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Following two human infections with the H10N3 avian influenza virus (AIV) in 2021 and 2022, a third case was discovered in Yunnan, China, in 2024, raising concerns about the potential for future pandemics. Recent studies have indicated that novel H10N3 viruses are highly pathogenic in mice and can be transmitted between guinea pigs via respiratory droplets without prior adaptation. However, the biological characteristics of novel H10N3 in poultry have not been fully elucidated. Our findings revealed that H10 subtype AIVs are predominantly prevalent in waterfowl. Notably, H10N8 and H10N3 viruses that have infected humans were primarily isolated from chickens. For the first time, double basic hemagglutinin cleavage sites (motif PEIKQGR↓GL) were identified in novel H10N3 AIVs, which exhibit enhanced replication in chickens, and can be transmitted between chickens through direct contact and respiratory droplets. Animal experimental studies demonstrated that ducks are also susceptible to H10N3 viruses and that the virus is transmissible through direct contact, suggesting a greater risk of transmission and recombination. Serological studies conducted among poultry workers suggest that while the human population was largely naïve to H10N3 infection, sporadic and undetected human infections did occur, indicating a potential increasing trend. These data further emphasize the growing threat to public health posed by zoonotic H10N3 subtype AIVs.IMPORTANCEExposure to poultry in live poultry markets (LPMs) is strongly associated with human infection with avian influenza viruses (AIVs), with chickens being the most common species found in these markets in China. The prevalence of AIVs in chickens, therefore, increases the risk of human infection. Notably, the main host of the novel H10N3 virus has shifted from waterfowl to chickens, and the virus can be transmitted between chickens via respiratory droplets, posing a potential risk of a pandemic within poultry populations. The novel H10N3 virus also remains sensitive to ducks and can be transmitted through direct contact, which means a greater risk of transmission and recombination. Significantly, the human population remains largely naïve to H10N3 infection, but sporadic seropositivity among poultry workers indicates previous exposure to H10 subtype AIVs. Therefore, a comprehensive surveillance of the novel H10N3 viruses in poultry is imperative. Effective control of the virus within poultry populations could significantly reduce the risk of emerging human infections.

Previous studies have shown that E6 interacts with the E6-associated protein (E6AP) ubiquitin-protein ligase and directs its ubiquitylation activity toward several specific cellular proteins, one of the most important of which is p53. Interestingly, E6AP not only aids in the E6-directed degradation of cellular substrates but also stabilizes the E6 protein by protecting it from proteasome-mediated degradation. However, there is no information available about the ubiquitin ligases that regulate the stability and activity of the human papillomavirus (HPV) E6 oncoprotein in the absence of E6AP. Therefore, to identify these novel ubiquitin ligases, we performed high-throughput human siRNA library screen against ubiquitin ligases in clustered regularly interspaced palindromic repeat (CRISPR)-edited E6AP-knockout human embryonic kidney (HEK) 293 cells, stably expressing green fluorescent protein (GFP)-tagged HPV-18E6. We found a number of ubiquitin ligases that increase the expression of GFP-tagged 18E6 upon their knockdown in the absence of E6AP. Upon validation of the interaction of 18E6 with these ubiquitin ligases in cervical cancer-derived cell lines, we found that the knockdown of ubiquitin ligase F-box protein 4 (FBXO4), together with E6AP knockdown, leads to a dramatic increase in the levels of endogenous HPV-18E6 oncoprotein. Furthermore, our data demonstrate that the combined knockdown of FBXO4 and E6AP not only rescues the protein levels of E6 but also induces high levels of cell death in a p53-dependent manner in the HPV-positive cervical cancer cell line, HeLa. These results indicate a close interplay between FBXO4, E6AP, and p53 in the regulation of cell survival in HPV-positive cervical tumor-derived cells.

Importance: E6-associated protein (E6AP)-mediated stabilization of human papillomavirus (HPV) E6 plays a crucial role in the development and progression of cervical and other HPV-associated cancers. This study, for the first time, identifies a novel ubiquitin ligase, FBXO4 that targets the degradation of HPV E6 oncoprotein in the absence of E6AP in cervical cancer-derived cell lines. This may have significant implications for our understanding of HPV-associated cancers by providing deeper insights into the intricate interplay between viral proteins and host cellular machinery and the development of targeted therapies.

