Utilization of a ts-sacB selection system for the generation of a Mycobacterium avium serovar-8 specific glycopeptidolipid allelic exchange mutant

Annals of Clinical Microbiology and Antimicrobials Utilization of a ts-sacB selection system for the generation of a Mycobacterium avium serovar-8 specific glycopeptidolipid allelic exchange mutant Vida R Irani 2 Sun-Hwa Lee 1 Torsten M Eckstein 0 Julia M Inamine 0 John T Belisle 0 Joel N Maslow 2 3 0 Mycobacterial Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University , Fort Collins CO 80523 , USA 1 Harvard Medical School, New England Regional Primate Center , Southborough, MA 01772 , USA 2 School of Medicine, Division of Infectious Diseases, University of Pennsylvania , Philadelphia, PA 19104 , USA 3 Section of Infectious Diseases, VA Medical Center , Philadelphia PA 19104 , USA Background: Mycobacterium avium are ubiquitous environmental organisms and a cause of disseminated infection in patients with end-stage AIDS. The glycopeptidolipids (GPL) of M. avium are proposed to participate in the pathogenesis of this organism, however, establishment of a clear role for GPL in disease production has been limited by the inability to genetically manipulate M. avium. Methods: To be able to study the role of the GPL in M. avium pathogenesis, a ts-sacB selection system, not previously used in M. avium, was employed as a means to achieve homologous recombination for the rhamnosyltransferase (rtfA) gene of a pathogenic serovar 8 strain of M. avium to prevent addition of serovar-specific sugars to rhamnose of the fatty acyl-peptide backbone of GPL. The genotype of the resultant rtfA mutant was confirmed by polymerase chain reaction and southern hybridization. Disruption in the proximal sugar of the haptenic oligosaccharide resulted in the loss of serovar specific GPL with no change in the pattern of non-serovar specific GPL moieties as shown by thin layer chromatography and gas chromatography/mass spectrometry. Complementation of wild type (wt) rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wt serovar 8 parent strain. Results: In this study, we affirm our results that rtfA encodes an enzyme responsible for the transfer of Rha to 6d-Tal and provide evidence of a second allelic exchange mutagenesis system suitable for M. avium. Conclusion: We report the second allelic exchange system for M. avium utilizing ts-sacB as doublenegative and xylE as positive counter-selection markers, respectively. This system of allelic exchange would be especially useful for M. avium strains that demonstrate significant isoniazid (INH) resistance despite transformation with katG. Through the construction of mutants in GPL or other mycobacterial components, their roles in M. avium pathogenesis, biosynthesis, or drug resistance can be studied in a consistent manner. - Background Mycobacterium avium is a frequent cause of disseminated infection among patients with end-stage AIDS [9,11,19]. M. avium can also present with a similar spectrum of pulmonary and extra pulmonary syndromes as Mycobacterium tuberculosis [27] including the establishment of latent infection that can reactivate concomitant with immune suppression [13]. While significant advances have been made in deciphering the host responses against M. avium infection, there is only a rudimentary understanding of the bacterial factors involved in the pathogenesis of infection [14,22]. Numerous studies have implicated the cell wall lipids in mycobacterial pathogenesis. For M. avium, there is evidence that the glycopeptidolipids (GPL), as the dominant lipid for this species, may negatively affect host immunity [4,25]. Study of GPL in M. avium pathogenesis has been limited by a lack of suitable genetic techniques to be able to create site directed knockouts. Further, as reviewed below, there is controversy as to which portion of GPL predominates in disease production. The GPLs are comprised of a lipopeptide (LP) core of Dphenylalanine-D-allo threonine-D-alanine-alaninol with a fatty acyl group N-linked to the phenylalanine residue and a methylated rhamnose modifying the terminal alaninol. The LP core is glycosylated at D-allo threonine with 6deoxytalose (6dTal) to form non-specific GPL (nsGPL) and is further glycosylated at 6dTal with a haptenic oligosaccharide to yield serovar-specific GPL (ssGPL). All serovars maintain a common -L-rhamnopyranosyl(12)-6dTal [6]. Historically, the predominance of serovars 1, 4, and 8, among patients with disseminated infection [10,26] has been suggested as evidence to support a role for the oligosaccharide moiety of GPL in pathogenesis, but may conversely represent the fact that a restricted set of clones are disease producing. More direct evidence of a role of the GPL oligosaccharide in pathogenesis is provided by the study of Minami that demonstrated that heat-killed Staphylococcus aureus coated with M. avium GPL promote phagocytosis and inhibit phagolysosomal fusion in relation to serovar [17]. Other studies have, however, suggested a dominant role for the lipopeptide core in pathogenesis [5]. Significantly limiting the development of a consistent framework of the role of GPL in mycobacterial pathogenesis has been the inability to construct isogenic strains differing in GPL structure, necessitating the comparison of genetically distinct strains of differing serotypes. To study the role of the serovar-specific oligosaccharide moiety of GPL in the pathogenesis of M. avium, an allelic exchange mutant in rtfA was created for a pathogenic serovar 8 strain to yield a strain deficient in ssGPL. Homologous recombination was performed using a novel allelic exchange vector that incorporated a temperature-sensitive mycobacterial origin of replication (ts-oriM) and sacB as counter selective markers [21] and xylE [7] as a positive selection marker. Complementation of rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wild type (wt) serovar 8 smooth opaque (SmO) parent strain. In addition to reaffirming our results for serovar 2 [15] that rtfA encodes an enzyme responsible only for the transfer of Rha to 6d-Tal to form the serovar-8 specific oligosaccharide, this study delineates a second system of allelic exchange mutagenesis for M. avium. Methods Bacterial strains and plasmids Escherichia coli strain DH5 was used as the host strain for plasmid construction and propagation. Wild type and recombinant M. avium and Mycobacterium smegmatis strains were grown in Middlebrook 7H9 broth or 7H11 agar supplemented with 10% OADC (Difco Laboratories, Detroit, MI) at 37C, except where indicated. M. smegmatis mc2155 [24] was employed as a test strain for mycobacterial shuttle vectors. M. avium 920A6 is a serovar 8 bloodstream isolate cultured from a patient with AIDS [1]. Transformation of E. coli and M. smegmatis was performed as described [23,24]. Transformation of M. avium was performed according to the protocol of Lee et al. [12]. For E. coli, selection was carried out us (...truncated)