Contribution of Salmonella Enteritidis virulence factors to intestinal colonization and systemic dissemination in 1-day-old chickens

Abstract

Salmonella enterica serovar Enteritidis is one of the most common serovars associated with poultry and poultry product contamination in the United States. We previously identified 14 mutant strains of Salmonella Enteritidis phage type 4 (PT4) with significantly reduced invasiveness in human intestinal epithelial cells (Caco-2), chicken macrophages (HD-11), and chicken hepatocellular epithelial cells (LMH). These included Salmonella Enteritidis mutants with transposon insertions in 6 newly identified Salmonella Enteritidis-specific genes (pegD and SEN1393), and genes or genomic islands common to most other Salmonella serovars (SEN0803, SEN0034, SEN2278, and SEN3503) along with 8 genes previously known to contribute to enteric infection (hilA, pipA, fliH, fljB, csgB, spvR, and rfbMN). We hypothesized that Salmonella Enteritidis employs both common Salmonella enterica colonization factors and Salmonella Enteritidis-specific traits to establish infection in chickens. Four Salmonella Enteritidis mutants (SEN0034::Tn5, fliH::Tn5, SEN1393::Tn5, and spvR::Tn5) were indistinguishable from the isogenic wild-type strain when orally inoculated in 1-d-old chickens, whereas 2 mutants (CsgB::Tn5 and PegD::Tn5) were defective for intestinal colonization (P < 0.05) and 8 mutants (hilA::Tn5, SEN3503::Tn5, SEN0803::Tn5, SEN2278::Tn5, fljB::Tn5, rfbM::Tn5, rfbN::Tn5, and pipA::Tn5) showed significant in vivo attenuation in more than one organ (P < 0.05). Complementation studies confirmed the role of rfbN and SEN3503 during infection. This study should contribute to a better understanding of the mechanisms involved in Salmonella Enteritidis pathogenesis, and the target genes identified here could potentially serve as targets for the development of live-attenuated or subunit vaccine.

Genetic Basis of Salmonella Enteritidis Pathogenesis in Chickens

“…Allen-Vercoe and Woodward (1999) also reported that afimbriate mutants of SE were not impaired in colonization of the GI tract or internal organs or fecal shedding in orally infected 1-day-old chickens. We reported that disruption of csgB in SE impaired invasiveness in chicken liver cells, but did not significantly impact intestinal colonization and internal organ invasiveness in orally challenged 1-day-old chickens (Addwebi et al, 2014;Shah et al, 2012b). Similarly, inactivation of stdA in SE was reported to result in reduced colonization of cecum as well as less invasion of the internal organs in orally inoculated 7-day-old chickens (Shippy et al, 2013).…”

“…We reported that impaired secretion of flagellar proteins (FlgK, FljB, and FlgL) among wild-type strains of SE was associated with a low-invasive phenotype in chicken macrophages (Shah et al, 2011). We also reported that disruption of the fljB results in reduced invasiveness of SE in cultured chicken liver cells and reduced colonization of the small intestine in orally inoculated day-old chickens (Addwebi et al, 2014;Shah et al, 2012b). Shippy et al (2014) reported that deletion of flgC, encoding the flagellum basal body protein, resulted in significantly reduced colonization of liver and spleen in orally infected 1-week-old chickens.…”

“…The role of SPI-1 and SPI-2 in pathogenesis of SE infection in chickens appears somewhat contradictory and poorly defined. Published data from our laboratory and others show that inactivation of hilA (SPI-1 invasion gene activator) in SE results in significantly reduced intestinal colonization (i.e., fewer bacteria recovered from the ceca) and invasion (i.e., few bacteria invade the GI tract to subsequently colonize internal organs such as liver and spleen) in orally inoculated day-old chickens (Addwebi et al, 2014;Bohez et al, 2006). In addition, suppression of hilA gene expression through supplementation of medium-chain fatty acids also results in reduced intestinal colonization and invasiveness of SE in orally challenged 5-day-old chicks (Van Immerseel et al, 2004).…”

“…We reported that a disruption of siiE gene of SPI-4 in SE resulted in significantly reduced survival in chicken macrophages; however, it is currently unknown if disruption of SPI-4 would result in similar effects in vivo (Shah et al, 2012b). We have also noted that disruption of pipA gene of SPI-5 significantly altered the ability to colonize the GI tract and internal organs of day-old chickens (Addwebi et al, 2014). In addition, several SPI-5 genes were upregulated in the GI tract of young chicks orally infected with SE (Dhawi et al, 2011).…”

“…Finally, Clayton et al (2008) systematically inactivated each of the 13 annotated fimbrial operons of SE and reported that only the inactivation of peg operon resulted in significant reduction in the intestinal colonization in orally inoculated 18-day-old chickens. The peg fimbriae is interesting because it is not found in other NTS, including S. Typhimurium, and has been identified as a pseudogene in an avian host adapted serotype S. Gallinarum (Addwebi et al, 2014;Clayton et al, 2008). It would be of interest to further dissect the role of peg fimbriae in SE pathogenesis in chickens.…”