Antigens of the type-three secretion system of Aeromonas salmonicida subsp. salmonicida prevent protective immunity in rainbow trout

Aeromonas salmonicida subsp. salmonicida is the etiologic agent of furunculosis, a frequent and significant disease of fisheries worldwide. The disease is largely controlled by commercial oil adjuvanted vaccines containing bacterins. However, the mechanisms leading to a protective immune response remain poorly understood. The type-three secretion system (T3SS) plays a central role in virulence of A. salmonicida subsp. salmonicida and thus may have an influence on the immune response of the host. The aim of this study was to evaluate the role of the T3SS antigens in mounting a protective immune response against furunculosis. Rainbow trout were intraperitoneally vaccinated in two independent experiments with bacterins prepared from a wild-type A. salmonicida strain and an isogenic strain carrying a deletion in the T3SS (ΔascV). Fish were challenged with the wt strain eight weeks after vaccination. In both trials, the survival rate of trout vaccinated with the ΔascV strain was significantly higher (23-28%) in comparison to the group vaccinated with the wt strain. High-throughput proteomics analysis of whole bacteria showed the ascV deletion in the mutant strain resulted in lower expression of all the components of the T3SS, several of which have a potential immunosuppressive activity. In a third experiment, fish were vaccinated with recombinant AcrV (homologous to the protective antigen LcrV of Yersinia) or S-layer protein VapA (control). AcrV vaccinated fish were not protected against a challenge while fish vaccinated with VapA were partially protected. The presence of T3SS proteins in the vaccine preparations decreased the level of protection against A. salmonicida infection and that AcrV was not a protective antigen. These results challenge the hypothesis that mounting specific antibodies against T3SS proteins should bring better protection to fish and demonstrate that further investigations are needed to better understand the mechanisms underlying effective immune responses against A. salmonicida infection.

Aeromonas salmonicida subsp. salmonicida in the light of its type-three secretion system

Aeromonas salmonicida subsp. salmonicida is an important pathogen in salmonid aquaculture and is responsible for the typical furunculosis. The type-three secretion system (T3SS) is a major virulence system. In this work, we review structure and function of this highly sophisticated nanosyringe in A. salmonicida. Based on the literature as well as personal experimental observations, we document the genetic (re)organization, expression regulation, anatomy, putative functional origin and roles in the infectious process of this T3SS. We propose a model of pathogenesis where A. salmonicida induces a temporary immunosuppression state in fish in order to acquire free access to host tissues. Finally, we highlight putative important therapeutic and vaccine strategies to prevent furunculosis of salmonid fish.

The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

BACKGROUND Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. RESULTS Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. CONCLUSIONS We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[(32)P]-ATP. Its crystal structure, determined to 2.9-Å resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β-rich domain at the C-terminus. On crystal soaking with ATP/Mg(2+), additional electron density indicated the presence of a PP(i) /Mg(2+) moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg(2+) and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the α-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel β-strand interactions between enzyme and target is suggested.

Characterization of the type I secretion system of the RTX toxin ApxII in "Actinobacillus porcitonsillarum"

Strains of Actinobacillus porcitonsillarum are regularly isolated from the tonsils of healthy pigs. A. porcitonsillarum is non pathogenic but phenotypically it strongly resembles the pathogenic species Actinobacillus pleuropneumoniae, thereby interfering with the diagnosis of the latter. A. porcitonsillarum is hemolytic but unlike A. pleuropneumoniae, it contains only apxII genes and not apxI or apxIII genes. In contrast to the truncated apxII operon of A. pleuropneumoniae, which lacks the type I secretion genes BD, characterization of the apxII operon in A. porcitonsillarum revealed that it contains an intact and complete apxII operon. This shows a typical RTX operon structure with the gene arrangement apxIICABD. The region upstream of the apxII operon is also different from that in A. pleuropneumoniae and contains an additional gene, aspC, encoding a putative aspartate aminotransferase. Trans-complementation experiments in Escherichia coli and A. pleuropneumoniae indicated that the entire apxII operon of A. porcitonsillarum is sufficient to express and secrete the ApxIIA toxin and that the ApxIIA toxin of A. pleuropneumoniae can be secreted by the type I secretion system encoded by apxIIBD. These findings suggest that the complete apxII operon found in A. porcitonsillarum might be an ancestor of the truncated homologue found in A. pleuropneumoniae. The genetic context of the apxII locus in A. porcitonsillarum and A. pleuropneumoniae suggests that in the latter, the contemporary truncated operon is the result of a recombination event within the species, rather than a horizontal transfer of an incomplete operon.

