Human Fungal Pathogen Cryptococcus Neoformans Research Articles

Calcineurin contributes to RNAi-mediated transgene silencing and small interfering RNA production in the human fungal pathogen Cryptococcus neoformans.

Adaptation to external environmental challenges at the cellular level requires rapid responses and involves relay of information to the nucleus to drive key gene expression changes through downstream transcription factors. Here, we describe an alternative route of adaptation through a direct role for cellular signaling components in governing gene expression via RNA interference-mediated small RNA production. Calcium-calcineurin signaling is a highly conserved signaling cascade that plays central roles in stress adaptation and virulence of eukaryotic pathogens, including the human fungal pathogen Cryptococcus neoformans. Upon activation in C. neoformans, calcineurin localizes to P-bodies, membraneless organelles that are also the site for RNA processing. Here, we studied the role of calcineurin and its substrates in RNAi-mediated transgene silencing. Our results reveal that calcineurin regulates both the onset and the reversion of transgene silencing. We found that some calcineurin substrates that localize to P-bodies also regulate transgene silencing but in opposing directions. Small RNA sequencing in mutants lacking calcineurin or its targets revealed a role for calcineurin in small RNA production. Interestingly, the impact of calcineurin and its substrates was found to be different in genome-wide analysis, suggesting that calcineurin may regulate small RNA production in C. neoformans through additional pathways. Overall, these findings define a mechanism by which signaling machinery induced by external stimuli can directly alter gene expression to accelerate adaptative responses and contribute to genome defense.

Proteomic Profiling of Samples Derived from a Murine Model Following Cryptococcus neoformans Infection.

Proteomic profiling provides in-depth information about the regulation of diverse biological processes, activation of and communication across signaling networks, and alterations to protein production, modifications, and interactions. For infectious disease research, mass spectrometry-based proteomics enables detection of host defenses against infection and mechanisms used by the pathogen to evade such responses. In this chapter, we outline protein extraction from organs, tissues, and fluids collected following intranasal inoculation of a murine model with the human fungal pathogen Cryptococcus neoformans. We describe sample preparation, followed by purification, processing on the mass spectrometer, and a robust bioinformatics analysis. The information gleaned from proteomic profiling of fungal infections supports the detection of novel biomarkers for diagnostic and prognostic purposes.

In Vitro Titan Cell Generation in Cryptococcus neoformans and Automated Cell Size Measurements.

A special feature of the human fungal pathogen Cryptococcus neoformans is its morphological changes triggered by the interaction with the host. During infection, a specific increase in cell size is observed, particularly in lung tissue, from a typical cell size of 5-7μm cells to cells larger than 10μm, dubbed titan cells (TCs). However, the study of this specific cell subpopulation was, until now, only possible via recovery of TCs from lungs of mice during experimental infections where stable and reproducible generation of TCs occurs.The protocol described here generates TCs using in vitro conditions and measures cell size using a rapid, automated method. TC generation in vitro is robust and reproducible, generating yeast cells harboring the same characteristics of TCs generated in vivo.

Histone acetyltransferase Gcn5-mediated histone H3 acetylation facilitates cryptococcal morphogenesis and sexual reproduction.

Eukaryotic gene transcription is typically regulated by a series of histone modifications, which play a crucial role in adapting to complex environmental stresses. In the ubiquitous human fungal pathogen Cryptococcus neoformans, sexual life cycle is a continuous intracellular differentiation process that strictly occurs in response to mating stimulation. Despite the comprehensive identification of the regulatory factors and genetic pathways involved in its sexual cycle, understanding of the epigenetic modifications involved in this process remains quite limited. In this research, we found that histone acetyltransferase Gcn5-mediated histone H3 acetylation plays a crucial role in completing the cryptococcal sexual cycle, including yeast-hyphae morphogenesis and the subsequent sexual reproduction. Furthermore, we demonstrated that Gcn5 participates in this process primarily through regulating the key morphogenesis regulator Znf2 and its targets. This study thus provided a comprehensive understanding of how histone acetylation modification impacts sexual life cycle in a high-risk human pathogenic fungus.

