mBio最新文献

The persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a key obstacle for HBV cure. This study aims to comprehensively assess the effect of interferon (IFN) and small-interfering RNA (siRNA) combination on the cccDNA minichromosome. Utilizing both cell and mouse cccDNA models, we compared the inhibitory effects of IFNα, siRNA, and their combination on cccDNA activity and assessed its epigenetic state. IFNα2 treatment alone reduced HBV RNAs, HBeAg, and HBsAg levels by approximately 50%, accompanied by a low-level reconstitution of SMC5/6-a chromatin modulator that restricts cccDNA transcription. HBx-targeting siRNA (siHBx) achieved significant suppression of viral antigens and reconstitution of SMC5/6, but this effect could be reversed by the deacetylase inhibitor Belinostat. The combination of IFN with siHBx resulted in over 95% suppression of virological markers, reduction in epigenetic activation modifications (H3Ac and H4Ac) on cccDNA, and further reduced cccDNA accessibility, with the effect not reversible by Belinostat. In an extracellular humanized IFNAR C57BL/6 mouse model harboring recombinant cccDNA, the effect of combination of clinically used pegylated IFNα2 and GalNac-siHBx was further clarified, indicating a higher and more durable suppression of cccDNA activity compared to either therapy alone. In conclusion, the combination of IFNα and siRNA achieves a more potent and durable epigenetic inhibition of cccDNA activity in cell and mouse models, compared to monotherapy. These findings deepen the understanding of cccDNA modulation and strengthen the scientific basis for the potential of combination therapy.

Importance: Since there are currently no approved drugs targeting and silencing covalently closed circular DNA (cccDNA), achieving a "functional cure" remains difficult. This study aims to comprehensively compare the effects of IFNα, small-interfering RNA targeting hepatitis B virus (HBV), and their combination on the activity, accessibility, and epigenetic modifications of cccDNA minichromosomes in cell models. A more durable and stable inhibition of HBV RNAs and antigens expression by IFNα and HBx-targeting siRNA (siHBx) synergy was observed, associated with augmented epigenetic repression of the cccDNA minichromosome. Besides, in an extracellular humanized IFNAR mouse model harboring recombinant cccDNA with an intact response to human IFNα, the synergistic effect of clinically used pegylated IFNα2 and in-house-developed GalNac-siHBx was further clarified.

Nectin cell adhesion molecule 1 (NECTIN1) is a cell adhesion molecule that belongs to the immunoglobulin superfamily. It has been considered the most ubiquitous receptor for herpesviruses. However, in the context of flavivirus infection, its role was previously unknown. In this study, we described an arrayed siRNA screen mainly targeting Ig-like proteins that showed NECTIN1-restricted bovine viral diarrhea virus (BVDV) infection. We demonstrated that the depletion of NECTIN1 could significantly enhance the infection of both biotypes and multiple genotypes of BVDV, including BVDV-1a, -1b, -1c, -1p, -1m, -1v, and -2a. Notably, the IgV of NECTIN1 has emerged as the key domain restricting BVDV infection. Moreover, NECTIN1 inhibited BVDV attachment without exerting a significant influence on BVDV translation or transcription. Furthermore, we demonstrated that both NECTIN1 and CD46 could bind to BVDV E2, while the binding affinity of NECTIN1 for BVDV E2 was greater than that for CD46. We further identified that the BVDV E2 domain DD was a key domain of BVDV interacting with NECTIN1. In addition, we showed that NECTIN1 inhibited infections by classical swine fever virus (CSFV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), which belong to the Flaviviridae family, but had limited effects on bluetongue virus (BTV), vesicular stomatitis virus (VSV), Akabane virus (AKAV), and Sindbis virus (SINV). Overall, our study has important implications for understanding the entry of BVDV and revealed a novel role for NECTIN1 as a restriction factor that inhibits flavivirus infection.

Importance: NECTIN1, also known as CD111 or PVRL1, has been recognized as the primary receptor for several alpha herpesviruses, including herpes simplex virus (HSV), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1). However, our study revealed a novel role for NECTIN1 in the virus life cycle by influencing BVDV infection. Contrary to its role as a receptor for alpha herpesviruses, NECTIN1 acts as a restriction factor for BVDV by inhibiting viral attachment via competition with CD46 for binding to the domain DD of BVDV E2. We further revealed that the replication of members of the Flaviviridae family was inhibited by NECTIN1, while the replication of other RNA viruses did not significantly differ. Our results demonstrate that NECTIN1 is a novel factor restricting Flaviviridae family virus replication and highlight the complexity of virus-host interactions and the multifaceted nature of host factors involved in viral infection.

