Roles Of Salmonella Enterica Serovar Pullorum T3SS2 And Its Effector CigR In Pathogenicity And Immunity

Salmonella enterica serovar Pullorum (S. Pullorum) is the causative agent of pullorum disease (PD) and results in severe economic losses in poultry. PD is an acute systemic disease more common in young chicks with high morbidity and mortality, and often results in weight loss, decreased fertility and hatchability, diarrhea, and reproductive tract abnormalities in infected adults with a chronic carrier state. In addition, S. Pullorum can be transmitted to chicks through eggs. In many developing countries, it remains a major threat and the incidence of this disease has been frequently reported in recent years. In many developed countries, PD is rare because of their modern poultry-rearing facilities and well-established disease control programs, but it is difficult to eliminate this disease. Vaccination is one of the most effective methods of preventing Salmonella infections. Killed vaccines are the most commonly used commercially available vaccines, as only a few Salmonella live vaccines are registered. These live vaccines offer more effective protection than killed vaccines because they can induce more comprehensive cellular immune responses.Salmonella pathogenicity island 2 (SPI2) which can encode type Ⅲ secretion system 2 (T3SS2) is a major virulence determinant of Salmonella species. T3SS2 is very important for the ability of Salmonella to survive and replicate inside host cells, and plays an important role in the development of systemic infection, it can translocate more than 30 effectors which can modify the host cell and modulate the intracellular environment, the delivery of T3SS2 effectors to the host cell cytosol is a precisely controlled process, and different effectors have different functions, numerous effectors are associated with Salmonella virulence. However, the T3SS2 effector genes are not present in all S. enterica serovars, some genes are pseudogenes. Deletion of cigR can increase the virulence of Salmonella Typhimurium. There are many papers describing the function of T3SS2 and its effectors in Salmonella Typhimurium and Salmonella Enteritidis, but only few reports described the function of T3SS2 and its effectors in Salmonella Pullorum.In the present study, in order to deepen the knowledge on the virulence mechanisms, prevention and control of Salmonella Pullorum, we analyzed the pathogenesis of Salmonella Pullorum based on T3SS2 and its effector CigR, and evaluated the efficacy of an attenuated S. Pullorum mutant as a candidate vaccine. This study laid a foundation for further study of the pathogenesis of Salmonella Pullorum based on T3SS2 and its effectors.1. Construction and characterization of SPI2 deletion mutant of Salmonella PullorumIn this study, a SPI2 (-40 kb) deletion mutant of Salmonella Pullorum strain S06004 was constructed using the λ-red. recombinant system in order to research the pathogenicity of SPI-2 deletion mutant of Salmonella Pullorum and preliminary explore the feasibility of developing safe attenuated Salmonella Pullorum candidate vaccine strain. Then the biological characteristics such as growth rate, biochemical properties, genetic stability and virulence were evaluated between the deletion mutant strain S06004△SPI2 and its parent strain S06004. The growth rate and biochemical properties of S06004ASPI2 were consistent with those of its parent strain S06004. The mutant was stable with the deletion of SPI2. The chicken lethal test showed that the LD50 of S06004ASPI2 was 252 times higher than that of its parent strain S06004. The virulence of S06004ASPI2 was obviously attenuated. These results provided basic data for further study of the functions of SPI2, and implied its potential to develop attenuated Salmonella vaccine.2. Evaluation of Salmonella enterica serovar Pullorum pathogenicity island 2 mutant as a candidate live attenuated vaccineThe immunogenicity and protective efficacy of S06004ASPI2, a Salmonella pathogenicity island 2 (SPI2) deleted mutant of S. Pullorum was evaluated in 2-day-old chickens using two vaccination routes (oral and intramuscular), respectively in order to develop a safe and immunogenic vaccine. Our results showed that those vaccinated chickens revealed no differences in body weight or clinical symptoms compared to control group. S06004ASPI2 bacteria could not be isolated from livers and spleens of immunized chickens after a short period of time, and specific humoral and cellular immune responses were significantly