Complete protection against P. berghei malaria upon heterologous prime/boost immunization against circumsporozoite protein employing Salmonella type III secretion system and Bordetella adenylate cyclase toxoid

Sterile immunity against malaria can be achieved by the induction of IFNgamma-producing CD8(+) T cells that target infected hepatocytes presenting epitopes of the circumsporozoite protein (CSP). In the present study we evaluate the protective efficacy of a heterologous prime/boost immunization protocol based on the delivery of the CD8(+) epitope of Plasmodium berghei CSP into the MHC class I presentation pathway, by either a type III secretion system of live recombinant Salmonella and/or by direct translocation of a recombinant Bordetella adenylate cyclase toxoid fusion (ACT-CSP) into the cytosol of professional antigen-presenting cells (APCs). A single intraperitoneal application of the recombinant ACT-CSP toxoid, as well as a single oral immunization with the Salmonella vaccine, induced a specific CD8(+) T cell response, which however conferred only a partial protection on mice against a subsequent sporozoite challenge. In contrast, a heterologous prime/boost vaccination with the live Salmonella followed by ACT-CSP led to a significant enhancement of the CSP-specific T cell response and induced complete protection in all vaccinated mice.

Detection of type III secretion genes as a general indicator of bacterial virulence

Type III secretion systems of Gram-negative bacteria are specific export machineries for virulence factors which allow their translocation to eukaryotic cells. Since they correlate with bacterial pathogenicity, their presence is used as a general indicator of bacterial virulence. By comparing the genetic relationship of the major type III secretion systems we found the family of genes encoding the inner-membrane channel proteins represented by the Yersinia enterocolitica lcrD (synonym yscV) and its homologous genes from other species an ideal component for establishing a general detection approach for type III secretion systems. Based on the genes of the lcrD family we developed gene probes for Gram-negative human, animal and plant pathogens. The probes comprise lcrD from Y. enterocolitica, sepA from enteropathogenic Escherichia coli, invA from Salmonella typhimurium, mxiA from Shigella sonnei, as well as hrcV from Erwinia amylovora. In addition we included as a control probe the flhA gene from E. coli K-12 to validate our approach. FlhA is part of the flagellar export apparatus which shows a high degree of similarity with type III secretions systems, but is not involved in pathogenicity. The probes were evaluated by screening a series of pathogenic as well as non-pathogenic bacteria. The probes detected type III secretion in pathogens where such systems were either known or were expected to be present, whereas no positive hybridization signals could be found in non-pathogenic Gram-negative bacteria. Gram-positive bacteria were devoid of known type III secretion systems. No interference due to the genetic similarity between the type III secretion system and the flagellar export apparatus was observed. However, potential type III secretion systems could be detected in bacteria where no such systems have been described yet. The presented approach provides therefore a useful tool for the assessment of the virulence potential of bacterial isolates of human, animal and plant origin. Moreover, it is a powerful means for a first safety assessment of poorly characterized strains intended to be used in biotechnological applications.

AsaGEI2b: a new variant of a genomic island identified in the Aeromonas salmonicida subsp. salmonicida JF3224 strain isolated from a wild fish in Switzerland.

Aeromonas salmonicida subsp. salmonicida is the causal agent of furunculosis in salmonids. We recently identified a group of genomic islands (AsaGEI) in this bacterium. AsaGEI2a, one of these genomic islands, has almost exclusively been identified in isolates from North America. To date, Aeromonas salmonicida subsp. salmonicida JF3224, a strain isolated from a wild brown trout (Salmo trutta) caught in Switzerland, was the only European isolate that appeared to bear AsaGEI2a. We analyzed the genome of JF3224 and showed that the genomic island in JF3224 is a new variant of AsaGEI, which we have called AsaGEI2b. While AsaGEI2b shares the same integrase gene and insertion site as AsaGEI2a, it is very different in terms of many other features. Additional genomic investigations combined with PCR genotyping revealed that JF3224 is sensitive to growth at 25°C, leading to insertion sequence-dependent rearrangement of the locus on the pAsa5 plasmid that encodes a type three secretion system, which is essential for the virulence of the bacterium. The analysis of the JF3224 genome confirmed that AsaGEIs are accurate indicators of the geographic origins of A. salmonicida subsp. salmonicida isolates and is another example of the susceptibility of the pAsa5 plasmid to DNA rearrangements.

InvB is required for type III-dependent secretion of SopA in Salmonella enterica serovar Typhimurium.

The Salmonella effector protein SopA is translocated into host cells via the SPI-1 type III secretion system (TTSS) and contributes to enteric disease. We found that the chaperone InvB binds to SopA and slightly stabilizes it in the bacterial cytosol and that it is required for its transport via the SPI-1 TTSS.

Classification of acute and delayed contrast media-induced reactions: proposal of a three-step system

Physicians and scientists use a broad spectrum of terms to classify contrast media (CM)-induced adverse reactions. In particular, the designation of hypersensitivity reactions is quite varied. Consequently, comparisons of different papers dealing with this subject are difficult or even impossible. Moreover, general descriptions may lead to problems in understanding reactions in patients with a history of adverse CM-reactions, and in efficiently managing these patients. Therefore, the goal of this paper is to suggest an easy system to clearly classify these reactions. The proposed three-step systems (3SS) is built up as follows: step 1 exactly describes the clinical features, including their severity; step 2 categorizes the time point of the onset (immediate or nonimmediate); and step 3 generally classifies the reaction (hypersensitivity or nonhypersensitivity reaction). The 3SS may facilitate better understanding of the clinical manifestations of adverse CM reactions and may support the prevention of these reactions on the basis of personalized medicine approaches.

