A short report in the October issue of Infection and Immunity reveals that the presence of cloning vectors or fluorescent proteins could be affecting the results of bacterial pathogenicity studies.

Leigh Knodler and colleagues wanted to test whether the presence of plasmids or fluorescent reporter proteins such as green fluorescent protein (GFP) per se had any effect on the ability of Salmonella enterica serovar Typhimurium to invade and replicate in eukaryotic host cells. To do so, they carried out a series of simple experiments involving commonly used plasmids of low-to-medium copy number carrying selectable markers.

Salmonella can enter cells either by an active, actin-dependent process using effectors encoded by Salmonella pathogenicity island 1 (SPI-1) or by SPI-1-independent phagocytosis. Cell-invasion experiments in non-phagocytic HeLa and phagocytic RAW264.7 cells demonstrated that some plasmids could decrease the efficiency of both SPI-1-mediated and non-SPI-1-mediated uptake, as well as affecting intracellular replication. Additionally, Knodler et al. also found that the presence of GFP or red fluorescent protein (RFP) impaired the ability of serovar Typhimurium to invade both cell lines, and RFP also impaired serovar Typhimurium intracellular replication in HeLa cells.

As mouse models are commonly used in Salmonella pathogenesis studies, Knodler et al. went on to investigate whether the presence of plasmid pACYC184, GFP or RFP had any effects on serovar Typhimurium infection in mice. No effect was seen for pACYC184, but the presence of GFP and RFP did reduce the competitive index of the bacteria expressing these protein markers.

So, this work shows that, depending on the route of uptake, the presence of simple plasmid cloning vectors or fluorescent protein markers can perturb the interaction of S. enterica with host cells. The authors conclude that “results from in vitro and in vivo experiments that rely exclusively on GFP- and RFP-expressing bacteria should be interpreted with some caution”.