In this study, a total of 1,340 DEGs were screened through quantitative gene analysis and were subjected to various analyses based on gene expression levels (principal component, correlation, differential gene screening). These DEGs were further analyzed by GO function significant enrichment analysis, pathway significant enrichment analysis, and clustering analysis, and most of the genes found to be enriched were classified into: cellular processes and catalytic activity, structural constituent of ribosome, structural molecule activity, pathogenesis, locomotion, bacterial chemotaxis, flagellar assembly, and Salmonella infection. These DEGs therefore appeared to be functionally closely related to the adaptation of Salmonella to in vivo microenvironments, ensuring the survival of bacteria, and promoting bacterial proliferation. On this basis, we optimized the screening conditions and screened 58 significantly upregulated genes (FPKM > 10). These DEGs mostly encoded type III secretion system (T3SS) proteins, some enzymes, and the SPI2 effector proteins. The T3SS effectors provide Gram-negative bacteria with a unique virulence mechanism enabling them to inject bacterial effector proteins directly into the host cell cytoplasm, bypassing the extracellular milieu [18]. Salmonella replicates within both nonphagocytic epithelial cells and macrophages in Salmonella-containing vacuoles (SCVs) [19]. T3SS secretion effectors alter vacuole positioning by acting on host cell actin filaments, microtubule motors, and components of the Golgi complex. Once positioned, maturation is stalled and bacterial replication is initiated. The T3SS effectors could block phagocytosis or promote bacterial invasion of non-phagocytic cells, altering membrane trafficking, and modulating innate and adaptive immune responses [20, 21]. The natural immune system is the first line of defense against bacterial disease [22]. These effectors, which are injected into host cells through a secretory system (such as T3SS), employ complex and sophisticated strategies to block and control the host's signal transduction pathways, particularly those that have important functions in host innate immunity. For example, Salmonella AvrA can be secreted into the host cytoplasm, where it inhibits inflammation and cellular pro-apoptotic responses [23, 24]. SseL protein can inhibit the intracellular NF-kB pathway and enhance the pathogenicity of Salmonella pullorum [16]. These activities enable bacteria to survive and replicate, causing disease.
In this study, most of the proteins in the T3SS are upregulated in vivo, and their secreted effector proteins such as SpiC, SseB, SseC, SseD, SseE, SseF, SseL, and STM1410 also showed significant high levels of expression. It has been confirmed that SifA is an SPI2 effector protein. It plays a vital role in maintaining the integrity of the SCV membrane, and is transported and localized in cells [25]. SseJ is necessary for bacterial survival, but sifA is not [26]. However, deletion of sifA in S. typhimurium reduces the proliferation of bacteria in cells and systemic toxicity in mice [27]. Although the role of sifs in the pathogenesis of Salmonella is unclear, sifA mutants are severely attenuated in virulence studies [28].
In the Sse family, in addition to SseA, which is essential for bacterial survival in cells and a key factor in determining bacterial virulence [29], SseJ also regulates SCV membrane dynamics and is associated with SCV and Sifs [28], and SseB, SseC, and SseD are located on the bacterial cell membrane and are also essential for the survival of bacteria in cells, being secreted by the SPI2-mediated T3SS and playing a toxic role outside of the bacterial membrane [30].
SsaM is essential for the secretion of SseB, SseC, and SseD. Following the deletion of SsaM, the bacteria cannot form Sifs and cannot transfer the effector protein SseJ to infected cells. The virulence of the deletion strain and its replication ability in infected cells are all decreased [31]; Spic also promotes SseB and SseC secretion. The decrease in virulence caused by Spic deletion is not due to a single effector deletion, but results from the deletion of all SPI2 effector proteins. Spic inhibits the interaction between SCVs and late endosomes and lysosomes, as well as the endocytosis and recycling of transferrin [32], so SsaM and Spic are also virulence genes of S. typhimurium [25].
