Exploring the genetic basis of natural resistance to microcins.

Enterobacteriaceae produce an arsenal of antimicrobial compounds including microcins, ribosomally produced antimicrobial peptides showing diverse structures and mechanisms of action. Microcins target close relatives of the producing strain to promote its survival. Their narrow spectrum of antibacterial activity makes them a promising alternative to conventional antibiotics, as it should decrease the probability of resistance dissemination and collateral damage to the host's microbiota. To assess the therapeutic potential of microcins, there is a need to understand the mechanisms of resistance to these molecules. In this study, we performed genomic analyses of the resistance to four microcins [microcin C, a nucleotide peptide; microcin J25, a lasso peptide; microcin B17, a linear azol(in)e-containing peptide; and microcin E492, a siderophore peptide] on a collection of 54 Enterobacteriaceae from three species: Escherichia coli, Salmonella enterica and Klebsiella pneumoniae. A gene-targeted analysis revealed that about half of the microcin-resistant strains presented mutations of genes involved in the microcin mechanism of action, especially those involved in their uptake (fhuA, fepA, cirA and ompF). A genome-wide association study did not reveal any significant correlations, yet relevant genetic elements were associated with microcin resistance. These were involved in stress responses, biofilm formation, transport systems and acquisition of immunity genes. Additionally, microcin-resistant strains exhibited several mutations within genes involved in specific metabolic pathways, especially for S. enterica and K. pneumoniae.

MRPA-independent mechanisms of antimony resistance in Leishmania infantum.

Control of both human and canine leishmaniasis is based on a very short list of chemotherapeutic agents, headed by antimonial derivatives (Sb). The utility of these molecules is severely threatened by high rates of drug resistance. The ABC transporter MRPA is one of the few key Sb resistance proteins described to date, whose role in detoxification has been thoroughly studied in Leishmania parasites. Nonetheless, its rapid amplification during drug selection complicates the discovery of other mechanisms potentially involved in Sb resistance. In this study, stepwise drug-resistance selection and next-generation sequencing were combined in the search for novel Sb-resistance mechanisms deployed by parasites when MRPA is abolished by targeted gene disruption. The gene mrpA is not essential in L. infantum, and its disruption leads to an Sb hypersensitive phenotype in both promastigotes and amastigotes. Five independent mrpA-/- mutants were selected for antimony resistance. These mutants displayed major changes in their ploidy, as well as extrachromosomal linear amplifications of the subtelomeric region of chromosome 23, which includes the genes coding for ABCC1 and ABCC2. Overexpression of ABCC2, but not of ABCC1, resulted in increased Sb tolerance in the mrpA-/- mutant. SNP analyses revealed three different heterozygous mutations in the gene coding for a serine acetyltransferase (SAT) involved in de novo cysteine synthesis in Leishmania. Overexpression of satQ390K, satG321R and satG325R variants led to a 2-3.2 -fold increase in Sb resistance in mrpA-/- parasites. Only satG321R and satG325R induced increased Sb resistance in wild-type parasites. These results reinforce and expand knowledge on the complex nature of Sb resistance in Leishmania parasites.

Genomewide Analysis of Mode of Action of the S-Adenosylmethionine Analogue Sinefungin in Leishmania infantum.

To further our understanding of one-carbon metabolism in the protozoan parasite Leishmania, we conducted genomic screens to study how the parasite responded to sinefungin (SNF) selection. SNF is a structural analogue of S-adenosylmethionine (AdoMet), a key methyl group donor to a number of biomolecules. One screen consisted of sequencing SNF-resistant mutants generated by stepwise selection with gradually increasing drug concentrations. These studies demonstrated deletion of the AdoMet transporter (AdoMetT1) by intergenic recombination as a crucial loss-of-function marker for SNF resistance. The second screen consisted of Cos-seq, a gain-of-function cosmid-based genomewide functional screen with increasing SNF concentration coupled to next-generation sequencing. Cosmids enriched in that screen and sequenced led to the identification of (i) the AdoMet synthetase (METK) as the major SNF target, (ii) an mRNA [(guanine-N7)-methyltransferase (CMT1)], (iii) a leucine carboxyl methyltransferase (LCMT), (iv) two tryparedoxin genes, and (v) two protein phosphatase regulatory genes. Further functional exploration indicated that LCMT interacts with one phosphatase catalytic subunit (PP2AC) and that mutation of the C-terminal leucine residue of PP2AC affects sinefungin susceptibility. These holistic screens led to the identification of transporters, biosynthetic genes, RNA and protein methyltransferases, as well as phosphatases linked to AdoMet-mediated functions in Leishmania IMPORTANCE The two main cellular metabolic one-carbon donors are reduced folates and S-adenosylmethionine, whose biosynthetic pathways have proven highly effective in chemotherapeutic interventions in various cell types. Sinefungin, a nucleoside analogue of S-adenosylmethionine, was shown to have potent activity against the protozoan parasite Leishmania Here, we studied resistance to sinefungin using whole-genome approaches as a way to further our understanding of the role of S-adenosylmethionine in this parasite and to reveal novel potential drug targets. These approaches allowed the characterization of novel features related to S-adenosylmethionine function in Leishmania which could further help in the development of sinefungin-like compounds against this pathogenic parasite.

