(Student contributor: Swarnav Bhakta, PG II-Biotechnology)

One of the biggest challenges of the current health care system is to detect and treat pathogens with developing resistances against the drugs, especially which manifest high antibiotic tolerance. In addition to the more well-known host-pathogen or drug-pathogen interaction mechanisms, development of persister cell populations in chronic infections is getting progressive importance due to its widespread association with intervention failures(Fischer et al., 2017), including Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Salmonella enterica and Staphylococcus aureus (Helaine& Kugelberg 2014, Harms et al., 2016, Michiels et al., 2017). Persistence of ‘dormancy’ is a phenomenon that describes the ability of a pathogenic subpopulation to survive against the treatment with the potential drug for an extended period. This evolutionarily conserved adaptive mechanism for drug tolerance is associated with non-heritable phenotypic variations (e.g. phenotypic switching in the bacterial populations, macrophage-induced mechanisms and dramatic change in metabolism, etc.) which make this adaptation different from other mechanisms that generate genetic resistance (Balaban et al., 2004, Adams et al., 2011, Amato et al., 2014). Interestingly, little after the discovery of antibiotics, Joseph W. Bigger, an Irish physician who was working in England back in 1944, recognized the presence of persister cells in the form of slow-growing, penicillin-tolerant populations of Staphylococcus aureus which survived high lethal doses of penicillin (Kim et al., 2016). He termed these cocci, with the phenotypic change of lack of growth, as persisters so to separate them from the resisters, found in bacteria due to heritable genetic mutations.   Such adaptation is prominently exemplified latent infection of Mycobacterium tuberculosis which can persist even for lifelong in a metabolically dormant state (Mandal et al., 2019).

Persister cell development is associated with developing a subset of the population that is metabolically quiescent and hence cannot be intervened by drug treatment (Fischer et al., 2017). Persisters though represent a subpopulation of the total cells, but their survival shows some kin selection and altruistic effect by allowing the whole population to survive at the time of high drug exposure and immunological stress. After the disappearance of stress-factors, persisters revert to normal proliferative mode, reinitiate growth and repopulate the local environment. This post-treatment sensitization phenomenon emulates ecological succession, where immunological stress and drug exposure represent bottlenecks events, with persisters acting as the founders of new environmental niches (Bhattacharya et al., 2020). Eukaryotic pathogens, including fungal and parasitic protozoans, are also akin to metabolic switching from proliferative to dormant state (Barrett et al., 2019). For a range of fungal pathogens including Candida albicans and C.auris nutrient depletion and stress renders metabolic drop off to circumvent fungicides like amphotericin B (Wuyts et al., 2018).The hypnozoite liver stages of Plasmodium that is often associated with the relapse of infection even years after successful therapeutic clearance is one such persister-like stage for Plasmodium vivax and is a marked threat for the eradication of malaria from the human populations (Markus 2017). In the case of lower eukaryotic pathogens like trypanosomatids, semi-quiescence to quiescence intracellular forms are detected for several species of Leishmania and in Trypanosomacruzi (Barrett et al., 2019). Persistence adaptations are particularly relevant clinically for Leishmania as a relapsing condition like Post kala-azar dermal leishmaniasis (PKDL). Post-treatment sensitization occurs several years after the treatment for visceral leishmaniasis(VL) and leishmaniasis recidivans, occurring after the treatment of cutaneous leishmaniasis, emerge from possible metabolically distinct parasites that circumvent drug treatment due to dormancy without acquiring resistance by signature genetic alterations (Rutte et al., 2019).

