SteE enhances the colonization of Salmonella Pullorum in chickens

Salmonella pullorum ( S . pullorum ) is the causative agent of pullorum disease and results in severe economic losses in poultry, and can long-term survival by colonizing host organs. steE is an effector protein secreted by Salmonella pathogenicity island 2. It is not clear in vivo for the colonization of Salmonella . To investigate the role of steE on the colonization of S. Pullorum in the principal organs of chicken, we used S. pullorum and S. pullorum Δ steE strains immunized chickens, respectively. The results of the virulence assay showed that the LD 50 of S. pullorum Δ steE was 22.8 times higher than that of S. pullorum in chickens. The colonization exper-iment of bacteria showed that the overall change trend of the number of S. pullorum and S. pullorum Δ steE strains were similar in chicken liver, spleen, heart, bursa, and cecum, which increased first and then decreased. However, the deletion of steE caused significantly reduced colonization, pathological change, and virulence of S . pullorum in a chicken infection model. Our findings provide exciting insights into the pathogenic mechanism and live attenuated vaccine associated with steE in S. pullorum .


Introduction
Salmonella enterica serovar Pullorum (S. pullorum) is an intracellular pathogen of host specificity (Li et al., 2018;Chechet et al., 2022).It mainly infects chickens within three weeks of age, resulting in acute systemic diseases and high mortality in poultry.The growth and production performance of young and adult chickens infected with S. pullorum will be badly affected (Li et al., 2019;Foster et al., 2021).S. pullorum can be transmitted vertically, and the hatching rate of contaminated breeding eggs will significantly reduce.Sick chickens and infected chickens are the primary sources of S. pullorum.The cocks carrying S. pullorum can also transmit to the hens through vertical transmission.One of the essential measures to control the occurrence and prevalence of S. pullorum is the purification of S. pullorum in breeding chickens and the disinfection of breeding eggs.At present, S. pullorum is still occurring and prevalent in many countries, especially in developing countries, including Brazil and India, which has caused substantial economic losses to the poultry industry (Geng et al., 2019;Xian et al., 2020).Therefore, further study of the pathogenic mechanism of S. pullorum is necessary.
The survival and proliferation of Salmonella are related to a particular cell membrane region formed in the host cell, namely the Salmonella-containing vacuole (SCV).The formation of SCV is inseparable from type III secretion system 2 (T3SS2) encoded by Salmonella pathogenic island 2 (SPI-2).Therefore, T3SS2 plays an essential role in the pathogenesis of S. pullorum (Figueira et al., 2013;Knuff-Janzen et al., 2021;Cohen et al., 2021).T3SS2 is an injection device in that the Salmonella-secreted effector is translocated into the host cells through the device to provide a favorable environment for Salmonella invasion (Greene et al., 2021;Fang & Méresse, 2021).As a novel effector protein of Salmonella T3SS2, steE is encoded in Salmonella prophage gifsy-1, which helps to survive and replicate in macrophages and plays a vital role in the evolution of Salmonella and the regulation of host innate immune response (Coombes et al., 2005).
In the present study, little work is reported about the steE of S. pullorum.Continuing to explore the relationship between steE and the virulence of S. pullorum contributes to revealing the pathogenic mechanism of S. pullorum.Therefore, chickens were infected with S. pullorum and S. pullorum ΔsteE strains as an animal models to analyze their potential role in the virulence and colonization of S. pullorum.

Strains and Animals
S. pullorum and S. pullorum ΔsteE strains were preserved and constructed in our laboratory.Our laboratory hatched healthy 2-day-old Jinghong laying hens.

Analysis of Clinical Symptoms and Autopsy
The clinical symptoms and morbidity of the chicks were observed every day after S. pullorum or S. pullorum ΔsteE strain infection and recorded and photographed.

