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The current situation with H5N1 highly pathogenic avian influenza virus (HPAI) is causing a worldwide concern due to multiple outbreaks in wild birds, poultry, and mammals. Moreover, multiple zoonotic infections in humans have been reported. Importantly, HPAI H5N1 viruses with genetic markers of adaptation to mammals have been detected. Together with HPAI H5N1, avian influenza viruses H7N9 (high and low pathogenic) stand out due to their high mortality rates in humans. This raises the question of how prepared we are serologically and whether seasonal vaccines are capable of inducing protective immunity against these influenza subtypes. An observational study was conducted in which sera from people born between years 1925-1967, 1968-1977, and 1978-1997 were collected before or after 28 days or 6 months post-vaccination with an inactivated seasonal influenza vaccine. Then, hemagglutination inhibition, viral neutralization, and immunoassays were performed to assess the basal protective immunity of the population as well as the ability of seasonal influenza vaccines to induce protective responses. Our results indicate that subtype-specific serological protection against H5N1 and H7N9 in the representative Spanish population evaluated was limited or nonexistent. However, seasonal vaccination was able to increase the antibody titers to protective levels in a moderate percentage of people, probably due to cross-reactive responses. These findings demonstrate the importance of vaccination and suggest that seasonal influenza vaccines could be used as a first line of defense against an eventual pandemic caused by avian influenza viruses, to be followed immediately by the use of more specific pandemic vaccines.IMPORTANCEInfluenza A viruses (IAV) can infect and replicate in multiple mammalian and avian species. Avian influenza virus (AIV) is a highly contagious viral disease that occurs primarily in poultry and wild water birds. Due to the lack of population immunity in humans and ongoing evolution of AIV, there is a continuing risk that new IAV could emerge and rapidly spread worldwide, causing a pandemic, if the ability to transmit efficiently among humans was gained. The aim of this study is to analyze the basal protection and presence of antibodies against IAV H5N1 and H7N9 subtypes in the population from different ages. Moreover, we have evaluated the humoral response after immunization with a seasonal influenza vaccine. This study is strategically important to evaluate the level of population immunity that is a major factor when assessing the impact that an emerging IAV strain would have, and the role of seasonal vaccines to mitigate the effects of a pandemic.

Acinetobacter baumannii is a Gram-negative opportunistic pathogen and is a common cause of nosocomial infections. The increasing development of antibiotic resistance in this organism is a global health concern. The A. baumannii clinical isolate AB307-0294 produces a type VI secretion system (T6SS) that delivers three antibacterial effector proteins that give this strain a competitive advantage against other bacteria in polymicrobial environments. Each effector, Tse15, Tde16, and Tae17, is delivered via a non-covalent interaction with a specific T6SS VgrG protein (VgrG15, VgrG16, and VgrG17, respectively). Here we define the regions of interaction between Tae17 and its cognate delivery protein VgrG17 and identify that amino acids G1069 and W1075 in VgrG17 are essential for Tae17 delivery via the T6SS, the first time such specific delivery determinants of T6SS cargo effectors have been defined. Furthermore, we determine that the Tae17 effector is a multidomain, bifunctional, peptidoglycan-degrading enzyme that has both amidase activity, which targets the sugar-peptide bonds, and lytic transglycosylase activity, which targets the peptidoglycan sugar backbone. Moreover, we show that the Tae17 transglycosylase activity is more important than amidase activity for the killing of Escherichia coli. This study provides molecular insight into how the T6SS allows A. baumannii strains to gain dominance in polymicrobial communities and thus improve their chances of survival and transmission.IMPORTANCEWe have shown that the Acinetobacter baumannii T6SS effector Tae17 is a modular, bifunctional, peptidoglycan-degrading enzyme that has both lytic transglycosylase and amidase activities. Both activities contribute to the ability to degrade peptidoglycan, but the transglycosylase activity was more important for the killing of Escherichia coli. We have defined the specific regions of Tae17 and its cognate delivery protein VgrG17 that are necessary for the non-covalent interactions and, for the first time, identified specific amino acids essential for T6SS cargo effector delivery. This work contributes to our molecular understanding of bacterial competition strategies in polymicrobial environments and may provide a window to design new therapeutic approaches for combating infection by A. baumannii.

The nickel-pincer nucleotide (NPN) cofactor is a modified pyridinium mononucleotide that tri-coordinates nickel and is crucial for the activity of certain racemases and epimerases. LarB, LarC, and LarE are responsible for NPN synthesis, with the cofactor subsequently installed into LarA homologs. Hurdles for investigating the functional properties of such proteins arise from the difficulty of obtaining the active, NPN cofactor-loaded enzymes and in assaying their diverse reactivities. Here, we show that when the Lactiplantibacillus plantarum lar genes are cloned into the Duet expression system and cultured in Escherichia coli, they confer lactate racemase activity to the cells. By replacing L. plantarum larA with related genes from other microorganisms, this system allows for the generation of active LarA homologs. Furthermore, the Duet system enables the functional testing of LarB, LarC, and LarE homologs from other microorganisms. In addition to applying the Duet expression system for synthesis of active, NPN cofactor-containing enzymes in E. coli, we demonstrate that circular dichroism spectroscopy provides a broadly applicable means of assaying these enzymes. By selecting a wavelength of high molar ellipticity and low absorbance for a given 2-hydroxy acid substrate enantiomer, the conversion of one enantiomer/epimer into the other can be monitored for LarA homologs without the need for any coupling enzymes or reagents. The methods discussed here further our abilities to investigate the unique activities of Lar proteins.

