Genetic validation of Leishmania genes essential for amastigote survival in vivo using N-myristoyltransferase as a model

BACKGROUND: Proving that specific genes are essential for the intracellular viability of Leishmania parasites within macrophages remains a challenge for the identification of suitable targets for drug development. This is especially evident in the absence of a robust inducible expression system or functioning RNAi machinery that works in all Leishmania species. Currently, if a target gene of interest in extracellular parasites can only be deleted from its genomic locus in the presence of ectopic expression from a wild type copy, it is assumed that this gene will also be essential for viability in disease-promoting intracellular parasites. However, functional essentiality must be proven independently in both life-cycle stages for robust validation of the gene of interest as a putative target for chemical intervention. METHODS: Here, we have used plasmid shuffle methods in vivo to provide supportive genetic evidence that N-myristoyltransferase (NMT) is essential for Leishmania viability throughout the parasite life-cycle. Following confirmation of NMT essentiality in vector-transmitted promastigotes, a range of mutant parasites were used to infect mice prior to negative selection pressure to test the hypothesis that NMT is also essential for parasite viability in an established infection. RESULTS: Ectopically-expressed NMT was only dispensable under negative selection in the presence of another copy. Total parasite burdens in animals subjected to negative selection were comparable to control groups only if an additional NMT copy, not affected by the negative selection, was expressed. CONCLUSIONS: NMT is an essential gene in all parasite life-cycle stages, confirming its role as a genetically-validated target for drug development

Interferon-γ-Producing CD4+ T Cells Drive Monocyte Activation in the Bone Marrow During Experimental Leishmania donovani Infection

Ly6Chi inflammatory monocytes develop in the bone marrow and migrate to the site of infection during inflammation. Upon recruitment, Ly6Chi monocytes can differentiate into dendritic cells or macrophages. According to the tissue environment they can also acquire different functions. Several studies have described pre-activation of Ly6Chi monocytes in the bone marrow during parasitic infection, but whether this process occurs during experimental visceral leishmaniasis and, if so, the mechanisms contributing to their activation are yet to be established. In wild type C57BL/6 (B6) mice infected with Leishmania donovani, the number of bone marrow Ly6Chi monocytes increased over time. Ly6Chi monocytes displayed a highly activated phenotype from 28 days to 5 months post infection (p.i), with >90% expressing MHCII and >20% expressing iNOS. In comparison, in B6.Rag2 -/- mice <10% of bone marrow monocytes were MHCII+ at day 28 p.i., an activation deficiency that was reversed by adoptive transfer of CD4+ T cells. Depletion of CD4+ T cells in B6 mice and the use of mixed bone marrow chimeras further indicated that monocyte activation was driven by IFNγ produced by CD4+ T cells. In B6.Il10 -/- mice, L. donovani infection induced a faster but transient activation of bone marrow monocytes, which correlated with the magnitude of CD4+ T cell production of IFNγ and resolution of the infection. Under all of the above conditions, monocyte activation was associated with greater control of parasite load in the bone marrow. Through reinfection studies in B6.Il10 -/- mice and drug (AmBisome®) treatment of B6 mice, we also show the dependence of monocyte activation on parasite load. In summary, these data demonstrate that during L. donovani infection, Ly6Chi monocytes are primed in the bone marrow in a process driven by CD4+ T cells and whereby IFNγ promotes and IL-10 limits monocyte activation and that the presence of parasites/parasite antigen plays a crucial role in maintaining bone marrow monocyte activation

Leishmania HASP and SHERP Genes are Required for In Vivo Differentiation, Parasite Transmission and Virulence Attenuation in the Host