High-throughput Cos-Seq screen with intracellular Leishmania infantum for the discovery of novel drug-resistance mechanisms.

Increasing drug resistance towards first line antimony-derived compounds has forced the introduction of novel therapies in leishmaniasis endemic areas including amphotericin B and miltefosine. However, their use is threatened by the emergence and spread of drug-resistant strains. In order to discover stage-dependent resistance genes, we have adapted the Cos-Seq approach through the introduction of macrophage infections in the pipeline. A L. infantum intracellular amastigote population complemented with a L. infantum cosmid library was submitted to increasing concentrations of miltefosine, amphotericin B and pentavalent antimonials in experimental infections of THP-1 cells. For each step of selection, amastigotes were extracted and cosmids were isolated and submitted to next-generation sequencing, followed by subsequent gene-enrichment analyses. Cos-Seq screen in amastigotes revealed four highly enriched loci for antimony, five for miltefosine and one for amphotericin B. Of these, a total of seven cosmids were recovered and tested for resistance in both promastigotes and amastigotes. Candidate genes within the pinpointed genomic regions were validated using single gene overexpression in wild-type parasites and/or gene disruption by means of a CRISPR-Cas9-based approach. This led to the identification and validation of a stage-independent antimony-resistance gene (LinJ.06.1010) coding for a putative leucine rich repeat protein and a novel amastigote-specific miltefosine-resistance gene (LinJ.32.0050) coding for a member of the SEC13 family of WD-repeat proteins. This study further reinforces the power of Cos-Seq approach to discover novel drug-resistance genes, some of which are life-stages specific.

Different Mutations in a P-type ATPase Transporter in Leishmania Parasites are Associated with Cross-resistance to Two Leading Drugs by Distinct Mechanisms.

Leishmania infantum is an etiological agent of the life-threatening visceral form of leishmaniasis. Liposomal amphotericin B (AmB) followed by a short administration of miltefosine (MF) is a drug combination effective for treating visceral leishmaniasis in endemic regions of India. Resistance to MF can be due to point mutations in the miltefosine transporter (MT). Here we show that mutations in MT are also observed in Leishmania AmB-resistant mutants. The MF-induced MT mutations, but not the AmB induced mutations in MT, alter the translocation/uptake of MF. Moreover, mutations in the MT selected by AmB or MF have a major impact on lipid species that is linked to cross-resistance between both drugs. These alterations include changes of specific phospholipids, some of which are enriched with cyclopropanated fatty acids, as well as an increase in inositolphosphoceramide species. Collectively these results provide evidence of the risk of cross-resistance emergence derived from current AmB-MF sequential or co-treatments for visceral leishmaniasis.

Engagement of the Aryl Hydrocarbon Receptor in Mycobacterium tuberculosis-Infected Macrophages Has Pleiotropic Effects on Innate Immune Signaling.

