GFP plasmid-induced defects in Salmonella invasion depend on plasmid architecture, not protein expression

Abstract

We have investigated the impact of plasmids and GFP expression on invasion of cultured epithelial cells by Salmonella enterica Typhimurium strain SL1344. The invasiveness of SL1344 carrying plasmids derived from pBR322, encoding promoterless GFP or constitutively expressed rpsM-GFP, was compared under optimal growth conditions with that of SL1344(pBR322), unmodified SL1344 and a strain with chromosome-integrated rpsM-GFP. The strain carrying pBR322 exhibited normal invasion, but the presence of modified plasmids impaired invasiveness, and impairment was exacerbated by plasmid-encoded chloramphenicol resistance (Cm R ). Using a different antibiotic resistance marker, kanamycin (Km R ), did not impair invasiveness. Despite the effect of plasmid-encoded Cm R , the strain containing chromosomally encoded GFP, also carrying a Cm R gene, was as invasive as the wild-type. To investigate the mechanism by which plasmid carriage decreases invasion, we monitored SPI-1 gene expression using prgH promoter activity as an index of SPI-1 activity. An SL1344 strain with a chromosome-integrated prgH : : gfp reporter construct exhibited lower GFP expression during exponential phase when carrying plasmids incorporating Cm R or gfp, mirroring invasion data. These data provide evidence that suppression of SPI-1 gene expression is a major factor in the loss of invasiveness associated with plasmid carriage. Our findings also indicate that some plasmids, especially those carrying Cm R , should be used with caution, as virulence traits and gene expression may be affected by their presence. Integration of reporter proteins into the bacterial chromosome, however, appears to circumvent the adverse effects observed with plasmids.

H-NS Silences gfp , the Green Fluorescent Protein Gene: gfp TCD Is a Genetically Remastered gfp Gene with Reduced Susceptibility to H-NS-Mediated Transcription Silencing and with Enhanced Translation

“…The introduction of AϩT-rich plasmid DNA can thus titrate H-NS away from native chromosomal locations, leading to a mild ⌬hns phenotype with pleiotropic effects, including reduced virulence and motility (3,6). We have shown that H-NS binds strongly in the gfpϩ gene, perhaps explaining a recent report of reduced invasiveness of Salmonella due solely to the presence of a promoterless gfpϩ gene on a multicopy plasmid (2). Over the years, many bacterial studies have relied on plasmid-based gfp fusions, and some of these studies may have experienced unrecognized pleiotropic effects due to H-NS titration.…”

Omics in Aid of Host-pathogen Interaction Studies: A Bacterial Perspective

“…Using microscopy, the number and the distribution or location of bacteria within host cells or cell-associated bacteria can be determined in a proteins from plasmids can affect Salmonella's virulence [28]. Evidence for this was provided by Clark et al [29] who observed a reduction in the expression of Salmonella pathogenic island 1 (SPI-1) genes and a reduced infectivity either in bacteria containing the GFP-expressing plasmid or the empty control plasmid. On the other hand, to avoid the expression of GFP protein from plasmids, the gfp gene can be directly cloned in the bacterial chromosome allowing for its expression from a constitutive promoter [30].…”

Enhanced recovery of Salmonella Typhimurium DT104 from exposure to stress at low temperature

“…The SPI-1 T3SS needle structure required for invasion of non-phagocytic cells is encoded by genes located within the prg/org operon (Klein et al, 2000), and expression of prgH, the first gene in the prg/org operon, is regulated by a graded input system that results in an all-or-nothing output (Temme et al, 2008). This can be exploited to produce a reporter system in which expression of GFP driven by the prgH promoter enables the proportion of Salmonella positive for PprgHgfp expression to be used as an index of SPI-1 expression status (Clark et al, 2009;Hautefort et al, 2003;Perrett et al, 2009). Salmonella populations have previously been described as demonstrating heterogeneity of gene expression for prgH, whereby maximal expression (53-70 % positive) occurs in late-exponential phase (Hautefort et al, 2003;Perrett et al, 2009).…”

“…Following infection, cells were washed thoroughly in PBS and fixed in 2 % (w/v) PFA overnight. Differential adhered/invaded staining and quantification were carried out as previously described (Clark et al, 2009;Perrett & Jepson, 2007).…”