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 which are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for the intracellular growth defect, colony morphology on LB (X-p) media, and virulence in mice. The locus consists of an apparent operon of two genes, designated mglAB, for macrophage growth locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 on LB (X-p) media. Sequencing of mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. Preliminary studies have also identified a convergently transcribed gene, tentatively designated mglC, immediately downstream of mglB. mglC null mutants are defective for intracellular growth and show the same phenotype on LB (X-p) agar as GB2. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Western blot analysis revealed the absence of MglA in an mglB null mutant, indicating MglB may influence MglA levels. Analysis of the regulation of mglA expression during growth in broth culture shows a decrease in expression upon entering late log-early stationary phase. mglA is also expressed during culture in macrophages. Cell fractionation studies revealed several differences in the protein profiles of mgl mutants compared with wild-type F. novicida, most notably the absence of a 70 kDa secreted protein. A candidate clone for the gene encoding this 70 kDa protein has been isolated. 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 control the expression of genes whose products contribute to survival and growth within macrophages. Roles for the putative MglC and possibly the 70 kDa secreted protein in this activity are also indicated.
Acid phosphatases capable of inhibiting the respiratory burst of neutrophils have been identified in certain intracellular pathogens. The gene encoding AcpA, a respiratory burst-inhibiting acid phosphatase of Francisella , was cloned and sequenced. The deduced amino acid sequence of AcpA showed limited similarity to phospholipase C proteins present in Pseudomonas aeruginosa and Mycobacterium tuberculosis. An F. novicida acpA null mutant was found to exhibit wild-type growth kinetics in both cell-line and inflammatory mouse macrophages as well as remaining virulent for mice. These data suggest that AcpA is not essential for the intracellular growth or virulence of F. novicida, and that any role it may play in virulence is subtle. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/8454 |
| Date | 24 August 2017 |
| Creators | Baron, Gerald Stephen |
| Contributors | Nano, Francis E. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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