SpiA of S. typhimurium is critical for the virulence of host cells. After deletion of this gene, phenotypic and biological analysis showed a reduction in growth rate, changes in morphology, and changes in adhesion and invasion of epithelial cells compared with wild-type strains. Similarly, the deletion strain showed a decrease in biofilm formation, as observed by scanning electron microscopy in a quantitative microtiter assay, and the proliferation of the deletion strain in cells was significantly decreased during the biofilm formation stage and the exponential growth period. This suggests that the spiA gene is involved in biofilm formation and the virulence of Salmonella [33].
To ensure intracellular survival, Salmonella must not only avoid removal by the host immune system but also strive to obtain nutrients, which requires the adjustment of gene expression levels to adapt to a rapidly changing environment [6], and these processes are mostly dependent on certain special biological macromolecules. At present, it is still unclear which regulatory networks are affected by effector proteins, the SPI, and transport processes, and their role in adapting to the SCV microenvironment in the host cell [6].
To date, the biotin synthesis pathway has been studied in detail in E. coli, Bacillus sphaericus, Bacillus subtilis, Saccharomyces cerevisiae, and plants [34]. Studies have shown that after S. typhimurium infection of macrophages, SPI2-mediated secretion of the T3SS proteins SsaQ and SseE and expression of the biotin biosynthesis proteins BioB and BioD increased. After loss of the bioB gene, the ability of the bacteria to proliferate in phagocytes was reduced, indicating that biotin plays a role in the survival of S. typhimurium in macrophages [35]. In our experiments, not only bioB and bioD were found to be upregulated, but also bioA, bioC, and bioF. BioF is an enzyme that catalyzes the first step in the biotin synthesis pathway. Its structure and function have been well studied in microorganisms such as E. coli, B. subtilis, and B. sphaericus [36, 37], but not in S. typhimurium [35].
In this study, we used homologous recombination to construct derivatives of S. typhimurium in which the bioF and invA genes were disrupted. S. typhimurium invA mutants (ΔinvA) were included as a control. Gene disruption had no evident effect on growth in vitro or on cellular morphology. The parent strains (WT) and their bioF or invA mutant derivatives were inoculated into mice. The ΔbioF strain was significantly attenuated compared with the WT strain. After knocking out the candidate gene bioF, the mortality of the mice was significantly reduced (P < 0.05). Unlike ΔinvA, there was no significant difference in the bacterial load in the spleen between the initial stage of ΔbioF infection and infection with the parental strain. Consistent with the ΔinvA strain, the bacterial load was significantly lower with the ΔbioF strain than with the parental strain as the infection time became prolonged. The reason for this may be that invA is a virulence gene that has been confirmed to be involved in the invasion of the T3SS. The invA gene plays a role in the process of bacterial invasion, and deletion of this gene directly reduces the number of invading cells [38]; by contrast, bioF is a gene involved in bacterial metabolism and its function does not affect the invasion stage of bacteria pathogenesis. Later on in the infection, the bacterial load was significantly lower with the mutant strain than with the reference strain, suggesting that in a microenvironment of intracellular nutrient deficiency, the loss of bioF affects the proliferation of the bacteria. These results indicated that the bioF gene plays an important role in the survival and proliferation of S. typhimurium in vivo.
