Characterization of a Novel Intracellularly Activated Gene from Salmonella enterica Serovar Typhi

Abstract

A Salmonella enterica serovar Typhi gene that is selectively up-regulated upon bacterial invasion of eukaryotic cells was characterized. The open reading frame encodes a 298-amino-acid hydrophobic polypeptide (30.8 kDa), which is predicted to be an integral membrane protein with nine membrane-spanning domains. The protein is closely related (87 to 94% reliability) to different transport and permease systems. Gene expression under laboratory conditions was relatively weak; however, sevenfold induction was observed in a high-osmolarity medium (300 mM NaCl). The growth pattern in a laboratory medium of a serovar Typhi strain Ty2 derivative containing a 735-bp in-frame deletion in this gene, named gaiA (for gene activated intracellularly), was not affected. In contrast, the mutant was partially impaired in intracellular survival in murine peritoneal macrophages, as well as in human monocyte-derived macrophages. However, in the case of human macrophages, this survival defect was modest and evident only at late infection times (24 h). Despite the distinct intracellular survival kinetics displayed in macrophages of different species, the gaiA null mutant was significantly affected in its potential to trigger apoptosis in both murine and human macrophages. Provision of the gaiA gene in trans resulted in complementation of these phenotypes. Interestingly, the absence of a functional gaiA gene caused a marked attenuation in the mouse mucin model, as shown by the increase (3 orders of magnitude) in the 50% lethal dose of the mutant strain over that of the parental strain Ty2 (P < 0.05). Altogether, these data indicate that the product encoded by the gaiA gene is required for triggering apoptosis and bacterial survival within murine macrophages, which is consistent with the in vivo results obtained in the mouse mucin model. However, gaiA was not required for initial intracellular survival in human cells, indicating that its role in the natural host might be more complex than is suggested by the studies performed in the murine system.

Apoptotic cell death of macrophages by iron-stressed Salmonella enterica serovar Typhimurium

“…Necrosis, a passive pattern of cell death, is affected by some physical, chemical and biological factors (Nagarathnamma et al 1997). The results of the present study are in agreement with many other studies conducted on apoptosis where apoptosis has been shown with these DNA binding dyes (Chen et al 1996;Basso et al 2002). Further, detailed characterization and localization of the stress-induced proteins/OMPs will help us to understand better the pathophysiology of the disease under conditions which are more closer to in vivo situations and the mechanisms involved in the pathogenesis.…”

“…Hoechst 33342 and PI (propidium iodide) can bind with DNA, causing a blue or red fluorescence respectively (Basso et al 2002). PI can only permeate through the membrane of dead cells, whereas Hoechst 33342 can permeate through intact membranes as well.…”

A novel PhoP-regulated locus encoding the cytolysin ClyA and the secreted invasin TaiA of Salmonella enterica serovar Typhi is involved in virulence

“…To generate nonpolar mutations of taiA and clyA, the overlap-extension PCR method described by Basso et al (2002) was used. For taiA, fragments were amplified with primers STY1499-BF and STY1499-BR (Table 2) for the 59 end of the gene, and primers STY1499-EF and STY1499-ER for the 39 end of the gene.…”

Role of the Salmonella enterica serovar Typhi Fur regulator and small RNAs RfrA and RfrB in iron homeostasis and interaction with host cells

“…Deletion of fur, rfrA or rfrB individually or in combination (fur/rfrA, fur/rfrB, fur/ rfrA/rfrB and rfrA/rfrB) was done by allelic exchange as described previously (Faucher et al, 2009) using the overlap-extension PCR method (Basso et al, 2002). Primers are listed in Table 2.…”