Persistersin Trypanosomatids – Cellular and Molecular Perspectives

Till 2015, there was a dearth of systemic study on persister development in lower eukaryotes, particularly in trypanosomatids, due to technical constraints, principally the absence of exclusive labeling methods for quiescent cells. However, this has now become one of the most emerging avenues of research after the detailed identification and characterization of semi-quiescent Leishmania parasites. The seminal work by Kloehn and colleagues(Kloehn et al., 2015), for the first time, has clearly demonstrated that intracellular amastigotes in the infected non-healing lesions of the BALB/c mice are in a metabolically quiescent stage which leads to ~3 fold increase in doubling time for the infected state. The partly tranquil metabolic state was also reflected by low de novo synthesis and turnovers of lipid and protein molecules which possibly are responses to complex growth restriction in the intracellular microenvironments of the granulomas. Interestingly, the study also found two distinct macrophage populations, representing two distinct metabolic varieties of amastigotes in the inflammatory lesions. Mandell et al., 2015, also identified a definite fraction of L. major amastigotes with sparse proliferation in C57BL/6J mice.  This population was observed to the harbor in less infected macrophages and constituted a third of amastigotes under the condition of persistent infection; a remaining subset of amastigotes retained the ability to replicate (Mandell and Beverley 2015).L. major, lacking the Golgi GDP-mannose transporter required for lipophosphoglycan synthesis (lpg2^-/-) persists in the absence of pathology and in mouse infections, this knocked outline attained persister like feature immediately after infection (Mandell and Beverley 2015). A comparative analysis between the L. braziliensis promastigotes and amastigotes depicted the “semi-quiescent characteristic features” of the amastigotes as they replicate at a negligible rate with minimal metabolic activity. Metabolomic profiles demarcated the amastigotes as a metabolically-restricted life stage compared to their rapidly dividing promastigote counterparts(Jara et al., 2017). Persistence adaptations gain further support for trypanosomatid infection as regimens including short term therapy or even 60-day long treatment for T.cruzi infection are not related to resistance development but possibly the parasite alleviates drug tolerance by adopting quiescence. After drug withdrawal, one or two dormant parasites are detectable in each infected cell in case of trypanosomatid and these dormant parasites can reinitiate proliferation.T. cruzi amastigotes regularly and spontaneously cease replication and become non-responsive to effective trypanocidal drugs like benznidazole and nifurtimox. The amastigotes, even after a long term of drug exposure, retain the ability to get converted to the infectious trypomastigotes for re-establishing new infections (Sánchez-Valdéz et al., 2018).

Exploring the intricacies of the alteration of physiological status for intracellular amastigotes in infected tissues by proteomic or transcriptomic approaches is impaired by the paucity of enrichment protocols. However, one significant difference between a viable cell and quiescent cell is the translational activity of ribosomal action, which is subsided with a concomitant decrease in the number of active ribosomes in the quiescent cell. Reduced transcription of rDNA loci can be a consistent marker for quiescence which in T. brucei is tightly regulated by the transcription factor TDP1 that strongly binds to the rDNA locus(Narayanan and Rudenko 2013). To better understand the role of rDNA loci, Jara and colleagues have developed an assay in which they checked for the expression of a reporter GFP gene under the control of the 18S ribosomal DNA locus; the GFP expression acts as a biosensor for quiescence in the laboratory and clinical strains of L. braziliensis and L. mexicana (Jara et al., 2019). The results revealed reduced expression of rGFPcoupled with the transition from promastigotes to amastigotes and this change in expression level was amicable with BrdU uptake which indicates proliferation. These outcomes together with the applicability of this assay in animal-models of latent inhibition, clearly demonstrate the association between the transcription status of the ribosomal RNA genes and particular life stages in trypanosomatids.

Clinical Significance

The concept of metabolic diversity in amastigotes with the coexistence of shallow and deep quiescent stages (Jara et al., 2019), it is now significant that quiescence is crucial for subclinical infections and transmissions with its potential role in drug tolerance (Bhattacharya et al., 2020). This phenomenon serves as reservoirs for transmission and to elicit protective response against subsequent infections in trypanosomatids, which warrants additional exploration (Mandell and Beverley 2017). For instance, about 2.5% to 20% of patients recovered from VL develop PKDL, sufficient to menace the success of the VL elimination program in the sub-continent by re-escalating spur of endemic (Gedda et al., 2020). Therefore, the identification of strategies to combat dormancy or exploit it in developing immunization strategy with some novel assay methods might expedite the success of elimination programs against lower eukaryotic pathogens en masse.

