Enhanced priming of adaptive immunity by Mycobacterium smegmatis mutants with high-level protein secretion

Abstract

Mycobacteria have features that make them attractive as potential vaccine vectors. The nonpathogenic and rapidly growing Mycobacterium smegmatis can express both Mycobacterium tuberculosis antigens and heterologous antigens from other pathogens, and it has been used as a viable vector for the development of live vaccines. In order to further improve antigen-specific immunogenicity of M. smegmatis, we screened a random transposon mutant library for mutants displaying enhanced efficiency of protein secretion ("high secretors") and isolated 61 mutants showing enhanced endogenic and transgenic protein secretion. Sequence analysis identified a total of 54 genes involved in optimal secretion of insert proteins, as well as multiple independent transposon insertions localized within the same genomic loci and operons. The majority of transposon insertions occurred in genes that have no known protein secretion function. These transposon mutants were shown to prime antigen-specific CD8(+) T cell responses better than the parental strain. Specifically, upon introducing the simian immunodeficiency virus (SIV) gag gene into these transposon mutant strains, we observed that they primed SIV Gag-specific CD8(+) T cell responses significantly better than the control prime immunization in a heterologous prime/boost regimen. Our results reveal a dependence on bacterial secretion of mycobacterial and foreign antigens for the induction of antigen-specific CD8(+) T cells in vivo. The data also suggest that these M. smegmatis transposon mutants could be used as novel live attenuated vaccine strains to express foreign antigens, such as those of human immunodeficiency virus type 1 (HIV-1), and induce strong antigen-specific T cell responses.

Fig 1

Map of the reporter plasmid and transposon mutagenesis strategy. (A) An efficient screening strategy was developed to isolate M. smegmatis transposon mutants with enhanced expression and secretion of transgene products, as described in the text. (B) A reporter plasmid, pSL300 contains the M. tuberculosis antigen 85 promoter (Ag85p) and the SecA1-dependent signal sequence from the same protein (Ag85ss) cloned upstream of the truncated E. coli PhoA (′phoA) in the mycobacterial integrating vector. (C) The parental strain (SLMS300), M. smegmatis expressing pSL300, was randomly mutated using a mariner-based Himar1 transposon (Tn5332). Among 35,000 transposon mutants, 72 dark-blue colonies on BCIP medium were selected. To demonstrate the difference in blue phenotype, wild-type mc²155 expressing empty vector and the intense-blue mutants resulting from transposon insertion into the pstS gene (pstS::Tn) were used as negative and positive controls, respectively. All other dark-blue colonies represent the individual 72 transposon mutants.

Fig 2

Significantly higher levels of protein secretion are detected in M. smegmatis transposon mutants. The parental strain and transposon mutants expressing M. tuberculosis LpqH protein tagged with the SIINFEKL epitope were grown to log phase in 7H9 medium without oleic acid-albumin-dextrose-catalase (OADC). Proteins were fractionated into a cell-associated pellet (PPT) fraction and a short-term culture filtrate (CF) fraction and analyzed by Western blotting for the indicated proteins. SIINFEKL polyclonal antibody for LpqH, Hsp65-specific monoclonal antibody for a protein loading control, and DnaK antibody for M. smegmatis secreted protein were used. Bolded transposon mutants are those evaluated in immunogenicity studies. The protein expression levels of the transposon mutants and the parental strain were compared by calculating the LpqH/Hsp65 ratio in PPTs.

Fig 3

Enhanced immunogenicity of transposon mutants with higher secretion of the transgene product. C57BL/6 mice were immunized intravenously with approximately 1 × 10⁷ CFU of either parental or transposon mutant strains expressing LpqH-SIINFEKL or Ag85B. (A) The mean (±standard error of the mean) percentages of SIINFEKL-specific tetramer positive CD8⁺ T cells from PBMCs collected at week 1 are shown (n = 5 per group). (B) MHC II-specific T cell responses in spleens were assessed by using IFN-γ ELISPOT assays to measure the recall response of CD4 T cells specific for p25 in spleen suspensions; mean (±standard error of the mean) results are shown. The experiments were repeated two times, and results of a representative experiment are shown. Statistical significance was assessed by comparing immunogenicity results for the parental strain group (MS-LpqH-SIINFEKL or MS-Ag85B) to those for recombinant transposon mutants expressing the transgenes (n = 5 per group) using ANOVA one-way analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to results for MS-SIINFEKL).

Fig 4

Improved SIV Gag-specific CD8⁺ T cell responses of M. smegmatis transposon mutants. Mice were immunized via the intravenous route with 1 × 10⁷ CFU of actively growing log-phase M. smegmatis transposon mutants, H10 and H24, expressing pSL7 (SIV Gag). Heterologous boost was accomplished by injecting 10⁶ PFU rAd5-SIV Gag via the intramuscular route. Recombinant transposon mutants, H10-pSL7 and H24-pSL7, were primed for higher Gag-specific CD8⁺ T cell responses following suboptimal rAd5-Gag (1 × 10⁶ PFU) boosting than the parental M. smegmatis strain expressing SIV Gag. The P values were determined by ANOVA. * and **, P < 0.05 and P < 0.01, respectively.

Fig 5

Construction of the lpqM mutant. (A) Maps of the lpqM genomic regions of wild-type M. smegmatis (Msmeg) and a representative lpqM mutant. The primer sites (LL, RR, Hyg1, and Hyg2) used for PCR confirmation of mutant clones are indicated as black boxes. (B) PCR products of genomic DNAs from wild-type M. smegmatis (lane 1), an allelic exchange plasmid construct (pSL330) for lpqM mutagenesis (lane 2), and two independent lpqM mutant clones from M. smegmatis (lanes 3 and 4). Primers LL/Hyg1, RR/Hyg2, and LL/RR were used, and the expected sizes of the PCR products are indicated. A molecular size marker is shown on the right.

Fig 6

Improved transgene secretion and CD8⁺ T cell responses by the M. smegmatis ΔlpqM mutant. (A) The cell-associated pellet (PPT) fraction and the short-term culture filtrate (CF) fraction of the gene deletion mutant (ΔlpqM-lpqH) and the complemented strain (ΔlpqM-C-lpqH) expressing LpqH-SIINFEKL were analyzed by Western blotting for the indicated proteins. As a control, wild-type M. smegmatis (MS) and M. smegmatis expressing LpqH-SIINFEKL (MS-lpqH) were included. (B and C) SIINFEKL-specific tetramer-positive CD8⁺ T cells from PBMCs (B) and the Ag85B-specific CD4⁺ T cell response determined by IFN-γ ELISPOT assay in splenocytes (C) of mice immunized with the indicated recombinant strains (1 × 10⁷ CFU). *, P < 0.05.