mSphere最新文献

The human microbiome varies extensively between individuals. While there are numerous studies investigating the effects of inter-individual differences on microbiome composition, there are few studies investigating inter-individual effects on microbial modulation of the host or host-specific effects. To address this knowledge gap, we colonized human bronchial epithelial air-liquid interface tissue cultures generated from six different adults with one of three phylogenetically diverse bacteria and compared how each microbe differentially modulated host gene expression in each of the six donors. Microbial treatment had the strongest effect on transcription, followed by donor-specific effects. Gene pathways differed markedly in their donor and microbe specificity; interferon expression was highly donor-dependent, while transcription of epithelial barrier and antibacterial innate immunity genes was predominantly microbially driven. Moreover, we evaluated whether microbial regulation of alternative splicing was modulated by the donor. Strikingly, we found significant nonredundant, donor-specific regulation of alternative splicing exclusively in the gram-positive commensal microbes. These findings highlight that microbial effects on the human airway epithelium are not only species-specific but also deeply individualized, underscoring the importance of the host context in shaping microbe-induced transcriptional and splicing responses.IMPORTANCEMicrobiota are integral regulators of host gene expression, utilizing diverse mechanisms that are shaped by the interplay between microbiome composition and inter-individual differences, i.e., host-specific factors. While previous studies have characterized inter-individual variation in microbiome composition and the effects of variable microbiome composition on the host, the extent to which host-specificity itself regulates host-microbe interactions remains poorly understood. In this study, we address this gap by characterizing changes in epithelial gene expression from six different human donors following colonization with one of three phylogenetically diverse bacteria. By systematically comparing donor-specific responses, we demonstrate that host specificity is a key determinant of the host transcriptional response to microbial colonization. Importantly, we demonstrate that the effects of host specificity are not uniform, but instead are dependent on the colonizing microbe. Our findings underscore the complexity of host-microbe relationships and establish host specificity as a significant factor shaping host-microbe interactions.

Tuberculosis (TB) remains a threat for human and livestock health. Mycobacteria causing TB are host-adapted pathogens that occasionally spill over into other species. Mycobacterium bovis causes bovine TB, a well-known zoonosis. Mycobacterium tuberculosis is adapted to humans and can occasionally trigger symptomatic infection in cattle. However, immunocompetent cattle are resistant to experimental infection with M. tuberculosis. Hallmarks of TB in susceptible hosts are organized multicellular tissue lesions termed granulomas. In the absence of suitable in vitro systems that enable investigations of bovine tuberculous granuloma, we developed a three-dimensional granuloma model using bovine leukocytes and magnetic cell labeling. This model was termed the in vitro granuloma-like structure (IVGLS). We generated stable IVGLS resembling TB granulomas at the innate stage, composed of macrophages, and at adaptive stages, containing lymphocytes in addition. M. bovis Bacillus Calmette-Guérin (BCG) replicated within IVGLS and triggered the progression of macrophages to foamy phenotypes. Within the IVGLS, the lymphocytes accelerated BCG-induced apoptotic cell death over time. IVGLS released abundant chemoattractants and Th1-associated cytokines and adopted a glycolytically polarized metabolism. Magnetic bioprinted bovine granulomas recapitulate features of TB granulomas and thus facilitate the study of immune responses to mycobacteria, including spatial and temporal mapping, as well as establishing precise cell death patterns within multicellular microenvironments. Deciphering protective immune responses within IVGLS can contribute to vaccine development for bovine TB, and elucidation of resistance mechanisms can facilitate the design of novel interventions for human TB.

