Development Of Molecular Genetic Tools Research Articles

Distinguishing recrudescence from reinfection in lymphatic filariasis

The Global Program to Eliminate Lymphatic Filariasis (GPELF) is the largest public health program based on mass drug administration (MDA). Despite decades of MDA, ongoing transmission in some countries remains a challenge. To optimise interventions, it is critical to differentiate between recrudescence and new infections. Since adult filariae are inaccessible in humans, deriving a method that relies on the offspring microfilariae (mf) is necessary. We developed a genome amplification and kinship analysis-based approach using Brugia malayi samples from gerbils, and applied it to analyse Wuchereria bancrofti mf from humans in Côte d'Ivoire. We examined the pre-treatment genetic diversity in 269 mf collected from 18 participants, and further analysed 1-year post-treatment samples of 74 mf from 4 participants. Hemizygosity of the male X-chromosome allowed for direct inference of haplotypes, facilitating robust maternal parentage inference. To enrich parasite DNA from samples contaminated with host DNA, a whole-exome capture panel was created for W.bancrofti. By reconstructing and temporally tracking sibling relationships across pre- and post-treatment samples, we differentiated between new and established maternal families, suggesting reinfection in one participant and recrudescence in three participants. The estimated number of reproductively active adult females ranged between 3 and 11 in the studied participants. Population structure analysis revealed genetically distinct parasites in Côte d'Ivoire compared to samples from other countries. Exome capture identified protein-coding variants with ∼95% genotype concordance rate. We have generated resources to facilitate the development of molecular genetic tools that can estimate adult worm burdens and monitor parasite populations, thus providing essential information for the successful implementation of GPELF. This work was financially supported by the Bill and Melinda Gates Foundation (https://www.gatesfoundation.org) under grant OPP1201530 (Co-PIs PUF & Gary J. Weil). B.malayi parasite material was generated with support of the Foundation for Barnes Jewish Hospital (PUF). In addition, the development of computational methods was supported by the National Institutes of Health under grants AI144161 (MM) and AI146353 (MM). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Harnessing the power of new genetic tools to illuminate Giardia biology and pathogenesis.

Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis.

Chlamydial Infection From Outside to Inside.

Chlamydia are obligate intracellular bacteria, characterized by a unique biphasic developmental cycle. Specific interactions with the host cell are crucial for the bacteria’s survival and amplification because of the reduced chlamydial genome. At the start of infection, pathogen-host interactions are set in place in order for Chlamydia to enter the host cell and reach the nutrient-rich peri-Golgi region. Once intracellular localization is established, interactions with organelles and pathways of the host cell enable the necessary hijacking of host-derived nutrients. Detailed information on the aforementioned processes will increase our understanding on the intracellular pathogenesis of chlamydiae and hence might lead to new strategies to battle chlamydial infection. This review summarizes how chlamydiae generate their intracellular niche in the host cell, acquire host-derived nutrients in order to enable their growth and finally exit the host cell in order to infect new cells. Moreover, the evolution in the development of molecular genetic tools, necessary for studying the chlamydial infection biology in more depth, is discussed in great detail.

The Use of Grafting to Study Systemic Signaling in Plants.

Grafting has long been an important technique in agriculture. Nowadays, grafting is a widely used technique also to study systemic long-distance signaling in plants. Plants respond to their surrounding environment, and at that time many aspects of their physiology are regulated systemically; these start from local input signals and are followed by the transmission of information to the rest of the plant. For example, soil nutrient conditions, light/photoperiod, and biotic and abiotic stresses affect plants heterogeneously, and plants perceive such information in specific plant tissues or organs. Such environmental cues are crucial determinants of plant growth and development, and plants drastically change their morphology and physiology to adapt to various events in their life. Hitherto, intensive studies have been conducted to understand systemic signaling in plants, and grafting techniques have permitted advances in this field. The breakthrough technique of micrografting in Arabidopsis thaliana was established in 2002 and led to the development of molecular genetic tools in this field. Thereafter, various phenomena of systemic signaling have been identified at the molecular level, including nutrient fixation, flowering, circadian clock and defense against pathogens. The significance of grafting is that it can clarify the transmission of the stimulus and molecules. At present, many micro- and macromolecules have been identified as mobile signals, which are transported through plant vascular tissues to co-ordinate their physiology and development. In this review, we introduce the various grafting techniques that have been developed, we report on the recent advances in the field of plant systemic signaling where grafting techniques have been applied and provide insights for the future.

