Gene deletion as a possible strategy adopted by New World Leishmania infantum to maximize geographic dispersion

  1. Monique Florêncio
  2. Marne Coimbra Chagas
  3. Anderson Guimarães-Costa
  4. Jullyanna Oliveira
  5. Ingrid Waclawiak
  6. Thamara K. F. Oliveira
  7. Elvira Maria Saraiva
  8. Anita Leocadio Freitas-Mesquita
  9. José Roberto Meyer-Fernandes
  10. Laura Aragão-Farias
  11. Camilly Enes Trindade
  12. Patricia Cuervo Escobar
  13. Renata Azevedo do Nascimento
  14. Otacilio C. Moreira
  15. Flávia Lima Ribeiro-Gomes
  16. Yara M. Traub-Csekö
  17. Erich Loza Telleria
  18. Slavica Vaselek
  19. Tereza Leštinová
  20. Petr Volf
  21. Gerald F. Späth
  22. Elisa Cupolillo
  23. Mariana C. Boité

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Abstract

Background

The present study investigates implications of a sub-chromosomal deletion in Leishmania infantum strains, the causative agent of American Visceral Leishmaniasis (AVL). Primarily found in New World strains, the deletion leads to the absence of the ecto-3’-nucleotidase/nuclease enzyme (3’NU/NT), impacting parasite virulence, pathogenicity, and drug susceptibility. The potential factors favoring prevalence and the widespread geographic distribution of these deleted mutant parasites (DEL) in the New World (NW) are discussed under the generated data.

Methods

We conducted phenotypic analyses of the parasites showing the sub- chromosomal deletion by applying in vitro assays of 3’NU/NT activity, metacyclic enrichment, and relative quantitation of transcripts abundance on axenic parasites. We further performed experimental infections in both in vitro and in vivo models of vertebrate and invertebrate hosts using geographically diverse mutant field isolates.

Results

Virulence assays, poorer ability to survive neutrophil traps (NETs) and murine model infection revealed reduced pathogenicity in vertebrate hosts by the DEL strains. Conversely, these parasites exhibit enhanced metacyclogenesis and colonization rates in sand flies, potentially facilitating transmission. This combination may represent a more efficient way to maintain and disperse the transmission cycle of DEL strains.

Conclusions

Phenotypic assessments reveal altered parasite fitness, with enhanced transmissibility at the population level. Reduced susceptibility of DEL strains to miltefosine, a key drug in VL treatment, further complicates control efforts. Our study underscores the importance of typing parasite genomes for surveillance and control and proposes the sub-chromosomal deletion as a molecular marker in AVL management.

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    Referee #3

    Evidence, reproducibility and clarity

    In this study, the authors investigate potential developmental changes in Leishmania infantum isolates from different regions of Brazil (plus a single isolate from Portugal) both in vitro and in vivo. The manuscript present interesting phenotypic comparisons between isolates with a 12-kb deletion of a subtelomeric region on Chr31 and isolates with a complete sequence. The authors suggest that distinctions associated with the presence or absence of that region may potentially be linked with fitness in the wild. A more detailed analysis of such valuable sample cohort would increase the significance of this research. Therefore, I highlight a few important concerns that need to be addressed, as well as a disconnect between the presented data and conclusions made.

    Major issues

    • The % of metacyclics in all the sand fly infection experiments was extremely low ( <6%). The only exception was NonDEL_PI_2972, which showed around 16% metacyclics. This tells me that the parasites did not develop properly inside any of the 3 vectors used. Does an increase from 2% to 4% make any difference in vector competence? If metacyclogenesis is not significantly higher in DEL isolates, then there will most likely not be an increase in fitness concerning transmission. Also, I assume that both the stomodeal valve infection and the metacyclic % were assessed at 192h p.i., but this must be clearly stated in the text. At what time-point were parasites per gut counted at Charles Univ.? The parameters used in all the different sand fly experiments should be clearly labeled.
    • If the metacyclic % data are available for the Fiocruz colony experiments, then it will be important to present those. Conversely, are the #s of parasites/gut available using another time point at Charles Univ.?
    • It is mentioned that at least 3 independent sand fly infections were performed, so the data points should be shown for each of the replicates where applicable. A bar plot without error bars is not ideal for representing the stomodeal valve and metacyclic % data either. Based on the current plots, it is difficult to infer biological significance.
    • The entire presentation of the sand fly infection experiments is confusing and does not provide consistent findings to support the conclusions, which should be toned down. The whole rationale for the different vector species should be better explained.
    • The 3'NT activity in two HTZ isolates is as high as in some NonDEL, while in one HTZ strain it is as low as the DEL isolates. Is there any genomic difference between the HTZ to justify this difference? Since 3254 HTZ was not included in the qPCR analysis presented in Schwabl, et al. 2021, it is difficult to see any correlation.
    • Pg. 16 ln.398: The authors conclude that DEL isolates cause effective, yet less pathogenic infection compared to NonDEL isolates. However, it is not possible to reach that conclusion based on the early-infection data presented. For that, i.v.-infected mice would have to be monitored for several weeks and assessed for visceralization and VL severity.
    • The most relevant data in this work suggest that metacyclics in nonDEL vs DEL might present important differences. The META2 gene expression, the different M0 infection% rather than different # of intracellular amastigotes, and increases in parasite #s in the draining lymph node at 13-16 hours after ear inoculation suggest to me that the metacyclics could have different infection competence. That possibility should be addressed. Do they display similar morphology? Do they differentiate at similar rates to axenic amastigotes? SHERP transcript levels should be quantified in metacyclics from the different isolates.

