Microbiology (1 994),140,3309-33 18 Printed in Great Britain Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 3P6, Canada INTRODUCTION Se r u m -se n s i t i ve m u t a t i o n of Francisella novicida: association with an ABC transporter gene Khisimuzi E. Mdluli, Lawrence 5. D. Anthony, Gerald S. Baron, Megan K. McDonald, Svetlana V. Myltseva and Francis E. Nan0 Author for correspondence: Khisimuzi Mdluli. Tel: + 1 604 721 7074. Fax: + 1 604 721 8855. e-mail : fnano@sol.uvic.ca Francisella novicida is a facultative intracellular pathogen that can survive and grow in macrophages by preventing phagolysosomal fusion. In this study in vitro cassette mutagenesis was used to generate a library of insertion mutants of F. novicida. Two related mutants, KM14 and KM145, initially identified as defective for growth in macrophages, were found to be sensitive to serum. These mutants were also found to grow approximately 1000-fold less well in the livers and spleens of infected mice. We cloned a genetic locus that was presumably mutagenized in these mutants and found that it included genes that had high similarity in their deduced amino acid sequence to those of msbA and orfE of Escherichia coli. The former is a member of the superfamily of ABC transporter proteins. We named the corresponding genes in F. novicida, valAB. Integration of a cloned valAB locus into the chromosome of KM14S partially restored the serum resistance phenotype found in wild-type F. novicida. Keywords : Francisella, tularaemia, complement, ABC transporter, macrophage Francisella tularensis is a facultative intracellular bacterial pathogen that causes a granulomatous, febrile illness in a wide range of animals. The pathology of human infection by virulent F. tzllarensis is limited in about two-thirds of the cases to a localized lymphadenopathy; in the re- mainder of the cases a typhoidal type disease results with pathological lesions similar to those seen in tuberculosis (Dienst, 1963; Evans etal., 1985; Goodpasture & House, 1928). Rodent studies with virulent and attenuated strains of F. tzllarensis suggest that the course of disease is dependent on the route of infection, which in turn may determine the type and rapidity of the immune response (Downs et al., 1947; Fortier et al., 1991). Animal studies and autopsy of human cases also show that F. tzllarensis grows in several organs in the body, especially the liver, spleen and, in typhoidal cases, the lungs. The normal course of disease in humans, without antibiotic treatment, runs approximately 32 d, with no long-term sequelae evident (Dienst, 1963). . , . , . . ,, . . , , ,,, . . ,, . . .. , . . . . . . . . . ... . . . . ... . . . . . . . . . . . . . . . ... . . . . . . . . .. . . . . , . , . , . . . . , . . . . .. . . . . .. . . . . . . .. . . . .. . . ... . . . . .. . . . . . . .. . . . .. . . ... . . . . . . . . . . . . . The GenBank accession number for the nucleotide sequence reported in this paper is L17003. In experimentally infected animals, F. tzllarensis is found both inside macrophages and extracellularly (White et al., 1964). Unlike macrophages, polymorphonuclear leucocytes (PMNs) are unable to phagocytose F. tzllarensis in the absence of antiserum (Proctor et al., 1975). Francisella can grow in vitro in rodent resident peritoneal, thioglycollate-elicited, or bone-marrow-derived macro- phages (Anthony et al., 1991a). There is some evidence that, in situ, F. tzllarensis can also invade non-professional phagocytic cells such as hepatocytes (Conlan & North, 1992). The growth of Francisella inside macrophages is similar to that of Mycobacterium tuberculosis and Legionella pnezlmophila in that they prevent phagolysosomal fusion (Anthony et al., 1991a). Consistent with intracellular replication is the observation that cell-mediated immunity is needed to clear F. tularensis infections (Anthony & Kongshavn, 1987 ; Allen, 1961 ; Eigelsbach et al., 1975; Kostiala et al., 1975; Claflin & Larson, 1972). The cellular response is H-2 restricted (Anthony & Kongshavn, 1988; Surcel et al., 1989), and involves the activation of macrophages via the action of the cytokines interferon-y and tumour necrosis factor-a (Anthony et al,, 1989; Fortier et al., 1992; Leiby et al., 1992). Growth of F. tzllarensis in murine macrophages in 0001-9149 0 1994 SGM 3309