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Investigating the intra-host diversity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for understanding its transmission and the emergence of new variants. However, there is limited insight into SARS-CoV-2 intra-host diversity and the extent to which shared intra-host single nucleotide variants (iSNVs) occur among samples without epidemiological links. To characterize intra-host diversity, we analyzed sequencing data from 803 samples across four Omicron transmission clusters. The potential co-mutation patterns formed by shared iSNVs contributed to regions in the genome with elevated iSNV density. Most samples did not share iSNVs. Even among the sample pairs that did share at least one iSNV, 24.4% originated from different transmission clusters. For shared iSNV sites that can become fixed as single nucleotide polymorphisms (SNPs), iSNVs cluster within the phylogenetic tree, with branches supporting the same variants as SNPs. This observation suggests that iSNVs likely serve as reservoirs for SNPs. Additionally, the BA.1.1 samples carried iSNVs identical to the characteristic mutations of BA.2 and BA.2.3. These findings provide important insights into the evolution and transmission inference of SARS-CoV-2.

Importance: Understanding the mechanisms behind viral evolution and transmission is crucial, as novel SARS-CoV-2 variants continue to emerge and spread worldwide. Viral evolution is driven not only by variants that circulate globally but also by mutations arising within individual hosts, resulting in the emergence of iSNVs. The role of iSNVs in shaping SARS-CoV-2 evolution and transmission remains poorly characterized. Our results showed a significant enrichment of shared iSNVs in high-density genomic regions, potentially contributing to the formation of co-mutation patterns. However, the presence of shared iSNVs in samples lacking epidemiological links indicates that they alone are insufficient for accurately reconstructing transmission routes. Instead, iSNVs may act as a reservoir for the emergence of single nucleotide polymorphisms. Our study offers new insights into the evolution of SARS-CoV-2 and the interpretation of transmission from sequencing data.

The CRISPR-Cas system is a bacterial adaptive immune system that protects against infection by phages: viruses that infect bacteria. To develop immunity, bacteria integrate spacers-fragments of the invading nucleic acids-into their CRISPR array to serve as the basis for sequence-targeted DNA cleavage. However, upon infection, a phage quickly takes over the metabolism of the bacterium, leaving little time for the bacterium to acquire new spacers, transcribe them, and use them to cut the invading DNA. To develop CRISPR immunity, bacteria must be safely exposed to phage DNA. Phage infection releases environmental DNA (eDNA) which could be involved in the development of CRISPR immunity. Using Streptococcus thermophilus and phages 2972 and 858 as a model for CRISPR immunity, we show that eDNA is involved in CRISPR immunity, as generation of phage-immune bacterial colonies decreases with eDNA digestion. Furthermore, it is phage eDNA specifically that impacts CRISPR immunity since only its addition increases the generation of phage-immune colonies. We also show that the effect of eDNA is phage-specific, sequence-specific, and can even be traced to a region of the genome covering the early-expressed genes, which differ between phages 2972 and 858. However, we also show that eDNA is not used as a source of genetic information for spacer acquisition. These results link eDNA to the CRISPR-Cas system, providing a better understanding of the context of the emergence of CRISPR immunity and could inform our understanding of the mechanisms through which bacteria detect phage infection.IMPORTANCEHow can a bacterial adaptive immune system (the CRISPR-Cas system) exist at all, when exposure to a virulent phage is so consistently lethal? We proposed that bacteria might actively sample their genetic environment for phage DNA through natural competence. In testing this hypothesis, we revealed that free phage DNA is important to CRISPR immunity-but not as the source of the immunological memory.