Type VI secretion system (T6SS) is utilized by many Gram-negative bacteria to eliminate competing bacterial species and manipulate host cells. Acinetobacter baumannii ATCC 17978 utilizes T6SS at the expense of losing pAB3 plasmid to induce contact-dependent killing of competitor microbes, resulting in the loss of antibiotic resistance carried by pAB3. However, the regulatory network associated with T6SS in A. baumannii remains poorly understood. Here, we identified an Mn²⁺-dependent post-transcriptional regulation of T6SS mediated by a bonafide small RNA, AbsR28. A. baumannii utilizes MumT, an Mn²⁺-uptake inner membrane transporter, for the uptake of extracellular Mn²⁺ during oxidative stress. We demonstrate that the abundance of intracellular Mn²⁺ enables complementary base pairing of AbsR28-tssM mRNA (that translates to TssM, one of the vital inner membrane components of T6SS), inducing RNase E-mediated degradation of tssM mRNA and resulting in T6SS repression. Thus, AbsR28 mediates a crosstalk between MumT and T6SS in A. baumannii.IMPORTANCESmall RNAs (sRNAs) are identified as critical components within the bacterial regulatory networks involved in fine regulation of virulence-associated factors. The sRNA-mediated regulation of type VI secretion system (T6SS) in Acinetobacter baumannii was unchartered. Previously, it was demonstrated that A. baumannii ATCC 17978 cells switch from T6- to T6+ phenotype, resulting in the loss of antibiotic resistance conferred by plasmid pAB3. Furthermore, the derivatives of pAB3 found in recent clinical isolates of A. baumannii harbor expanded antibiotic resistance genes and multiple determinants for virulence factors. Hence, the loss of this plasmid for T6SS activity renders A. baumannii T6+ cells susceptible to antibiotics and compromises their virulence. Our findings show how A. baumannii tends to inactivate T6SS through an sRNA-mediated regulation that relies on Mn²⁺ and retains pAB3 during infection to retain antibiotic resistance genes carried on the plasmid.

The unfolded protein response (UPR) is a cell-autonomous stress response aimed at restoring homeostasis due to the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses often hijack the host cell machinery, leading to an accumulation of misfolded proteins in the ER. The cell-autonomous UPR is the immediate response of an infected cell to this stress, aiming to restore normal function by halting protein translation, degrading misfolded proteins, and activating signaling pathways that increase the production of molecular chaperones. The cell-non-autonomous UPR involves the spreading of UPR signals from initially stressed cells to neighboring unstressed cells that lack the stressor. Though viruses are known modulators of cell-autonomous UPR, recent advancements have highlighted that cell-non-autonomous UPR plays a critical role in elucidating how local infections cause systemic effects, thereby contributing to disease symptoms and progression. Additionally, by utilizing cell-non-autonomous UPR, viruses have devised novel strategies to establish a pro-viral state, promoting virus spread. This review discusses examples that have broadened the understanding of the role of UPR in virus infections and disease progression by looking beyond cell-autonomous to non-autonomous processes and mechanistic details of the inducers, spreaders, and receivers of UPR signals.