References

Barrett MP, Kyle DE, Sibley LD, Radke JB, Tarleton RL. Protozoan persister-like cells and drug treatment failure. Nat Rev Microbiol. 2019;17(10):607-620. doi:10.1038/s41579-019-0238-x

Bhattacharya A, Corbeil A, do Monte-Neto RL, Fernandez-Prada C. Of Drugs and Trypanosomatids: New Tools and Knowledge to Reduce Bottlenecks in Drug Discovery. Genes (Basel). 2020;11(7):E722. Published 2020 Jun 29. doi:10.3390/genes11070722

Fisher RA, Gollan B, Helaine S. Persistent bacterial infections and persister cells. Nat Rev Microbiol. 2017;15(8):453-464. doi:10.1038/nrmicro.2017.42

Gedda MR, Singh B, Kumar D, et al. Post kala-azar dermal leishmaniasis: A threat to elimination program. PLoSNegl Trop Dis. 2020;14(7):e0008221. Published 2020 Jul 2. doi:10.1371/journal.pntd.0008221

Harms A, Maisonneuve E, Gerdes K. Mechanisms of bacterial persistence during stress and antibiotic exposure. Science. 2016;354(6318):aaf4268. doi:10.1126/science.aaf4268

Helaine S, Kugelberg E. Bacterial persisters: formation, eradication, and experimental systems. Trends Microbiol. 2014;22(7):417-424. doi:10.1016/j.tim.2014.03.00

Jara M, Berg M, Caljon G, et al. Macromolecular biosynthetic parameters and metabolic profile in different life stages of Leishmaniabraziliensis: Amastigotes as a functionally less active stage. PLoS One. 2017;12(7):e0180532. Published 2017 Jul 25. doi:10.1371/journal.pone.0180532

Jara M, Maes I, Imamura H, Domagalska MA, Dujardin JC, Arevalo J. Tracking of quiescence in Leishmania by quantifying the expression of GFP in the ribosomal DNA locus. Sci Rep. 2019;9(1):18951. Published 2019 Dec 12. doi:10.1038/s41598-019-55486-z

Kim JS, Wood TK. Persistent Persister Misperceptions. Front Microbiol. 2016;7:2134. Published 2016 Dec 27. doi:10.3389/fmicb.2016.02134

Kloehn J, Saunders EC, O’Callaghan S, Dagley MJ, McConville MJ. Characterization of metabolically quiescent Leishmania parasites in murine lesions using heavy water labeling. PLoSPathog. 2015;11(2):e1004683. Published 2015 Feb 25. doi:10.1371/journal.ppat.1004683

Le Rutte EA, Zijlstra EE, de Vlas SJ. Post-Kala-Azar Dermal Leishmaniasis as a Reservoir for Visceral Leishmaniasis Transmission. Trends Parasitol. 2019;35(8):590-592. doi:10.1016/j.pt.2019.06.007

Mandal S, Njikan S, Kumar A, Early JV, Parish T. The relevance of persisters in tuberculosis drug discovery. Microbiology. 2019;165(5):492-499. doi:10.1099/mic.0.000760

Mandell MA, Beverley SM. Continual renewal and replication of persistent Leishmania major parasites in concomitantly immune hosts. ProcNatlAcadSci U S A. 2017;114(5):E801-E810. doi:10.1073/pnas.1619265114

Markus MB. Malaria Eradication and the Hidden Parasite Reservoir. Trends Parasitol. 2017;33(7):492-495. doi:10.1016/j.pt.2017.03.002

Michiels JE, Van den Bergh B, Verstraeten N, Michiels J. Molecular mechanisms and clinical implications of bacterial persistence. Drug Resist Updat. 2016;29:76-89. doi:10.1016/j.drup.2016.10.002

Narayanan MS, Rudenko G. TDP1 is an HMG chromatin protein facilitating RNA polymerase I transcription in African trypanosomes. Nucleic Acids Res. 2013;41(5):2981-2992. doi:10.1093/nar/gks1469

Sánchez-Valdéz FJ, Padilla A, Wang W, Orr D, Tarleton RL. Spontaneous dormancy protects Trypanosomacruzi during extended drug exposure. Elife. 2018;7:e34039. Published 2018 Mar 26. doi:10.7554/eLife.34039

Wuyts J, Van Dijck P, Holtappels M. Fungal persister cells: The basis for recalcitrant infections?.PLoSPathog. 2018;14(10):e1007301. Published 2018 Oct 18. doi:10.1371/journal.ppat.1007301