A fungal lytic polysaccharide monooxygenase is required for cell wall integrity, thermotolerance, and virulence of the fungal human pathogen Cryptococcus neoformans.

Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment.

Importance of Clinical Isolates in Cryptococcus neoformans Research.

The human pathogenic fungus Cryptococcus neoformans is a global health concern. Previous research in the field has focused on studies using reference strains to identify virulence factors, generate mutant libraries, define genomic structures, and perform functional studies. In this review, we discuss the benefits and drawbacks of using reference strains to study C. neoformans, describe how the study of clinical isolates has expanded our understanding of pathogenesis, and highlight how studies using clinical isolates can further develop our understanding of the host-pathogen interaction during C. neoformans infection.

Drug Target Elucidation Through Isolation and Analysis of Drug-Resistant Mutants in Cryptococcus neoformans.

Drug target identification is an essential component to antifungal drug development. Many methods, including large chemical library screening, natural product screening, and drug repurposing efforts, can identify compounds with favorable in vitro antifungal activity. However, these approaches will often identify compounds with no known mechanism of action. Herein, we describe a method utilizing the human fungal pathogen Cryptococcus neoformans to identify antifungal drug targets through the isolation of spontaneous resistant mutants, antifungal testing, whole-genome sequencing, and variant analysis.

An Atypical ABC Transporter Is Involved in Antifungal Resistance and Host Interactions in the Pathogenic Fungus Cryptococcus neoformans.

ABSTRACTATP-binding cassette (ABC) transporters represent one of the largest protein superfamilies. Functionally diverse, ABC transporters have been implicated in many aspects of microbial physiology. The genome of the human fungal pathogen Cryptococcus neoformans encodes 54 putative ABC transporters and most of them remain uncharacterized. In a previous genetic screen for fungal regulators of phagocytosis, we identified an uncharacterized gene, CNAG_06909, that modulates host interactions. This gene encoded a half-size ABC transporter of the PDR-type, and phenotypic studies of a strain with this gene deleted revealed an altered antifungal susceptibility profile, including hypersensitivity to fluconazole (FLC). This gene, which we named PDR6, localized to the endoplasmic reticulum (ER) and plasma membrane (PM), and when absent, less ergosterol was observed in the PM. Additionally, we observed that the pdr6Δ strain displayed a reduction in secreted polysaccharide capsular material. These changes to the cellular surface may explain the observed increased uptake by macrophages and the reduced intracellular survival. Finally, studies in mice demonstrated that Pdr6 function was required for the normal progression of cryptococcal infection. Taken together, this study demonstrates a novel dual role for PDR transporters in C. neoformans, which could represent a potential target for antifungal therapeutics. Furthermore, the atypical half-size transporter encoded by PDR6 is conserved in many fungal pathogens, but absent in model nonpathogenic fungi. Hence, this study provided a function for this unique group of fungal half-size PDR transporters that, although conserved, remain largely understudied.

Identification and characterisation of sPEPs in Cryptococcus neoformans

Short open reading frame (sORF)-encoded peptides (sPEPs) have been found across a wide range of genomic locations in a variety of species. To date, their identification, validation, and characterisation in the human fungal pathogen Cryptococcus neoformans has been limited due to a lack of standardised protocols. We have developed an enrichment process that enables sPEP detection within a protein sample from this polysaccharide-encapsulated yeast, and implemented proteogenomics to provide insights into the validity of predicted and hypothetical sORFs annotated in the C. neoformans genome. Novel sORFs were discovered within the 5′ and 3′ UTRs of known transcripts as well as in “non-coding” RNAs. One novel candidate, dubbed NPB1, that resided in an RNA annotated as “non-coding”, was chosen for characterisation. Through the creation of both specific point mutations and a full deletion allele, the function of the new sPEP, Npb1, was shown to resemble that of the bacterial trans-translation protein SmpB.

Long Non-Coding RNAs in Cryptococcus neoformans: Insights Into Fungal Pathogenesis.