Differentiation of extracellular Leishmania promastigotes within their sand fly vector, termed metacyclogenesis, is considered to be essential for parasites to regain mammalian host infectivity. Metacyclogenesis is accompanied by changes in the local parasite environment, including secretion of complex glycoconjugates within the promastigote secretory gel and colonization and degradation of the sand fly stomodeal valve. Deletion of the stage-regulated HASP and SHERP genes on chromosome 23 of Leishmania major is known to stall metacyclogenesis in the sand fly but not in in vitro culture. Here, parasite mutants deficient in specific genes within the HASP/SHERP chromosomal region have been used to investigate their role in metacyclogenesis, parasite transmission and establishment of infection. Metacyclogenesis was stalled in HASP/SHERP mutants in vivo and, although still capable of osmotaxis, these mutants failed to secrete promastigote secretory gel, correlating with a lack of parasite accumulation in the thoracic midgut and failure to colonise the stomodeal valve. These defects prevented parasite transmission to a new mammalian host. Sand fly midgut homogenates modulated parasite behaviour in vitro, suggesting a role for molecular interactions between parasite and vector in Leishmania development within the sand fly. For the first time, stage-regulated expression of the small HASPA proteins in Leishmania (Leishmania) has been demonstrated: HASPA2 is expressed only in extracellular promastigotes and HASPA1 only in intracellular amastigotes. Despite its lack of expression in amastigotes, replacement of HASPA2 into the null locus background delays onset of pathology in BALB/c mice. This HASPA2-dependent effect is reversed by HASPA1 gene addition, suggesting that the HASPAs may have a role in host immunomodulation

TNF signalling drives expansion of bone marrow CD4+ T cells responsible for HSC exhaustion in experimental visceral leishmaniasis

Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150+ CD34- CD48- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4+ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4+ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ+CD4+ T cells that promote the irreversible loss of BM function. These finding provide new insights into the pathogenic potential of CD4+ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously

Spatial Point Pattern Analysis Identifies Mechanisms Shaping the Skin Parasite Landscape in Leishmania donovani Infection

Increasing evidence suggests that in hosts infected with parasites of the Leishmania donovani complex, transmission of infection to the sand fly vector is linked to parasite repositories in the host skin. However, a detailed understanding of the dispersal (the mechanism of spread) and dispersion (the observed state of spread) of these obligatory-intracellular parasites and their host phagocytes in the skin is lacking. Using endogenously fluorescent parasites as a proxy, we apply image analysis combined with spatial point pattern models borrowed from ecology to characterize dispersion of parasitized myeloid cells (including ManR+ and CD11c+ cells) and predict dispersal mechanisms in a previously described immunodeficient model of L. donovani infection. Our results suggest that after initial seeding of infection in the skin, heavily parasite-infected myeloid cells are found in patches that resemble innate granulomas. Spread of parasites from these initial patches subsequently occurs through infection of recruited myeloid cells, ultimately leading to self-propagating networks of patch clusters. This combination of imaging and ecological pattern analysis to identify mechanisms driving the skin parasite landscape offers new perspectives on myeloid cell behavior following parasitism by L. donovani and may also be applicable to elucidating the behavior of other intracellular tissue-resident pathogens and their host cells

CD4⁺ T cells drive HSC exhaustion in an IFNγ-dependent manner.

<p>(A) Diagram of experimental layout, RAG2 KO mice were adoptively transferred with sorted CD4⁺ T cells from naive mice, and then infected in the following day with <i>L</i>. <i>donovani</i>. At day 28 p.i., we analysed the distribution of hematopoietic progenitors in the BM in naïve RAG mice, infected RAG2 KO mice and infected RAG mice that receive adoptively transferred CD4⁺ T cells: (B-E) Number of HSPCs: LT-HSCs (B), LSK CD150⁺CD34^-CD48⁺ cells (C), LSK CD150⁺CD34⁺ cells (D) and quiescent LT-HSCs LSK in BM (E) (n = 12–17 per group, from three independent experiments). (F) Frequency of HSPCs populations within Lineage negative cells in naïve RAG mice with and without adoptive CD4⁺ T cell transfer; and (F) number of quiescent LT-HSCs (n = 9–5 per group). (H-J) Frequency of progenitor cells within Lineage negative cells in infected RAG mice without or with adoptive transfer of IFNγ sufficient or IFNγ-deficient CD4⁺ T cells (G); Number of quiescent LT-HSCs (n = 4–5) (H). (J) Parasites per 1000 nuclei in the spleen. Data presented as presented as scatter plot and mean bar; *p ≤ 0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001; one-way Anova and Tukey’s multiple comparisons test.</p

Intrinsic IFNγ receptor signaling is required for expansion of BM T cells following infection.

