-to-cell spread characterizing the lifestyle of Shigella spp. Here, we review the structure, assembly, function, and methods to measure the activity of the injectisome with a focus on Shigella, but complemented with data from other T3SS if required. We also present the regulatory cascade that controls the expression of T3SS genes in Shigella. Finally, we describe the function of translocators and effectors during cell-to-cell spread, particularly during escape from the vacuole, a key element of Shigella’s pathogenesis that has yet to reveal all of its secrets. Full article

(This article belongs to the Special Issue Type III Secretion Systems in Human/Animal Pathogenic Bacteria)

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24 pages, 2878 KiB

Open AccessReview

Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity

by Juan Luis Araujo-Garrido, Joaquín Bernal-Bayard and Francisco Ramos-Morales

Microorganisms 2020, 8(3), 357; https://doi.org/10.3390/microorganisms8030357 - 02 Mar 2020

Cited by 16 | Viewed by 3942

Abstract

Type III secretion systems are used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, into the cytosol of host cells. These virulence factors interfere with a diverse array of host signal transduction pathways and cellular processes. Many effectors have catalytic activities to promote post-translational modifications of host proteins. This review focuses on a family of effectors with glycosyltransferase activity that catalyze addition of N-acetyl-d-glucosamine to specific arginine residues in target proteins, leading to reduced NF-κB pathway activation and impaired host cell death. This family includes NleB from Citrobacter rodentium, NleB1 and NleB2 from enteropathogenic and enterohemorrhagic Escherichia coli, and SseK1, SseK2, and SseK3 from Salmonella enterica. First, we place these effectors in the general framework of the glycosyltransferase superfamily and in the particular context of the role of glycosylation in bacterial pathogenesis. Then, we provide detailed information about currently known members of this family, their role in virulence, and their targets. Full article

(This article belongs to the Special Issue Type III Secretion Systems in Human/Animal Pathogenic Bacteria)

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