Infection In Drosophila Research Articles

Activation of immune defences against parasitoid wasps does not underlie the cost of infection.

Parasites reduce the fitness of their hosts, and different causes of this damage have fundamentally different consequences for the evolution of immune defences. Damage to the host may result from the parasite directly harming its host, often due to the production of virulence factors that manipulate host physiology. Alternatively, the host may be harmed by the activation of its own immune defences, as these can be energetically demanding or cause self-harm. A well-studied model of the cost of infection is Drosophila melanogaster and its common natural enemy, parasitoid wasps. Infected Drosophila larvae rely on humoral and cellular immune mechanisms to form a capsule around the parasitoid egg and kill it. Infection results in a developmental delay and reduced adult body size. To disentangle the effects of virulence factors and immune defences on these costs, we artificially activated anti-parasitoid immune defences in the absence of virulence factors. Despite immune activation triggering extensive differentiation and proliferation of immune cells together with hyperglycaemia, it did not result in a developmental delay or reduced body size. We conclude that the costs of infection do not result from these aspects of the immune response and may instead result from the parasite directly damaging the host.

Hunting Drosophila viruses from wild populations: A novel isolation approach and characterisation of viruses

Metagenomic studies have demonstrated that viruses are extremely diverse and abundant in insects, but the difficulty of isolating them means little is known about the biology of these newly discovered viruses. To overcome this challenge in Drosophila, we created a cell line that was more permissive to infection and detected novel viruses by the presence of double-stranded RNA. We demonstrate the utility of these tools by isolating La Jolla virus (LJV) and Newfield virus (NFV) from several wild Drosophila populations. These viruses have different potential host ranges, with distinct abilities to replicate in five Drosophila species. Similarly, in some species they cause high mortality and in others they are comparatively benign. In three species, NFV but not LJV caused large declines in female fecundity. This sterilization effect was associated with differences in tissue tropism, as NFV but not LJV was able to infect Drosophila melanogaster follicular epithelium and induce follicular degeneration in the ovary. We saw a similar effect in the invasive pest of fruit crops Drosophila suzukii, where oral infection with NFV caused reductions in the fecundity, suggesting it has potential as a biocontrol agent. In conclusion, a simple protocol allowed us to isolate new viruses and demonstrate that viruses identified by metagenomics have a large effect on the fitness of the model organism D. melanogaster and related species.

Multifunctional Protein A Is the Only Viral Protein Required for Nodavirus RNA Replication Crown Formation.

Positive-strand RNA virus RNA genome replication occurs in membrane-associated RNA replication complexes (RCs). Nodavirus RCs are outer mitochondrial membrane invaginations whose necked openings to the cytosol are "crowned" by a 12-fold symmetrical proteinaceous ring that functions as the main engine of RNA replication. Similar protein crowns recently visualized at the openings of alphavirus and coronavirus RCs highlight their broad conservation and functional importance. Using cryo-EM tomography, we earlier showed that the major nodavirus crown constituent is viral protein A, whose polymerase, RNA capping, membrane interaction and multimerization domains drive RC formation and function. Other viral proteins are strong candidates for unassigned EM density in the crown. RNA-binding RNAi inhibitor protein B2 co-immunoprecipitates with protein A and could form crown subdomains that protect nascent viral RNA and dsRNA templates. Capsid protein may interact with the crown since nodavirus virion assembly has spatial and other links to RNA replication. Using cryoelectron tomography and complementary approaches, we show that, even when formed in mammalian cells, nodavirus RC crowns generated without B2 and capsid proteins are functional and structurally indistinguishable from mature crowns in infected Drosophila cells expressing all viral proteins. Thus, the only nodaviral factors essential to form functional RCs and crowns are RNA replication protein A and an RNA template. We also resolve apparent conflicts in prior results on B2 localization in infected cells, revealing at least two distinguishable pools of B2. The results have significant implications for crown structure, assembly, function and control as an antiviral target.

