Qin Liu

Caspase-11 Requires the Pannexin-1 Channel and the Purinergic P2X7 Pore to Mediate Pyroptosis and Endotoxic Shock

The noncanonical inflammasome induced by intracellular lipopolysaccharide (LPS) leads to caspase-11-dependent pyroptosis, which is critical for induction of endotoxic shock in mice. However, the signaling pathway downstream of caspase-11 is unknown. We found that cytosolic LPS stimulation induced caspase-11-dependent cleavage of the pannexin-1 channel followed up by ATP release, which in turn activated the purinergic P2X7 receptor to mediate cytotoxicity. In the absence of P2X7 or pannexin-1, pyroptosis induced by cytosolic LPS was abrogated. Cleavage of pannexin-1 required the catalytic activity of caspase-11 and was essential for ATP release and P2X7-mediated pyroptosis. Priming the caspase-11 pathway inxa0vivo with LPS or Toll-like receptor-3 (TLR3) agonist resulted in high mortality inxa0wild-type mice after secondary LPS challenge, but not in Casp11(-/-), Panx1(-/-), or P2x7(-/-) mice. These results reveal a critical role for pannexin-1 and P2X7 downstream of caspase-11 for pyroptosis and susceptibility to sepsis induced by the noncanonical inflammasome.

LuxO controls extracellular protease, haemolytic activities and siderophore production in fish pathogen Vibrio alginolyticus

Aims:u2002 To characterize the luxO gene in fish pathogen Vibrio alginolyticus MVP01 and investigate its roles in regulation of extracellular products (ECP) and siderophore production.

Regulation of Vibrio alginolyticus virulence by the LuxS quorum-sensing system

Quorum sensing (QS) is a bacterial intercommunication system that controls the expression of multiple genes in response to population density. The LuxS QS system regulates the expression of several virulence factors in a wide variety of pathogenic bacteria. LuxS has been characterized to be responsible for producing a type of autoinducer, AI-2, which stimulates the expression of the luciferase operon in Vibrio harveyi. Vibrio alginolyticus is established as an opportunistic pathogen of several marine animals, and its LuxS QS system remains undefined. To investigate the pathogenic role of luxS in V. alginolyticus, the luxS mutants of both the standard strain ATCC 33787 and a fish-clinical isolate MVP01, named MYJS and MYJM, respectively, were constructed. The mutation resulted in reduced lethality to Pagrus major. Intraperitoneal LD(50) of MYJS and MYJM increased by 15- and 93-fold, respectively. The two luxS mutants exhibited a lower growth rate and defective flagellar biosynthesis. They also showed a significant decrease in protease production and an increase in both extracellular polysaccharide production and biofilm development. The results suggest that the LuxS QS system plays an important role in regulating the expression of virulence factors in V. alginolyticus.

Novel Bacterial Surface Display Systems Based on Outer Membrane Anchoring Elements from the Marine Bacterium Vibrio anguillarum

ABSTRACT Surface display of heterologous peptides and proteins such as receptors, antigens, and enzymes on live bacterial cells is of considerable value for various biotechnological and industrial applications. In this study, a series of novel cell surface display systems were examined by using Vibrio anguillarum outer membrane protein and outer membrane lipoprotein as anchoring motifs. These display systems consist of (i) the signal sequence and first 11 N-terminal amino acids of V. anguillarum outer membrane lipoprotein Wza, or the signal sequence and first 9 N-terminal amino acids of the mature major Escherichia coli lipoprotein Lpp, and (ii) transmembrane domains of V. anguillarum outer membrane proteins Omporf1, OmpU, or Omp26La. In order to assay the translocation efficiency of constructed display systems in bacteria, green fluorescent protein (GFP) was inserted to the systems and the results of GFP surface localization confirmed that four of the six surface display systems could successfully display GFP on the E. coli surface. For assaying its potential application in live bacteria carrier vaccines, an excellent display system Wza-Omporf1 was fused with the major capsid protein (MCP) of large yellow croaker iridovirus and introduced into attenuated V. anguillarum strain MVAV6203, and subsequent analysis of MCP surface localization proved that the novel display system Wza-Omporf1 could function as a strong tool in V. anguillarum carrier vaccine development.

Search for live attenuated vaccine candidate against edwardsiellosis by mutating virulence‐related genes of fish pathogen Edwardsiella tarda

Aims:u2002 The aims of this study were to construct and evaluate the live attenuated vaccine against edwardsiellosis on zebra fish model.