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen.

Chlamydia species infect millions of individuals worldwide and are important etiological agents of sexually transmitted disease, infertility, and blinding trachoma. Historically, the genetic intractability of this intracellular pathogen has hindered the molecular dissection of virulence factors contributing to its pathogenesis. The obligate intracellular life cycle of Chlamydia and restrictions on the use of antibiotics as selectable markers have impeded the development of molecular tools to genetically manipulate these pathogens. However, recent developments in the field have resulted in significant gains in our ability to alter the genome of Chlamydia, which will expedite the elucidation of virulence mechanisms. In this review, we discuss the challenges affecting the development of molecular genetic tools for Chlamydia and the work that laid the foundation for recent advancements in the genetic analysis of this recalcitrant pathogen.

Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis.

ABSTRACTAlthough progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation.

Selection of reference genes for quantitative real time RT-PCR during dimorphism in the zygomycete Mucor circinelloides.

Mucor circinelloides is a dimorphic fungal model for studying several biological processes including cell differentiation (yeast-mold transitions) as well as biodiesel and carotene production. The recent release of the first draft sequence of the M. circinelloides genome, combined with the availability of analytical methods to determine patterns of gene expression, such as quantitative Reverse transcription-Polymerase chain reaction (qRT-PCR), and the development of molecular genetic tools for the manipulation of the fungus, may help identify M. circinelloides gene products and analyze their relevance in different biological processes. However, no information is available on M. circinelloides genes of stable expression that could serve as internal references in qRT-PCR analyses. One approach to solve this problem consists in the use of housekeeping genes as internal references. However, validation of the usability of these reference genes is a fundamental step prior to initiating qRT-PCR assays. This work evaluates expression of several constitutive genes by qRT-PCR throughout the morphological differentiation stages of M. circinelloides; our results indicate that tfc-1 and ef-1 are the most stable genes for qRT-PCR assays during differentiation studies and they are proposed as reference genes to carry out gene expression studies in this fungus.

Herpèsviroses des équidés: intérêts et limites des outils de génétique moléculaire

At least five equine herpesviruses are known to infect horses : three alpha herpesviruses (equine herpesvirus-1, -3 and -4) and two gamma herpesviruses (equine herpesvirus-2 and -5). Equine herpesvirus-1 is responsible for three clinical presentations : respiratory, abortigenic and neurological. This latter, widely known as EHV-1 associated, myeloencephalopathy has been thoroughly investigated over the past years. Most of the studies focused on the relation between the presence of a mutation in ORF 30 coding for DNA polymerase and the expression of the paralytic (neurological) form. Even if this mutation seems to play a role, other factors related to the host and the environment are certainly involved in the development of the disease. Equine herpesvirus-4 is better known as the rhinopneumonia virus and is responsible for a large part of respiratory disorders in young foals. Equine herpesvirus-3 is responsible for equine coital exanthema, whose transmission through the respiratory tract is now proven. Equine herpesvirus-2 and -5 are associated with subclinical respiratory diseases in equine athletes, even though their pathogenic role has yet to be clarified as they were detected in all continents only relatively recently. The development of molecular genetic tools lead to major progress in the detection of these viruses, but the differentiation between latent and infectious forms is still unresolved. Although studies are currently performed on the typing of strains responsible for different clinical presentations involving equine herpesviruses as well as in other equine viruses (influenza, equine viral arteritis), there is no international consensus on a phylogenic tree for any of the 5 equine herpesviruses. Further studies are definitely required to monitor and characterise the different strains of EHV.