The Hxk1 protein of Candida albicans phosphorylates N-acetylglucosamine (GlcNAc) which is necessary for various cellular functions, including energy production and chitin synthesis. Further, this protein also regulates morphogenesis independently of its role in GlcNAc catabolism. When HXK1 is deleted, cells are hyperfilamentous on serum-containing medium. Furthermore, Hxk1 translocates to the nucleus in the presence of glucose. To gain a broad understanding of the effect of Hxk1 on gene expression in C. albicans, we performed genome-wide transcriptional profiling of the hxk1 mutant strain by RNA-Seq. The analysis of these RNA-Seq data showed that Hxk1 affects gene expression in both a carbon source-dependent and -independent manner. However, the effect on gene expression occurs via an indirect mechanism, as genome-wide CUT&RUN binding experiments demonstrated that Hxk1 does not bind to the upstream intergenic regions of the differentially expressed genes. Deletion of HXK1 not only resulted in differences in gene expression of genes present in the GlcNAc and galactose regulons, but also in glucose transporter genes, including HGT13. Hxk1 also negatively influences the expression of virulence-associated genes, including HWP1, BRG1, and UME6. Consequently, an hxk1 mutant strain showed higher toxicity toward gut epithelial cells compared to the WT strain. Furthermore, the hxk1 mutant strain had higher expression levels of SOD4 and SOD5 and showed higher resistance toward H₂O₂. These findings highlight the multiple functions of Hxk1 in different cellular processes.IMPORTANCECandida albicans is a fungus that lives in the human body but does not cause any harm in healthy individuals. However, when the immune system is weakened, C. albicans can spread via the bloodstream all over the body and can lead to severe illness and even death. To infect the human body, multiple proteins hold distinct functions. Hxk1 is one of these proteins. This protein is involved in N-acetylglucosamine (GlcNAc) phosphorylation, as well as hyphae formation and glucose transport. To obtain a complete view of the processes regulated by Hxk1, we performed an RNA-Seq experiment. These data revealed that Hxk1 influences the regulation of genes involved in metabolic and virulence-related processes, such as GlcNAc metabolism, sterol metabolism, and oxidative stress resistance. These findings are important to better understand how C. albicans adapted itself to infect the host.

Viruses of microorganisms impact microbial population dynamics, community structure, nutrient cycling, gene transfer, and genomic innovation. In wetlands, root-associated microbial communities mediate key biogeochemical processes important for plants involved in ecosystem maintenance. Nonetheless, the presence and role of microbial viruses in salt marshes remain poorly understood. In this study, we analyzed 24 metagenomes retrieved from the root zone of Spartina alterniflora, a foundation plant in salt marshes of the eastern and Gulf coasts of the U.S. The samples span three plant compartments-bulk sediment, rhizosphere, and root-and two cordgrass plant phenotypes: short and tall. We observed differentiation between phenotypes and increased similarity in viral communities between the root and rhizosphere, indicating that plant compartment and phenotype shape viral community composition. The majority of viral populations characterized are novel at the genus level, with a subset predicted to target microorganisms known to carry out key biogeochemical functions. The findings contribute to ongoing efforts to understand plant-associated viral diversity and community composition and to identify potential targets for exploring viral modulation of microbially mediated ecosystem functioning in intertidal wetlands.IMPORTANCESalt marshes are vital coastal ecosystems. Microbes in these environments drive nutrient cycling and support plant health, with Spartina alterniflora serving as a foundation species. This study explores viral communities associated with S. alterniflora, revealing how plant compartments and phenotypes shape viral composition. The discovery of numerous novel viruses, some potentially influencing microbes involved in key biogeochemical processes, highlights their ecological significance. Given the increasing pressures on coastal ecosystems, understanding virus-microbe-plant interactions is essential for predicting and managing ecosystem responses to environmental change.