Long non-coding RNAs (lncRNAs) are highly expressed and can modulate multiple cellular processes including transcription, splicing, translation, and many diverse signaling events. LncRNAs can act as sponges for miRNAs, RNA and DNA binding proteins, functioning as competitive endogenous RNAs. The contribution of lncRNAs to microbial pathogenesis is largely neglected in eukaryotic pathogens despite the abundance of RNA sequencing datasets encompassing conditions of stress, gene deletions and conditions that mimic the host environment. The human fungal pathogen Cryptococcus neoformans encodes 6975 (84%) protein-coding and 1359 (16%) non-protein-coding RNAs, of which 1182 (14.2%) are lncRNAs defined by a threshold of greater than 200 nucleotides in length. Here, we discuss the current state of knowledge in C. neoformans lncRNA biology. Utilizing existing RNA seq datasets, we examine trends in lncRNA expression and discuss potential implications for pathogenesis.

Dynamic genome plasticity during unisexual reproduction in the human fungal pathogen Cryptococcus deneoformans.

Genome copy number variation occurs during each mitotic and meiotic cycle and it is crucial for organisms to maintain their natural ploidy. Defects in ploidy transitions can lead to chromosome instability, which is a hallmark of cancer. Ploidy in the haploid human fungal pathogen Cryptococcus neoformans is exquisitely orchestrated and ranges from haploid to polyploid during sexual development and under various environmental and host conditions. However, the mechanisms controlling these ploidy transitions are largely unknown. During C. deneoformans (formerly C. neoformans var. neoformans, serotype D) unisexual reproduction, ploidy increases prior to the onset of meiosis, can be independent from cell-cell fusion and nuclear fusion, and likely occurs through an endoreplication pathway. To elucidate the molecular mechanisms underlying this ploidy transition, we identified twenty cell cycle-regulating genes encoding cyclins, cyclin-dependent kinases (CDK), and CDK regulators. We characterized four cyclin genes and two CDK regulator genes that were differentially expressed during unisexual reproduction and contributed to diploidization. To detect ploidy transition events, we generated a ploidy reporter, called NURAT, which can detect copy number increases via double selection for nourseothricin-resistant, uracil-prototrophic cells. Utilizing this ploidy reporter, we showed that ploidy transition from haploid to diploid can be detected during the early phases of unisexual reproduction. Interestingly, selection for the NURAT reporter revealed several instances of segmental aneuploidy of multiple chromosomes, which conferred azole resistance in some isolates. These findings provide further evidence of ploidy plasticity in fungi with significant biological and public health implications.

Genetic analysis of Hsp90 function in Cryptococcus neoformans highlights key roles in stress tolerance and virulence.

The opportunistic human fungal pathogen Cryptococcus neoformans has tremendous impact on global health, causing 181,000 deaths annually. Current treatment options are limited, and the frequent development of drug resistance exacerbates the challenge of managing invasive cryptococcal infections. In diverse fungal pathogens, the essential molecular chaperone Hsp90 governs fungal survival, drug resistance, and virulence. Therefore, targeting this chaperone has emerged as a promising approach to combat fungal infections. However, the role of Hsp90 in supporting C. neoformans pathogenesis remains largely elusive due to a lack of genetic characterization. To help dissect the functions of Hsp90 in C. neoformans, we generated a conditional expression strain in which HSP90 is under control of the copper-repressible promoter CTR4-2. Addition of copper to culture medium depleted Hsp90 transcript and protein levels in this strain, resulting in compromised fungal growth at host temperature; increased sensitivity to stressors, including the azole class of antifungals; altered C. neoformans morphology; and impaired melanin production. Finally, leveraging the fact that copper concentrations vary widely in different mouse tissues, we demonstrated attenuated virulence for the CTR4-2p-HSP90 mutant specifically in an inhalation model of Cryptococcus infection. During invasion and establishment of infection in this mouse model, the pathogen is exposed to the relatively high copper concentrations found in the lung as compared to blood. Overall, this work generates a tractable genetic system to study the role of Hsp90 in supporting the pathogenicity of C. neoformans and provides proof-of-principle that targeting Hsp90 holds great promise as a strategy to control cryptococcal infection.

Zinc-binding domain mediates pleiotropic functions of Yvh1 in Cryptococcus neoformans.

