The Francisella pathogenicity island protein IglA localizes to the bacterial cytoplasm and is needed for intracellular growth

Abstract

Background: Francisella tularensis is a gram negative, facultative intracellular bacterium that is the etiological agent of tularemia. F. novicida is closely related to F. tularensis but has low virulence for humans while being highly virulent in mice. IglA is a 21 kDa protein encoded by a gene that is part of an iglABCD operon located on the Francisella pathogenicity island (FPI).

Results: Bioinformatics analysis of the FPI suggests that IglA and IglB are components of a newly described type VI secretion system. In this study, we showed that IglA regulation is controlled by the global regulators MglA and MglB. During intracellular growth IglA production reaches a maximum at about 10 hours post infection. Biochemical fractionation showed that IglA is a soluble cytoplasmic protein and immunoprecipitation experiments demonstrate that it interacts with the downstream-encoded IglB. When the iglB gene was disrupted IglA could not be detected in cell extracts of F. novicida, although IglC could be detected. We further demonstrated that IglA is needed for intracellular growth of F. novicida. A non-polar iglA deletion mutant was defective for growth in mouse macrophage-like cells, and in cis complementation largely restored the wild type macrophage growth phenotype.

Conclusion: The results of this study demonstrate that IglA and IglB are interacting cytoplasmic proteins that are required for intramacrophage growth. The significance of the interaction may be to secrete effector molecules that affect host cell processes.

Figure 1

Similarity of the FPI to other virulence gene clusters. Homologues of Francisella pathogenicity island proteins IglA and IglB are found on a conserved gene clusters known as IcmF associated homologous proteins (IAHP), which, in some cases encode a proposed type VI secretion system. In the FPI the IcmF motif appears at the C-terminus of PdpB. Downstream of the pdpB gene is an ORF designated "vgr" that encodes a protein with similarity the Vgr family, one of which is secreted by the proteins encoded by an IAHP cluster in Vibrio cholerae. Homologues of the Legionella dotU gene are often associated with IAHP clusters. A very weak similarity to dotU is seen in an ORF that is sixth downstream of pdpB.

Figure 2

IglA regulation by MglA and MglB. Western blot showing lack of IglA in mglA and mglB mutants but present in the wild type strain U112. All samples were normalized to 6 μg protein per lane.

Figure 3

IglA expression in J774 macrophages. Western blot showing expression of IglA during infection of macrophages. J774 macrophages were infected with parent strain U112 (m.o.i 300:1) and lysed at the indicated time post infection. Loading was normalized according to the number of viable bacteria (CFU) in each sample as determined by plating on TSA-C plates. Lane J774, uninfected macrophages. TSB, broth grown U112 grown to indicated optical density (600 nm). All samples were normalized to 10⁷ CFU by viable counts. The macrophage cell lysates altered the appearance of the IglA bands, but control experiments showed that the cell lysates did not mask IglA reactivity with antibody.

Figure 4

Subcellular localization of IglA. Anti-IglA was used to probe Western immunoblot of subcellular fractions of F. novicida. The sarkosyl insoluble fraction represents an enrichment of outer membrane protein and the sarkosyl soluble fraction contains largely inner membrane protein. Samples were prepared as outlined in Methods and normalized to 10 μg protein per lane before separation on a 12% SDS-PAGE gel. Results are representative of three independent experiments.

Figure 5

Co-immunoprecipitation of a 60 kDa protein with IglA. Panel A. Anti-IglA serum co-immunoprecipitates a circa 60 kDa soluble protein (arrow, lanes 1 and 4). The band is absent in control reactions with non-specific antibody (lane 3) and in immunoprecipitations with an iglA mutant (lane 2). Numbers shown indicate molecular mass standards. Results are representative of those of three experiments. Panel B. MALDI-TOF identified the 60 kDa protein as IglB. Underlined sequences indicate peptides identified by MALDI-TOF. The second and third regions each represent two peptides (break after the "R"). Of 25 queries submitted, 9 showed significant identity with rabbit heavy chain and 9 showed significant identity with IglB of F. novicida. No other significant hits were found in the MSDB 20060224 databank.

Figure 6

Deletion mutagenesis of iglA. Panel A. Diagram of steps used to construct an iglA deletion mutant. A fragment of pdpD was joined to iglB and these two fragments were ligated to an Em^R-sacB cassette. After transformation the recombinant construct integrated into the F. novicida chromosome. Plating the strain with the integrated fragment on sucrose selected for strains that had undergone an excision of the sacB and neighboring regions. Panel B. PCR confirmation of the deletion of iglA. The small arrows indicate the location of the primers used in the reactions.

Figure 7

In cis complementation of iglA. Panel A. Diagram of complementation scheme. A PCR amplicon containing the iglA and neighboring regions was ligated to a Km^R cassette and used to transform a ΔiglA strain. Integration of the recombinant construct resulted in a strain with a chromosomally-integrated iglA. Panel B. PCR reactions demonstrating the presence of iglA in the complemented strain. Arrows in lower part of diagram indicate the location of the PCR primers used in the reactions.

Figure 8

An iglA mutant lacks the expression of a 21 kDa protein. Western blot showing the lack of an anti-IglA serum reactive 21 kDa protein in the ΔiglA strain (top panel). Wild type levels of IglC are retained in the ΔiglA strain (bottom panel). In contrast, the iglA::Em mutant lacks expression of IglC. The expression of IglC is threefold lower in an iglB::Em strain than in JLO and ΔiglA. Fluorescence intensity was used to quantify relative amounts of protein.

Figure 9

IglA is required for intracellular growth. Growth of ΔiglA strain in J774 mouse macrophage-like cells. Filled squares, parental strain JLO; open squares, ΔiglA; triangles, in cis complementation strain; diamonds, iglC transposon insertion mutant CG62. The experiments were done in triplicate and standard errors are shown by bars. This graph shows data from one of three independent experiments.

Figure 10

ΔiglA mutant is less virulent in chicken embryos. Infection of chicken embryos with 600 CFU of wild type (JL0) F. novicida lead to death of 7/7 embryos in 5 days (Panel A), whereas infection with 4,500 CFU of the ΔiglA strain (ODB2) lead to the death of 1/7 embryos in 6 days (Panel B).