Salmonella is a common causative agent of infectious intestinal and systemic disease and has been extensively studied for several decades. Yet, much of Salmonella pathogenicity remains a mystery due in part to the highly complex virulence and adaptation strategies at the pathogen's disposal. One of the more influential tools within the field, an attenuated aroA-deficient Salmonella strain, has been used for many years to probe the host immune response that would otherwise be impossible with a fully virulent strain. Now, new work by Rooke et al. (J. L. Rooke, E. C. A. Goodall, K. Pullela, R. Da Costa, et al., mBio 15:e03319-23, 2024, https://doi.org/10.1128/mbio.03319-23) utilizes in-depth transposon-directed insertion-site sequencing to elucidate the contribution of genes to Salmonella fitness within isogenic wild-type and aroA-deficient strains. Specifically, Rooke et al. demonstrate that the deletion of the aroA gene leads to iron-dependent membrane instability, raising several exciting new ideas surrounding Salmonella biology and therapeutic strategies.

Bordetella pertussis is the causative agent of whooping cough in humans, a disease that has recently experienced a resurgence. In contrast, Bordetella bronchiseptica infects the respiratory tract of various mammalian species, causing a range of symptoms from asymptomatic chronic carriage to acute illness. Both pathogens utilize type III secretion system (T3SS) to deliver the effector protein BteA into host cells. Once injected, BteA triggers a cascade of events leading to caspase 1-independent necrosis through a mechanism that remains incompletely understood. We demonstrate that BteA-induced cell death is characterized by the fragmentation of the cellular endoplasmic reticulum and mitochondria, the formation of necrotic balloon-like protrusions, and plasma membrane permeabilization. Importantly, genome-wide CRISPR-Cas9 screen targeting 19,050 genes failed to identify any host factors required for BteA cytotoxicity, suggesting that BteA does not require a single nonessential host factor for its cytotoxicity. We further reveal that BteA triggers a rapid and sustained influx of calcium ions, which is associated with organelle fragmentation and plasma membrane permeabilization. The sustained elevation of cytosolic Ca²⁺ levels results in mitochondrial calcium overload, mitochondrial swelling, cristolysis, and loss of mitochondrial membrane potential. Inhibition of calcium channels with 2-APB delays both the Ca²⁺ influx and BteA-induced cell death. Our findings indicate that BteA exploits essential host processes and/or redundant pathways to disrupt calcium homeostasis and mitochondrial function, ultimately leading to host cell death.IMPORTANCEThe respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica exhibit cytotoxicity toward a variety of mammalian cells, which depends on the type III secretion effector BteA. Moreover, the increased virulence of B. bronchiseptica is associated with enhanced expression of T3SS and BteA. However, the molecular mechanism underlying BteA cytotoxicity is elusive. In this study, we performed a CRISPR-Cas9 screen, revealing that BteA-induced cell death depends on essential or redundant host processes. Additionally, we demonstrate that BteA disrupts calcium homeostasis, which leads to mitochondrial dysfunction and cell death. These findings contribute to closing the gap in our understanding of the signaling cascades targeted by BteA.

Many antibiotics that are used in healthcare, farming, and aquaculture end up in environments with different spatial structures that might promote heterogeneity in the emergence of antibiotic resistance. However, the experimental evolution of microbes at sub-inhibitory concentrations of antibiotics has been mainly carried out at the population level which does not allow capturing single-cell responses to antibiotics. Here, we investigate and compare the emergence of resistance to ciprofloxacin in Escherichia coli in well-mixed and structured environments using experimental evolution, genomics, and microfluidics-based time-lapse microscopy. We discover that resistance to ciprofloxacin and cross-resistance to other antibiotics is stronger in the well-mixed environment due to the emergence of target mutations, whereas efflux regulator mutations emerge in the structured environment. The latter mutants also harbor sub-populations of persisters that survive high concentrations of ciprofloxacin that inhibit bacterial growth at the population level. In contrast, genetically resistant bacteria that display target mutations also survive high concentrations of ciprofloxacin that inhibit their growth via population-level antibiotic tolerance. These resistant and tolerant bacteria keep doubling while shrinking in size in the presence of ciprofloxacin and regain their original size after antibiotic removal, which constitutes a newly discovered phenotypic response. This new knowledge sheds light on the diversity of strategies employed by bacteria to survive antibiotics and poses a stepping stone for understanding the link between mutations at the population level and phenotypic single-cell responses.