Yvh1 is a dual-specificity phosphatase (DUSP) that is evolutionarily conserved in eukaryotes, including yeasts and humans. Yvh1 is involved in the vegetative growth, differentiation, and virulence of animal and plant fungal pathogens. All Yvh1 orthologs have a conserved DUSP catalytic domain at the N-terminus and a zinc-binding (ZB) domain with two zinc fingers (ZFs) at the C-terminus. Although the DUSP domain is implicated in the regulation of MAPK signaling in humans, only the ZB domain is essential for most cellular functions of Yvh1 in fungi. This study aimed to analyze the functions of the DUSP and ZB domains of Yvh1 in the human fungal pathogen Cryptococcus neoformans, whose Yvh1 (CnYvh1) contains a DUSP domain at the C-terminus and a ZB domain at the N-terminus. Notably, CnYvh1 has an extended internal domain between the two ZF motifs in the ZB domain. To elucidate the function of each domain, we constructed individual domain deletions and swapping strains by complementing the yvh1Δ mutant with wild-type (WT) or mutated YVH1 alleles and examined their Yvh1-dependent phenotypes, including growth under varying stress conditions, mating, and virulence factor production. Here, we found that the complementation of the yvh1Δ mutant with the mutated YVH1 alleles having two ZFs of the ZB domain, but not the DUSP and extended internal domains, restored the WT phenotypic traits in the yvh1Δ mutant. In conclusion, the ZB domain, but not the N-terminal DUSP domain, plays a pivotal role in the pathobiological functions of cryptococcal Yvh1.

Interactions between fungal hyaluronic acid and host CD44 promote internalization by recruiting host autophagy proteins to forming phagosomes

SummaryPhagocytosis and autophagy play critical roles in immune defense. The human fungal pathogen Cryptococcus neoformans (Cn) subverts host autophagy-initiation complex (AIC)-related proteins, to promote its phagocytosis and intracellular parasitism of host cells. The mechanisms by which the pathogen engages host AIC-related proteins remain obscure. Here, we show that the recruitment of host AIC proteins to forming phagosomes is dependent upon the activity of CD44, a host cell surface receptor that engages fungal hyaluronic acid (HA). This interaction elevates intracellular Ca2+ concentrations and activates CaMKKβ and its downstream target AMPKα, which results in activation of ULK1 and the recruitment of AIC components. Moreover, we demonstrate that HA-coated beads efficiently recruit AIC components to phagosomes and CD44 interacts with AIC components. Taken together, these findings show that fungal HA plays a critical role in directing the internalization and productive intracellular membrane trafficking of a fungal pathogen of global importance.

Puf4 Mediates Post-transcriptional Regulation of Cell Wall Biosynthesis and Caspofungin Resistance in Cryptococcus neoformans.

The human fungal pathogen Cryptococcus neoformans is intrinsically resistant to the echinocandin antifungal drug caspofungin, which targets the β-1,3-glucan synthase encoded by FKS1 Echinocandins have been on the market for 20 years, yet they are the newest class of antifungal drugs. Analysis of a C. neoformanspuf4Δ mutant, lacking the pumilio/FBF RNA binding protein family member Puf4, revealed exacerbated caspofungin resistance. In contrast, overexpression of PUF4 resulted in caspofungin sensitivity. The FKS1 mRNA contains three Puf4-binding elements (PBEs) in its 5' untranslated region. Puf4 binds with specificity to this region of FKS1 The FKS1 mRNA was destabilized in the puf4Δ mutant, and the abundance of the FKS1 mRNA was reduced compared to wild type, suggesting that Puf4 is a positive regulator of FKS1 mRNA stability. In addition to FKS1, the abundance of additional cell wall biosynthesis genes, including chitin synthases (CHS3, CHS4, and CHS6) and deacetylases (CDA1, CDA2, and CDA3) as well as a β-1,6-glucan synthase gene (SKN1), was regulated by Puf4. The use of fluorescent dyes to quantify cell wall components revealed that the puf4Δ mutant had increased chitin content, suggesting a cell wall composition that is less reliant on β-1,3-glucan. Overall, our findings suggest a mechanism by which caspofungin resistance, and more broadly, cell wall biogenesis, is regulated post-transcriptionally by Puf4.IMPORTANCECryptococcus neoformans is an environmental fungus that causes pulmonary and central nervous system infections. It is also responsible for 15% of AIDS-related deaths. A significant contributor to the high morbidity and mortality statistics is the lack of safe and effective antifungal therapies, especially in resource-poor settings. Yet, antifungal drug development has stalled in the pharmaceutical industry. Therefore, it is essential to understand the mechanism by which C. neoformans is resistant to caspofungin to design adjunctive therapies to potentiate the drug's activity toward this important pathogen.