A mannose-sensing AraC-type transcriptional activator regulates cell–cell aggregation of Vibrio cholerae

In addition to catalyzing coupled transport and phosphorylation of carbohydrates, the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulates various physiological processes in most bacteria. Therefore, the transcription of genes encoding the PTS is precisely regulated by transcriptional regulators depending on substrate availability. As the distribution of the mannose-specific PTS (PTSMan) is limited to animal-associated bacteria, it has been suggested to play an important role in host-bacteria interactions. In Vibrio cholerae, mannose is known to inhibit biofilm formation. During host infection, the transcription level of the V. cholerae gene encoding the putative PTSMan (hereafter referred to as manP) significantly increases, and mutations in this gene increase host survival rate. Herein, we show that an AraC-type transcriptional regulator (hereafter referred to as ManR) acts as a transcriptional activator of the mannose operon and is responsible for V. cholerae growth and biofilm inhibition on a mannose or fructose-supplemented medium. ManR activates mannose operon transcription by facilitating RNA polymerase binding to the promoter in response to mannose 6-phosphate and, to a lesser extent, to fructose 1-phosphate. When manP or manR is impaired, the mannose-induced inhibition of biofilm formation was reversed and intestinal colonization was significantly reduced in a Drosophila melanogaster infection model. Our results show that ManR recognizes mannose and fructose in the environment and facilitates V. cholerae survival in the host.

Sodium Selenite Enhances Antibiotics Sensitivity of Pseudomonas aeruginosa and Deceases Its Pathogenicity by Inducing Oxidative Stress and Inhibiting Quorum Sensing System.

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is commonly found in clinical settings and immuno-compromised patients. It is difficult to be eradicated due to its strong antibiotic resistance, and novel inactivation strategies have yet to be developed. Selenium is an essential microelement for humans and has been widely used in dietary supplement and chemoprevention therapy. In this study, the physiological and biochemical effects of sodium selenite on P. aeruginosa PAO1 were investigated. The results showed that 0~5 mM sodium selenite did not impact the growth of PAO1, but increased the lethality rate of PAO1 with antibiotics or H2O2 treatment and the antibiotics susceptibility both in planktonic and biofilm states. In addition, sodium selenite significantly reduced the expression of quorum sensing genes and inhibited various virulence factors of this bacterium, including pyocyanin production, bacterial motilities, and the type III secretion system. Further investigation found that the content of ROS in cells was significantly increased and the expression levels of most genes involved in oxidative stress were up-regulated, which indicated that sodium selenite induced oxidative stress. The RNA-seq result confirmed the phenotypes of virulence attenuation and the expression of quorum sensing and antioxidant-related genes. The assays of Chinese cabbage and Drosophila melanogaster infection models showed that the combination of sodium selenite and antibiotics significantly alleviated the infection of PAO1. In summary, the results revealed that sodium selenite induced oxidative stress and inhibited the quorum sensing system of P. aeruginosa, which in turn enhanced the antibiotic susceptibility and decreased the pathogenicity of this bacterium. These findings suggest that sodium selenite may be used as an effective strategy for adjunct treatment of the infections caused by P. aeruginosa.

Deciphering of Candida parapsilosis induced immune response in Drosophila melanogaster

ABSTRACT Candida infections are the most prevalent cause of serious human mycoses and are the third most common pathogens isolated from bloodstream infections in hospitalized patients. C. parapsilosis is a member of the non-albicans spp., which have a predilection for causing life-threatening disease in neonates and hospitalized pediatric patients. In this study, we utilized a Drosophila melanogaster infection model to analyze the immunological responses to C. parapsilosis. Our results demonstrate that the Toll pathway in Drosophila controls C. parapsilosis proliferation as the Toll signaling mutant MyD88−/ − flies are highly susceptible to C. parapsilosis. We also confirmed that the MyD88−/ − fly is a convenient invertebrate animal model to analyze virulence properties of different species and strains from the C. parapsilosis sensu lato complex as C. orthopsilosis, C. metapsilosis proved to be less virulent than C. parapsilosis sensu stricto and the N-mannan deficient C. parapsilosis och1Δ/Δ strain showed attenuated pathogenicity in this immunodeficient Drosophila background. We also found that Persephone protease is not required for detection and activation of Toll pathway during C. parapsilosis infection. Furthermore, we observed that Drosophila β-glucan receptor deficient flies where more sensitive to C. parapsilosis compared to wild-type flies; however, we could not find a clear dependence on the recognition of this receptor and the cell wall β-glucan exposure-induced host response. These studies establish this D. melanogaster infection model as an efficient tool in deciphering immune responses to C. parapsilosis as well as for assessing virulence factors produced by this emerging fungal predator.

Deletion of the PA4427-PA4431 Operon of Pseudomonas aeruginosa PAO1 Increased Antibiotics Resistance and Reduced Virulence and Pathogenicity by Affecting Quorum Sensing and Iron Uptake.

