Edwardsiella tarda T6SS component evpP is regulated by esrB and iron, and plays essential roles in the invasion of fish.

Aims: To characterize the luxO gene in fish pathogen Vibrio alginolyticus MVP01 and investigate its roles in regulation of extracellular products (ECP) and siderophore production.



Methods and Results:  The luxO gene was cloned from V. alginolyticus MVP01. Genetic analysis revealed that it encoded a protein with high similarity to other LuxO homologues. The luxO in-frame deletion mutant and rpoN null mutant were constructed with suicide plasmids. We demonstrated that sole deletion in LuxO increased the secretion of extracellular protease and haemolytic products, but decreased siderophore production for V. alginolyticus MVP01. Mutants with null rpoN displayed significantly enhanced protease level and siderophore production while notable reduction in haemolytic activities of ECP.



Conclusions: Vibrio alginolyticus harbours functional luxO gene that regulates the secretion of extracellular protease and haemolytic materials as well as siderophore production in either σ54 dependent or independent manners.



Significance and Impact of the Study:  The current study demonstrated that V. alginolyticus MVP01 produces extracellular protease and haemolytic activity material as well as siderophore, which may be characteristics of the virulence of the strain. Revelations that secretion of these products is under the regulation of LuxO and σ54 as well as the potential quorum sensing systems in V. alginolyticus MVP01 will expedite the understanding of vibriosis pathogenesis.

https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2672.2007.03380.x

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

The current seventh pandemic of cholera, caused by serogroup O1, El Tor biotype, has now involved almost the entire developing world. The ongoing dynamic epidemiology of cholera, involving evolution of new strains, prolonged and more frequent epidemics, increased antimicrobial resistance, and awareness of the role of climate change upon the global burden has returned cholera to the forefront of global public health discussions. Improved water and sanitation should continue to be the mainstays of cholera-prevention efforts, but major improvements are a far-off goal for much of the cholera-affected developing world. The advent of safe and effective, new-generation oral vaccines against cholera has created renewed interest in the use of vaccines as a tool to control cholera.

/pdf/oral-vaccines-against-cholera-18zand0w5v.pdf

Oral Vaccines Against Cholera

In marine Vibrio species, the Vibrio harveyi-type LuxR protein, a key player in a quorum-sensing system, controls the expression of various genes. In this study, the luxR homologue in Vibrio alginolyticus was identified and named luxRval, whose expression was greatly induced by the increase of cell number. The luxRval in-frame deletion mutant showed a significant downregulation of total extracellular protease activity, and especially caused a 70% decrease in the transcript levels of extracellular alkaline serine protease A (proA), which was an important virulent factor of V. alginolyticus. Complementation in trans with luxRval could restore the expression of proA to the level of the wild-type strain. Deletion of the luxRval gene also resulted in changes of colony morphology, extracellular polysaccharide production and mobility. Therefore, another member of the V. harveyi-type LuxR regulator family has been characterized in V. alginolyticus.

Roles of LuxR in regulating extracellular alkaline serine protease A, extracellular polysaccharide and mobility of Vibrio alginolyticus.

In this study, rpoS gene was identified from Edwardsiella tarda EIB202 and its functional role was analyzed by using an in-frame deletion mutant rpoS and the complemental strain rpoS (+). Compared with the wild type and rpoS (+), rpoS was impaired in terms of the ability to survive under oxidative stress and nutrient starvation, as well as the resistance to 50% serum of Scophthalmus maximus in 3 h, demonstrating essential roles of RpoS in stress adaptation. The rpoS mutant also displayed markedly increased chondroitinase activity and biofilm formation. Real-time polymerase chain reaction revealed that the expression level of quorum sensing autoinducer synthetase genes luxS and edwI was increased by 3.7- and 2.5-fold in the rpoS mutant strain. Those results suggested that rpoS might be involved in the negative or positive regulation of chondroitinase and biofilm formation, or quorum sensing networks in E. tarda, respectively. Although there were no obvious differences between the wild-type and the rpoS mutant in adherence of epithelioma papulosum cyprini (EPC) cell and in the lethality on fish model, rpoS deletion leads to the drastically reduced capacity for E. tarda to internalize in EPC cells, indicating that RpoS was, while not the main, the factor required for the virulence network of E. tarda.

Characterization of Edwardsiella tarda rpoS: effect on serum resistance, chondroitinase activity, biofilm formation, and autoinducer synthetases expression.

IEM101, a Vibrio cholerae O1 El Tor Ogawa strain naturally deficient in CTXPhi, was previously selected as a live cholera vaccine candidate. To make a better and safer vaccine that can induce protective immunity against both the bacteria and cholera toxin (CT), a new vaccine candidate, IEM108, was constructed by introducing a ctxB gene and an El Tor-derived rstR gene into IEM101. The ctxB gene codes for the protective antigen CTB subunit, and the rstR gene mediates phage immunity. The stable expression of the two genes was managed by a chromosome-plasmid lethal balanced system based on the housekeeping gene thyA. Immunization studies indicate that IEM108 generates good immune responses against both the bacteria and CT. After a single-dose intraintestinal vaccination with 10(9) CFU of IEM108, both anti-CTB immunoglobulin G and vibriocidal antibodies were detected in the immunized-rabbit sera. However, only vibriocidal antibodies are detected in rabbits immunized with IEM101. In addition, IEM108 but not IEM101 conferred full protection against the challenges of four wild-type toxigenic strains of V. cholerae O1 and 4 micro g of CT protein in a rabbit model. By introducing the rstR gene, the frequency of conjugative transfer of a recombinant El Tor-derived RS2 suicidal plasmid to IEM108 was decreased 100-fold compared to that for IEM101. This indicated that the El Tor-derived rstR cloned in IEM108 was fully functional and could effectively inhibit the El Tor-derived CTXPhi from infecting IEM108. Our results demonstrate that IEM108 is an efficient and safe live oral cholera vaccine candidate that induces antibacterial and antitoxic immunity and CTXPhi phage immunity.

https://iai.asm.org/content/iai/71/10/5498.full.pdf

Construction and Evaluation of a Safe, Live, Oral Vibrio cholerae Vaccine Candidate, IEM108

The invention provided a novel vibrio cholerae strain from which vaccine can be prepared, the said strain has lost the toxic factor structure group, but retains its surface antigen structure, it is non-toxic for humanbody and animal, it has effect of improving immunocompetence. It also provided oral live vaccine prepared from the said strain.

Shixia Wang

Papers

Construction and Evaluation of a Safe, Live, Oral Vibrio cholerae Vaccine Candidate, IEM108

New strain of ribrio comma and its live vaccine