    Minor issues

    • Gene IDs of the transcripts measured should be listed somewhere.
    • Fig.1C, Fig2A&B: missing p-value labels.
    • Fig.1B needs more detail on the statistical test used. Is it One-way ANOVA? What post-hoc test was used?
    • Figs. 2 & 3: "Mann Whitney Rank Sum", "Mann Whitney t-test". The name of the statistical test is either Mann Whitney U test or Wilcoxon rank-sum test.
    • Figs. 4 & 5: information missing on statistical tests used in both.
    • Pg.8 ln.212: The NT1 median FC in the NonDEL group does not seem to be 0.8 in Fig.2B.

    Typos:

    • Fig.2 title: "promastigotes harvest from" -> harvested
    • Fig2. legend: "Paraflagellar". Is this a paraflagellar rod protein (PFR)? If so, it should be specified throughout the text.
    • Fig 2 & 3: "ns = no significant" -> not significant
    • Different parts of the text: "alfa-tubulin" -> alpha-tubulin
    • Fig.4A: Y-axis reads "Inspected sand flies"

    Significance

    Despite constituting the same species, significant genetic differences exist in Leishmania infantum variants found in the American continent compared to those in Europe, Africa and Asia. Phenotypic comparison studies such as reported here are relevant to the Parasitology field and may lead to new insights on the pathogenesis of specific clinical outcomes of leishmaniasis disease. The authors attempt to associate a major genetic deletion found in specific neotropical Leishmania infantum strains, isolated in Brazil, with phenotypic changes that could potentially lead to fitness increase during cyclical transmission in the wild.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript the authors have investigated the role of sub chromosomal deletion found in new world Leishmania infantum species. The deletion (12 kb) spans over across the four copies of tetrasomic chromosome 31 which includes the loss of four open reading frames (ORF) LinJ.31.2370 (ecto-3ʹ-nucleotidase/nuclease), LinJ.31.2380 (ecto-3'-nucleotidase precursor), LinJ.31.2390 (helicase-like protein) and LinJ.31.2400 (3,2-trans-enoyl-CoA isomerase). The ecto-3'-nucleotidase/nuclease (3'NU/NT) activity has been shown to have an important role in trypanosomatids in the purine salvage pathway implicated as virulence factor affecting the parasite's ability to infect macrophages. The authors showed that having such deletions enhances the metacyclogenesis in vitro but are highly susceptible to killing by neutrophils and macrophages. In addition, they are also less virulent in vivo. The authors claim that enhanced metacyclogenesis increases its transmissibility in the invertebrate host but may not be highly infectious in the vertebrate host. They speculate that that such parasites may provide immune response that may control the infection in endemic population by having large group of asymptomatic individuals. These outcomes are highly speculative. This study is thought provoking but needs to be studied thoroughly.

    Following are the comments:

    1. Leishmania 3"NU/NT plays an important role in virulence of the parasite and its survival,

    a. how do 3'-NU/NT DEL parasites survive in the host which lack all the 4 copies of NT?

    b. what is the advantage of having such parasites circulating in the endemic areas?

    1. Since metacyclics are important for the pathogenesis of the parasites,

    a. What is the mechanism of increased metacyclogenesis of L. Infantum 3'-NU/NT DEL parasites in the absence of 3'-NU/NT activity which is essential for the virulence?

    1. How does lack of 3'NU/NT enhance transmissibility since such metacyclics from 3'-NU/NT DEL parasites barely survive in vivo?

    a. the parasites are less resistant to NET and are killed easily.

    b. there is reduced recruitment of neutrophils (NT) and monocytes in the ear.

