Functional dissection of translocon proteins of the <Emphasis Type="Italic">Salmonella</Emphasis> Pathogenicity Island 2-encoded type III secretion system

Background  

Type III secretion systems (T3SS) are essential virulence factors of most Gram-negative bacterial pathogens. T3SS deliver effector proteins directly into the cytoplasm of eukaryotic target cells and for this function, the insertion of a subset of T3SS proteins into the target cell membrane is important. These proteins form hetero-oligomeric pores acting as translocon for the delivery of effector proteins. Salmonella enterica is a facultative intracellular pathogen that uses the Salmonella Pathogenicity Island 2 (SPI2)-encoded T3SS to manipulate host cells in order to survive and proliferate within the Salmonella-containing vacuole of host cells. Previous work showed that SPI2-encoded SseB, SseC and SseD act to form the translocon of the SPI2-T3SS.     

The <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1

Background  

Pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) share a virulence plasmid encoding a type three secretion system (T3SS). This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins), the specific Yop chaperones (Sycs), and the Ysc (Yop secretion) proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study.     

Direct and negative regulation of the sycO-ypkA-ypoJ operon by cyclic AMP receptor protein (CRP) in Yersinia pestis

Background  

Pathogenic yersiniae, including Y. pestis, share a type III secretion system (T3SS) that is composed of a secretion machinery, a set of translocation proteins, a control system, and six Yop effector proteins including YpkA and YopJ. The cyclic AMP receptor protein (CRP), a global regulator, was recently found to regulate the laterally acquired genes (pla and pst) in Y. pestis. The regulation of T3SS components by CRP is unknown.     

Secretion of flagellin by the LEE-encoded type III secretion system of enteropathogenic Escherichia coli

Background  

Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen that possesses a type III secretion system (T3SS) encoded within the locus of enterocyte effacement (LEE). The LEE is essential for A/E lesion formation and directs the secretion and translocation of multiple LEE-encoded and non-LEE encoded effector proteins into the cytosol of infected cells. In this study we used proteomics to compare proteins exported to the culture supernatant by wild type EPEC E2348/69, a ΔespADB mutant and a ΔescF T3SS mutant.     

Presence of genes for type III secretion system 2 in <Emphasis Type="Italic">Vibrio</Emphasis><Emphasis Type="Italic">mimicus</Emphasis> strains

Background  

Vibrios, which include more than 100 species, are ubiquitous in marine and estuarine environments, and several of them e.g. Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus, are pathogens for humans. Pathogenic V. parahaemolyticus strains possess two sets of genes for type III secretion system (T3SS), T3SS1 and T3SS2. The latter are critical for virulence of the organism and be classified into two distinct phylogroups, T3SS2α and T3SS2β, which are reportedly also found in pathogenic V. cholerae non-O1/non-O139 serogroup strains. However, whether T3SS2-related genes are present in other Vibrio species remains unclear.     

Crystal structure of the C‐terminal domain of the Salmonella type III secretion system export apparatus protein InvA

InvA is a prominent inner‐membrane component of the Salmonella type III secretion system (T3SS) apparatus, which is responsible for regulating virulence protein export in pathogenic bacteria. InvA is made up of an N‐terminal integral membrane domain and a C‐terminal cytoplasmic domain that is proposed to form part of a docking platform for the soluble export apparatus proteins notably the T3SS ATPase InvC. Here, we report the novel crystal structure of the C‐terminal domain of Salmonella InvA which shows a compact structure composed of four subdomains. The overall structure is unique although the first and second subdomains exhibit structural similarity to the peripheral stalk of the A/V‐type ATPase and a ring building motif found in other T3SS proteins respectively.     

Extracellular overexpression of recombinant <Emphasis Type="Italic">Thermobifida fusca</Emphasis> cutinase by alpha-hemolysin secretion system in <Emphasis Type="Italic">E. coli</Emphasis> BL21(DE3)

Background  

Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.     

High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium

Background  

During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of β-lactam antibiotics were systematically improved.     

Two distinct regions in the model protein Peb1 are critical for its heterologous transport out of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli is frequently the first-choice host organism in expression of heterologous recombinant proteins in basic research as well as in production of commercial, therapeutic polypeptides. Especially the secretion of proteins into the culture medium of E. coli is advantageous compared to intracellular production due to the ease in recovery of the recombinant protein. Since E. coli naturally is a poor secretor of proteins, a few strategies for optimization of extracellular secretion have been described. We have previously reported efficient secretion of the diagnostically interesting model protein Peb1 of Campylobacter jejuni into the growth medium of Escherichia coli strain MKS12 (ΔfliCfliD). To generate a more detailed understanding of the molecular mechanisms behind this interesting heterologous secretion system with biotechnological implications, we here analyzed further the transport of Peb1 in the E. coli host.     

Development of an improved <Emphasis Type="Italic">Pseudoalteromonas haloplanktis</Emphasis> TAC125 strain for recombinant protein secretion at low temperature

Background  

In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in Pseudoalteromonas haloplanktis TAC125. This system makes use of the psychrophilic α-amylase from P. haloplanktis TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, Pseudoalteromonales are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a P. haloplanktis TAC125 mutant strain with reduced extra-cellular proteolytic activity.     

