More Like this

1. Analysis of five near-complete genome assemblies of the tomato pathogen Cladosporium fulvum uncovers additional accessory chromosomes and structural variations induced by transposable elements effecting the loss of avirulence genes

   https://doi.org/10.1186/s12915-024-01818-z  

   Zaccaron, Alex Z. ; Stergiopoulos, Ioannis ( January 2024 , BMC Biology)

   Fungal plant pathogens have dynamic genomes that allow them to rapidly adapt to adverse conditions and overcome host resistance. One way by which this dynamic genome plasticity is expressed is through effector gene loss, which enables plant pathogens to overcome recognition by cognate resistance genes in the host. However, the exact nature of these loses remains elusive in many fungi. This includes the tomato pathogenCladosporium fulvum, which is the first fungal plant pathogen from which avirulence (Avr) genes were ever cloned and in which loss ofAvrgenes is often reported as a means of overcoming recognition by cognate tomatoCfresistance genes. A recent near-complete reference genome assembly ofC. fulvumisolate Race 5 revealed a compartmentalized genome architecture and the presence of an accessory chromosome, thereby creating a basis for studying genome plasticity in fungal plant pathogens and its impact on avirulence genes.

   Here, we obtained near-complete genome assemblies of four additionalC. fulvumisolates. The genome assemblies had similar sizes (66.96 to 67.78 Mb), number of predicted genes (14,895 to 14,981), and estimated completeness (98.8 to 98.9%). Comparative analysis that included the genome of isolate Race 5 revealed high levels of synteny and colinearity, which extended to the density and distribution of repetitive elements and of repeat-induced point (RIP) mutations across homologous chromosomes. Nonetheless, structural variations, likely mediated by transposable elements and effecting the deletion of the avirulence genesAvr4E,Avr5, andAvr9, were also identified. The isolates further shared a core set of 13 chromosomes, but two accessory chromosomes were identified as well. Accessory chromosomes were significantly smaller in size, and one carried pseudogenized copies of two effector genes. Whole-genome alignments further revealed genomic islands of near-zero nucleotide diversity interspersed with islands of high nucleotide diversity that co-localized with repeat-rich regions. These regions were likely generated by RIP, which generally asymmetrically affected the genome ofC. fulvum.

   Our results reveal new evolutionary aspects of theC. fulvumgenome and provide new insights on the importance of genomic structural variations in overcoming host resistance in fungal plant pathogens.

    

   more » « less
2. An expanded toolkit of drug resistance cassettes for Candida glabrata , Candida auris , and Candida albicans leads to new insights into the ergosterol pathway

   https://doi.org/10.1128/msphere.00311-23  

   Gregor, Justin B. ; Gutierrez-Schultz, Victor A. ; Hoda, Smriti ; Baker, Kortany M. ; Saha, Debasmita ; Burghaze, Madeline G. ; Vazquez, Cynthia ; Burgei, Kendra E. ; Briggs, Scott D. ( December 2023 , mSphere)

   Mitchell, Aaron P. (Ed.)

   The World Health Organization recently published the first list of priority fungal pathogens highlighting multipleCandidaspecies, includingCandida glabrata,Candida albicans, andCandida auris. However, prior studies in these pathogens have been mainly limited to the use of two drug resistance cassettes,NatMXandHphMX, limiting genetic manipulation capabilities in prototrophic laboratory strains and clinical isolates. In this study, we expanded the toolkit forC. glabrata,C. auris, andC. albicansto includeKanMXandBleMXwhen coupled with anin vitroassembled CRISPR-Cas9 ribonucleoprotein (RNP)-based system. Repurposing these drug resistance cassettes forCandida, we were able to make single gene deletions, sequential and simultaneous double gene deletions, epitope tags, and rescue constructs. We applied these drug resistance cassettes to interrogate the ergosterol pathway, a critical pathway for both the azole and polyene antifungal drug classes. Using our approach, we determined for the first time that the deletion ofERG3inC. glabrata,C. auris,andC. albicansprototrophic strains results in azole drug resistance, which further supports the conservation of the Erg3-dependent toxic sterol model. Furthermore, we show that anERG5deletion inC. glabratais azole susceptible at subinhibitory concentrations, suggesting that Erg5 could act as an azole buffer for Erg11. Finally, we identified a synthetic growth defect when bothERG3andERG5are deleted inC. glabrata,which suggests the possibility of another toxic sterol impacting growth. Overall, we have expanded the genetic tools available to interrogate complex pathways in prototrophic strains and clinical isolates.

   The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies inCandida glabrata,Candida auris, andCandida albicanshave been mainly limited to the use ofNatMX/SAT1andHphMX/CaHygfor genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated thatNatMX/SAT1, HphMX, KanMX,and/orBleMXdrug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway ofC. glabrata,C. auris, andC. albicans. Moreover, the utility of these tools has provided new insights intoERGgenes and their relationship to azole resistance inCandida. Overall, we have expanded the toolkit forCandidapathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis.

