Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transposition biases and strategies for novel mutant discovery

“…The conditionally replicating (temperature-sensitive) transposon donor phagemid WMycoMarT7 was propagated at 30 uC on Mycobacterium smegmatis, ultimately delivering the Himar1-derived MycoMarT7 transposon into MAP (Sassetti et al, 2001). The Tn5367 mutants were randomly picked from a large mutant collection as previously described (Rathnaiah et al, 2014).…”

“…These cultures were infected with WMycoMarT7 phage at a nominal m.o.i. (ratio of viable phage particles to bacterial cells) of 10, incubated for 6 h at 37 uC, and treated with stop buffer (MP buffer containing 20 mM sodium citrate and 0.2 % (v/v) Tween 80) as described previously (Bardarov et al, 1997;Rathnaiah et al, 2014). Five independent transductions were performed, plating each on four MOADC agar plates without Tween 80, supplemented with 50 mg ml 21 kanamycin.…”

“…Genomic DNA was isolated from the reduced pool of mutants, and a nested PCR to amplify transposon-chromosomal junctions for sequencing was performed in two rounds as previously described, to locate the MycoMarT7 transposon insertion site in MAP (McAdam et al, 2002). In the first-round 30 ml PCR, the transposonspecific primer MMP1 (0.1 mM) and the arbitrarily degenerate primer AMT38 (1.0 mM) were used as previously described (Rathnaiah et al, 2014) to amplify the chromosomal sequence flanking the transposon insertion site. Thermocycler settings were 94 uC for 5 min, followed by 40 cycles of 94 uC for 60 s, 55 uC for 30 s, 72 uC for 90 s, and a final extension at 72 uC for 7 min.…”

“…Five or six colonies per plate were picked and further grown to prepare 109 glycerol stocks, each one representing an individual transductant. To analyse the mutants generated from Tn5367 transposon mutagenesis, we randomly picked 108 mutants (one mutant per plate) from a previously described collection of 13 536 stored individually in 96-well plates as an indexed library (Rathnaiah et al, 2014). This collection was generated using the phagemid phAE94, which yielded a lower transduction frequency of 1.1|10…”

“…27 (Harris et al, 1999;Rathnaiah et al, 2014). This procedure differs from prior screens where mutants were picked based on phenotypes that may be associated with virulence or libraries that were not comprehensive (Cavaignac et al, 2000;Harris et al, 1999;Scandurra et al, 2009;Shin et al, 2006;Wang et al, 2014).…”