Identification and characterization of a spore-like morphotype in chronically starved Mycobacterium avium subsp. paratuberculosis cultures

Abstract

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP). We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat-treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated.

Figure 1. MAP morphotype induction is dependent upon temperature.

One year old MB7H9 MAP broth cultures were inoculated on A) A–K agar and C) BHI agar for 72 h at 37°C and 39°C. MAP showed growth only at 39°C compared with B. subtilis and E. coli K-12 controls, which had substantial growth at both temperatures (A). Spore enrichment was determined by malachite green staining (B). In order to confirm purity of MAP culture, the year old MAP culture was grown on BHI agar and determined to be free of any contaminating organisms (C).

Figure 2. Ultrastructural characterization of MAP morphologies.

Fine MAP morphotype structure was determined by TEM (A). TEM images were taken of log-phase, dormant and A–K MAP cultures. While the dormant MAP culture showed a mix of vegetative cells and spores, A–K MAP cultures displayed typical spore characteristics, including a cortex, plasma membrane and coat layers. (B) All MAP cultures were assessed for contamination of duplex and normal PCR of IS900, spoIVA and Clostridium 16SrDNA. Only MAP samples contained the IS900 element and did not amplify Bacillus and Clostridium related genes. (C) Spore formation was confirmed by the detection of dipicolnic acid (DPA) using a colorimetric assay. DPA is a chemical found within the spore core of endospores. Intact and autoclaved mycobactin J (250.0 µg/mL) were used as controls. Each sample was conducted in triplicate.

Figure 3. Sporulation is reproducible on traditional sporulation medium.

Spore enrichment was independently conducted by the National Animal Disease Center (Ames, IA) using a separate MAP K-10 culture inoculated on A–K agar. MAP K-10 year old MB7H9 broth culture, B. subtilis and E. coli K-12 were inoculated on B) Potato extract agar with mycobactin J at 37°C and 39°C. MAP K-10 growth was observed after two weeks of incubation at 39°C in comparison to overnight growth of B. subtilis and E. coli K-12 controls. Biomasses were collected similarly to A–K cultures and processed for TEM (B). MAP K-10 TEM images showed similar structures as those observed in Figure 2 . Furthermore, biomasses were streaked on blood agar and incubated at 37°C and 39°C for 4 weeks to confirm purity (C). MAP K-10 failed to grow for the entire duration of incubation in comparison to B. subitlis and E. coli K-12 controls.

Figure 4. Sporulation occurs in multiple MAP strains.

MAP strains 7565, Ben and Linda were inoculated on A–K agar. Biomasses were collected and processed for TEM. All strains show characteristic spore structures.

Figure 5. MAP morphotypes survive 70°C and are positive for MAP 16SrRNA and IS900.

(A) MAP K-10 log phase and spores, B. subtilis and C. perfringens were heat treated at 70°C for 30 min and subsequently treated with 2% lysozyme, PK, or kanamycin. Heat treated samples were plated on MB7H9 or blood agar and incubated at 37°C under aerobic or anaerobic conditions. (B) Heat treated cultures were plated on blood agar to determine growth of any contaminates. (C) 16SrRNA sequences of germinated heat treated MAP spores compared to reference sequences from MAP, Bacillus spp., Streptomyces spp. and Clostridium spp.. Ten colonies from each plate were selected for sequences. Sequences shown are a consensus from the ten colonies. (D) IS900 duplex PCR of germinated heat treated MAP spores.

Figure 6. Dormant MAP cultures upregulate spore-related transcripts.

(A) A BLAST comparison and reciprocal BLAST searches were conducted between known sporulation genes from Bacillus spp.^† and Streptomyces spp.^‡ against MAP. Percent similarity was determined by protein alignment. (B) Quantitative real-time PCR was performed on dormant MAP cultures to determine the presence of carD (MAP0475 and MAP0987) and spo0A (MAP1002c). All three genes are upregulated in comparison to log-phase MAP K-10 culture. All samples were conducted in triplicate. C) Multiple sequence alignment of selected CarD proteins. CarD has recently been shown by Stallings et al. to be necessary component of stringency regulation in mycobacteria. Other studies indicate that the stringency response is also necessary for the initiation of sporulation. A multiple sequence alignment of CarD amino acid sequences from mycobacteria and sporulating bacteria was conducted using CLUSTALW.

Figure 7. MAP spores retain infectivity and germinate into acid-fast bacilli in a bovine MDM model.

MAP spores, MAP log-phase and Bacillus subtilis spores were allowed to infect MDMs for 0.5, 2, 6, 24, and 48 h p.i.. MAP spores readily infected MDMs and germinated by 24 h p.i.. Upon 48 h p.i., MDMs were lysed and MAP spores successfully germinated into acid fast bacilli.