Secreted glyceraldehyde-3-phosphate dehydrogenase as a broad spectrum vaccine candidate against microbial infection in aquaculture.

Aims:u2002 Subcellullar localizations and cross‐immunities of GAPDHs from six common pathogenic bacteria in aquaculture were investigated.

The Bacterial T6SS Effector EvpP Prevents NLRP3 Inflammasome Activation by Inhibiting the Ca2+-Dependent MAPK-Jnk Pathway

Inflammasome activation is an important innate immune defense mechanism against bacterial infection, and in return, bacteria express virulence determinants that counteract inflammasome activation. Many such effectors are secreted into host cells viaxa0specialized bacterial secretion systems. Here, the intracellular pathogenic bacterium Edwardsiella tarda was demonstrated to activate NLRC4 and NLRP3 inflammasomes via a type III secretion system (T3SS), and to inhibit NLRP3 inflammasome via a type VI secretion system (T6SS), indicating thexa0antagonistic roles of these systems in inflammasome signaling. Furthermore, a non-VgrG T6SS effector, EvpP, was identified that significantly inhibited NLRP3 inflammasome activation. Subsequent studies revealed that EvpP significantly suppressed Jnk activation, thus impairing oligomerization of the inflammasome adaptor ASC. Moreover, EvpP counteracted cytoplasmic Ca2+ increase, which works upstream of Jnk activation to regulate the NLRP3 inflammasome. Finally, EvpP-mediated inflammasome inhibition promoted bacterial colonization inxa0vivo. This work expands our understanding of bacterial T6SS in counteracting host immune responses.

Identification and functional characterization of EseH, a new effector of the type III secretion system of Edwardsiella piscicida

Edwardsiella piscicida, a bacterial pathogen in fish and humans, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. However, little is known about the associated effectors and their functional importance. In this study, we identified the ETAE_1757 encoded protein, termed here E. piscicida secretion effector H (EseH) as a novel T3SS effector. We found that upon infection with E. piscicida, EseH is translocated into nucleus of host cells which required the T3SS. Homology modelling analysis suggests that EseH is an enzyme that belongs to the family of phosphothreothine lyases. Consistently, EseH inhibited phosphorylation of ERK1/2, p38α and JNK MAPK pathways in host cells, but had no effect on the NF‐kB pathway. Furthermore, mutation of the critical amino acid residues predicted to confer phosphothreonine lyase activity abolished the ability of EseH to inhibit phosphorylation of ERK1/2, p38α and JNK MAPK pathways in host cells. In addition, we found an increase in transcript levels of TNF‐α, IL‐12, IL‐10 and IFN‐γ in zebrafish infected with the eseH mutant when compared with the wild type bacterium. Importantly, the virulence of E. piscicida deficient in EseH was highly attenuated in the zebrafish infection model which correlated with decreased loads of the mutant bacterium in both liver and kidney. Complementation of the E. piscicida mutant strain with EseH restored virulence in zebrafish. These results identified EseH as a critical T3SS effector that contributes to virulence by targeting MAPK signalling during E. piscicida infection.

Genetic relationships of Edwardsiella strains isolated in China aquaculture revealed by rep‐PCR genomic fingerprinting and investigation of Edwardsiella virulence genes

Aims:u2002 The aim of this study was to classify Edwardsiella strains isolated from China aquaculture based on biochemical and molecular methods.

A new target for the old regulator: H-NS suppress T6SS secretory protein EvpP, the major virulence factor in the fish pathogen Edwardsiella tarda.

The evpP gene in fish pathogen Edwardsiella tarda, coding the T6SS secretory protein EvpP and carrying an evpA‐evpO independent promoter region, was crucial for host cell invasion. The transcription of evpP was positively regulated by either the two‐component system EsrA‐EsrB or iron concentration, and its overexpression was known to enhance the invasion ability in our previous study. This work demonstrated that the H‐NS protein, a pleiotropic regulator of gene expression, was a new transcriptional modulator of evpP gene. The results showed that in vivo the transcriptional level of evpP was downregulated by H‐NS and in vitro this global regulator interacted directly with evpP promoter region. Moreover, DNase I footprinting experiments mapping the interaction regions of H‐NS and evpP revealed that this global regulator bound to evpP promoter and neighbouring areas at multiple sites. We provided a new insight into evpP regulation network and demonstrated the repression of H‐NS to the transcription of evpP gene.