induced. Immunized chickens were challenged with S. Pullorum strain S06004 and S. Gallinarum strain SG9 at 10 days post-immunization (dpi), and efficient protection against the challenges was observed based on mortality and clinical symptoms compared to control group. These findings suggest that S06004ASPI2 appears to be a highly immunogenic and efficient live attenuated vaccine candidate.3. Influence of Salmonella enterica serovar Pullorum pathogenicity island 2 on T3SS2 effetor gene expression in chicken macrophageWe analyzed 20 T3SS2 effector genes in the chromosome of Salmonella Pullorum strain S06004 by sequence analysis, sifB, sspH2 and steC were pseudogenes. Here, the influence of Salmonella enterica serovar Pullorum pathogenicity island 2 on these T3SS2 effetor genes expression in chicken macrophage HD11 cells was performed with quantitative real-time PCR (qRT-PCR), the mRNA transcription level of these genes of S06004 and S06004△SPI2 were detected at 2.5 hour post-infection (hpi),7.5 hpi and 15 hpi compared to that of S06004 at 1.5 hpi. Our results showed that all the 20 genes (including pseudogenes sifB, sspH2 and steC) can express in chicken macrophage HD11 cells of Salmonella Pullorum infection. SPI2 plays significantly roles in expression of genes pipB2, sifB, sopD2, sseJ, sseL, sspH2 and steD at three time points, steA at 2.5 hpi, pipB at 7.5 hpi, sifA at 7.5 hpi and 15 hpi. Pseudogene can also express in the process of Salmonella Pullorum infection. The mRNA transcription level of genes sseF, sseG and spiC of S06004△SPI2 could not be detected because they are located within SPI2. These findings provided important clues for further study of the functions of T3SS2 and its effectors.4. Distribution of T3SS2 effector genes among Salmonella Pullorum isolatesIn this study, we determined the distribution of 25 T3SS2 effector genes among 237 Salmonella Pullorum isolates in China from 1962 to 2013 by PCR, in order to investigate the distribution of T3SS2 effector genes among Salmonella Pullorum isolates. Meanwhile, we also analyzed the epidemic characteristics of these genes in 18 different serovars Sallmonella strains whose genome sequences were downloaded from GenBank. PCR determination of 25 T3SS2 effector genes demonstrated the presence of cigR, pipB, pipB2, slrP, sopD, sopD2, spiC, sseF, sseG, sseJ, sseKl, sseL, steA, steB, steC and steD genes and the absence of gogB and sspHl genes in these isolates, the frequency values of other genes gtgA, gtgE, sifA, sifB, ssel, sseK2 and sspH2 were 99.6%,45.2%,98.7%,99.2%,5.9%,15.2% and 97.5%, respectively. The distribution levels of gtgE, ssel and sseK2 in the period 1962-1979 were significantly higher than that in 2000-2013. Sequence analysis revealed that 15 genes cigR, pipB, pipB2, sifA, sifB, slrP, sopD, sopD2, spiC, sseF, sseG, sseL, steA, steC and steD exist in all of the 18 different serovars strains; sifB, sseKl, sspH2 and steC exist as pseudogenes in some of the 18 strains. Our results indicated that gtgE, ssel and sseK2 genes have chronological change of prevalence among the 237 Salmonella Pullorum isolates; most of the 25 T3SS2 effector genes have extensive distribution; some genes are pseudogenes during microevolution process.5. Function analysis of Salmonella Pullorum T3SS2 effector CigRAs a T3SS2 effector and predicted membrane protein, CigR is encoded by cigR gene within SPI3. In order to research the influence of the cigR gene on S. Pullorum, a cigR mutant of S. Pullorum S06004 was constructed by λ-red recombinant system, and then its characterization were analyzed. Lack of cigR did not affect the growth and biochemical properties, but resulted in decreased biofilm formation, the mutant strain was stable with deletion of cigR. Macrophage infection assay and in vivo competition assay showed that the mutant strain increased the replication and/or survival ability in HD11 cell line and in chickens compared to that of the parent strain, the LD50 of the mutant strain was one fifth of the parent strain for 2-day-old chickens when injected intramuscularly. In addition, the mRNA expression levels of CXCLI1, CXCLI2, IFN-y, IL-1β, IL-6, IL-10 and iNOS were significantly down-regulated because of the deletion of cigR, but it did not affect the mRNA expression of IL-4, IL-18, NF-kB1, TGF-(34 and TNF-α. These results demonstrate that deletion of cigR can decrease significantly biofilm formation, increase significantly virulence and affect the mRNA expression of some chicken cytokines, chemokines and other immune-related genes.