Genetic contribution to high temperature tolerance in Cryptococcus neoformans.

The human fungal pathogen Cryptococcus neoformans relies on a complex signaling network for the adaptation and survival at the host temperature. Protein phosphatase calcineurin is central to proliferation at 37°C but its exact contributions remain ill-defined. To better define genetic contributions to the C. neoformans temperature tolerance, 4031 gene knockouts were screened for genes essential at 37°C and under conditions that keep calcineurin inactive. Identified 83 candidate strains, potentially sensitive to 37°C, were subsequently subject to technologically simple yet robust assay, in which cells are exposed to a temperature gradient. This has resulted in identification of 46 genes contributing to the maximum temperature at which C. neoformans can proliferate (Tmax). The 46 mutants, characterized by a range of Tmax on drug-free media, were further assessed for Tmax under conditions that inhibit calcineurin, which led to identification of several previously uncharacterized knockouts exhibiting synthetic interaction with the inhibition of calcineurin. A mutant that lacked septin Cdc11 was among those with the lowest Tmax and failed to proliferate in the absence of calcineurin activity. To further define connections with calcineurin and the role for septins in high temperature growth, the 46 mutants were tested for cell morphology at 37°C and growth in the presence of agents disrupting cell wall and cell membrane. Mutants sensitive to calcineurin inhibition were tested for synthetic lethal interaction with deletion of the septin-encoding CDC12 and the localization of the septin Cdc3-mCherry. The analysis described here pointed to previously uncharacterized genes that were missed in standard growth assays indicating that the temperature gradient assay is a valuable complementary tool for elucidating the genetic basis of temperature range at which microorganisms proliferate.

RELATe enables genome-scale engineering in fungal genomics.

CRISPR-Cas9-based screening with single-guide RNA (sgRNA) libraries has emerged as a revolutionary tool for comprehensive analysis of genetic elements. However, genome-scale sgRNA libraries are currently available only in a few model organisms. The traditional approach is to synthesize thousands to tens of thousands of sgRNAs, which is laborious and expensive. We have developed a simple method, RELATe (restriction/ligation coupled with Agrobacterium-mediated transformation), to generate sgRNA libraries from 10 μg of genomic DNA, targeting over 98% of the protein-coding genes in the human fungal pathogen Cryptococcus neoformans Functional screens identified 142 potential C. neoformans genes contributing to blood-brain barrier penetration. We selected two cryptococcal genes, SFP1 and WDR1, for a proof-of-concept demonstration that RELATe-identified genes are relevant to C. neoformans central nervous system infection. Our RELATe method can be used in many other fungal species and is powerful and cost-effective for genome-wide high-throughput screening for elucidating functional genomics.

A Transcriptional Regulatory Map of Iron Homeostasis Reveals a New Control Circuit for Capsule Formation in Cryptococcus neoformans.

