Identification of essential genes in Mycobacterium avium subsp. paratuberculosis genome for persistence in dairy calves

Abstract

To cause disease Mycobacterium avium subsp. paratuberculosis needs to enter mammalian cells, arrest phagosomal maturation and manipulate the host immune system. The genetic basis of the bacterial capacity to achieve these outcomes remains largely unknown. Identifying these genes would allow us to gain a deeper understanding of MAP’s pathogenesis and potentially develop a live attenuated Johne’s disease vaccine by knocking out these genes. MAP genes demonstrated to be essential for colonization in the natural host, ruminants, are unknown. Genome-wide transposon mutagenesis and high-throughput sequencing were combined to evaluate the essentiality of each coding region in the bacterial genome to survive in dairy calves. A saturated library of 3,852 MAP Tn mutants, with insertions in 56% of TA sites, interrupting 88% of genes, was created using a MycoMarT7 phagemid containing a mariner transposon. Six calves were inoculated with a high dose of a library of MAP mutants, 1011 CFUs, (input) at 2 weeks of age. Following 2 months of incubation, MAP cells were isolated from the ileum, jejunum, and their associated lymph nodes of calves, resulting in approximately 100,000 colonies grown on solid media across 6 animals (output). Targeted next-generation sequencing was used to identify the disrupted genes in all the mutants in the input pool and the output pool recovered from the tissues to identify in vivo essential genes. Statistical analysis for the determination of essential genes was performed by a Hidden Markov Model (HMM), categorizing genes into essential genes that are devoid of insertions and growth-defect genes whose disruption impairs the growth of the organism. Sequence analysis identified 430 in vivo essential and 260 in vivo growth-defect genes. Gene ontology enrichment analysis of the in vivo essential and growth-defect genes with the highest reduction in the tissues revealed a high representation of genes involved in metabolism and respiration, cell wall and cell processing, virulence, and information pathway processes. This study has systematically identified essential genes for the growth and persistence of MAP in the natural host body.

Evaluation of Mycobacterium avium subsp. paratuberculosis isocitrate lyase (IcL) and ABC transporter (BacA) knockout mutants as vaccine candidates

“…paratuberculosis and their efficacy to induce cell-mediated immune responses by a comparative approach in mouse and natural host calf models. A previous study identified genes necessary for MAP survival in dairy calves and permitted us to select a gene to knockout that is essential for MAP survival in vivo (MAP1643, IcL ) or one which is required for intracellular growth (MAP1531c, BacA ) ( EshraghiSamani et al., 2022 ). The results of the current study confirmed the attenuation of these mutants in calves and indicated that disruption of these genes affect MAP's intracellular survival without affecting its immunogenicity.…”

“…The wild-type is able to advert immune system by having all these properties and this will delay the activation of macrophages and the downstream signaling pathways ( Arsenault et al., 2014 )and delay the proinflammatory responses. In addition, the deletion of BacA has been found to impair MAP growth in vivo ( EshraghiSamani et al., 2022 ), these might suggest that Δ BacA is not over-attenuated to get cleared before inducing immune responses, and it is not under-attenuated to persist in tissue and maintain a chronic infection.…”

“…Screening a library of MAP transposon mutants in calf infection model categorized MAP coding regions into 683 essential, 304 growth-defect, 3277 non-essential and 92 growth-advantage coding regions ( EshraghiSamani et al., 2022 ). Allelic exchange mutagenesis was employed to create two MAP live attenuated strains by knocking out one essential gene ( IcL , MAP1643) and one growth-defect gene ( BacA , MAP1531c) ( Figure 1A ).…”

The pan-genome of Mycobacterium avium subsp. paratuberculosis (Map) confirms ancestral lineage and reveals gene rearrangements within Map Type S

“…Analysis of the larger Mac and Map genome dataset in this study identified four copies of the PE-PGRS gene in Map Type B, Mah and M. chimaera and two copies in Type C, Type S, Maa and M. intracellular. There are four PPE proteins (PPE4, PPE65, PPE25 and PPE26), all members of the ESX-5 and ESX-3 systems that are essential for virulence in Mycobacteria [ 33 ]. The PPE4 protein was found in only two out of the 52 Type S strains.…”

Immunogenicity and efficacy of an oral live-attenuated vaccine for bovine Johne’s disease

“…The BacA gene is essential for the optimal growth of Mycobacterium avium subsp. paratuberculosis (MAP) in calf tissue (20,21), and it is involved in the import of hydrophilic compounds such as vitamin B12 and iron metabolism. Mycobacterium tuberculosis mutant of BacA indicated diminished capacity to maintain a chronic infection (22).…”