<p>(A) Experimental design for competitive mixed BM chimeras using wild-type (WT) and <i>Ifnγr2</i> knockout (IFNγR2 KO). Analyses were performed 12 weeks after BMT from CD45.2 <i>Ifnr2</i>^-/- mice and CD45.1 WT mice (50:50) to lethally irradiated CD45.1 recipient mice, subsequently infected with <i>L</i>. <i>donovani</i> for 28 days. (B) Frequency of donor cells in BM. (C) Frequency of BM LSK CD150⁺ CD48^- cells (enriched for LT-HSCs) and LSK CD150⁺ CD48⁺ cells within donor cells. (D) Frequency of LT-HSCs in G0 (Ki67^-) within donor cells. (E) Frequency of donor cells in spleen. (F) Frequency of T cells within donor cells in spleen. (G) Number of donor T cells in spleen. (H) Frequency of BM T cells within donor cells. (I) Number of donor T cells in the BM. Data presented as scatter plot and mean bar (n = 4–8); *p ≤ 0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001; One-way Anova followed by Tukey’s multiple comparisons test.</p

<i>L</i>. <i>donovani</i> infection expands the population of BM T cells expressing IFNγ and TNF.

<p>Comparison of BM T cells in naïve and d28-infected mice (Ld28). (A) Number of T cells in BM. (B) Frequency of total CD44^high and CD44^high subsets within total BM CD4⁺ T cell population. (C) Frequency of IFNγ⁺ subsets in total BM cells. (D) Frequency of TNF⁺ subsets in total BM cells. (E) Frequency of IFNγ⁺ within BM CD4⁺ T cell population following stimulation <i>in vitro</i>. (F) Frequency of IFNγ⁺ within BM CD4⁺ T cell population directly <i>ex vivo</i>. (G) Frequency of TNF⁺ within BM CD4⁺ T cell population following stimulation <i>in vitro</i>. (H) Frequency of TNF⁺ within BM CD4⁺ T cell population directly ex vivo. (I) Mean Fluorescence Intensity (MFI) of IFNγ in IFNγ⁺CD4⁺ T cells. (J) MFI of TNF in TNF⁺CD4⁺ T cells. Data from at least two independent experiments (n = 6–14 per group) presented as scatter plot and mean bar; *p ≤ 0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001; unpaired t test. (K) Representative dot plots for IFNγ expression by stimulated BM CD4⁺ T cells (top) or <i>ex vivo</i> (bottom).</p

Loss of quiescent LT-HSCs following <i>L</i>. <i>donovani</i> infection.

<p>(A and B) Frequency (A) and number (B) of Ki67⁺ within HSPCs populations in BM of naïve and d28-infected B6 mice. (C) Number of LT-HSCs in G0 (Ki67^-) in BM in naïve and d28-infected mice (n = 12 per group; three independent experiments). Data presented as scatter plot and mean bar; unpaired t test; *p ≤ 0.05, **p ≤0.01 ****p ≤0.0001. (D) Representative dot plots for Ki67 expression on LSK CD150⁺CD34^-CD48^- cells.</p

Quiescent LT-HSCs are retained in infected <i>Rag2</i>^-/- mice.

<p>(A) Number of HSPCs in BM of naïve B6.WT (light squares), naïve B6.<i>Rag2</i>^-/- (dark grey triangle), d28 infected B6 (dark grey squares) and d28 <i>Rag2</i>^-/- (light grey circles) mice. (B) Number of BM LT-HSCs in G0 (Ki67^-). (C) Spleen parasite burden in B6.WT and B6 <i>Rag2</i>^-/-, presented as number of parasites per 1000 nuclei. Data from three independent experiments presented as scatter plot and mean bar (n = 9–16 per group); *p ≤ 0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001; unpaired t test.</p