Importance: Mycobacterial infections, including bovine tuberculosis (TB), have a profound impact on global health. This is exemplified by zoonotic TB in humans and animal TB, which is a life-threatening disease in livestock and wildlife. Mycobacteria cause the formation of granulomas, which significantly impact disease progression. Therefore, decoding granulomas is essential for an in-depth understanding of immune responses to mycobacteria. Conventional mouse models frequently fail to develop organized granulomas, and the procurement of samples from granulomatous lesions in cattle and humans is challenging, offering limited insights into the course of infection. Most in vitro TB research is confined to two-dimensional cell cultures, which neglect the spatial characteristics and cellular architecture of granulomas in vivo. To address this gap in knowledge, we have developed a novel multicellular in vitro model for TB. Our spheroid granuloma model, derived from bovine leukocytes using nanotechnologies, offers an adaptable platform for deciphering immune events within granulomas.

Members of the Fusarium solani species complex (FSSC) are major causes of keratitis in humans. The underlying mechanisms leading to this disease are not well understood, partially due to the absence of more complex in vitro infection models. Here, we compared the pathogenicity of keratitis-causing FSSC members F. falciforme, F. keratoplasticum, and F. petroliphilum in a 2D monolayer infection model using a human corneal epithelial cell line and a newly established 3D human cornea infection model which comprises the multilayer epithelium and the stroma. In both models, F. keratoplasticum emerged as the most virulent species, showing extensive invasion and host cell damage and, in rare cases, even inducing the formation of transcellular tunnels. While F. falciforme exhibited strong adhesion to corneal epithelial cells, its capacity for invasion and damage was limited. F. petroliphilum was the least virulent among the FSSC species with low adhesion and invasion. The use of the 3D model allowed the investigation of fungal infection in a more physiologically relevant context and revealed that all three FSSC species disseminated deeper into the cornea than C. albicans under identical conditions. This may partly explain the unfavorable clinical outcome of Fusarium keratitis in patients, as the deep penetration of Fusarium hyphae complicates the accessibility of antifungal drugs to the pathogen. Our data indicate species-specific infection routes which might avoid recognition by the host cell defense system and could contribute to the overall high virulence of the species.IMPORTANCEFusarium keratitis is a rare fungal infection of the human eye. The outcome for affected patients is often poor, with loss of eyesight or even the entire eye being common. Investigation of this disease is challenging due to the absence of established in vitro complex infection models that go beyond a simple 2D monolayer of a single cell type. Here, we performed a comparative analysis of three Fusarium species in a classic 2D infection model and a newly established 3D human cornea model which comprised the epithelium and the stroma. Our experiments revealed that F. keratoplasticum shows a higher potential for invasion and host cell damage when compared to related species. The 3D human cornea model could be a helpful tool for future investigations of fungal pathogenicity and antifungal drug susceptibility during cornea infections.

Inhalation of dust is significant and relevant to health effects. As pollution and climate change worsen in dryland regions, wind currents entrain loose sediment and dust. This potentially disperses toxic geochemical and microbial burdens throughout the region. When inhaled environmental dust and host-associated microbiomes mingle, they pose exposure risks to host respiratory health. The Salton Sea, California's largest lake, is shrinking, thus exposing nearby communities to playa dust. Therefore, we analyze the effect of Salton Sea dust exposure in murine models to relate lung microbial communities and respiratory health. We used an environmental chamber to expose mice to dust filtrate or ambient air and examined the effects of those exposures on lung microbiomes. We found that lung microbial composition varied by dust exposure. Furthermore, dust elicited neutrophil recruitment and immune responses more than mice exposed to ambient air. Sources of dust differentially affected the composition of the lung core microbiome. Lung microbial diversity correlated with neutrophil recruitment as lungs associated with inflammatory responses harbored more diverse microbiomes. Although Salton Sea dust influences dust microbiomes and prevalent taxa, these responses are variable. The composition of lungs exposed to dust collected further from the Salton Sea was more similar to lungs from ambient air exposures; in contrast, dust collected near the Salton Sea yielded lung microbiomes that clustered further from lungs exposed to ambient air. As lakes continue to dry out, we expect greater public health risks in proximal dryland regions, which may correlate with dust microbial dispersal-related changes to lung microbiomes.

