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

Abstract

Salmonella enterica serovar Typhi ( S . Typhi) is a human-restricted pathogen that replicates in macrophages. In this study, we investigated the roles of the S . Typhi type 3 secretion systems (T3SSs) encoded on Salmonella pathogenicity islands (SPI)-1 (T3SS-1) and SPI-2 (T3SS-2) during human macrophage infection. We found that mutants of S . 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 S . 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 S . 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 S . Typhi T3SSs during its replication within human macrophages and during systemic infection of humanized mice. IMPORTANCE Salmonella enterica serovar Typhi is a human-restricted pathogen that causes typhoid fever. Understanding the key virulence mechanisms that facilitate S . Typhi replication in human phagocytes will enable rational vaccine and antibiotic development to limit the spread of this pathogen. While S . Typhimurium replication in murine models has been studied extensively, there is limited information available about S . Typhi replication in human macrophages, some of which directly conflict with findings from S . Typhimurium murine models. This study establishes that both of S . Typhi’s two type 3 secretion systems (T3SS-1 and T3SS-2) contribute to intramacrophage replication and virulence.

Transcriptional profiling links unique human macrophage phenotypes to the growth of intracellular Salmonella enterica serovar Typhi

“…Briefly, pFCcGi contains a constitutively expressed mCherry and an L-arabinose inducible gfp . Prior to infection, S. Typhi pFCcGi is grown in the presence of L-arabinose whereas during infection L-arabinose is removed so that GFP is ‘diluted’ as bacteria replicate 14 , 15 . This plasmid also indicates the viability of intracellular S .…”

The Salmonella Typhi SPI-2 injectisome enigma

“…Typhi are not consistent. Some have shown that it is required for replication in human epithelial cells [13] and in human-derived macrophages [14–16]. However, other work has led to the conclusion that it does not contribute to replication in such macrophages [13, 17, 18].…”

“…[ 16 ] used the same strains and found that in two separate comparisons, the SPI-2 injectisome accounted for a difference in intracellular growth of at least fourfold over 48 h. More recently, Hamblin et al . [ 15 ] measured the replication of SPI-2 null mutants in primary human and a human-derived THP-1 macrophage cell line. While no differences were detected by c.f.u., a more sensitive method based on replication-associated dilution of a fluorescent protein revealed a replication defect of SPI-2 mutant between 12 and 24 h.…”

“…However, in similar tests done by Hamblin et al . but involving a lower dose of mixed inoculum and a 5 day infection period, fewer SPI-2 mutants than wild-type bacteria were recovered from spleens (but not livers) of mice [ 15 ].…”

“…Regardless, Hamblin et al . [ 15 ] detected a replication defect of S . Typhi pipB2 and sifA mutants at 24 h post-uptake, indicating that the encoded proteins contribute to replication of S .…”