Understanding the mechanisms of host macrophage responses to Mycobacterium tuberculosis is essential for uncovering potential avenues of intervention to boost host resistance to infection. Macrophage transcriptome profiling revealed that M. tuberculosis infection strongly induced the expression of several enzymes controlling tryptophan catabolism. These included IDO1 and tryptophan 2,3-dioxygenase, which catalyze the rate-limiting step in the kynurenine pathway, producing ligands for the aryl hydrocarbon receptor (AHR). The AHR and heterodimeric partners AHR nuclear translocator and RELB are robustly expressed, and AHR and RELB levels increased further during infection. Infection enhanced AHR/AHR nuclear translocator and AHR/RELB DNA binding and stimulated the expression of AHR target genes, including that encoding the inflammatory cytokine IL-1ß. AHR target gene expression was further enhanced by exogenous kynurenine, and exogenous tryptophan, kynurenine, or synthetic agonist indirubin reduced mycobacterial viability. Comparative expression profiling revealed that AHR ablation diminished the expression of numerous genes implicated in innate immune responses, including several cytokines. Notably, AHR depletion reduced the expression of IL23A and IL12B transcripts, which encode subunits of IL-23, a macrophage cytokine that stimulates production of IL-22 by innate lymphoid cells. AHR directly induced IL23A transcription in human and mouse macrophages through near-upstream enhancer regions. Taken together, these findings show that AHR signaling is strongly engaged in M. tuberculosis-infected macrophages and has widespread effects on innate immune responses. Moreover, they reveal a cascade of AHR-driven innate immune signaling, because IL-1ß and IL-23 stimulate T cell subsets producing IL-22, another direct target of AHR transactivation.

Next Generation Vaccine Biomarkers workshop October 30-31, 2014--Ottawa, Canada.

Vaccine biomarkers are critical to many aspects of vaccine development and licensure, including bridging findings in pre-clinical studies to clinical studies, predicting potential adverse events, and predicting vaccine efficacy. Despite advances in our understanding of various biological pathways, and advances in systems analyses of the immune response, there remains much to learn about qualitative and quantitative aspects of the human host response to vaccination. To stimulate discussion and identify opportunities for collaborative ways to advance the field of vaccine biomarkers, A Next Generation Vaccine Biomarker workshop was held in Ottawa. The two day workshop, sponsored by the National Research Council Canada, Canadian Institutes of Health Research, Public Health Agency of Canada, Pfizer, and Medicago, brought together stakeholders from Canadian and international industry, government and academia. The workshop was grouped in themes, covering vaccine biomarker challenges in the pre-clinical and clinical spaces, veterinary vaccines, regulatory challenges, and development of biomarkers for adjuvants and cancer vaccines. The use of case studies allowed participants to identify the needs and gaps requiring innovation. The workshop concluded with a discussion on opportunities for vaccine biomarker discovery, the Canadian context, and approaches for moving forward. This article provides a synopsis of these discussions and identifies steps forward for advancing vaccine biomarker research in Canada.

The impact of distinct culture media in Leishmania infantum biology and infectivity.

An ideal culture medium for Leishmania promastigotes should retain the basic characteristics of promastigotes found in sandflies (morphology and infectivity). Furthermore, the media should not create a bias in experimental settings, thus enabling the proper extrapolation of results. To assess this we studied several established media for promastigote growth. We analysed morphology, viability, cell cycle progression, metacyclic profile, capacity to differentiate into axenic amastigotes and infectivity. Furthermore, using a rational approach from the evaluated media we developed a simple serum-free medium (cRPMI). We report that parasites growing in different media present different biological characteristics and distinct in vitro and in vivo infectivities. The developed medium, cRPMI, proved to be a less expensive substitute for traditional serum-supplemented media for the in vitro maintenance of promastigotes. In fact, cRPMI is ideal for the maintenance of parasites in the laboratory, diminishing the expected loss of virulence over time typical of the parasite cultivation. Ultimately this report is a clear warning that the normalization of culture media should be a real concern in the field as media-specific phenomena are sufficient to induce biological bias with consequences in infectivity and general parasite biology.

Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes.

Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV-1 co-infections. We have delineated the mechanism of cell death induced by the HIV-1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir-Leishmania interactions, we selected Nelfinavir-resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related Leishmania species were used as a screening tool to compare Nelfinavir-resistant and -sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of Nelfinavir-resistant and -sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time-dependent manner. Furthermore, high-resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir-resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in drug-induced intracellular vesicles.

Telomeric gene deletion and intrachromosomal amplification in antimony-resistant Leishmania.

Antimonials are still the mainstay of treatment against leishmaniasis but drug resistance is increasing. We carried out short read next-generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony-resistant mutants. Copy number variations were consistently detected with both NGS and CGH. A major attribute of antimony resistance was a novel terminal deletion of variable length (67 kb to 204 kb) of the polyploid chromosome 31 in the three mutants. Terminal deletions in two mutants occurred at the level of inverted repeated sequences. The AQP1 gene coding for an aquaglyceroporin was part of the deleted region and its transfection into resistant mutants reverted resistance to SbIII. We also highlighted an intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded for ascorbate-dependent peroxidase (APX) and glucose-6-phosphate dehydrogenase (G6PDH). Overexpression of these genes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS). Generation of a G6PDH null mutant in one revertant exhibited SbIII sensitivity and a decreased protection of ROS. Our genomic analyses and functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania.