Polymorphic microsatellites in the human bloodfluke, Schistosoma japonicum, identified using a genomic resource

Re-emergence of schistosomiasis in regions of China where control programs have ceased requires development of molecular-genetic tools to track gene flow and assess genetic diversity of Schistosoma populations. We identified many microsatellite loci in the draft genome of Schistosoma japonicum using defined search criteria and selected a subset for further analysis. From an initial panel of 50 loci, 20 new microsatellites were selected for eventual optimization and application to a panel of worms from endemic areas. All but one of the selected microsatellites contain simple tri-nucleotide repeats. Moderate to high levels of polymorphism were detected. Numbers of alleles ranged from 6 to 14 and observed heterozygosity was always >0.6. The loci reported here will facilitate high resolution population-genetic studies on schistosomes in re-emergent foci.

Leptospira: a spirochaete with a hybrid outer membrane

Leptospira is a genus of spirochaetes that includes organisms with a variety of lifestyles ranging from aquatic saprophytes to invasive pathogens. Adaptation to a wide variety of environmental conditions has required leptospires to acquire a large genome and a complex outer membrane with features that are unique among bacteria. The most abundant surface-exposed outer membrane proteins are lipoproteins that are integrated into the lipid bilayer by amino-terminal fatty acids. In contrast to many spirochaetes, the leptospiral outer membrane also includes lipopolysaccharide and many homologues of well-known beta-barrel transmembrane outer membrane proteins. Research on leptospiral transmembrane outer membrane proteins has lagged behind studies of lipoproteins because of their aberrant behaviour by Triton X-114 detergent fractionation. For this reason, transmembrane outer membrane proteins are best characterized by assessing membrane integration and surface exposure. Not surprisingly, some outer membrane proteins that mediate host-pathogen interactions are strongly regulated by conditions found in mammalian host tissues. For example, the leptospiral immunoglobulin-like (Lig) repeat proteins are dramatically induced by osmolarity and mediate interactions with host extracellular matrix proteins. Development of molecular genetic tools are making it possible to finally understand the roles of these and other outer membrane proteins in mechanisms of leptospiral pathogenesis.

An improved and secreted luciferase reporter for schistosomes

Schistosomes are multicellular parasites of humans exhibiting interesting biological adaptations to their parasitic lifestyle. Concerted and in depth analyses of these adaptations and their cell and molecular biology requires further development of molecular genetic tools in schistosomes. In the current study, we demonstrate that a Gaussia luciferase reporter leads to significantly higher levels of luciferase activity in schistosomes compared to other tested luciferases. In addition, Gaussia luciferase can be secreted into culture media enabling non-invasive analysis of reporter activity. The secretion of Gaussia luciferase should allow a variety of new experimental paradigms for schistosome studies. Comparison of biolistic and electroporation transfection methods using luciferase RNA reporters and the luciferase acitivty produced indicates that electroporation of sporocysts and schistosomula is the most efficient transfection method for the four stages analyzed. These data should facilitate additional studies in schistosomes and provide a framework for further development of DNA transfection and gene expression analysis.

Prevention of Infectious Diseases by Neonatal and Early Infantile Immunization: Prospects for the New Millennium*

Newborns and young infants are at increased risk of severe or prolonged infections. Recent advances in our understanding of the host immune response in this age group, coupled with the development of molecular genetic tools, have paved the way for a new generation of preventive vaccines.

Prevention of infectious diseases by neonatal and early infantile immunization: prospects for the new millennium.

Newborns and young infants are at increased risk of severe or prolonged infections. Recent advances in our understanding of the host immune response in this age group, coupled with the development of molecular genetic tools, have paved the way for a new generation of preventive vaccines.

Applying in vivo expression technology (IVET) to the fungal pathogen Histoplasma capsulatum

Understanding how pathogens survive within the host cell is of paramount importance in the development of vaccines and therapeutic agents. This task has been particularly daunting in the study of fungal pathogens due to the lack of easily manipulated genetic systems. In recent years several molecular genetic reporter systems have been developed to identify genes expressed during the infection process and potential virulence determinants. The development of one method in particular,in vivo expression technology (IVET), has led to the discovery of several genes from various bacterial pathogens necessary for survival during infection. The recent development of molecular genetic tools for Histoplasma capsulatum has enabled us to adapt the IVET technology for this pathogenic fungus utilizing the URA5 gene, which is essential for H. capsulatum survival in mice and in cultured macrophages, as a reporter of in vivo gene expression. We report the first successful application of IVET screening of a fungal pathogen for genes expressed exclusively during infection.