The Functional and Molecular Effects of Doxycycline Treatment on Borrelia burgdorferi Phenotype
John R. Caskey1,2†, Nicole R. Hasenkampf1, Dale S. Martin1, Vladimir N. Chouljenko2, Ramesh Subramanian2, Mercedes A. Cheslock1 and Monica E. Embers1*
2019https://www.frontiersin.org/articles/10 ... 00690/full
Recent studies have shown that Borrelia burgdorferi can form antibiotic-tolerant persisters in the presence of microbiostatic drugs such as doxycycline. Precisely how this occurs is yet unknown. Our goal was to examine gene transcription by B. burgdorferi following doxycycline treatment in an effort to identify both persister-associated genes and possible targets for antimicrobial intervention. To do so, we performed next-generation RNA sequencing on doxycycline-treated spirochetes and treated spirochetes following regrowth, comparing them to untreated B. burgdorferi. A number of genes were perturbed and most of those which were statistically significant were down-regulated in the treated versus the untreated or treated/re-grown. Genes upregulated in the treated B. burgdorferi included a number of Erp genes and rplU, a 50S ribosomal protein. Among those genes associated with post-treatment regrowth were bba74 (Oms28), bba03, several peptide ABC transporters, ospA, ospB, ospC, dbpA and bba62. Studies are underway to determine if these same genes are perturbed in B. burgdorferi treated with doxycycline in a host environment.
The results of the bioassay in mice demonstrate that after treatment with the MBC of doxycycline, the spirochetes were able to host adapt, and evade immune pressure to establish an infection. The number of spirochetes injected into each mouse was held constant, but viability was not assessed. Therefore, the number of actively growing B. burgdorferi was likely significantly reduced after doxycycline treatment. What is most interesting about the result is that no significant differences were observed in the spirochete infectivity when immunocompetent versus immunodeficient mice were used. This result suggests that exit from dormancy may occur very rapidly in vivo, allowing the immunoevasive phenotype to become established.
We have previously demonstrated that the persister cell phenotype appears to be generated stochastically and driven by slowed growth (Caskey and Embers, 2015). In this study, we aimed to identify gene expression patterns associated with the survival and re-growth of B. burgdorferi in the antibiotic environment. While a similar study in which RNASeq was applied to antibiotic treatment of B. burgdorferi was conducted (Feng et al., 2015), several important distinctions between that study and ours should be made. In that report by Feng et al, in vitro-cultured Bb were treated with either doxycycline or amoxicillin (50 μg/mL) for 6 days and then subjected to RNASeq, with comparison to the untreated control. Genes that were up-regulated by 2-fold or more were ascribed significant and the pathways affected were elucidated. In our study, we treated a slightly denser culture (5 × 107 versus 1 × 107) with doxycycline at the same dose (50 μg/ml) for 5 days. An aliquot of the treated cells was allowed to re-grow, such that we had 3 treatment groups. In addition, we performed RNASeq on duplicate samples and ascribed significance using both fold-change and p-values to account for variation between samples. Both studies shed light on the mechanisms that Bb may use to establish persistence and re-grow. The Feng study identified a large number of genes as upregulated following treatment, whereas we found that the vast majority of genes were down-regulated, likely owing to a global decline in transcription. In the Feng study, the ClpP protease was indicated as the most highly up-regulated gene in treated B. burgdorferi. It was up-regulated in our screen as well (0.43-fold), but not determined to be significant. The genes found to be significantly increased in treated versus control encoded several Erp proteins and a 50S ribosomal protein (bb0778). This was verified by standard RT-PCR, as shown in Figure 4. The results in Figure 4 are derived from amplifying transcript from an equal quantity of input RNA. We did not have a good constitutively transcribed gene that is consistently expressed in all groups equally to be used as a housekeeping gene control, based on the RNASeq data. Thus, these results are not fully quantitative but only meant for validation of the RNASeq results
We fully expected that genes categorized by involvement in the stress response (Bugrysheva et al., 2003; Drecktrah et al., 2015; Cabello et al., 2017) and DNA repair mechanisms (Fisher et al., 2017) would be significantly up-regulated with antibiotic treatment. However, this did not appear to be the case with either the Feng study or our own. The mechanisms governing the development of persister cells are not easily discerned by these RNASeq analyses, perhaps because the spirochetes stochastically enter dormancy (Caskey and Embers, 2015) and regrowth is determined by non-heritable traits. One supposition may be that instead, post-transcriptional, or even post-translational events such as lysine acetylation (Fisher et al., 2017; Bontemps-Gallo et al., 2018) govern the entry and exit from dormancy in the antibiotic environment. For example, in the toxin-mediated growth reduction within S. typhimurium, the toxins (TacT) add a post-translational modification (acetylation) to tRNA, which is reversed by a peptidyl tRNA hydrolase (Cheverton et al., 2016). A toxin-antitoxin system for Borrelia persister development has not been identified, but enzymatic modification of translational components could be involved in formation of persister cells and their re-growth. Nonetheless, we have identified a number of surface proteins which may be antigenic and serve as targets for novel immunotherapeutic strategies.