Bacterial Colonization in Organs
S. pullorum and S. pullorum ΔsteE strains were cultured in LB broth for 12 h.The bacteria cultures were washed three times with PBS and suspended to adjust the bacterial concentration.Sixty chickens were randomly divided into three groups (n = 20).Chicken from each group was infected orally with 1 × 10 8 CFU of S. pullorum or S. pullorum ΔsteE strain in 100 μl of PBS, according to the LD 50 assay as mentioned previously.Twenty chickens received 100 μL of PBS as a control group.Chickens were deprived of food and water for 12 hours before and after chicken immunization.At 12 h, 24 h, 36 h, 2 d, 3 d, 4 d, and 7 d postchallenge, the cecum, liver, spleen, bursa, and heart organs were harvested from each chicken.After weighing, organs from each group were homogenized mechanically, and diluted serially for the subsequent cultivation on XLD agar plates at 37 °C for 12−16 h.The bacterial number was counted and displayed as log10 CFU/g.The dynamic distribution of S. pullorum and S. pullorum ΔsteE strains in various organs was analyzed by plating on XLD agar.The bacterial number was counted and expressed as log10 CFU/g at 12 h, 24 h, 36 h, 2 d, 3 d, 4 d, and 7 d.

Clinical Symptoms and Changes of Autopsy
One day after the chickens were infected with S. pullorum or S. pullorum ΔsteE strain, the chicks were depressed and had a poor appetite, accompanied by the phenomenon of gathering together.Some chicks could not stand steadily, discharged white sticky feces, and the feathers around the anus were covered with feces.As shown in Fig. 1, the pathological change of chicken organs infected with S. pullorum was more severe than that of the S. pullorum ΔsteE and control groups as follows: there were dark red needle tips and large bleeding spots at the edge of the liver; splenomegaly, dark red; bursal enlargement; the cecum has puffed and bleeding; heart congestion, swelling with blood filaments.

steE Enhances the Colonization of S. Pullorum in Chicken Cecum
The results of bacterial colonization showed that the bacterial number of S. pullorum and S. pullorum ΔsteE strains in the cecum were increased from 12 h to 3 d but decreased from 3 d to 7 d (Fig. 2).At three days post-challenge, the colonization of S. Pullorum and S. pullorum ΔsteE stains reached the peak in the chicken cecum, and the settlement amount of S. pullorum and S. pullorum ΔsteE strains reached the lowest at 7 d.The changing trend of S. Pullorum and S. pullorum ΔsteE strains was increased at first and then decreased in the whole process of the infection.Still, the settled quantity of S. pullorum ΔsteE was consistently lower than that of S. pullorum.

steE Enhances the colonization of S. pullorum in chicken liver
The results of bacterial colonization showed that the bacterial number of S. Pullorum was increased from 12 h to 4 d in the whole process of the infection and decreased significantly from 4 d to 7 d (Fig. 3).The total amount of S. pullorum ΔsteE was increased from 12 h to 3 d after inoculation, and decreased from 4 d to 7 d.The colonization of S. Pullorum and S. pullorum ΔsteE strains reached the peak r about four days after chicken inoculation.The changing trend of S. pullorum and S. pullorum ΔsteE strains increased first and then decreased throughout the experiment.However, the settled quantity of S. pullorum ΔsteE was consistently lower than that of S. pullorum.

steE Enhances the Colonization of S. pullorum in Chicken Spleen
The colonization of S. pullorum and S. pullorum ΔsteE strains increased from 12 h to 3d after chicken inoculation, and decreased significantly from 3 d to 7 d in the chicken spleen (Fig. 4).The colonization of S. pullorum and S. pullorum ΔsteE strains reached the peak about 3 d in the chicken spleen after chicken inoculation.The changing trend of S. pullorum and S. Pullorum ΔsteE strains increased initially and then decreased throughout the infection.Still, the settled quantity of S. pullorum ΔsteE was consistently lower than that of S. pullorum.

steE Enhances the Colonization of S. pullorum in Chicken Brusa
The colonization of S. Pullorum was increased from 12 h to 3 d in chicken bursa, increased slowly at 36 h, and decreased from 3 d to 7 d.The results showed that S. pullorum reached the peak of about 3 d in the bursa after chicken inoculation (Fig. 5).The changing trend of S. pullorum ΔsteE in chicken bursa is consistent with that of S. pullorum.At the same time, the settled number of S. pullorum ΔsteE was consistently lower than that of S. pullorum.

steE Enhances the Colonization of S. pullorum in Chicken Heart
The colonization of S. Pullorum and S. pullorum ΔsteE strains was increased from 12 h to 4 d in the heart after chicken inoculation and decreased from 4 d to 7 d.As shown in Fig. 6, the bacterial number of S. pullorum and S. pullorum ΔsteE stains peaked about four days in the heart after chicken inoculation.The settlement amount of S. pullorum and S. pullorum ΔsteE strains reached the lowest at 7 d.The changing trend of S. pullorum and S. pullorum ΔsteE strains was increased at first and then decreased in the whole process of the infection, but the settled quantity of S. pullorum ΔsteE was consistently lower than that of S. pullorum in chicken heart.