The respiratory chain is very important for bacterial survival and pathogenicity, yet the roles of the respiratory chain in P. aeruginosa remain to be fully elucidated. Here, we not only proved experimentally that the operon PA4427-PA4431 of Pseudomonas aeruginosa PAO1 encodes respiratory chain complex III (cytobc1), but also found that it played important roles in virulence and pathogenicity. PA4429–31 deletion reduced the production of the virulence factors, including pyocyanin, rhamnolipids, elastase, and extracellular polysaccharides, and it resulted in a remarkable decrease in pathogenicity, as demonstrated in the cabbage and Drosophila melanogaster infection models. Furthermore, RNA-seq analysis showed that PA4429–31 deletion affected the expression levels of the genes related to quorum-sensing systems and the transport of iron ions, and the iron content was also reduced in the mutant strain. Taken together, we comprehensively illustrated the function of the operon PA4427–31 and its application potential as a treatment target in P. aeruginosa infection.

Baicalin Represses Type Three Secretion System of Pseudomonas aeruginosa through PQS System.

Therapeutics that target the virulence of pathogens rather than their viability offer a promising alternative for treating infectious diseases and circumventing antibiotic resistance. In this study, we searched for anti-virulence compounds against Pseudomonas aeruginosa from Chinese herbs and investigated baicalin from Scutellariae radix as such an active anti-virulence compound. The effect of baicalin on a range of important virulence factors in P. aeruginosa was assessed using luxCDABE-based reporters and by phenotypical assays. The molecular mechanism of the virulence inhibition by baicalin was investigated using genetic approaches. The impact of baicalin on P. aeruginosa pathogenicity was evaluated by both in vitro assays and in vivo animal models. The results show that baicalin diminished a plenty of important virulence factors in P. aeruginosa, including the Type III secretion system (T3SS). Baicalin treatment reduced the cellular toxicity of P. aeruginosa on the mammalian cells and attenuated in vivo pathogenicity in a Drosophila melanogaster infection model. In a rat pulmonary infection model, baicalin significantly reduced the severity of lung pathology and accelerated lung bacterial clearance. The PqsR of the Pseudomonas quinolone signal (PQS) system was found to be required for baicalin’s impact on T3SS. These findings indicate that baicalin is a promising therapeutic candidate for treating P. aeruginosa infections.

Tornadic Shear Stress Induces a Transient, Calcineurin-Dependent Hypervirulent Phenotype in Mucorales Molds.

Trauma-related necrotizing myocutaneous mucormycosis (NMM) has a high morbidity and mortality in victims of combat-related injuries, geometeorological disasters, and severe burns. Inspired by the observation that several recent clusters of NMM have been associated with extreme mechanical forces (e.g., during tornados), we studied the impact of mechanical stress on Mucoralean biology and virulence in a Drosophila melanogaster infection model. In contrast to other experimental procedures to exert mechanical stress, tornadic shear challenge (TSC) by magnetic stirring induced a hypervirulent phenotype in several clinically relevant Mucorales species but not in Aspergillus or Fusarium Whereas fungal growth rates, morphogenesis, and susceptibility to noxious environments or phagocytes were not altered by TSC, soluble factors released in the supernatant of shear-challenged R. arrhizus spores rendered static spores hypervirulent. Consistent with a rapid decay of TSC-induced hypervirulence, minimal transcriptional changes were revealed by comparative RNA sequencing analysis of static and shear-challenged Rhizopus arrhizus However, inhibition of the calcineurin/heat shock protein 90 (hsp90) stress response circuitry by cyclosporine and tanespimycin abrogated the increased pathogenicity of R. arrhizus spores following TSC. Similarly, calcineurin loss-of-function mutants of Mucor circinelloides displayed no increased virulence capacity in flies after undergoing TSC. Collectively, these results establish that TSC induces hypervirulence specifically in Mucorales and point out the calcineurin/hsp90 pathway as a key orchestrator of this phenotype. Our findings invite future studies of topical calcineurin inhibitor treatment of wounds as an adjunct mitigation strategy for NMM following high-energy trauma.IMPORTANCE Given the limited efficacy of current medical treatments in trauma-related necrotizing mucormycosis, there is a dire need to better understand the Mucoralean pathophysiology in order to develop novel strategies to counteract fungal tissue invasion following severe trauma. Here, we describe that tornadic shear stress challenge transiently induces a hypervirulent phenotype in various pathogenic Mucorales species but not in other molds known to cause wound infections. Pharmacological and genetic inhibition of calcineurin signaling abrogated hypervirulence in shear stress-challenged Mucorales, encouraging further evaluation of (topical) calcineurin inhibitors to improve therapeutic outcomes of NMM after combat-related blast injuries or violent storms.

