Abstract
Intracellular pathogens and other organisms have evolved mechanisms to exploit host cells for their life cycles. Virulence genes of some intracellular bacteria responsible for these mechanisms are located in pathogenicity islands, such as secretion systems that secrete effector proteins. The Francisella pathogenicity island is required for phagosomal escape, intracellular replication, evasion of host immune responses, virulence, and encodes a type 6 secretion system. We hypothesize that some Francisella novicida pathogenicity island proteins are secreted during infection of host cells. To test this hypothesis, expression plasmids for all Francisella novicida FPI-encoded proteins with C-terminal and N-terminal epitope FLAG tags were developed. These plasmids expressed their respective epitope FLAG-tagged proteins at their predicted molecular weights. J774 murine macrophage-like cells were infected with Francisella novicida containing these plasmids. The FPI proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of the respective genes that were deficient for intramacrophage growth. Using these expression plasmids, the localization of the Francisella pathogenicity island proteins were examined via immuno-fluorescence microscopy within infected macrophage-like cells. Several Francisella pathogenicity island encoded proteins (IglABCDEFGHIJ, PdpACE, DotU and VgrG) were detected extracellularly and they were co-localized with the bacteria, while PdpBD and Anmk were not detected and thus remained inside bacteria. Proteins that were co-localized with bacteria had different patterns of localization. The localization of IglC was dependent on the type 6 secretion system. This suggests that some Francisella pathogenicity island proteins were secreted while others remain within the bacterium during infection of host cells as structural components of the secretion system and were necessary for secretion.
Highlights
Pathogenicity islands exist in many pathogenic bacteria, are acquired via horizontal gene transfer, and encode genes that facilitate interactions with host cells [1]
IglG-FLAG expression was lower than the other Francisella pathogenicity island (FPI) proteins and was not visible here (Fig. 1C); expression of IglG-FLAG was confirmed with longer exposure times causing over exposure with the other proteins
Intramacrophage Growth Complementation Since several FPI genes are needed for intracellular growth, the C-FLAG and N-FLAG-tagged proteins ability to complement respective knock out mutant strains were assessed (Table 1)
Summary
Pathogenicity islands exist in many pathogenic bacteria, are acquired via horizontal gene transfer, and encode genes that facilitate interactions with host cells [1]. Protein secretion is an important mechanism for bacteria to adapt and survive in their environment, including within an infected host [2]. Effector proteins are enzymes or toxins that facilitate infection and are secreted by these secretion systems [3]. The FPI is found in all Francisella species and strains, and is duplicated in all humanvirulent biovars of F. tularensis. The molecular mechanisms contributing to the intracellular survival of Francisella are poorly understood, and FPI mutagenesis approaches are useful in identifying genes required for intracellular replication and virulence [4,6,7,8,9,10,11]
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