Generation of Leishmania hybrids by whole genomic DNA transformation.

Genetic exchange is a powerful tool to study gene function in microorganisms. Here, we tested the feasibility of generating Leishmania hybrids by electroporating genomic DNA of donor cells into recipient Leishmania parasites. The donor DNA was marked with a drug resistance marker facilitating the selection of DNA transfer into the recipient cells. The transferred DNA was integrated exclusively at homologous locus and was as large as 45 kb. The independent generation of L. infantum hybrids with L. major sequences was possible for several chromosomal regions. Interfering with the mismatch repair machinery by inactivating the MSH2 gene enabled an increased efficiency of recombination between divergent sequences, hence favouring the selection of hybrids between species. Hybrids were shown to acquire the phenotype derived from the donor cells, as demonstrated for the transfer of drug resistance genes from L. major into L. infantum. The described method is a first step allowing the generation of in vitro hybrids for testing gene functions in a natural genomic context in the parasite Leishmania.

Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum.

In most organisms, the primary function of homologous recombination (HR) is to allow genome protection by the faithful repair of DNA double-strand breaks. The vital step of HR is the search for sequence homology, mediated by the RAD51 recombinase, which is stimulated further by proteins mediators such as the tumor suppressor BRCA2. The biochemical interplay between RAD51 and BRCA2 is unknown in Leishmania or Trypanosoma. Here we show that the Leishmania infantum BRCA2 protein possesses several critical features important for the regulation of DNA recombination at the genetic and biochemical level. A BRCA2 null mutant, generated by gene disruption, displayed genomic instability and gene-targeting defects. Furthermore, cytological studies show that LiRAD51 can no longer localize to the nucleus in this mutant. The Leishmania RAD51 and BRCA2 interact together and the purified proteins bind single-strand DNA. Remarkably, LiBRCA2 is a recombination mediator that stimulates the invasion of a resected DNA double-strand break in an undamaged template by LiRAD51 to form a D-loop structure. Collectively, our data show that LiBRCA2 and LiRAD51 promote HR at the genetic and biochemical level in L. infantum, the causative agent of visceral leishmaniasis.

Discovery of factors linked to antimony resistance in Leishmania panamensis through differential proteome analysis.

The rate of treatment failure to antileishmanial chemotherapy in Latin America is up to 64%. Parasite drug resistance contributes to an unknown proportion of treatment failures. Identification of clinically relevant molecular mechanisms responsible for parasite drug resistance is critical to the conservation of available drugs and to the discovery of novel targets to reverse the resistant phenotype. We conducted comparative proteomic-based analysis of Leishmania (Viannia) panamensis lines selected in vitro for resistance to trivalent antimony (Sb(III)) to identify factors associated with antimony resistance. Using 2-dimensional gel electrophoresis, two distinct sub-proteomes (soluble in NP-40/urea and Triton X-114, respectively) of promastigotes of WT and Sb(III)-resistant lines were generated. Overall, 9 differentially expressed putative Sb-resistance factors were detected and identified by mass spectrometry. These constituted two major groups: (a) proteins involved in general stress responses and (b) proteins with highly specific metabolic and transport functions, potentially directly contributing to the Sb-resistance mechanism. Notably, the sulfur amino acid-metabolizing enzymes S-adenosylmethionine synthetase (SAMS) and S-adenosylhomocysteine hydrolase (SAHH) were over-expressed in Sb(III)-resistant lines and Sb(III)-resistant clinical isolates. These enzymes play a central role in the upstream synthesis of precursors of trypanothione, a key molecule involved in Sb-resistance in Leishmania parasites, and suggest involvement of epigenetic regulation in response to drug exposure. These data re-enforce the importance of thiol metabolism in Leishmania Sb resistance, reveal previously unrecognized steps in the mechanism(s) of Sb tolerance, and suggest a cross-talk between drug resistance, metabolism and virulence.

Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species.

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage.

Intrachromosomal tandem duplication and repeat expansion during attempts to inactivate the subtelomeric essential gene GSH1 in Leishmania.