Importance: Dust inhalation can lead to health effects, especially when toxic chemicals and microbes mix in with the dust particles. As California's Salton Sea dries up, it exposes lake bottom sediments to wind, which disperses the dried sediments. To mimic the effect of inhaling Salton Sea dust, we collected and filtered airborne dust to use in exposure experiments with mice in environmental chambers. We predicted that inhaling small dust particles, chemicals, and microbial residues found in this dust would affect mouse respiratory health or change the microbes found inside their lungs. We found that inhaling dust led to lung inflammation, and the dust source influenced the type of microbes found inside mouse lungs. As lakes continue to dry out, we expect greater health risks and changes to lung microbiomes.

Certain strains of Bifidobacterium adolescentis inhabiting the gastrointestinal tract have been shown to possess properties that are purported to improve host health. However, given the genetic and phenotypic diversity that has been reported among B. adolescentis strains, it is unclear if and to what extent these traits are conserved across the species. Accordingly, we examined potentially beneficial properties using a combination of genomic and in vitro approaches. Phylogeny inferred from core genomes of 148 B. adolescentis isolates revealed five major lineages. At the functional genomic level, the strains were separated into three groups based on carbohydrate-active enzymes (CAZymes) that were predicted to act on a diverse range of substrates. Fifteen different B. adolescentis strains representing the major phylogenetic groups were tested in vitro for several metabolic traits. The ability to metabolize lactose (all strains; 2-232 β-galactosidase Miller units) and resistant starch (66% of strains; 26-74% utilization) and to produce γ-aminobutyric acid (80% of strains; 0.3-14.4 mM) and folate (all strains; 23-281 ng/mL) was common but showed substantial quantitative variation across strains under the conditions tested. Genes for the formation of antimicrobial compounds (7% of genomes) and antibiotic resistance (23% of genomes) were less frequent. While individual traits did not exhibit strong phylogenetic clustering, B. adolescentis strains had higher CAZyme gene counts relative to other comparison species, suggesting a broader adaptation for carbohydrate metabolism. Further studies on the distribution of beneficial traits and their genetic basis will provide insights into the contributions of B. adolescentis to health.IMPORTANCEBifidobacterium adolescentis is a gut commensal that is prevalent among healthy adults and centenarian populations, potentially contributing to host health through diverse functional properties. Here, through genomic and phenotypic analyses, we advanced our understanding of the prevalence of multiple potentially beneficial properties of B. adolescentis, including those associated with improving lactose tolerance, metabolic health, and mood, and supplying vitamins and inhibiting pathogens. Our findings revealed substantial quantitative variation in metabolic activities and production of relevant end-products across strains, highlighting the importance of strain-level differences and the health benefits they may confer. In addition, while the presence of specific genes was partially predictive of the magnitude of traits, the associations between genetics and phenotypes established here provide a foundation for improving future predictions.

Nasal colonization by Staphylococcus aureus or Streptococcus pneumoniae is associated with an increased risk of infection by these pathobionts, whereas nasal colonization by Dolosigranulum species is associated with health. Human nasal epithelial organoids (HNOs) differentiated at air-liquid interface (ALI) physiologically recapitulate human nasal respiratory epithelium with a robust mucociliary blanket. Due to their natural stem-like properties, HNO lines are a long-term experimental resource that offers genetic diversity based on the different donors. To develop HNOs as a new model system for bacterial nasal colonization, we reproducibly monocolonized HNOs differentiated at ALI with S. aureus, S. pneumoniae, or Dolosigranulum pigrum for up to 48 h with varying kinetics across species. HNOs tolerated bacterial monocolonization with localization of bacteria to the mucus layer and with minimal cytotoxicity compared to uncolonized HNOs. Human nasal epithelium exhibited both species-specific and general cytokine responses, without induction of type I interferons, which is consistent with colonization rather than infection. Only live S. aureus colonization robustly induced epithelial cell production of interleukin-1 family cytokines, suggestive of inflammasome signaling. D. pigrum and live S. aureus decreased CXCL10, whereas S. pneumoniae increased CXCL11, chemokines involved in antimicrobial responses to both viruses and bacteria. Overall, HNOs are a new model system for uncovering microbe-epithelial cell dynamics at the human nasal mucosa.