Heat Shock Protein DnaJ in Pseudomonas aeruginosa Affects Biofilm Formation via Pyocyanin Production.

Heat shock proteins (HSPs) play important biological roles, and they are implicated in bacterial response to environmental stresses and in pathogenesis of infection. The role of HSPs in P. aeruginosa, however, remains to be fully elucidated. Here, we report the unique role of HSP DnaJ in biofilm formation and pathogenicity in P. aeruginosa. A dnaJ mutant produced hardly any pyocyanin and formed significantly less biofilms, which contributed to decreased pathogenicity as demonstrated by reduced mortality rate in a Drosophila melanogaster infection model. The reduced pyocyanin production in the dnaJ mutant was a result of the decreased transcription of phenazine synthesis operons including phzA1, phzA2, phzS, and phzM. The reduction of biofilm formation and initial adhesion in the dnaJ mutant could be reversed by exogenously added pyocyanin or extracellular DNA (eDNA). Consistent with such observations, absence of dnaJ significantly reduced the release of eDNA in P. aeruginosa and addition of exogenous pyocyanin could restore eDNA release. These results indicate dnaJ mutation caused reduced pyocyanin production, which in turn caused the decreased eDNA, resulting in decreased biofilm formation. DnaJ is required for pyocyanin production and full virulence in P. aeruginosa; it affects biofilm formation and initial adhesion via pyocyanin, inducing eDNA release.

Spaceflight and simulated microgravity conditions increase virulence of Serratia marcescens in the Drosophila melanogaster infection model

While it has been shown that astronauts suffer immune disorders after spaceflight, the underlying causes are still poorly understood and there are many variables to consider when investigating the immune system in a complex environment. Additionally, there is growing evidence that suggests that not only is the immune system being altered, but the pathogens that infect the host are significantly influenced by spaceflight and ground-based spaceflight conditions. In this study, we demonstrate that Serratia marcescens (strain Db11) was significantly more lethal to Drosophila melanogaster after growth on the International Space Station than ground-based controls, but the increased virulence phenotype of S. marcescens did not persist after the bacterial cultures were passaged on the ground. Increased virulence was also observed in bacteria that were grown in simulated microgravity conditions on the ground using the rotating wall vessel. Increased virulence of the space-flown bacteria was similar in magnitude between wild-type flies and those that were mutants for the well-characterized immune pathways Imd and Toll, suggesting that changes to the host immune system after infection are likely not a major factor contributing towards increased susceptibility of ground-reared flies infected with space-flown bacteria. Characterization of the bacteria shows that at later timepoints spaceflight bacteria grew at a greater rate than ground controls in vitro, and in the host. These results suggest complex physiological changes occurring in pathogenic bacteria in space environments, and there may be novel mechanisms mediating these physiological effects that need to be characterized.

Combination of Repurposed Drug Diosmin with Amoxicillin-Clavulanic acid Causes Synergistic Inhibition of Mycobacterial Growth

Effective therapeutic regimens for the treatment of tuberculosis (TB) are limited. They are comprised of multiple drugs that inhibit the essential cellular pathways in Mycobacterium tuberculosis (Mtb). The present study investigates an approach which enables a combination of Amoxicillin-Clavulanic acid (AMC) and a repurposed drug for its synergistic effect towards TB treatment. We identified Diosmin (DIO), by targeting the active site residues of L,D-transpeptidase (Ldt) enzymes involved in Mtb cell wall biosynthesis by using a structure-based drug design method. DIO is rapidly converted into aglycone form Diosmetin (DMT) after oral administration. Binding of DIO or DMT towards Ldt enzymes was studied using molecular docking and bioassay techniques. Combination of DIO (or DMT) and AMC exhibited higher mycobactericidal activity against Mycobacterium marinum as compared to individual drugs. Scanning electron microscopy study of M. marinum treated with AMC-DIO and AMC-DMT showed marked cellular leakage. M. marinum infected Drosophila melanogaster fly model showed an increased fly survival of ~60% upon treatment with a combination of AMC and DIO (or DMT). Finally, the enhanced in vitro antimicrobial activity of AMC-DIO was validated against Mtb H37Ra and a MDR clinical isolate. Our results demonstrate the potential for AMC and DIO (or DMT) as a synergistic combination for the treatment of TB.

