Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2

Abstract

Background: Francisella tularensis is a zoonotic intracellular bacterial pathogen that causes tularemia. The subspecies tularensis is highly virulent and is classified as a category A agent of biological warfare because of its low infectious dose by an aerosol route, and its ability to cause severe disease. In macrophages F. tularensis exhibits a rather novel intracellular lifestyle; after invasion it remains in a phagosome for three to six hours before escaping to, and replicating in the cytoplasm. The molecular mechanisms that allow F. tularensis to invade and replicate within a host cell have not been well defined.

Methods: We constructed a stable transposon mutagenesis library of virulent strain Schu S4 using a derivative of the EZ::TN transposon system. Approximately 2000 mutants were screened for the inability to invade, and replicate in the hepatic carcinoma cell line HepG2. These mutants were also tested for replication within the J774.1 macrophage-like cell line.

Results: Eighteen mutants defective in intracellular replication in HepG2 cells were identified. Eight of these mutants were auxotrophs; seven had mutations in nucleotide biosynthesis pathways. The remaining mutants had insertions in genes that were predicted to encode putative transporters, enzymes involved in protein modification and turnover, and hypothetical proteins. A time course of the intracellular growth of a pyrB mutant revealed that this mutant was only able to grow at low levels within HepG2 cells but grew like wild-type bacteria in J774.1 cells. This pyrB mutant was also attenuated in mice.

Conclusion: This is the first reported large-scale mutagenesis of a type A strain of F. tularensis and the first identification of mutants specifically defective in intracellular growth in a hepatic cell line. We have identified several genes and pathways that are key for the survival and growth of F. tularensis in a hepatic cell line, and a number of novel intracellular growth-defective mutants that have not been previously characterized in other pathogens. Further characterization of these mutants will help provide a better understanding of the pathogenicity of F. tularensis, and may have practical applications as targets for drugs or attenuated vaccines.

Figure 1

Map of transposon EZ::TN<rpsL^p Rparr-2> that was used to create transposon-insertion library in Schu S4. Rparr-2 is a gene that encodes resistance to rifampin by inactivation of the drug. Arrows indicate the location of various primers that were used to for PCR amplification of the transposon or sequencing the flanking DNA. ME-mosaic ends (specifically recognized by the EZ::TN transposase), R6Kγori is an origin of replication that is used to rescue the transposon after it is inserted into the genome.

Figure 2

Chromosomal location of transposon EZ::TN<rpsL^p Rparr-2> in selected strains from the transposon-insertion library of Schu S4. A circular map of the Schu S4 chromosome showing the location of transposon insertion sites

Figure 3

Identification of mutants defective in intracellular growth in HepG2 cells. Individual transposon-insertion mutants were incubated with HepG2 cells for two hours, treated with gentamicin, incubated for an additional 24 hrs, then the HepG2 cells were lysed. The lysates were transferred to MHA plates with a 48-pin replicator and incubated for 48 hours. Representative mutants that showed no or reduced growth after 48 hrs are indicated by arrowheads.

Figure 4

Southern blot of transposon insertion mutants of Schu S4 that were defective in intracellular growth in HepG2 cells. Chromosomal DNA from mutants digested with BclI was subjected Southern hybridization with a PCR product amplified from the EZ::TN<rpsL^pRparr-2> DNA template. Lanes 1–15 contain DNA from rifampin-resistant mutants of Schu S4. Lane 16 contains wild-type Schu S4 DNA and Lane 17 contains the probe DNA. Each mutant contains a single copy of the transposon.

Figure 5

Predicted operon (5A) and simplified pyrimidine nucleotide synthesis pathway (5B) in F. tularensis. The direction of transcription of the putative operon is from left to right.

Figure 6

Uracil auxotrophy of pyrB mutant BJM1001. Schu S4 mutant BJM1001 with transposon insertion in pyrB was spotted on CDM minimal media, containing supplements as indicated, and grown for 72 hours.

Figure 7

Invasion and intracellular survival of BJM1001 and Schu S4 in HepG2 cells. HepG2 cells were infected with BJM1001 at an MOI to 50:1 in microtiter wells. After 2 hrs the media was replaced with media containing gentamicin. Sets of three wells were lysed at 3, 8, 24, 48 and 72 hrs, diluted, and spread on MHA plates to determine the number of intracellular colony forming units (CFUs). Each time point represents the average and standard deviation (S.D.) of three wells.