When the monolayer is not disrupted, the recovered CFU mL−1 should remain essentially constant over the same
time course. The S. Typhimurium 14028s (black diamonds) and S. Typhimurium 14028s ΔsopD2∷FRT (NT060) (white circles) showed a slight decline over the time course of the assay, suggesting that the monolayer integrity was not significantly affected by these strains (Fig. 3). In contrast, CFU mL−1 of S. Typhi STH2370 abruptly decreased until they became undetectable, strongly suggesting that gentamicin leaked due to a monolayer disruption (white squares). When S. Typhi was complemented with sopD2STM gene (in the pNT007 plasmid, see Materials and methods) and used to infect the monolayer, we observed that the corresponding ABT-263 chemical structure CFU mL−1 showed a sharp difference with the otherwise isogenic wild-type strain resembling the S. R428 supplier Typhimurium phenotype (black triangles). The CFU mL−1 numbers from infected cells with S. Typhi carrying the empty plasmid (pCC1) showed no differences with respect to the wild-type strain (data not shown). It has been reported that sopD2 contributes to the synthesis of Sifs, lipid filaments essential for S. Typhimurium intracellular
proliferation (Brumell et al., 2003; Jiang et al., 2004; Birmingham et al., 2005). When we performed a gentamicin protection assay, we observed that S. Typhi sopD2STM showed a significant decrease of CFU recovered from HEp-2-infected monolayers compared with the wild-type strain (Fig. 4). In contrast, S. Typhi sopD2STM showed similar invasion levels compared with S. Typhimurium 14028s ΔsopD2∷FRT (NT060) (P=0.13749). The results suggest that loss of SopD2 function in the serovar Typhi contributes to the bacterial intracellular proliferation in human epithelial cells. In the process of adaptation to humans, bacterial genes no longer compatible with the lifestyle of facultative
pathogens within the host are selectively inactivated. These inactivated genes are called ‘antivirulence genes’ and their loss of function results in the adaptation to a given host (Maurelli, 2007). Salmonella enterica serovar Typhi is a facultative bacterial pathogen that has accumulated a large number of pseudogenes (approximately 5% of the genome), over 75% of which have completely lost their function (McClelland et al., 2004; selleck chemical Dagan et al., 2006). Compared with free-living organism genomes, facultative pathogens harbor several pseudogenes and a gene population structure that promotes the maintenance of specific mutations. In contrast to free-living bacteria (large genomes, a great diversity of functional genes and low percentage of laterally transferred genes) and obligate parasites (extremely reduced genomes), S. Typhi represents an intermediate step exhibiting some genome erosion directed to inactivation and loss of detrimental or nonessential functions for its environment, i.e. the host (Ochman & Moran, 2001).