    

   more » « less
3. Evolution of drug resistance in an antifungal-naive chronic Candida lusitaniae infection

   https://doi.org/10.1073/pnas.1807698115  

   Demers, Elora G. ; Biermann, Amy R. ; Masonjones, Sawyer ; Crocker, Alex W. ; Ashare, Alix ; Stajich, Jason E. ; Hogan, Deborah A. ( November 2018 , Proceedings of the National Academy of Sciences)

   Management of the limited number of antimicrobials currently available requires the identification of infections that contain drug-resistant isolates and the discovery of factors that promote the evolution of drug resistance. Here, we report a single fungal infection in which we have identified numerous subpopulations that differ in their alleles of a single gene that impacts drug resistance. The diversity at this locus was markedly greater than the reported heterogeneity of alleles conferring antibiotic resistance in bacterial infections. Analysis of genomes from hundreds ofClavispora(Candida)lusitaniaeisolates, through individual and pooled isolate sequencing, from a single individual with cystic fibrosis revealed at least 25 nonsynonymous mutations inMRR1, which encodes a transcription factor capable of inducing fluconazole (FLZ) resistance inCandidaspecies. Isolates with high-activity Mrr1 variants were resistant to FLZ due to elevated expression of theMDR1-encoded efflux pump. We found that high Mrr1-regulated Mdr1 activity protected against host and bacterial factors, suggesting drug resistance can be selected for indirectly and perhaps explaining the Mrr1 heterogeneity in this individual who had no prior azole exposure. Regional analysis ofC. lusitaniaepopulations from the upper and lower lobes of the right lung suggested intermingling of subpopulations throughout. Our retrospective characterization of sputum and lung populations by pooled sequencing found that alleles that confer FLZ resistance were a minority in each pool, possibly explaining why they were undetected before unsuccessful FLZ therapy. New susceptibility testing regimes may detect problematical drug-resistant subpopulations in heterogeneous single-species infections.

    

   more » « less
4. Insertion sequences and other mobile elements associated with antibiotic resistance genes in Enterococcus isolates from an inpatient with prolonged bacteraemia

   https://doi.org/10.1099/mgen.0.000855  

   Udaondo, Zulema ; Abram, Kaleb Z. ; Kothari, Atul ; Jun, Se-Ran ( August 2022 , Microbial Genomics)

   Insertion sequences (ISs) and other transposable elements are associated with the mobilization of antibiotic resistance determinants and the modulation of pathogenic characteristics. In this work, we aimed to investigate the association between ISs and antibiotic resistance genes, and their role in the dissemination and modification of the antibiotic-resistant phenotype. To that end, we leveraged fully resolved Enterococcus faecium and Enterococcus faecalis genomes of isolates collected over 5 days from an inpatient with prolonged bacteraemia. Isolates from both species harboured similar IS family content but showed significant species-dependent differences in copy number and arrangements of ISs throughout their replicons. Here, we describe two inter-specific IS-mediated recombination events and IS-mediated excision events in plasmids of E. faecium isolates. We also characterize a novel arrangement of the ISs in a Tn1546-like transposon in E. faecalis isolates likely implicated in a vancomycin genotype–phenotype discrepancy. Furthermore, an extended analysis revealed a novel association between daptomycin resistance mutations in liaSR genes and a putative composite transposon in E. faecium , offering a new paradigm for the study of daptomycin resistance and novel insights into its dissemination. In conclusion, our study highlights the role ISs and other transposable elements play in the rapid adaptation and response to clinically relevant stresses such as aggressive antibiotic treatment in enterococci. 

   more » « less
5. Historical Contingency Drives Compensatory Evolution and Rare Reversal of Phage Resistance

   https://doi.org/10.1093/molbev/msac182  

   Debray, Reena ; De Luna, Nina ; Koskella, Britt ( September 2022 , Molecular Biology and Evolution)

   Hendrickson, Heather (Ed.)

   Abstract Bacteria and lytic viruses (phages) engage in highly dynamic coevolutionary interactions over time, yet we have little idea of how transient selection by phages might shape the future evolutionary trajectories of their host populations. To explore this question, we generated genetically diverse phage-resistant mutants of the bacterium Pseudomonas syringae. We subjected the panel of mutants to prolonged experimental evolution in the absence of phages. Some populations re-evolved phage sensitivity, whereas others acquired compensatory mutations that reduced the costs of resistance without altering resistance levels. To ask whether these outcomes were driven by the initial genetic mechanisms of resistance, we next evolved independent replicates of each individual mutant in the absence of phages. We found a strong signature of historical contingency: some mutations were highly reversible across replicate populations, whereas others were highly entrenched. Through whole-genome sequencing of bacteria over time, we also found that populations with the same resistance gene acquired more parallel sets of mutations than populations with different resistance genes, suggesting that compensatory adaptation is also contingent on how resistance initially evolved. Our study identifies an evolutionary ratchet in bacteria–phage coevolution and may explain previous observations that resistance persists over time in some bacterial populations but is lost in others. We add to a growing body of work describing the key role of phages in the ecological and evolutionary dynamics of their host communities. Beyond this specific trait, our study provides a new insight into the genetic architecture of historical contingency, a crucial component of interpreting and predicting evolution. 

   more » « less