Importance: The evolution of antimicrobial resistance poses a pressing challenge to global health with an estimated 5 million deaths associated with antimicrobial resistance every year globally. Here, we investigate the diversity of strategies employed by bacteria to survive antibiotics. We discovered that bacteria evolve genetic resistance to antibiotics while simultaneously displaying tolerance to very high doses of antibiotics by doubling while shrinking in size.

Acarbose is a type 2 diabetes medicine that prevents dietary starch breakdown into glucose by inhibiting host amylase and glucosidase enzymes. Numerous gut species in the Bacteroides genus enzymatically break down starch and change in relative abundance within the gut microbiome in acarbose-treated individuals. To mechanistically explain this observation, we used two model starch-degrading Bacteroides, Bacteroides ovatus (Bo), and Bacteroides thetaiotaomicron (Bt). Bt growth on starch polysaccharides is severely impaired by acarbose, whereas Bo growth is much less affected by the drug. The Bacteroides use a starch utilization system (Sus) to grow on starch. We hypothesized that Bo and Bt Sus enzymes are differentially inhibited by acarbose. Instead, we discovered that although acarbose primarily targets the Sus periplasmic GH97 enzymes in both organisms, the drug affects starch processing at multiple other points. Acarbose competes for transport through the TonB-dependent SusC proteins and binds to the Sus transcriptional regulators. Furthermore, Bo expresses a non-Sus GH97 (BoGH97D) when grown in starch with acarbose. The Bt homolog, BtGH97H, is not expressed in the same conditions, nor can overexpression of BoGH97D complement the Bt growth inhibition in the presence of acarbose. This work informs us about unexpected complexities of Sus function and regulation in Bacteroides, including variation between related species. Furthermore, this indicates that the gut microbiome may be a source of variable response to acarbose treatment for diabetes.

Importance: Acarbose is a type 2 diabetes medication that works primarily by stopping starch breakdown into glucose in the small intestine. This is accomplished by the inhibition of host enzymes, leading to better blood sugar control via reduced ability to derive glucose from dietary starches. The drug and undigested starch travel to the large intestine where acarbose interferes with the ability of some bacteria to grow on starch. However, little is known about how gut bacteria interact with acarbose, including microbes that can use starch as a carbon source. Here, we show that two gut species, Bacteroides ovatus (Bo) and Bacteroides thetaiotaomicron (Bt), respond differently to acarbose: Bt growth is inhibited by acarbose, while Bo growth is less affected. We reveal a complex set of mechanisms involving differences in starch import and sensing behind the different Bo and Bt responses. This indicates the gut microbiome may be a source of variable response to acarbose treatment for diabetes via complex mechanisms in common gut microbes.

Staphylococcus epidermidis, a common commensal bacterium, is a leading cause of nosocomial catheter-associated bloodstream infections. S. epidermidis sequence type 2 (ST2) is specifically recognized globally for causing invasive disease. In this study, we identified a novel putative integrated conjugative element, pICE-Sepi-ST2, unique to the genomes of S. epidermidis ST2. Our investigation identified pICE-Sepi-ST2 in all ST2 isolates from bloodstream infections. Meanwhile, ST2 isolates from other infection sources, such as catheters, prosthetic joints, and fracture fixations, showed variable pICE-Sepi-ST2 prevalence. pICE-Sepi-ST2 encodes two putative cell wall anchored proteins that we have designated SesX and SesY. Biochemical characterization of SesY revealed that it binds both plasminogen (Plg) and plasmin (Pln) and inhibits Pln's ability to cleave a chromogenic substrate and degrade fibrin clots. Furthermore, all ST2 isolates containing a pICE-Sepi-ST2 also have a mutated sdrG gene. Thus, all ST2 isolates have two genetic modifications that target distinct steps in the hemostatic pathway. SdrG, which inhibits coagulation, is inactivated, and SesY, which inhibits fibrin, is introduced. These findings suggest that the hemostasis pathway is a strategic target for ST2 S. epidermidis bloodstream pathogenesis.