Discussion
S. pullorum has brought substantial economic losses to the poultry industry.The pathogenic mechanism of S. Pullorum needs to be further studied.After the host cell is infected with Salmonella, Salmonella can form a Salmonellacontaining vacuole (SCV) in the host cell and survive in it for immune escape (Walch et al., 2021).The maintenance of SCV function is inseparable from the participation of a series of Salmonella virulence factors, which play a significant role in the T3SS2 encoded by SPI-2 and its secreted effector protein (Röder et al., 2021;Morrison et al., 2022).SteE is an effector protein deeply involved in regulating the secretion of Salmonella pathogenic island two, and its role is significant for Salmonella virulence (Gibbs et al., 2020).
This study evaluated the virulence and dynamic distribution of S. pullorum and S. pullorum ΔsteE strains in chicken organs after chicken immunization.The results of the virulence assay showed that the deletion of steE caused a decrease in the pathogenicity of S. pullorum in chickens, which proved the critical role of steE in the virulence of S. Pullorum (Fig. 1).At the same time, the colonization of bacteria in chicken organs showed that the overall change trend of S. Pullorum was similar to that of S. pullorum ΔsteE.The colonization of S. Pullorum and S. pullorum ΔsteE strains in the chicken cecum, spleen, and bursa peaked at 3d, while the number of bacteria in the liver peaked at 4 d.From the whole infection process, the number of S. pullorum ΔsteE in various organs at the initial infection stage after chicken infection first increased, peaked at the middle infection stage, and decreased at the later infection stage.At the same time, the number of S. pullorum ΔsteE in chicken organs was always significantly lower than that of S. pullorum.In addition, the colonization number of S. pullorum ΔsteE in chicken organs was consistently lower than that of S. pullorum.These results show that the steE can promote the colonization of S. pullorum in chicken organs and contribute to the virulence of S. pullorum.
Recent studies demonstrated that the deletion of SPI-2 reduced the virulence of Salmonella, which is related to the fact that SPI-2 is a DNA fragment obtained by the superficial level in the process of Salmonella evolution.Some reported that the deletion of steE significantly attenuated colonization of mouse spleens and caused decreased virulence of S. typhimurium (Niemann et al., 2011).SteE had been considered an essential factor in host organs for persistent Salmonella infection (Pham et al., 2020).steE is secreted by Salmonella (Ruan et al., 2017;Stapels et al., 2018;Panagi et al., 2020), which reported activating the signal transducer and activator of transcription three signaling pathways and then produces anti-inflammatory cytokine IL-10, thereby promoting Salmonella replication in cells and increasing bacterial colonization in vivo (Jaslow et al., 2018).In this study, many factors affect the accuracy of the final results.
On the one hand, the interval between organ collections is relatively long, and the accurate time of bacteria invading various organs needs to be better determined.On the other hand, we can only determine when bacteria settle in organs from chickens to reach the peak by reducing the interval between collecting organs.In addition, other factors affect the number of bacteria, such as the cleanliness of equipment, reagents, Petri dishes, and so on in the test, and the errors caused by the operation in the test process.

Conclusions
In this study, the deletion of steE caused significantly decreased colonization and pathological change of S. pullorum in a chicken infection model, and its virulence was also considerably reduced.Altogether, our work shows that steE is closely related to the pathogenicity of S. pullorum, which provides exciting insights into the roles of steE in the pathogenic mechanism and the development of the live attenuated vaccine of S. pullorum.

Fig. 4 .
Fig. 4. The change of bacterial colonization in the chicken spleen

Fig. 5 .
Fig. 5.The change of bacterial colonization in the chicken bursa

Fig. 6 .
Fig. 6.The change of bacterial colonization in the chicken heart