Many fungal species can utilize N-acetylglucosamine (GlcNAc) as a carbon source. Studies in the pathogenic yeast Candida albicans have revealed that GlcNAc utilization and the induction of GlcNAc catabolic genes depend on the Ndt80 family transcription factor CaRep1 and the histone acetyltransferase CaNgs1. Additionally, GlcNAc induces filamentation via both alkalinization of the medium and CaNgs1 signaling. However, the roles of YlRep1 and YlNgs1 in GlcNAc catabolism and filamentous growth are not clear in the dimorphic yeast Yarrowia lipolytica. In this study, we demonstrate that YlRep1 and YlNgs1 are essential for the induction of GlcNAc catabolic genes in Y. lipolytica, which is similar to the function of CaRep1 and CaNgs1. YlRep1 and YlNgs1 interact physically and exhibit transcriptional activation activity on a reporter gene. Interestingly, unlike in C. albicans, GlcNAc inhibits filamentation in Y. lipolytica. This inhibition requires YlRep1-YlNgs1 but does not depend on the alteration of ambient pH. We show that YlRep1 and YlNgs1 co-repress a set of transcription factor and cell wall protein genes, some of which are associated with filamentation. Notably, this repression is independent of GlcNAc catabolism but requires the GlcNAc kinase, YlNag5.IMPORTANCEGlcNAc has been used previously to induce filamentation in Yarrowia lipolytica, but often in combination with a citrate buffer at near-neutral pH. The exact role of GlcNAc in regulating filamentous growth is unclear. In this study, we report that GlcNAc inhibits rather than promotes filamentation in Y. lipolytica, and this function does not require GlcNAc catabolism or the alteration of ambient pH by GlcNAc catabolism. We show that YlRep1-YlNgs1 signaling, which activates GlcNAc catabolic genes, represses a set of filamentation-related genes and is a key regulator in the inhibition of filamentation by GlcNAc. This finding indicates that YlRep1-YlNgs1 has dual roles, functioning both in the activation of GlcNAc catabolic genes and the repression of filamentation-related genes in response to GlcNAc. These findings provide new insights into the regulatory mechanisms of GlcNAc catabolism and signaling in Y. lipolytica.

Candida auris is an emerging fungus notable for its high drug resistance and persistent colonization of human hosts and environmental surfaces. However, its role in vulvovaginal candidiasis (VVC), a common form of superficial candidiasis, remains poorly understood. In this study, we investigated the colonization capacity of C. auris and vaginal defense mechanisms in a VVC model. Using an estrogenized VVC mouse model, we evaluated fungal burden, inflammatory cell counts, and S100A8 concentrations in vaginal lavages of wild-type (WT) and IL-17A knockout (Il17a^-/-) C57BL/6J mice following C. auris inoculation. Histopathological examination and flow cytometry analysis of vaginal immune cells were also conducted. Additionally, an in vitro adhesion assay was performed using VK2/E6E7 vaginal epithelial cells under aerobic and microaerobic conditions mimicking the vaginal environment. Persistent colonization by C. auris, particularly clades I, III, and IV, with minimal infiltration of inflammatory cells, was confirmed in Il17a^-/- mice. These findings were also supported by histopathological analysis. S100A8 concentration analysis revealed significant differences between WT and Il17a^-/- mice, with lower levels detected in the Il17a^-/- group. Furthermore, S100A8 levels showed positive correlations with inflammatory cell count and negative correlations with vaginal fungal burden. Flow cytometry analysis demonstrated a reduced number of vaginal neutrophils in Il17a^-/- mice. Additionally, in vitro adhesion assay revealed increased C. auris adherence to vaginal epithelial cells under microaerobic conditions. C. auris exhibits a strong affinity for the vaginal epithelium, and IL-17A appears to play a protective role in C. auris-associated VVC.

Importance: Candida auris is an emerging fungal species, and several reports have recently identified C. auris in patients with vulvovaginal candidiasis (VVC), although few studies have investigated the relationship between C. auris and VVC or the associated host factors. Our study, using the VVC mouse model, confirmed persistent vaginal colonization by C. auris, especially clades I, III, and IV, along with reduced neutrophil infiltration and lower S100A8 secretion under interleukin-17A-deficient conditions. In addition, in vitro assays demonstrated enhanced C. auris adhesion to vaginal epithelial cells, especially microaerobic conditions imitating human vaginal microenvironments. Our findings suggest that C. auris exhibits strong vaginal tropism, and IL-17A plays a critical role in controlling C. auris-associated VVC.