Bacteriophages, known for their ability to kill bacteria, are hampered in their effectiveness because bacteria are able to rapidly develop resistance, thereby posing a significant challenge for the efficacy of phage therapy. The impact of evolutionary trajectories on the long-term success of phage therapy remains largely unclear. Herein, we conducted evolutionary experiments, genomic analysis, and CRISPR-mediated gene editing, to illustrate the evolutionary trajectory occurring between phages and their hosts. Our results illustrate the ongoing "arms race" between a lytic phage and its host, a carbapenem-resistant Klebsiella pneumoniae clinical strain Kp2092, suggesting their respective evolutionary adaptations that shape the efficacy of phage therapy. Specifically, Kp2092 rapidly developed resistance to phages through mutations in a key phage receptor (galU) and bacterial membrane defenses such as LPS synthesis, however, this evolution coincides with unexpected benefits. Evolved bacterial clones not only exhibited increased sensitivity to clinically important antibiotics but also displayed a loss of virulence in an in-vivo model. In contrast, phages evolved under the selection pressure against Kp2092 mutants and exhibited enhanced bacterial killing potency, targeting mutations in phage tail proteins gp12 and gp17. These parallel evolutionary trajectories suggest a common genetic mechanism driving adaptation, ultimately favoring the efficacy of phage therapy. Overall, our findings highlight the potential of phages not only as agents for combating bacterial resistance, but also a driver of evolution outcomes that could lead to more favorable clinical outcomes in the treatment of multidrug resistance pathogens.IMPORTANCECarbapenem-resistant Klebsiella pneumoniae represents one of the leading pathogens for infectious diseases. With traditional antibiotics often being ineffective, phage therapy has emerged as a promising alternative. However, phage predation imposes a strong evolutionary pressure on the rapid evolution of bacteria, challenging treatment efficacy. Our findings illustrate how co-evolution enhances phage lytic capabilities through accumulated mutations in the tail proteins gp12 and gp17, while simultaneously reducing bacterial virulence and antibiotic resistance. These insights advance our understanding of phage-host interactions in clinical settings, potentially inspiring new approaches akin to an "arms race" model to combat multidrug-resistant crises effectively.

Diverse marine animals undergo a metamorphic larval-to-juvenile transition in response to surface-bound bacteria. Although this host-microbe interaction is critical to establishing and maintaining marine animal populations, the functional activity of bacterial products and how they activate the host's metamorphosis program has not yet been defined for any animal. The marine bacterium Pseudoalteromonas luteoviolacea stimulates the metamorphosis of a tubeworm called Hydroides elegans by producing a molecular syringe called metamorphosis-associated contractile structures (MACs). MACs stimulate metamorphosis by injecting a protein effector termed metamorphosis-inducing factor 1 (Mif1) into tubeworm larvae. Here, we show that MACs bind to tubeworm cilia and form visible pores on the cilia membrane surface, which are smaller and less numerous in the absence of Mif1. In vitro, Mif1 associates with eukaryotic lipid membranes and possesses phospholipase activity. MACs can also deliver Mif1 to human cell lines and cause parallel phenotypes, including cell surface binding, membrane disruption, calcium flux, and mitogen-activated protein kinase activation. Finally, MACs can also stimulate metamorphosis by delivering two unrelated membrane-disrupting proteins, MLKL and RegIIIɑ. Our findings demonstrate that membrane disruption by MACs and Mif1 is necessary for Hydroides metamorphosis, connecting the activity of a bacterial protein effector to the developmental transition of a marine animal.

Importance: This research describes a mechanism wherein a bacterium prompts the metamorphic development of an animal from larva to juvenile form by injecting a protein that disrupts membranes in the larval cilia. Specifically, results show that a bacterial contractile injection system and the protein effector it injects form pores in larval cilia, influencing critical signaling pathways like mitogen-activated protein kinase and calcium flux, ultimately driving animal metamorphosis. This discovery sheds light on how a bacterial protein effector exerts its activity through membrane disruption, a phenomenon observed in various bacterial toxins affecting cellular functions, and elicits a developmental response. This work reveals a potential strategy used by marine organisms to respond to microbial cues, which could inform efforts in coral reef restoration and biofouling prevention. The study's insights into metamorphosis-associated contractile structures' delivery of protein effectors to specific anatomical locations highlight prospects for future biomedical and environmental applications.

