Yeeun Kim, Seohyeon Lee, Kyungah Park, and Hyunjin Yoon. J. Microbiol. Biotechnol. 2022;32:602-11. https://doi.org/10.4014/jmb.2201.01034

Outer membrane vesicles (OMVs) are spherical membranous structures released by Gram-negative bacteria. Several bacterial pathogens utilize OMVs as vehicles for the delivery of virulence factors into host cells. Results of our previous study on proteomic analysis revealed that OMVs isolated from Salmonella enterica serovar Typhimurium had virulence effectors that are known to be translocated by Salmonella pathogenicity island 1 (SPI-1)-encoded type III secretion system (T3SS1) into the host cell. In the present study, immunoblot analysis confirmed the secretion of the six T3SS1 effector proteins, namely SipB and SipC (translocators of T3SS1), and SipA, SopA, SopB, and SopE2 (effectors translocated by T3SS1), by OMVs. Results of proteinase K treatment revealed the localization of these T3SS1 effector proteins on the outer surface of OMVs. SipC and SopE2 were secreted by OMVs independent of the three secretion systems T3SS1, T3SS2, and flagella, signifying OMVs to be an alternative delivery system to T3SSs. T3SS1 effectors SipA, SipC, and SopE2 were internalized into the cytoplasm of the host cell by OMVs independent of cellular Salmonella–host cell contact. In epithelial cells, addition of OMVs harboring T3SS1 effectors stimulated the production of F-actin, thereby complementing the attenuated invasion of ΔsopE2 into host cells. These results suggest that S. Typhimurium might exploit OMVs as a long-distance vehicle to deliver T3SS1 effectors into the cytoplasm of the host cell independent of bacteria–host cell interaction.

Non-typhoidal Salmonella (NTS) causes gastrointestinal infection, which is commonly self-limiting in healthy humans but may lead to invasive infection at extraintestinal sites, leading to bacteremia and focal systemic infections in the immunocompromised. However, a prophylactic vaccine against invasive NTS has not yet been developed. In this work, we explored the potential of a ΔyjeK mutant strain as a live attenuated vaccine against invasive NTS infection. YjeK in combination with YjeA is required for the post-translational modification of elongation factor P (EF-P), which is critical for bacterial protein synthesis. Therefore, malfunction of YjeK and YjeA-mediated EF-P activation might extensively influence protein expression during Salmonella infection. Salmonella lacking YjeK showed substantial alterations in bacterial motility, antibiotics resistance, and virulence. Interestingly, deletion of the yjeK gene increased the expression levels of Salmonella pathogenicity island (SPI)-1 genes but decreased the transcription levels of SPI-2 genes, thereby influencing bacterial invasion and survival abilities in contact with host cells. In a mouse model, the ΔyjeK mutant strain alleviated the levels of splenomegaly and bacterial burdens in the spleen and liver in comparison with the wild-type strain. However, mice immunized with the ΔyjeK mutant displayed increased Th1- and Th2-mediated immune responses at 28 days post-infection, promoting cytokines and antibodies production. Notably, the Th2-associated antibody response was highly induced by administration of the ΔyjeK mutant strain. Consequently, vaccination with the ΔyjeK mutant strain protected 100% of the mice against challenge with lethal invasive Salmonella and significantly relieved bacterial burdens in the organs. Collectively, these results suggest that the ΔyjeK mutant strain can be exploited as a promising live attenuated NTS vaccine.

Rapid detection of Salmonella serovars is important for the effective control and monitoring of food industries. In this study, we evaluate the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the rapid detection of three serovars, Enteritidis, Typhimurium, and Thompson, that are epidemiologically important in Korea. All strains were identified at the genus level, with a mean score of 2.319 using the BioTyper database, and their protein patterns were confirmed to be similar by principal component analysis and main spectrum profile dendrograms. Specific peaks for the three serovars were identified by analyzing 65 reference strains representing 56 different serovars. Specific mass peaks at 3018 ± 1 and 6037 ± 1, 7184 ± 1, and 4925 ± 1 m/z were uniquely found in the reference strains of serovars Enteritidis, Typhimurium, and Thompson, respectively, and they showed that the three serovars can be differentiated from each other and 53 other serovars. We verified the reproducibility of these mass peaks in 132 isolates, and serovar classification was achieved with 100% accuracy when compared with conventional serotyping through antisera agglutination. Our method can rapidly detect a large number of strains; hence, it will be useful for the high-throughput screening of Salmonella serovars.