The mating competence of geographically diverse Leishmania major strains in their natural and unnatural sand fly vectors

Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4-6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed '2n' DNA contents, but with a significant number of '3n' and one '4n' offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable 'mating types' that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating

TLR2 signaling in skin non-hematopoietic cells induces early neutrophil recruitment in response to Leishmania major infection

Neutrophils are rapidly recruited to the mammalian skin in response to infection with the cutaneous Leishmania pathogen. The parasites use neutrophils to establish the disease, however, the signals driving early neutrophil recruitment are poorly known. Here, we identified the functional importance of TLR2 signaling in this process. Using bone-marrow chimeras and immunohistology we identified the TLR2-expressing cells involved in this early neutrophil recruitment to be of non-hematopoietic origin. Keratinocytes are damaged and briefly in contact with the parasites during infection. We show that TLR2 triggering by L. major is required for their secretion of neutrophil-attracting chemokines. Furthermore, TLR2 triggering by L. major phosphoglycans is critical for neutrophil recruitment impacting negatively on disease development, as shown by better control of lesion size and parasite load in Tlr2-/- compared to wild type infected mice. Conversely, restoring early neutrophil presence in Tlr2-/- mice through injection of wild type neutrophils or CXCL1 at the onset of infection resulted in delayed disease resolution comparable to that observed in wild type mice. Taken together, our data demonstrate a new role for TLR2-expressing non-hematopoietic skin cells in the recruitment of the first wave of neutrophils following L. major infection, a process delaying disease control

Induction of mitochondrial recycling reverts age-associated decline of the hematopoietic and immune systems

Aging compromises hematopoietic and immune system functions, making older adults especially susceptible to hematopoietic failure, infections and tumor development, and thus representing an important medical target for a broad range of diseases. During aging, hematopoietic stem cells (HSCs) lose their blood reconstitution capability and commit preferentially toward the myeloid lineage (myeloid bias) <sup>1,2</sup> . These processes are accompanied by an aberrant accumulation of mitochondria in HSCs <sup>3</sup> . The administration of the mitochondrial modulator urolithin A corrects mitochondrial function in HSCs and completely restores the blood reconstitution capability of 'old' HSCs. Moreover, urolithin A-supplemented food restores lymphoid compartments, boosts HSC function and improves the immune response against viral infection in old mice. Altogether our results demonstrate that boosting mitochondrial recycling reverts the aging phenotype in the hematopoietic and immune systems

Proportion of promastigote developmental stages at the time of hybrid recovery in <i>P. duboscqi</i>.

<p>Midguts were dissected on the indicated times post-feeding and scored for the proportion of each developmental stage. Fn/Sat×Lv39c5 and Fn/Sat×Sd/Hyg hybrids are designated as FL and FS, respectively, followed by the day of hybrid recovery.</p

Summary of Ry/Sat×LV39c5/Hyg hybrid genotypes generated in <i>P. duboscqi</i>.

<p>H, hybrid.</p

Infection levels at time of dissection in <i>P. duboscqi</i> midguts with and without hybrids.

<p>Midguts were dissected on the indicated times post-feeding and scored for the number of viable promastigotes. Bars represent the median numbers of promastigotes per midgut.</p

Summary of Fn/Sat×Lv39c5/Hyg crosses in <i>Lu. Longipalpis</i>.

<p>Summary of Fn/Sat×Lv39c5/Hyg crosses in <i>Lu. Longipalpis</i>.</p

Summary of hybrid genotypes generated in <i>Lu. Longipalpis</i>.

<p>H, hybrid; ND, not determined.</p