Importance: This study uncovers a new virulence mechanism in Staphylococcus epidermidis ST2 bloodstream isolates. We identify a mobile genetic element (MGE) characteristic of an integrated conjugated element (ICE). pICE-Sepi-ST2 carries the genetic information needed to produce a cell wall-anchored (CWA) protein called SesY. The results indicate that SesY binds to plasminogen (Plg) and plasmin (Pln) and inhibits Pln's degradation of fibrin clots. Genetic analysis showed that all ST2 bloodstream isolates can express the plasmin inhibitor SesY and carry a mutation in the SdrG gene, resulting in the expression of inactive SdrG. Thus, we describe a molecular pathway targeting the coagulation pathway that may be required for S. epidermidis ST2 to cause bloodstream infections.

The acquisition of new capabilities by horizontal gene transfer (HGT) shapes the distribution of traits during microbial diversification. In the Chlorophyll (Chl) d-producing cyanobacterium Acaryochloris marina, the genes involved in the production and disassembly of the light-harvesting phycobiliprotein phycocyanin (PC) were lost in the A. marina common ancestor but then subsequently regained via HGT in A. marina strain MBIC11017. However, it remains unknown how the HGT-acquired PC genes in MBIC11017 have been reintegrated into its existing regulatory network after tens of millions of years since their loss. Here, we investigated potential mechanisms of regulatory assimilation of PC genes by comparing the transcriptomes of A. marina strain MBIC11017 and a PC-lacking close relative under both low irradiance far-red light and high irradiance white light. We found that PC assembly and degradation processes have been re-assimilated into a conserved ancestral response to high light. Further, we identified putative regulatory elements that were likely co-transferred with PC genes and could be recognized by A. marina's pre-existing light response machinery. This study offers insights into how HGT-acquired genes can be reintegrated into an existing transcriptional regulatory network that has evolved in their absence.IMPORTANCEHorizontal gene transfer, the asymmetric movement of genetic information between donor and recipient organisms, is an important mechanism for acquiring new traits. In order for newly acquired gene content to be retained, it must be integrated into the genetic repertoire and regulatory networks of the recipient cell. In a strain of the Chlorophyll d-producing cyanobacterium Acaryochloris marina, the recent reacquisition of the genes required to produce the light-harvesting pigment phycocyanin offers a rare opportunity to understand the mechanisms underlying the regulatory assimilation of an acquired complex trait in bacteria. The significance in our research is in characterizing how an ancestrally lost, complex trait can be reintegrated into a conserved regulatory network, even after millions of years.

Pathogenic fungi pose a significant threat to human health, especially given the rising incidence of invasive fungal infections and the emergence of drug-resistant strains. This requires the development of vaccines and the advancement of antifungal strategies. Recent studies have focused on the roles of fungal extracellular vesicles (EVs) in intercellular communication and host-pathogen interactions. EVs are nanosized, lipid membrane-bound particles that facilitate the transfer of proteins, lipids, and nucleic acids. Here, we review the multifaceted functions of EVs produced by different human fungal pathogens, highlighting their importance in the response of fungal cells to different environmental cues and their interactions with host immune cells. We summarize the current state of research on EVs and how leveraging this knowledge can lead to innovative approaches in vaccine development and antifungal treatment.

The polysaccharide hyaluronan (HA) is an important component of lung extracellular matrix that increases following infection with influenza or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hellman et al. (U. Hellman, E. Rosendal, J. Lehrstrand, J. Henriksson, et al., mBio 15:e01303-24, https://doi.org/10.1128/mbio.01303-24) show that fragmented HA accumulates in the lungs of coronavirus disease 2019 (COVID-19) patients, with systemic levels of HA being associated with reduced lung function 3-6 months after infection. This study provides novel insights into HA's role in COVID-19 pathology and its potential utility as a biomarker for disease severity. However, much remains to be understood about the lung HA matrix in COVID-19 and how it compares to other lung conditions. In particular, the role of HA-binding proteins in organizing HA into a crosslinked network is yet to be fully determined at a molecular level. This knowledge is crucial in understanding the inter-relationships between the structure of the HA matrix and the regulation of the immune response, and thus our ability to target HA therapeutically for improved outcomes in COVID-19.