AopP, a type III effector protein of Aeromonas salmonicida, inhibits the NF-kappaB signalling pathway

Aeromonas salmonicida subsp. salmonicida contains a functional type III secretion system that is responsible for the secretion of the ADP-ribosylating toxin AexT. In this study, the authors identified AopP as a second effector protein secreted by this system. The aopP gene was detected in both typical and atypical A. salmonicida isolates and was found to be encoded on a small plasmid of approximately 6.4 kb. Sequence analysis indicates that AopP is a member of the YopJ family of effector proteins, a group of proteins that interfere with mitogen-activated protein kinase (MAPK) and/or nuclear factor kappa B (NF-kappaB) signalling pathways. AopP inhibits the NF-kappaB pathway downstream of IkappaB kinase (IKK) activation, while a catalytically inactivated mutant, AopPC177A, does not possess this inhibitory effect. Unlike other effectors of the YopJ family, such as YopJ and VopA, AopP does not inhibit the MAPK signalling pathway.

Outbreaks of an ulcerative and haemorrhagic disease in Arctic char Salvelinus alpinus caused by Aeromonas salmonicida subsp. smithia

Arctic char Salvelinus alpinus farmed in different places in Austria and free of the viral diseases viral haemorrhagic septcaemia (VHS), infectious haematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) experienced disease and mortality. Diseased fish showed skin ulceration and pathological signs of sepsis. Aeromonas sp. was isolated as pure culture from the kidney of freshly euthanized diseased fish. Three independent isolates from outbreaks that occurred on 2 of the affected farms were analyzed phylogenetically by DNA sequence analysis of the rrs and gyrB genes and phenotypically with biochemical reactions. All 3 isolates were identified as Aeromonas salmonicida subsp. smithia. Analysis of virulence genes in these isolates revealed the presence of a Type III secretion system as well as several related virulence effector genes including aexT, encoding the Aeromonas exotoxin AexT, aopP and aopH. These genes are characteristic for virulent strains of typical and atypical subspecies of A. salmonicida.

The Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms.

Salmonella typhimurium can colonize the gut, invade intestinal tissues, and cause enterocolitis. In vitro studies suggest different mechanisms leading to mucosal inflammation, including 1) direct modulation of proinflammatory signaling by bacterial type III effector proteins and 2) disruption or penetration of the intestinal epithelium so that penetrating bacteria or bacterial products can trigger innate immunity (i.e., TLR signaling). We studied these mechanisms in vivo using streptomycin-pretreated wild-type and knockout mice including MyD88(-/-) animals lacking an adaptor molecule required for signaling via most TLRs. The Salmonella SPI-1 and the SPI-2 type III secretion systems (TTSS) contributed to inflammation. Mutants that retain only a functional SPI-1 (M556; sseD::aphT) or a SPI-2 TTSS (SB161; DeltainvG) caused attenuated colitis, which reflected distinct aspects of the colitis caused by wild-type S. typhimurium: M556 caused diffuse cecal inflammation that did not require MyD88 signaling. In contrast, SB161 induced focal mucosal inflammation requiring MyD88. M556 but not SB161 was found in intestinal epithelial cells. In the lamina propria, M556 and SB161 appeared to reside in different leukocyte cell populations as indicated by differential CD11c staining. Only the SPI-2-dependent inflammatory pathway required aroA-dependent intracellular growth. Thus, S. typhimurium can use two independent mechanisms to elicit colitis in vivo: SPI-1-dependent and MyD88-independent signaling to epithelial cells and SPI-2-dependent intracellular proliferation in the lamina propria triggering MyD88-dependent innate immune responses.

Positive and negative mutant selection in the human histone hairpin-binding protein using the yeast three-hybrid system.

We have used the yeast three-hybrid system in a positive selection for mutants of the human histone hairpin-binding protein (HBP) capable of interacting with non-canonical hairpins and in a negative selection for loss-of-binding mutants. Interestingly, all mutations from the positive selection are located in the N- and C-terminal regions flanking a minimal RNA-binding domain (RBD) previously defined between amino acids 126 and 198. Further, in vitro binding studies demonstrate that the RBD, which shows no obvious similarity to other RNA-binding motifs, has a relaxed sequence specificity compared to full-length HBP, allowing it to bind to mutant hairpin RNAs not normally found in histone genes. These findings indicate that the sequences flanking the RBD are important for restricting binding to the highly conserved histone hairpin structure. Among the loss-of-binding mutations, about half are nonsense mutations distributed throughout the N-terminal part and the RBD whereas the other half are missense mutations restricted to the RBD. Whereas the nonsense mutations permit a more precise definition of the C-terminal border of the RBD, the missense mutations identify critical residues for RNA binding within the RBD.