Importance: Human nasal microbiota often includes highly pathogenic members, many of which are antimicrobial resistance threats, e.g., methicillin-resistant Staphylococcus aureus and drug-resistant Streptococcus pneumoniae. Preventing colonization by nasal pathobionts decreases infections and transmission. In contrast, nasal microbiome studies identify candidate beneficial bacteria that might resist pathobiont colonization, e.g., Dolosigranulum pigrum. Learning how these microbionts interact with the nasal epithelium and identifying new means to reduce pathobiont colonization are key goals in the field. As a tool to advance this research, we developed human nasal epithelial organoids (HNOs) differentiated at an air-liquid interface as a new model system of bacterial nasal colonization. HNOs accurately represent the mucosal surface of the human nasal passages, enabling exploration of bacterial-epithelial interactions, which is important since the epithelium is an instigator of the initial innate immune response to bacteria. Here, we identified differential epithelial cytokine responses to these three bacteria, setting the stage for future research.

In our 2021 mSphere of Influence article (M. A. Brindley, mSphere 6:e00034-21, 2021, https://doi.org/10.1128/mSphere.00034-21), we discussed the concept of viral apoptotic mimicry and how the seminal study by Mercer and Helenius proposed a new paradigm in how viruses can gain entry into cells (J. Mercer and A. Helenius, Science 320:531-535, 2008, https://doi.org/10.1126/science.1155164). Building on the observation that enveloped viral lipids can mediate viral entry, subsequent studies have expanded these observations and further defined the lipids that can initiate infection, the cellular receptors involved, as well as started to explore how viruses obtain their membrane and potentially alter the lipid environment to enhance infection. Exploring the role viral apoptotic mimicry plays in the host has proven more difficult, as many of the receptors that interact with viral lipids also play key roles in immune signaling. This Full Circle review summarizes the lipids and receptors that are involved with viral apoptotic mimicry, the viruses that use them, as well as examines the studies that attempt to explore the role apoptotic mimicry plays in a host.

The tier 1 bioterrorism agent Burkholderia pseudomallei causes melioidosis, a tropical disease with fatality rates that can exceed 40% despite antibiotic therapy. Antibiotic failure is likely to be, at least in part, due to biofilm-dwelling B. pseudomallei, and therefore, an improved understanding of how this pathogen regulates biofilm formation could reveal new opportunities for clinical intervention. The antimicrobial radical nitric oxide (NO) plays a key role in host immune defenses against bacteria, and the ability of B. pseudomallei to sense and mitigate NO toxicity is vital for establishing infection. NO-sensing proteins (NosPs), which have recently emerged as key regulators of biofilm formation in many bacterial species, use a FIST domain to sense NO via a bound heme. We hypothesized that the NosP homolog in B. pseudomallei would regulate biofilm formation and mediate NO-protective responses. We used [γ-³²P]ATP autophosphorylation assays to show that NosP of B. pseudomallei controls the autophosphorylation rate of an associated histidine kinase protein (NosK) in an NO-dependent manner. NosK was found to phosphorylate a response regulator protein (NosR) with an HD-GYP output domain, which is associated with c-di-GMP signaling, therefore implicating NosP in modulating c-di-GMP-regulated phenotypes. Unmarked, in-frame deletion of either nosP or nosK caused significant changes in B. pseudomallei biofilm formation and increased sensitivity to nitrosative stress, in addition to affecting other virulence traits such as growth and swimming motility. These results indicate that NosP and NosK signaling control a range of infection-relevant phenotypes and may serve as targets for novel therapeutic intervention.IMPORTANCEMelioidosis is an emerging, potentially life-threatening infection caused by the bacterium Burkholderia pseudomallei, killing ~89,000 people per year globally. Antibiotic therapy fails in ~10%-40% of cases, and hence, an improved understanding of the molecular mechanisms that control B. pseudomallei virulence could reveal new approaches for improving melioidosis treatment. Biofilm formation and resistance to the antimicrobial radical NO are virulence traits that help bacteria establish infections. Here, we show that two proteins in B. pseudomallei, NosP and NosK, work together to detect NO and regulate a suite of virulence traits, including NO resistance, biofilm formation, growth, and swimming motility. This work, therefore, improves our understanding of the molecular mechanisms that control infection-related phenotypes in B. pseudomallei.