Stretching the arms of the type VI secretion sheath protein

The bacterial type VI secretion system (T6SS) is a multicomponent complex responsible for the translocation of effector proteins into the external milieu. The T6SS consists of an external sheath, an internal rigid tube, a baseplate, and a T6SS‐specific membrane complex. Secretion is accomplished by the contraction of the sheath, which expels the effector‐loaded tube. In this issue of EMBO reports, Brackmann et al 1 show how modifications of the sheath subunits can lock the T6SS assembly in the extended state. These findings allowed Wang et al 2 and Nazarov et al 3 to purify the T6SS sheath–tube–baseplate complex in the extended pre‐secretion state and to analyze its structure using cryo‐electron microscopy (cryoEM).     

Identification of Anaplasma marginale type IV secretion system effector proteins

Background

Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.

Results

By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141) of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system.

Conclusions

The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work.     

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

Genetically distinct pathways guide effector export through the type VI secretion system

Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), haemolysin co‐regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone‐like quality of Hcp. Application of this approach to the Hcp secretion island I‐encoded T6SS (H1‐T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (t ype VI s ecretion e xported 4), subsequently shown to act as a potent intra‐specific H1‐T6SS‐delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1‐T6SS effectors, Tse5 and Tse6, which differ from Hcp‐stabilized substrates by the presence of toxin‐associated PAAR‐repeat motifs and genetic linkage to members of the valine‐glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp‐stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1‐T6SS‐exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.     

VirB10 vaccination for protection against <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis>

Background

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the etiologic agent Anaplasma phagocytophilum. HGA was designated a nationally notifiable disease in the United States in 1998. Currently there are no vaccines available against HGA. Conserved membrane proteins that are subdominant in Anaplasma species, such as VirB9 and VirB10, may represent better vaccine targets than the variable immunodominant surface proteins. VirB9 and VirB10 are constituents of the Type 4 secretion system (T4SS) that is conserved amongst many intracellular bacteria and performs essential functions for invasion and survival in host cells.

Results

Immunogenicity and contribution to protection, provided after intramuscular vaccination of plasmid DNA encoding VirB9-1, VirB9-2, and VirB10 followed by inoculation of homologous recombinant proteins, in a prime-boost immunization strategy was evaluated in a murine model of HGA. Recombinant VirB9-1-, VirB9-2-, and VirB10-vaccinated mice developed antibody responses that specifically reacted with A. phagocytophilum organisms. However, only the mice vaccinated with VirB10 developed a significant increase in IFN-γ CD4⁺ T cells and partial protection against challenge with A. phagocytophilum.

Conclusions

This work provides evidence that A. phagocytophilum T4SS VirB10 is partially protective in a murine model against infection in an IFN-γ-dependent fashion and suggests that this protein may be a potential vaccine candidate against this and possibly other pathogenic bacteria with a T4SS.

     

Regulation of Vibrio parahaemolyticus T3SS2 gene expression and function of T3SS2 effectors that modulate actin cytoskeleton

Vibrio parahaemolyticus is a leading causative agent of seafood‐borne gastroenteritis worldwide. Most clinical isolates from patients with diarrhoea possess two sets of genes for the type III secretion system (T3SS) on each chromosome (T3SS1 and T3SS2). T3SS is a protein secretion system that delivers effector proteins directly into eukaryotic cells. The injected effectors modify the normal cell functions by altering or disrupting the normal cell signalling pathways. Of the two sets of T3SS genes present in V. parahaemolyticus, T3SS2 is essential for enterotoxicity in several animal models. Recent studies have elucidated the biological activities of several T3SS2 effectors and their roles in virulence. This review focuses on the regulation of T3SS2 gene expression and T3SS2 effectors that specifically target the actin cytoskeleton.     

Amino‐terminal residues dictate the export efficiency of the Campylobacter jejuni filament proteins via the flagellum

Bacterial flagella play an essential role in the pathogenesis of numerous enteric pathogens. The flagellum is required for motility, colonization, and in some instances, for the secretion of effector proteins. In contrast to the intensively studied flagella of Escherichia coli and Salmonella typhimurium, the flagella of Campylobacter jejuni, Helicobacter pylori and Vibrio cholerae are less well characterized and composed of multiple flagellin subunits. This study was performed to gain a better understanding of flagellin export from the flagellar type III secretion apparatus of C. jejuni. The flagellar filament of C. jejuni is comprised of two flagellins termed FlaA and FlaB. We demonstrate that the amino‐termini of FlaA and FlaB determine the length of the flagellum and motility of C. jejuni. We also demonstrate that protein‐specific residues in the amino‐terminus of FlaA and FlaB dictate export efficiency from the flagellar type III secretion system (T3SS) of Yersinia enterocolitica. These findings demonstrate that key residues within the amino‐termini of two nearly identical proteins influence protein export efficiency, and that the mechanism governing the efficiency of protein export is conserved among two pathogens belonging to distinct bacterial classes. These findings are of additional interest because C. jejuni utilizes the flagellum to export virulence proteins.     

Identification of tomato plant as a novel host model for <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis>

Background  

Burkholderia pseudomallei is the causative agent for melioidosis, a disease with significant mortality and morbidity in endemic regions. Its versatility as a pathogen is reflected in its relatively huge 7.24 Mb genome and the presence of many virulence factors including three Type Three Secretion Systems known as T3SS1, T3SS2 and T3SS3. Besides being a human pathogen, it is able to infect and cause disease in many different animals and alternative hosts such as C. elegans.