Salmonella-containing vacuole development in avian cells and characteristic of cigR in Salmonella enterica serovar Pullorum replication within macrophages

Salmonella enterica serovar Pullorum (S. Pullorum) is one of the host-restricted serotypes causing systemic infection in poultry. Survival in macrophages is one of the mechanisms underlying the persistent infection of S. Pullorum in hosts. Formation of Salmonella-containing vacuole (SCV) is essential for bacteria to be concealed in macrophages. In this study, confocal microscopy was applied to detect the SCV development by S. Pullorum in the chicken hepatocellular carcinoma cell line LMH and the macrophage cell line HD-11, respectively. The results showed that macrophages were more appropriate for the SCV maturation during S. Pullorum infection compared to epithelial cells. We evaluated the role of the CigR effector protein in the formation of SCVs. CigR is a membrane-binding protein, which is one of the type III secretion system 2 (T3SS2) effectors encoded within Salmonella pathogenicity island 3 (SPI3). The deletion of cigR in S. Typhimurium and S. Pullorum enhanced bacterial virulence to both mice and chickens. To analyze the influence of CigR on the SCV development during S. Pullorum infection, this study compared the formation of SCVs by using S. Pullorum C79-13 and the cigR deleted strain C79-13 Delta cigR. Compared to the wild type strain, the loss of cigR gene in strain C79-13 Delta cigR caused a four-fold increase in the recruitment ability of the SCV marker protein Rab7 in infected macrophages after 30 min, 5 h, and 16 h post-infection. In addition, infection and proliferation of strain C79-13 Delta cigR in the avian macrophage cell line HD-11 was higher than that of the wild type strain. Our findings suggest that CigR is an anti-virulence effector inhibiting replication of S. Pullorum and SCV development in macrophages.