The management of tuberculosis (TB), particularly drug-resistant variants, presents enduring clinical challenges characterized by complex therapeutic regimens, prolonged treatment durations, suboptimal success rates, and significant adverse effects, issues that have persisted as critical concerns in global healthcare. Current TB drug development predominantly focuses on novel compounds and combination therapies targeting pathogen-specific pathways while overlooking the influence of different drugs on host immunity, which is indeed a key factor affecting treatment-related tissue damage and treatment time. In this study, we evaluated the effects of important anti-TB drugs and candidate drugs on host innate immunity and found that PBTZ169 showed potent innate immunity activator, which is a promising drug for the treatment of drug-sensitive and -resistant TB. The expression of cytokines and type I interferon was strongly upregulated by PBTZ169 under lipopolysaccharide (LPS) stimulation and PBTZ169-resistant strain infection, and the innate immune activation enhanced antibacterial activity in macrophages. Mechanistically, PBTZ169 upregulated the NF-kB and MAPK signaling pathways by activating the phosphorylation of TAK1. TAK1 knockdown abrogated PBTZ169-mediated immune activation and antibacterial effects. We thus demonstrate for the first time that PBTZ169 up-regulates NF-κB and MAPK innate immune signaling pathways via activating TAK1 phosphorylation, which may inform clinical deployment strategies and patient selection.IMPORTANCEMaintaining immune homeostasis is paramount for efficient Mycobacterium tuberculosis (Mtb) clearance and tissue repair. Current therapeutic strategies, however, predominantly focus on achieving maximal bacterial suppression within compressed timelines while overlooking the immunomodulatory consequences of anti-tuberculosis agents. This critical knowledge gap underscores the urgent need for mechanistic investigations to establish evidence-based frameworks for optimizing drug combinations and integrating therapies with host-directed approaches.

Actin is highly conserved across eukaryotes. This versatile protein builds cytoskeletal networks central to diverse cellular processes, including cell division and cell motility. The most potent and broadly used reagents to detect polymerized actin distribution in fixed cells are fluorescently conjugated derivatives of the basidiomycete-derived toxin, phalloidin. However, despite its conservation, actin in many ascomycete fungi fails to bind phalloidin. Here, we trace the failure to bind phalloidin to a single amino acid change in a phalloidin-binding residue in actin. Reverting this change in the fungi Aureobasidium pullulans and Aspergillus nidulans by introducing the point mutation act1^V75I at the native ACT1 locus confers phalloidin binding while retaining actin function. This strategy should enable characterization of F-actin in a wider range of fungi.IMPORTANCEHigh-resolution tools to visualize filamentous actin networks are critical to the investigation of organisms' cell biology. The gold standard tool is fluorescent phalloidin, a mushroom toxin. However, several fungi have actin that fails to stain with phalloidin. Here, we describe a way to reverse that failure, rendering the invisible actin visible.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that causes healthcare-, community-, and livestock-associated infections. The methicillin resistance gene mecA is embedded in the mobile genetic element termed Staphylococcal Cassette Chromosome (SCCmec). SCCmec is shared among staphylococci inhabiting human and animal hosts, which are recognized epidemiologically as the genetic reservoir of SCCmec. However, the ability of diverse methicillin-resistant staphylococci (MRS) to serve as SCCmec donors for S. aureus has not been tested experimentally. Here, we investigated the ability of 157 MRS isolates from pets, meat, livestock, and humans to transfer SCCmec to methicillin-sensitive S. aureus strains using a recently developed natural transformation protocol in mixed biofilms. We found that 25 out of 157 isolates were able to transfer SCCmec to S. aureus. The most effective donor species were S. epidermidis (~33% of the tested isolates), S. felis (40%), and S. capitis (30%). Isolates from meat and livestock (collected in Vietnam and Thailand) had lower transfer rates of SCCmec (5% and 3%, respectively), compared to human and pet isolates from Japan (35% and 25%, respectively). The SCCmec transfer depended on site-specific integration/excision mediated by an intact attB site, which is recognized by the SCC recombinase Ccr. Our study experimentally demonstrates the presence of SCCmec donors in our living environments, highlighting the importance of specific staphylococcal species.IMPORTANCEHow MRSA emerges has long been the pivotal question regarding the ever-increasing burden of antimicrobial resistance (AMR) issues for over half a century. Extensive research efforts in bacteriology, epidemiology, genome biology, and healthcare fields have led to the common understanding that SCCmec is transmitted among distinct staphylococcal species. However, global efforts to provide empirical evidence for intercellular SCCmec transmission have yielded limited results. We recently established the mixed-biofilm transformation assay to evaluate intercellular and interspecies SCCmec transmission. This novel assay system allows us to gain insight into the question "How MRSA emerges," and here, we provide the first experimental results about the potential donor species and habitats. This is the first report to show the ability of staphylococci from distinct sources to transfer SCC to S. aureus. Moreover, the new finding of S. felis as an effective donor that is not commensal to humans reinforces the importance of the One Health concept.