As a major cause of infectious diarrheal diseases, non-typhoidal Salmonella (NTS), such as S. enterica subsp. enterica serovar Typhimurium, are of global health concern for both human and veterinary medicine. Besides causing usually self-limiting gastroenteritis in immunocompetent adult individuals, infection of neonates with this pathogen can induce invasive and potentially life-threatening conditions. Invasive infection caused by NTS are predominantly observed in developing countries with poor hygiene standards, where the pathogen is frequently isolated from cases of neonatal sepsis and meningitis. The pathogenicity of Salmonella is conferred by certain virulence factors encoded on chromosomal regions acquired by horizontal gene transfer, called Salmonella pathogenicity islands (SPIs). Delivery of effector proteins of SPI-1 and SPI-2 into the host cell is enabled via own type three secretion systems (T3SSs). The role of SPI effectors in host-pathogen interactions has been extensively studied in vitro and it is widely accepted that SPI-2 is crucial for the establishment of an intracellular compartment, the Salmonella-containing vacuole (SCV), which allows the bacteria to survive and replicate inside the host cell. Besides this, the translocated effector proteins allow the pathogen to modulate and manipulate the host cell system in various respects. Knowledge about effector contribution and their distinct role in pathogenesis of invasive NTS infections in vivo is rather scant. Therefore, we applied the neonate mouse model, which, in contrast to the commonly used streptomycin adult mouse infection model, allows the formation of SCVs inside infected enterocytes, followed by mucosal barrier penetration and subsequent bacterial dissemination without previous antibiotic depletion of the resident gut microbiota. In this study, we used this infection model to extend our knowledge on the role of individual SPI-2 effector proteins in establishment and progression of systemic Salmonella infections in the neonate host. Oral infection of neonates with wildtype and ΔSPI-2 Salmonella resulted in similar bacterial loads of the gastrointestinal tract, but re-isolation rates of SPI-2-deficient mutants from systemic organs, such as liver and spleen, and expression of pro-inflammatory cytokines were significantly decreased. Interestingly, in contrast to the general understanding of SPI-2 as prerequisite for SCV formation in vitro, mutants were able to establish and maintain SCVs in neonatal intestinal enterocytes. In fact, SPI-2-deficient bacteria grow to high numbers inside SCVs, nonetheless, like in the adult host, are attenuated in virulence and hampered in disease progression. The establishment of an SCV is not only feasible in the absence of the SPI-2 effector protein subset, but also without the host adaptor protein myeloid differentiation primary response 88 (MYD88). The observation of enlarged SCV formation in the absence of SPI-2, however, is strictly limited to the neonatal period. This is due to the immature intestinal tract constituting a unique environment with pending epithelial renewal on the one hand, whereas constant nutrient supply is ensured by fusion of the bacterial vacuole with transport vesicles on the other hand. By evaluating isogenic single SPI-2 effector protein mutants, we detected that SPI-2 effectors enabling interaction with the host cell microtubule network and positioning of the SCV inside the host cell partly phenocopy the SPI-2 dependent phenotype in vivo. Results achieved in this study suggest that depletion of SPI-2 effectors involved in the interaction with the host cell transport machinery still allows bacterial replication in the neonate intestine. However, the absence of these effectors prevents SCV transmigration from the apical to the basolateral side of the enterocytes. Mutants are therefore “stuck” inside their SCVs, which ultimately diminishes systemic spread to liver and spleen and complete pathogenesis of Salmonella in vivo. Thereby, SPI-2 effector proteins are needless in terms of early steps in NTS pathogenesis in the neonate host, but become urgent for the pathogen in order to overcome the epithelial barrier and achieve systemic dissemination.