Preexposure to Isavuconazole Increases the Virulence of Mucorales but Not Aspergillus fumigatus in a Drosophila melanogaster Infection Model.

Breakthrough mucormycosis in patients receiving isavuconazole prophylaxis or therapy has been reported. We compared the impact of isavuconazole and voriconazole exposure on the virulence of clinical isolates of Aspergillus fumigatus and different Mucorales species in a Drosophila melanogaster infection model. In contrast to A. fumigatus, a hypervirulent phenotype was found in all tested Mucorales upon preexposure to either voriconazole or isavuconazole. These findings may contribute to the explanation of breakthrough mucormycosis in isavuconazole-treated patients.

In Vivo Efficacy of Ellagic Acid against Candida albicans in a Drosophila melanogaster Infection Model.

The aim of this study was to evaluate the antifungal activity and the toxicity of ellagic acid (EA) using a Drosophila melanogaster model. Candida albicans bacteria were inoculated into Toll heterozygous flies. Survival curves were obtained for the evaluation of the antimicrobial effect and toxicity of EA. A protective effect of EA against fungal infection in Drosophila melanogaster was observed at nontoxic concentrations. This study showed that EA is a promising tool for the treatment of candidiasis.

Dicer-2-Dependent Generation of Viral DNA from Defective Genomes of RNA Viruses Modulates Antiviral Immunity in Insects

SummaryThe RNAi pathway confers antiviral immunity in insects. Virus-specific siRNA responses are amplified via the reverse transcription of viral RNA to viral DNA (vDNA). The nature, biogenesis, and regulation of vDNA are unclear. We find that vDNA produced during RNA virus infection of Drosophila and mosquitoes is present in both linear and circular forms. Circular vDNA (cvDNA) is sufficient to produce siRNAs that confer partially protective immunity when challenged with a cognate virus. cvDNAs bear homology to defective viral genomes (DVGs), and DVGs serve as templates for vDNA and cvDNA synthesis. Accordingly, DVGs promote the amplification of vDNA-mediated antiviral RNAi responses in infected Drosophila. Furthermore, vDNA synthesis is regulated by the DExD/H helicase domain of Dicer-2 in a mechanism distinct from its role in siRNA generation. We suggest that, analogous to mammalian RIG-I-like receptors, Dicer-2 functions like a pattern recognition receptor for DVGs to modulate antiviral immunity in insects.

Culex pipiens crossing type diversity is governed by an amplified and polymorphic operon of Wolbachia

Culex pipiens mosquitoes are infected with Wolbachia (wPip) that cause an important diversity of cytoplasmic incompatibilities (CIs). Functional transgenic studies have implicated the cidA-cidB operon from wPip and its homolog in wMel in CI between infected Drosophila males and uninfected females. However, the genetic basis of the CI diversity induced by different Wolbachia strains was unknown. We show here that the remarkable diversity of CI in the C. pipiens complex is due to the presence, in all tested wPip genomes, of several copies of the cidA-cidB operon, which undergoes diversification through recombination events. In 183 isofemale lines of C. pipiens collected worldwide, specific variations of the cidA-cidB gene repertoires are found to match crossing types. The diversification of cidA-cidB is consistent with the hypothesis of a toxin–antitoxin system in which the gene cidB co-diversifies with the gene cidA, particularly in putative domains of reciprocal interactions.

Fucoidan coated ciprofloxacin loaded chitosan nanoparticles for the treatment of intracellular and biofilm infections of Salmonella

