Identification of common highly expressed genes of Salmonella Enteritidis by in silico prediction of gene expression and in vitro transcriptomic analysis.

Chickens are the reservoir host of Salmonella Enteritidis. Salmonella Enteritidis colonizes the gastro-intestinal tract of chickens and replicates within macrophages without causing clinically discernable illness. Persistence of S. Enteritidis in the hostile environments of intestinal tract and macrophages allows it to disseminate extra-intestinally to liver, spleen, and reproductive tract. Extra-intestinal dissemination into reproductive tract leads to contamination of internal contents of eggs, which is a major risk factor for human infection. Understanding the genes that contribute to S. Enteritidis persistence in the chicken host is central to elucidate the genetic basis of the unique pathobiology of this public health pathogen. The aim of this study was to identify a succinct set of genes associated with infection-relevant in vitro environments to provide a rational foundation for subsequent biologically-relevant research. We used in silico prediction of gene expression and RNA-seq technology to identify a core set of 73 S. Enteritidis genes that are consistently highly expressed in multiple S. Enteritidis strains cultured at avian physiologic temperature under conditions that represent intestinal and intracellular environments. These common highly expressed (CHX) genes encode proteins involved in bacterial metabolism, protein synthesis, cell-envelope biogenesis, stress response, and a few proteins with uncharacterized functions. Further studies are needed to dissect the contribution of these CHX genes to the pathobiology of S. Enteritidis in the avian host. Several of the CHX genes could serve as promising targets for studies towards the development of immunoprophylactic and novel therapeutic strategies to prevent colonization of chickens and their environment with S. Enteritidis.

Dimethyl adenosine transferase (KsgA) contributes to cell-envelope fitness in Salmonella Enteritidis.

We previously reported that inactivation of a universally conserved dimethyl adenosine transferase (KsgA) attenuates virulence and increases sensitivity to oxidative and osmotic stress in Salmonella Enteritidis. Here, we show a role of KsgA in cell-envelope fitness as a potential mechanism underlying these phenotypes in Salmonella. We assessed structural integrity of the cell-envelope by transmission electron microscopy, permeability barrier function by determining intracellular accumulation of ethidium bromide and electrophysical properties by dielectrophoresis, an electrokinetic tool, in wild-type and ksgA knock-out mutants of S. Enteritidis. Deletion of ksgA resulted in disruption of the structural integrity, permeability barrier and distorted electrophysical properties of the cell-envelope. The cell-envelope fitness defects were alleviated by expression of wild-type KsgA (WT-ksgA) but not by its catalytically inactive form (ksgAE66A), suggesting that the dimethyl transferase activity of KsgA is important for cell-envelope fitness in S. Enteritidis. Upon expression of WT-ksgA and ksgAE66A in inherently permeable E. coli cells, the former strengthened and the latter weakened the permeability barrier, suggesting that KsgA also contributes to the cell-envelope fitness in E. coli. Lastly, expression of ksgAE66A exacerbated the cell-envelope fitness defects, resulting in impaired S. Enteritidis interactions with human intestinal epithelial cells, and human and avian phagocytes. This study shows that KsgA contributes to cell-envelope fitness and opens new avenues to modulate cell-envelopes via use of KsgA-antagonists.

Erratum to: The Salmonella pathogenicity island 13 contributes to pathogenesis in streptomycin pre-treated mice but not in day-old chickens.

[This corrects the article DOI: 10.1186/s13099-016-0098-0.].

The Salmonella pathogenicity island 13 contributes to pathogenesis in streptomycin pre-treated mice but not in day-old chickens.

BACKGROUND: Salmonella enterica serovar Enteritidis (S. Enteritidis) is a human and animal pathogen that causes gastroenteritis characterized by inflammatory diarrhea and occasionally an invasive systemic infection. Salmonella pathogenicity islands (SPIs) are horizontally acquired genomic segments known to contribute to Salmonella pathogenesis. The objective of the current study was to determine the contribution of SPI-13 to S. Enteritidis pathogenesis. METHODS: We deleted the entire SPI-13 (∆SPI-13) from the genome of S. Enteritidis CDC_2010K_0968 strain isolated from a human patient during the 2010 egg-associated outbreak in the US. The kinetics of infection of the wild-type parent and the ∆SPI-13 were compared in orally challenged day-old chickens and streptomycin pre-treated mice. The degree of intestinal inflammation and the survival of mutant strain within the avian (HD11) and murine (RAW264.7) macrophages were also determined. RESULTS: The deletion of the SPI-13 resulted in impaired infection kinetics of S. Enteritidis in streptomycin pre-treated mice which was characterized by significantly lower (P < 0.05) viable counts in the ceca, liver and spleen, impaired ability to induce intestinal inflammation and reduced survival within murine macrophages. Conversely, there were no significant differences in the infection kinetics of ∆SPI-13 in day-old chickens in any of the organs tested and the survival of ∆SPI-13 within chicken macrophages remained unaltered. CONCLUSIONS: The results of this study show that SPI-13 contributes to the pathogenesis of S. Enteritidis in streptomycin pre-treated mice but not in day-old chickens and raises the possibility that SPI-13 may play a role in pathogenesis and the host adaptation/restriction of Salmonella serovars.

Dimethyl adenosine transferase (KsgA) deficiency in Salmonella enterica Serovar Enteritidis confers susceptibility to high osmolarity and virulence attenuation in chickens.

Dimethyl adenosine transferase (KsgA) performs diverse roles in bacteria, including ribosomal maturation and DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that a ksgA mutation in Salmonella enterica serovar Enteritidis results in impaired invasiveness in human and avian epithelial cells. In this study, we tested the virulence of a ksgA mutant (the ksgA::Tn5 mutant) of S. Enteritidis in orally challenged 1-day-old chickens. The ksgA::Tn5 mutant showed significantly reduced intestinal colonization and organ invasiveness in chickens compared to those of the wild-type (WT) parent. Phenotype microarray (PM) was employed to compare the ksgA::Tn5 mutant and its isogenic wild-type strain for 920 phenotypes at 28°C, 37°C, and 42°C. At chicken body temperature (42°C), the ksgA::Tn5 mutant showed significantly reduced respiratory activity with respect to a number of carbon, nitrogen, phosphate, sulfur, and peptide nitrogen nutrients. The greatest differences were observed in the osmolyte panel at concentrations of ≥6% NaCl at 37°C and 42°C. In contrast, no major differences were observed at 28°C. In independent growth assays, the ksgA::Tn5 mutant displayed a severe growth defect in high-osmolarity (6.5% NaCl) conditions in nutrient-rich (LB) and nutrient-limiting (M9 minimum salts) media at 42°C. Moreover, the ksgA::Tn5 mutant showed significantly reduced tolerance to oxidative stress, but its survival within macrophages was not impaired. Unlike Escherichia coli, the ksgA::Tn5 mutant did not display a cold-sensitivity phenotype; however, it showed resistance to kasugamycin and increased susceptibility to chloramphenicol. To the best of our knowledge, this is the first report showing the role of ksgA in S. Enteritidis virulence in chickens, tolerance to high osmolarity, and altered susceptibility to kasugamycin and chloramphenicol.