Iron is essential for the growth of the human fungal pathogen Cryptococcus neoformans within the vertebrate host, and iron sensing contributes to the elaboration of key virulence factors, including the formation of the polysaccharide capsule. C. neoformans employs sophisticated iron acquisition and utilization systems governed by the transcription factors Cir1 and HapX. However, the details of the transcriptional regulatory networks that are governed by these transcription factors and connections to virulence remain to be defined. Here, we used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and transcriptome analysis (RNA-seq) to identify genes directly regulated by Cir1 and/or HapX in response to iron availability. Overall, 40 and 100 genes were directly regulated by Cir1, and 171 and 12 genes were directly regulated by HapX, under iron-limited and replete conditions, respectively. More specifically, we found that Cir1 directly controls the expression of genes required for iron acquisition and metabolism, and indirectly governs capsule formation by regulating specific protein kinases, a regulatory connection not previously revealed. HapX regulates the genes responsible for iron-dependent pathways, particularly under iron-depleted conditions. By analyzing target genes directly bound by Cir1 and HapX, we predicted the binding motifs for the transcription factors and verified that the purified proteins bind these motifs in vitro Furthermore, several direct target genes were coordinately and reciprocally regulated by Cir1 and HapX, suggesting that these transcription factors play conserved roles in the response to iron availability. In addition, biochemical analyses revealed that Cir1 and HapX are iron-containing proteins, implying that the regulatory networks of Cir1 and HapX may be influenced by the incorporation of iron into these proteins. Taken together, our identification of the genome-wide transcriptional networks provides a detailed understanding of the iron-related regulatory landscape, establishes a new connection between Cir1 and kinases that regulate capsule, and underpins genetic and biochemical analyses that reveal iron-sensing mechanisms for Cir1 and HapX in C. neoformans.

Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans

Ergosterol is the most important membrane sterol in fungal cells and a component not found in the membranes of human cells. We identified the ERG6 gene in the AIDS-associated fungal pathogen, Cryptococcus neoformans, encoding the sterol C-24 methyltransferase of fungal ergosterol biosynthesis. In this work, we have explored its relationship with high-temperature growth and virulence of C. neoformans by the construction of a loss-of-function mutant. In contrast to other genes involved in ergosterol biosynthesis, C. neoformans ERG6 is not essential for growth under permissive conditions in vitro. However, the erg6 mutant displayed impaired thermotolerance and increased susceptibility to osmotic and oxidative stress, as well as to different antifungal drugs. Total lipid analysis demonstrated a decrease in the erg6Δ strain membrane ergosterol content. In addition, this mutant strain was avirulent in an invertebrate model of C. neoformans infection. C. neoformans Erg6 was cyto-localized in the endoplasmic reticulum and Golgi complex. Our results demonstrate that Erg6 is crucial for growth at high temperature and virulence, likely due to its effects on C. neoformans membrane integrity and dynamics. These pathogen-focused investigations into ergosterol biosynthetic pathway components reinforce the multiple roles of ergosterol in the response of diverse fungal species to alterations in the environment, especially that of the infected host. These studies open perspectives to understand the participation of ergosterol in mechanism of resistance to azole and polyene drugs. Observed synergistic growth defects with co-inhibition of Erg6 and other components of the ergosterol biosynthesis pathway suggests novel approaches to treatment in human fungal infections.

Centromere scission drives chromosome shuffling and reproductive isolation

A fundamental characteristic of eukaryotic organisms is the generation of genetic variation via sexual reproduction. Conversely, significant large-scale genome structure variations could hamper sexual reproduction, causing reproductive isolation and promoting speciation. The underlying processes behind large-scale genome rearrangements are not well understood and include chromosome translocations involving centromeres. Recent genomic studies in the Cryptococcus species complex revealed that chromosome translocations generated via centromere recombination have reshaped the genomes of different species. In this study, multiple DNA double-strand breaks (DSBs) were generated via the CRISPR/Cas9 system at centromere-specific retrotransposons in the human fungal pathogen Cryptococcus neoformans The resulting DSBs were repaired in a complex manner, leading to the formation of multiple interchromosomal rearrangements and new telomeres, similar to chromothripsis-like events. The newly generated strains harboring chromosome translocations exhibited normal vegetative growth but failed to undergo successful sexual reproduction with the parental wild-type strain. One of these strains failed to produce any spores, while another produced ∼3% viable progeny. The germinated progeny exhibited aneuploidy for multiple chromosomes and showed improved fertility with both parents. All chromosome translocation events were accompanied without any detectable change in gene sequences and thus suggest that chromosomal translocations alone may play an underappreciated role in the onset of reproductive isolation and speciation.