Salmonella infections and their gallstone associated biofilm infections are difficult to treat due to poor penetration of antibiotics into the intracellular compartments of macrophages and within biofilms. Here we developed ciprofloxacin loaded chitosan nanoparticles (cCNPs) and fucoidan (Fu) coated cCNPs (Fu-cCNPs). Characterizations of these nanoparticles were carried out using Dynamic Light Scattering‎, Transmission electron microscopy and Fourier transform infrared spectroscopy. The prepared cCNPs and Fu-cCNPs have the size range of 124±7nm and 320±18nm, respectively. Both nanoparticles were found to be non-hemolytic and cytocompatible. In vitro sustained release of ciprofloxacin was observed from both cCNPs and Fu-cCNPs over a period of 2 weeks. The antimicrobial activity of cCNPs and Fu-cCNPs was tested under in vitro and in vivo conditions. The intracellular anti-Salmonella activity of Fu-cCNPs was 2 fold higher than cCNPs and 6 fold higher than ciprofloxacin alone. Fluorescence microscopic images confirmed enhanced delivery of Fu-cCNPs than the cCNPs within the intracellular compartment of macrophages. Both cCNPs and Fu-cCNPs are found to be equally effective in dispersing Salmonella Paratyphi A gallstone biofilms. The in vivo antibacterial activities of Fu-cCNPs were superior to cCNPs which we have validated using Salmonella Paratyphi A infected Drosophila melanogaster fly model. Our overall results showed that (1) Fu-cCNPs are more effective in eradicating Salmonella infections than cCNPs; (2) both cCNPs and Fu-cCNPs were equally effective in dispersing Salmonella gallstone biofilms.

Hairpin structures with conserved sequence motifs determine the 3′ ends of non-polyadenylated invertebrate iridovirus transcripts

Previously, we observed that the transcripts of Invertebrate iridescent virus 6 (IIV6) are not polyadenylated, in line with the absence of canonical poly(A) motifs (AATAAA) downstream of the open reading frames (ORFs) in the genome. Here, we determined the 3′ ends of the transcripts of fifty-four IIV6 virion protein genes in infected Drosophila Schneider 2 (S2) cells. By using ligation-based amplification of cDNA ends (LACE) it was shown that the IIV6 mRNAs often ended with a CAUUA motif. In silico analysis showed that the 3′-untranslated regions of IIV6 genes have the ability to form hairpin structures (22–56 nt in length) and that for about half of all IIV6 genes these 3′ sequences contained complementary TAATG and CATTA motifs. We also show that a hairpin in the 3′ flanking region with conserved sequence motifs is a conserved feature in invertebrate-infecting iridoviruses (genus Iridovirus and Chloriridovirus).

Host and Bacterial Factors Control Susceptibility of Drosophila melanogaster to Coxiella burnetii Infection.

Coxiella burnetii is the causative agent of Q fever, a zoonotic disease that threatens both human and animal health. Due to the paucity of experimental animal models, little is known about how host factors interface with bacterial components and affect pathogenesis. Here, we used Drosophila melanogaster, in conjunction with the biosafety level 2 (BSL2) Nine Mile phase II (NMII) clone 4 strain of C. burnetii, as a model to investigate host and bacterial components implicated in infection. We demonstrate that adult Drosophila flies are susceptible to C. burnetii NMII infection and that this bacterial strain, which activates the immune deficiency (IMD) pathway, is able to replicate and cause mortality in the animals. We show that in the absence of Eiger, the only known tumor necrosis factor (TNF) superfamily homolog in Drosophila, Coxiella-infected flies exhibit reduced mortality from infection. We also demonstrate that the Coxiella type 4 secretion system (T4SS) is critical for the formation of the Coxiella-containing vacuole and establishment of infection in Drosophila Altogether, our data reveal that the Drosophila TNF homolog Eiger and the Coxiella T4SS are implicated in the pathogenesis of C. burnetii in flies. The Drosophila/NMII model mimics relevant aspects of the infection in mammals, such as a critical role of host TNF and the bacterial T4SS in pathogenesis. Our work also demonstrates the usefulness of this BSL2 model to investigate both host and Coxiella components implicated in infection.

Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system.

Among Candida species, the opportunistic fungal pathogen Candida glabrata has become the second most common causative agent of candidiasis in the world and a major public health concern. Yet, few molecular tools and resources are available to explore the biology of C. glabrata and to better understand its virulence during infection. In this study, we describe a robust experimental strategy to generate loss-of-function mutants in C. glabrata. The procedure is based on the development of three main tools: (i) a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system, (ii) an online program facilitating the selection of the most efficient guide RNAs for a given C. glabrata gene, and (iii) the identification of mutant strains by the Surveyor technique and sequencing. As a proof-of-concept, we have tested the virulence of some mutants in vivo in a Drosophila melanogaster infection model. Our results suggest that yps11 and a previously uncharacterized serine/threonine kinase are involved, directly or indirectly, in the ability of the pathogenic yeast to infect this model host organism.