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MglA and MglB are required for the intramacrophage growth of Francisella novicida
1Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Abstract:
Francisella novicida is a facultative intracellular pathogen capable of growing in macrophages. A spontaneous mutant of F. novicida defective for growth in macrophages was isolated on LB media containing the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (X-p) and designated GB2. Using an in cis complementation strategy, four strains were isolated that are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for both the intracellular growth defect and the colony morphology on LB (X-p) media. The locus consists of an apparent operon of two genes, designated mgIAB, for Macrophage Growth Locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 or LB (X-p) media. Sequencing on mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Cell fractionation studies revealed several differences in the protein profiles of mgI mutants compared with wild-type F. novicida. The deduced amino acid sequences of mglA and mglB show similarity to the SspA and SspB proteins of Escherichia coli and Haemophilus spp. In E. coli, SspA and/or SspB influence the levels of multiple proteins under conditions of nutritional stress, and SspA can associate with the RNA polymerase holoenzyme. Taken together, these observations suggest that in Francisella MglA and MglB may affect the expression of genes whose products contribute to survival and growth within macrophages.
Insights
Francisella novicida requires the MglA and MglB proteins for growth within macrophages. Mutations in these genes impair intracellular survival and alter colony morphology, suggesting a role in regulating gene expression for macrophage survival.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Francisella novicida is an intracellular pathogen that can survive and replicate within macrophages.
- Understanding the genetic factors governing intracellular growth is crucial for deciphering Francisella pathogenesis.
Purpose of the Study:
- To identify and characterize genes essential for the intramacrophage growth of Francisella novicida.
- To elucidate the function of the identified Macrophage Growth Locus (mgIAB) in bacterial survival within host cells.
Main Methods:
- Isolation of a spontaneous mutant (GB2) defective for intracellular growth.
- Complementation analysis to identify the responsible genetic locus (mgIAB).
- Genetic manipulation (mutagenesis, sequencing) and protein expression analysis.
Main Results:
- A two-gene operon, mgIAB, was identified as crucial for intramacrophage growth.
- Mutants lacking mglA or mglB exhibited defects in intracellular growth and altered colony morphology.
- Sequence analysis revealed a missense mutation in mglA from the GB2 mutant.
- MglA and MglB show homology to E. coli SspA/SspB, suggesting a role in gene regulation.
Conclusions:
- The MglA and MglB proteins are essential for Francisella novicida's ability to grow within macrophages.
- MglA and MglB likely regulate the expression of genes involved in bacterial survival and adaptation inside host cells.