Dimethyl Adenosine Transferase (KsgA) Deficiency in Salmonella enterica Serovar Enteritidis Confers Susceptibility to High Osmolarity and Virulence Attenuation in Chickens

Abstract

bDimethyl 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.

Studies on Aminoglycoside Susceptibility Identify a Novel Function of KsgA To Secure Translational Fidelity during Antibiotic Stress

“…KsgA is a methyltransferase that dimethylates A1518 and A1519 of 16S rRNA. KsgA deficiency has pleiotropic effects, including resistance to kasugamycin killing (27), increased susceptibility to cold stress and oxidative stress (20), and compromised virulence (20,41). In addition, KsgA deficiency was found to increase the sensitivities to inhibition by kanamycin, gentamicin, and paromomycin, as demonstrated by the MIC 50 s (28)(29)(30).…”

“…The determination of the important role of KsgA during aminoglycoside killing raises the possibility of developing a KsgA inhibitor as a potentiator for aminoglycoside chemotherapy against bacterial infection. An additional advantage of such a potentiator might be that it disarms the virulence of pathogenic bacteria, including Salmonella enterica serovar Enteritidis (20), Yersinia pseudotuberculosis (45), Staphylococcus aureus (41), and A. baumannii, a WHO priority 1 pathogen for new antibiotic development. Thus, such a potentiator might have the ability to "kill two birds with one stone.…”

“…A deficiency in methyltransferase activity leads to bacterial resistance to kasugamycin (18). Second, KsgA is important for ribosomal biogenesis under cold stress (19,20). The deletion of ksgA leads to the accumulation of immature 30S ribosomal subunits, which is believed to be the main reason for the increased sensitivity to cold stress.…”

Comparative transcriptomes and development of expressed sequence tag‐simple sequence repeat markers for two closely related oak species

“…Moreover, we found that some of the pairs of orthologs have higher expression levels in response to drought, including qm9540 (Sinha et al, ), qm3901 (Scharf et al, ), qm27995 (Sun et al, ), qm20708 (Luo et al, ), qm8971 (Giarola et al, ), and qm13577 (Padma et al, ). In particular, one positively selected gene (qm16912), which is related to the response to cold (Chiok et al, ), showed higher expression levels in Q. mongolica compared with Q. liaotungensis . This could provide an insight into the origin of the differences between Q. mongolica and Q. liaotungensis .…”

Genetic Basis of Salmonella Enteritidis Pathogenesis in Chickens

“…The ribosomal maturation factor ksgA is under investigation in our laboratory for its contribution to pathogenicity in chickens. We have previously reported that inactivation of ksgA in SE results in reduced invasiveness in chicken liver cells (Shah et al, 2012b) and impaired intestinal and internal organ colonization in orally inoculated day-old chickens (Chiok et al, 2013). One study identified a few SE-specific genetic factors (SEN1001, SEN1140, SEN1970-SEN1999, and SEN4290-SEN4292) that contribute to pathogenicity in mice, but their role remains to be tested in a chicken model (Silva et al, 2012;Vishwakarma et al, 2012).…”