In host SCVs [39], a variety of metabolic changes, including bacterial oxidation and nitrification, are initiated to accommodate changes in the microenvironment [40]. Physiological, metabolic, and effector protein-mediated adaptation strategies allow the bacteria to replicate within the SCVs, and to form persister cells [39, 41]. Sulfur is an essential element for microorganisms that can be obtained from a variety of compounds, with sulfate being the preferred source [42]. Sulfate uptake is carried out by sulfate permease belonging to the SulT (CysPTWA), SulP, CysP / (PiT), and CysZ families. The main proteins of the sulfur metabolism transport pathway are significantly upregulated in cells, which may be an adaptation to the SCV microenvironment. Among them, CysA is a sulfate/thiosulfate transfer ATP-binding protein, which is involved in the formation of the ABC transporter complex CysA WTP located inside the cell membrane, and is responsible for energy coupling with the transport system. Within the bacterial cytoplasm, CysH is a phosphate adenosine phosphate reductase (thioredoxin), which is a member of the PAPS reductase family. The three substrates of the CysH enzyme are adenosine 3', 5'-diphosphate, sulfite, and thioredoxin disulfide, and the two products are 3'-adenosyl sulfate and thioredoxin(https://www.prospecbio.com/cysh_ecoli). In a study of the intracellular pathogenic bacteria Mycobacterium tuberculosis, it was confirmed that cysA deficiency leads to sulfate auxotrophy [43]. The activity of cysH can protect bacteria against free radicals during chronic infection, and nitrosation and oxidation exert excitatory resistance, which may be the mechanism that guarantees chronic persistent infection [44]. Nitrosation and oxidative stress play a key regulatory role in inflammation and the immune response. Nitric oxide produced by the host's several immune cell NO synthases has a severe impact on the major carbon metabolic pathways of Salmonella [45]. Following Salmonella infection in mice, both cysA and cysH were upregulated, suggesting that Salmonella has a similar mechanism of action to M. tuberculosis and plays an important role in regulating host cell nitrosation and oxidative stress.
Clusters of antibiotic resistance-related genes were significantly upregulated, such as those encoding the UgtL, VirK, Mig-14, and PagP proteins. The mig-14 gene regulates bacterial gene expression during the infection of cells and is an important virulence gene of S. typhimurium [46]. VirK is a gene adjacent to mig-14 on the Salmonella chromosome. It contributes to the regeneration of the bacterial outer membrane in response to the host environment in the late stage of Salmonella infection. Like mig-14, deletion of virK changes the resistance of the bacteria to antimicrobial peptides, such as bacteriocin B, but does not significantly reduce the proliferation of Salmonella in infected cells [47]. The synergy between pagP and ugtL facilitates the continued colonization of Salmonella in the gut and the construction of a robust outer membrane barrier [48].
The body temperature of the host can also affect the virulence of the pathogen. Moderate fever can reduce the severity of bacterial and fungal infections; however, Salmonella can regulate the expression of its own genes to adapt to a variety of rapidly changing temperatures, thereby allowing it to multiply in various ecosystems. MgtC and MgtB are Mg2+ transporters that have been shown to have complex functions and are required for bacterial virulence, flagellar independence, and cellulose synthesis. However, when the host temperature rises, the expression of mgtC increases, but mgtB is not necessary gene for the cell to adapt to temperature changes [49]. Both PipB and PipB2 are effector proteins that bind to the host cell membrane. Although neither of these genes are required for the proliferation of Salmonella in cells, their function is not completely redundant, and PipB2 is a known virulence factor. Studies have shown that pipB2 deletion strains show reduced virulence after infection in mice [28]. The Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that use and disrupt the eukaryotic ubiquitination pathway, a central eukaryotic regulatory mechanism that controls a variety of biological processes, such as programmed cell death, cell cycle progression, and signal transduction, and when deleted, can lead to cancer and neurodegenerative diseases [50].
Through our literature review of these genes, we obtained a total of 26 DEGs that have not yet been reported to be related to intracellular survival and proliferation. These genes are highly likely to be associated with such functions and are candidate key proteins for intracellular survival and proliferation. In future studies, we will further elucidate the expression of these proteins in host cells and their possible interaction with host cell proteins, enabling us to explore the immune signaling pathways that are involved in vivo. This will help us to clarify key issues such as the pathogenesis of intracellular bacteria and the mechanisms of evading host immune responses. Furthermore, such studies may provide target proteins for vaccine development and drug development, which may aid efforts to control infections by Salmonella as well as other important intracellular pathogens.