Gamma-glutamylcysteine synthetase encoded by GSH1 is the rate-limiting enzyme in the biosynthesis of glutathione and trypanothione in Leishmania. Attempts to generate GSH1 null mutants by gene disruption failed in Leishmania infantum. Removal of even a single allele invariably led to the generation of an extra copy of GSH1, maintaining two intact wild-type alleles. In the second and even third round of inactivation, the markers integrated at the homologous locus but always preserved two intact copies of GSH1. We probed into the mechanism of GSH1 duplication. GSH1 is subtelomeric on chromosome 18 and Southern blot analysis indicated that a 10-kb fragment flanked by 466-bp direct repeated sequences was duplicated in tandem on the same chromosomal allele each time GSH1 was targeted. Polymerase chain reaction analysis and sequencing confirmed the generation of novel junctions created at the level of the 466-bp repeats consequent to locus duplication. In loss of heterozygosity attempts, the same repeated sequences were utilized for generating extrachromosomal circular amplicons. Our results are consistent with break-induced replication as a mechanism for the generation of this regional polyploidy to compensate for the inactivation of an essential gene. This chromosomal repeat expansion through repeated sequences could be implicated in locus duplication in Leishmania.

Genome annotation and intraviral interactome for the Streptococcus pneumoniae virulent phage Dp-1.

Streptococcus pneumoniae causes several diseases, including pneumonia, septicemia, and meningitis. Phage Dp-1 is one of the very few isolated virulent S. pneumoniae bacteriophages, but only a partial characterization is currently available. Here, we confirmed that Dp-1 belongs to the family Siphoviridae. Then, we determined its complete genomic sequence of 56,506 bp. It encodes 72 open reading frames, of which 44 have been assigned a function. We have identified putative promoters, Rho-independent terminators, and several genomic clusters. We provide evidence that Dp-1 may be using a novel DNA replication system as well as redirecting host protein synthesis through queuosine-containing tRNAs. Liquid chromatography-mass spectrometry analysis of purified phage Dp-1 particles identified at least eight structural proteins. Finally, using comprehensive yeast two-hybrid screens, we identified 156 phage protein interactions, and this intraviral interactome was used to propose a structural model of Dp-1.

High affinity S-Adenosylmethionine plasma membrane transporter of Leishmania is a member of the folate biopterin transporter (FBT) family.

S-Adenosylmethionine (AdoMet) is an important methyl group donor that plays a central role in many essential biochemical processes. The parasite Leishmania can both synthesize and transport AdoMet. Leishmania cells resistant to the antifolate methotrexate due to a rearrangement in folate biopterin transporter (FBT) genes were cross-resistant to sinefungin, an AdoMet analogue. FBT gene rearrangements were also observed in Leishmania major cells selected for sinefungin resistance. One of the rearranged FBT genes corresponded to the main AdoMet transporter (AdoMetT1) of Leishmania as determined by gene transfection and gene inactivation experiments. AdoMetT1 was determined to be a high affinity plasma membrane transporter expressed constitutively throughout the growth phases of the parasite. Leishmania cells selected for resistance or naturally insensitive to sinefungin had lower expression of AdoMetT1. A new function in one carbon metabolism, also a pathway of interest for chemotherapeutic interventions, is described for a novel class of membrane proteins found in diverse organisms.

The gamma-glutamylcysteine synthetase gene of Leishmania is essential and involved in response to oxidants.

Gamma-glutamylcysteine synthetase, encoded by the GSH1 gene, is the rate-limiting enzyme in the biosynthesis of glutathione and of trypanothione in Leishmania. The importance of GSH1 was assessed by generating GSH1 null mutants in Leishmania infantum. Removal of even a single wild-type allelic copy of GSH1 invariably led to the generation of an extra copy of GSH1, maintaining two intact wild-type alleles. However, by first supplementing the parasites with a rescue plasmid, we succeeded in obtaining both a single and null chromosomal GSH1 mutants. Parasites with one intact GSH1 chromosomal allele lost the rescuing plasmid but not the double knockout, when grown in the absence of antibiotic, indicating the essentiality of the GSH1 gene in Leishmania. Heterozygous mutants with one allele-inactivated transcribed less GSH1 mRNA and synthesized less glutathione and trypanothione. These mutants were more susceptible to oxidative stresses in vitro as promastigotes and showed decreased survival inside activated macrophages producing reactive oxygen or nitrogen species. These mutants showed a significant decreased survival in the presence of antimony (SbV) compared with control cells. All phenotypes were reverted in the add-back mutant, thus proving the importance of thiols in dealing with oxidants including the action of antimonials.