Nick Lennemann studies the intracellular interactions of viral and host proteins. In this mSphere of Influence article, he discusses his employment history and the mentors that promoted his training and transition to an independent research program focused on proteolytic determinants of virus infection. He highlights how "A novel interaction between dengue virus nonstructural protein 1 and the NS4A-2K-4B precursor is required for viral RNA replication but not for formation of the membranous replication organelle" by A. Płaszczyca, P. Scaturro, C. J. Neufeldt, M. Cortese, et al. (PLoS Pathog 15:e1007736, 2019, https://doi.org/10.1371/journal.ppat.1007736) and "Species-specific disruption of STING-dependent antiviral cellular defenses by the Zika virus NS2B3 protease" by Q. Ding, J. M. Gaska, F. Douam, L. Wei, et al. (Proc Natl Acad Sci USA 115: E6310-E6318, 2018, https://doi.org/10.1073/pnas.1803406115) demonstrate the importance of viral protease activity for the establishment of a productive intracellular environment for infection.

Diet influences ruminal methane emissions by modulating the composition and activity of the rumen microbiome. However, how diet shapes the functional capacity of the rumen microbiome in Nelore cattle (Bos indicus), a key tropical beef breed, remains unclear. This study used metatranscriptomics to investigate how dietary supplementation with agro-industrial by-products affects the active rumen microbiome and its association with residual methane emissions. Rumen samples from 50 Nelore cattle fed either a conventional or by-product-based diet revealed that the active microbiome was dominated by bacteria (88.4% ± 3.16%) and archaea (11.6% ± 3.16%), with no significant taxonomic differences between diets. Despite this, functional profiling identified genes from 193 pathways and 3,512 gene families, with distinct metabolic signatures between diets. Specifically, six pathways and 87 gene families were unique to the conventional diet, while seven pathways and 210 gene families were unique to the by-product diet. The associations between gene families enriched under each diet with residual methane emission revealed that the expression of two gene families exhibited negative correlations, while five were positively correlated with methane emission under conventional diet. In the by-product diet, we identified five gene families positively associated with methane emissions and 14 negatively associated. These results demonstrate that diet alters rumen microbial functions with methane mitigation potential, without affecting taxonomic composition.

Importance: Understanding how diet modulates the functional activity of the rumen microbiome is essential for developing strategies to mitigate methane emissions in cattle. This study provides novel insights into how feeding agro-industrial by-products to Nelore cattle (Bos indicus), a key tropical beef breed, reshapes the functional profile of the rumen microbiome. Although no taxonomic shifts were detected, animals fed the by-product diet exhibited a greater number of microbial functions associated with lower methane production potential. These findings suggest that diet-driven modulation of microbial metabolism could contribute to strategies aimed at reducing methane emissions. Moreover, the use of by-products supports circular economy principles, enhancing the sustainability and economic resilience of tropical livestock systems. This work emphasizes the importance of examining the active microbiome through RNA rather than solely profiling taxonomic composition without considering microbial activity. It also contributes to unveiling microbial functions to support future methane mitigation and sustainable feeding strategies.