Pseudovibrio α-Proteobacteria have been repeatedly isolated from marine sponges and proposed to be beneficial to the host. Bacterial motility is known to contribute to host colonization. We have previously identified pseudovibriamides A and B, produced in culture by Pseudovibrio brasiliensis Ab134, and shown that pseudovibriamide A promotes flagellar motility. Pseudovibriamides are encoded in a hybrid nonribosomal peptide synthetase-polyketide synthase gene cluster that also includes several accessory genes. Pseudovibriamide A is a linear heptapeptide and pseudovibriamide B is a nonadepsipeptide derived from pseudovibriamide A. Here, we define the borders of the pseudovibriamides gene cluster, assign function to biosynthetic genes using reverse genetics, and test the hypothesis that pseudovibriamides impact motility by modulating gene transcription. RNA-sequencing transcriptomic analyses of strains having different compositions of pseudovibriamides suggested that both pseudovibriamides A and B affect genes potentially involved in motility, and that a compensatory mechanism is at play in mutants that produce only pseudovibriamide A, resulting in comparable flagellar motility as the wild type. The data gathered suggest that pseudovibriamides A and B have opposite roles in modulating a subset of genes, with pseudovibriamide B having a primary effect in gene activation, and pseudovibriamide A on inhibition. Finally, we observed many differentially expressed genes (up to 29% of the total gene number) indicating that pseudovibriamides have a global effect on transcription that goes beyond motility.IMPORTANCEMarine sponges are found throughout the oceans from tropical coral reefs to polar sea floors, playing crucial roles in marine ecosystems. Pseudovibrio bacteria have been proposed to contribute to sponge health. We have previously shown that pseudovibriamides produced by Pseudovibrio brasiliensis promote bacterial motility, a behavior that is beneficial to bacterial survival and host colonization. The gene cluster that encodes pseudovibriamide biosynthesis is found in two-thirds of Pseudovibrio genomes. This gene cluster is also present in Pseudomonas bacteria that interact with terrestrial plants and animals. Here, we first assign functions to pseudovibriamide biosynthetic genes using reverse genetics. We then show that pseudovibriamides play a major role in transcriptional regulation, affecting up to 29% of P. brasiliensis genes, including motility genes. Thus, this work gives insights into pseudovibriamide biosynthesis and provides evidence that they are signaling molecules relevant to bacterial motility and to other yet-to-be-identified phenotypes.

Candida albicans, the most frequently isolated fungal pathogen in humans, forms biofilms that enhance resistance to antifungal drugs and host immunity, leading to frequent treatment failure. Understanding the molecular mechanisms governing biofilm formation is crucial for developing anti-biofilm therapies. In this study, we conducted a genetic screen to identify novel genes that regulate biofilm formation in C. albicans. One identified gene is ORF19.6693, a homolog of the Saccharomyces cerevisiae SDD3 gene. The sdd3∆/∆ mutant exhibited severe defects in biofilm formation and significantly reduced chitin content in the cell wall. Overexpression of the constitutively active version of the Rho1 GTPase Rho1^G18V, an upstream activator of the protein kinase C (PKC)-mitogen-activated protein kinase (MAPK) cell-wall integrity pathway, rescued these defects. Affinity purification, mass spectrometry, and co-immunoprecipitation revealed Sdd3's physical interaction with Bem2, the GTPase-activating protein of Rho1. Deletion of SDD3 significantly reduced the amount of the active GTP-bound form of Rho1, thereby diminishing PKC-MAPK signaling and downregulating chitin synthase genes CHS2 and CHS8. Taken together, our studies identify a new biofilm regulator, Sdd3, in C. albicans that modulates Rho1 activity through its inhibitory interaction with Bem2, thereby regulating the PKC-MAPK pathway to control chitin biosynthesis, which is critical for biofilm formation. As an upstream component of the pathway and lacking a homolog in mammals, Sdd3 has the potential to serve as an antifungal target for biofilm infections.IMPORTANCEThe human fungal pathogen Candida albicans is categorized as a critical priority pathogen on the World Health Organization's Fungal Priority Pathogens List. A key virulence attribute of this pathogen is its ability to form biofilms on the surfaces of indwelling medical devices. Fungal cells in biofilms are highly resistant to antifungal drugs and host immunity, leading to treatment failure. This study conducted a genetic screen to discover novel genes that regulate biofilm formation. We found that deletion of the SDD3 gene caused severe biofilm defects. Sdd3 negatively regulates the Rho1 GTPase, an upstream activator of the protein kinase C-mitogen-activated protein kinase pathway, through direct interaction with Bem2, the GTPase-activating protein of Rho1, resulting in a significant decrease in chitin content in the fungal cell wall. This chitin synthesis defect leads to biofilm formation failure. Given its essential role in biofilm formation, Sdd3 could serve as an antifungal target for biofilm infections.