    1. Fig.7C: what is the reason for higher parasite load of DEL in dLN?
    2. Do you think there is reduced recruitment of NT in the infected site which would have controlled the parasites, hence they migrate quickly in the dLN? To test this possibility the authors should perform an in vitro NT recruitment assay.
    3. Line 417: How are the 3'-NU/NT DEL parasites continuous source for infection in sand flies, If such parasites are not infectious and will be cleared by the host?
    4. Line 400: Is it possible that having 3'-NU/NT DEL parasites in circulation dampens the infectivity of the NON-DEL and thus over all infection rate in the population goes down?
    5. Could the 3'-NU/NT DEL parasites be the source of asymptomatic infections?
    6. Is there literature evidence for such a possibility in the endemic region?

    Significance

    The authors claim that enhanced metacyclogenesis increases transmissibility of Leishmania in the invertebrate host but may not be highly infectious in the vertebrate host. They speculate that that such parasites may provide immune response that may control the infection in endemic population by having large group of asymptomatic individuals. These outcomes are highly speculative. This study is thought provoking but needs to be studied thoroughly.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Florencio et al., presented a detailed analysis of Brazilian L. infantum isolates with about half with a 4 gene deletion on chromosome 31 and the other half without that deletion. This deletion was first observed in the context of miltefosine responsiveness by the Mottram group. Here a multinational team is concluding that this deletion is associated with increased metacyclogenesis, increase parasite load in some sandfly vector species factors that contribute to their prevalence in South American. Yet the deleted strains show decreased survival invitro including in macrophages, but from what I understand increased load in an animal model, possibly in line with their reduced efficacy in attracting/activating key immune cells.

    The authors provide an interesting hypothesis on the equilibrium between pathogenesis (virulence) and capacity to transmit. The perfect parasite would like to infect as many hosts as possible while not killing them. I will try to provide the pro and cons.

    1. Obviously (and as acknowledged on p. 344-346) the conclusive experiment would be to add the 4 gene locus (the authors believe that it is the 3'NU/NT gene, so a single gene would even be simpler) in a DEL strain or alternatively KO the locus in a nonDEL strain. This would prove that the phenotype observed is indeed due to the loss of this locus. I understand that this is additional work but it would provide a definite answer. Now we have to rely on associations but alas the associations are not as tight as one would wish.
    2. Not sure if it is a dichotomy but they show decreased survival in the presence of NET (Fig. 6A) in vitro but reducing the attraction of immune cells including neutrophils in vivo (Fig. 7). For the nonDEL strain they observe no effect of NET in vitro (Fig. 6) while they seem to attract more immune cells (Fig. 7) in vivo. The in vitro data seems to reach significance for the three group of strains but not for the in vivo work (one out of three).
    3. The authors can be lauded for adding more pair of strains but this also adds to the complexity and frankly raises questions. The two strains derived from the PI and MT location differs in many aspects compared to the MS-Rj comparators.
    4. It is remarkable that the authors have tested other sandfly vector species (Fig. 5) but what is the conclusion and does it help in our understanding? I see variations in the figure between strains and vectors but not sure what it means.

    Secondary points

    1. Please confirm that the data in Fig. 1D and 1E are all the DEL and nonDEL strains. The number of dots does not match with the number of strains tested.
    2. Do we really need Fig. 2 and 3? Not part of the main message.
    3. Why is Fig. 4A only parasite count and 4B,C parasite count (please specify the number of hours) and additional information about stomodeal valve and metacyclics?
    4. Please explain sentence on p. 374-375. I thought that DEL is associated with increased metacyclics and increase parasite count in the sandfly
    5. Just for curiosity, have the authors try to rescue the phenotype in vitro by adding purines in the medium with the DEL strains?

    Significance

    Florencio et al., presented a detailed analysis of Brazilian L. infantum isolates with about half with a 4 gene deletion on chromosome 31 and the other half without that deletion. This deletion was first observed in the context of miltefosine responsiveness by the Mottram group. Here a multinational team is concluding that this deletion is associated with increased metacyclogenesis, increase parasite load in some sandfly vector species factors that contribute to their prevalence in South American. Yet the deleted strains show decreased survival invitro including in macrophages, but from what I understand increased load in an animal model, possibly in line with their reduced efficacy in attracting/activating key immune cells.

    The authors provide an interesting hypothesis on the equilibrium between pathogenesis (virulence) and capacity to transmit. The perfect parasite would like to infect as many hosts as possible while not killing them.

  5. Version published to 10.1101/2024.05.22.595165v1 on bioRxiv