Salmonella enterica serovar Typhi uses two type 3 secretion systems to replicate in human macrophages and colonize humanized mice.

serovar Typhi (. Typhi) is a human-restricted pathogen that replicates in macrophages. In this study, we investigated the roles of the . Typhi type 3 secretion systems (T3SSs) encoded on pathogenicity islands (SPI)-1 (T3SS-1) and SPI-2 (T3SS-2) during human macrophage infection. We found that mutants of . Typhi deficient for both T3SSs were defective for intramacrophage replication as measured by flow cytometry, viable bacterial counts, and live time-lapse microscopy. T3SS-secreted proteins PipB2 and SifA contributed to . Typhi replication and were translocated into the cytosol of human macrophages through both T3SS-1 and T3SS-2, demonstrating functional redundancy for these secretion systems. Importantly, an . Typhi mutant strain that is deficient for both T3SS-1 and T3SS-2 was severely attenuated in the ability to colonize systemic tissues in a humanized mouse model of typhoid fever. Overall, this study establishes a critical role for . Typhi T3SSs during its replication within human macrophages and during systemic infection of humanized mice. IMPORTANCE serovar Typhi is a human-restricted pathogen that causes typhoid fever. Understanding the key virulence mechanisms that facilitate . Typhi replication in human phagocytes will enable rational vaccine and antibiotic development to limit the spread of this pathogen. While . Typhimurium replication in murine models has been studied extensively, there is limited information available about . Typhi replication in human macrophages, some of which directly conflict with findings from . Typhimurium murine models. This study establishes that both of . Typhi's two type 3 secretion systems (T3SS-1 and T3SS-2) contribute to intramacrophage replication and virulence.