Importance: Enzymes containing the nickel-pincer nucleotide (NPN) cofactor are prevalent in a wide range of microorganisms and catalyze various critical biochemical reactions, yet they remain underexplored due, in part, to limitations in current research methodologies. The two significant advancements described here, the heterologous production of active NPN-cofactor containing enzymes in Escherichia coli and the use of a circular dichroism-based assay to monitor enzyme activities, expand our capacity to analyze these enzymes. Such additional detailed characterization will deepen our understanding of the diverse chemistry catalyzed by the NPN cofactor and potentially uncover novel roles for this organometallic species in microbial metabolism.

Many bacteria metabolize ethanolamine as a nutrient source through cytoplasmic organelles named bacterial microcompartments (BMCs). Here we investigated the molecular assembly, regulation, and function of BMCs in Fusobacterium nucleatum-a Gram-negative oral pathobiont that is associated with adverse pregnancy outcomes. The F. nucleatum genome harbors a conserved ethanolamine utilization (eut) locus with 21 genes that encode several putative BMC shell proteins and a two-component signal transduction system (TCS), in addition to the enzymes for ethanolamine transport and catabolism. We show that the expression of most of these genes and BMC formation are highly increased in wild-type fusobacteria when cultured in the presence of ethanolamine as a nutrient source. Deletion of the response regulator EutV eliminated this induction of eut mRNAs and BMCs, thus demonstrating that BMC formation is transcriptionally regulated by the TCS EutV-EutW in response to ethanolamine. Mass spectrometry of isolated BMCs unveiled the identity of the constituent proteins EutL, EutM₁, EutM₂, and EutN. Consistent with the role of these proteins in BMC assembly and metabolism, deletion of eutN, eutL/eutM₁/eutM₂, or eutL/eutM₁/eutM₂/eutN not only affected BMC formation but also ethanolamine utilization, causing cell growth defects with ethanolamine as a nutrient. BMCs are also assembled in fusobacteria cultured with placental cells or the culture media, a process that is dependent on the BMC shell proteins. Significantly, we show that the eutN mutant is defective in inducing preterm birth in a mouse model. Together, these results establish that the BMC-mediated metabolism of ethanolamine is critical for fusobacterial virulence.

Importance: The oral anaerobe Fusobacterium nucleatum can spread to distal internal organs, such as the colon and placenta, thereby promoting the development of colorectal cancer and inducing preterm birth, respectively. Yet, how this opportunistic pathogen adapts to the various metabolically distinct host cellular niches remains poorly understood. We demonstrated here that this microbe assembles specialized metabolic organelles, termed bacterial microcompartments (BMCs), to utilize environmental ethanolamine (EA) as a key environmental nutrient source. The formation of F. nucleatum BMCs, containing BMC shell proteins EutLM1M2N, is controlled by a two-component system, EutV-EutW, responsive to EA. Significantly, this ability of F. nucleatum to form BMCs in response to EA is crucial for its pathogenicity evidenced by the fact that the genetic disruption of BMC formation reduces fusobacterial virulence in a mouse model of preterm birth.

Snow algae darken the surface of snow, reducing albedo and accelerating melt. However, the impact of subsurface snow algae (e.g., when cells are covered by recent snowfall) on albedo is unknown. Here, we examined the impact of subsurface snow algae on surface energy absorption by adding up to 2 cm of clean snow to surface algal blooms and measuring reflectivity. Surprisingly, snow algae still absorb significant energy across an array of wavelengths when snow-covered. Furthermore, the scale of this effect correlates with algal cell densities and chlorophyll-a concentrations. Collectively, our results suggest that darkening by subsurface snow algae lowers albedo and thus potentially accelerates snowmelt even when the algae is snow-covered. Impacts of subsurface algae on melt await assessment. This implies that snow algae play a larger role in cryosphere melt than investigations of surface-only reflectance would suggest.

Importance: This study addresses a gap in research by examining the impact of subsurface snow algae on snow albedo, which affects snowmelt rates. Previous studies have focused on visible surface blooms, leaving the effects of hidden algae unquantified. Our findings reveal that snow algae beneath the surface can still absorb energy across various wavelengths, accelerating melt even when not visible to the naked eye. This suggests that spectral remote sensing can detect these hidden algae, although their biomass might be underestimated. Understanding how subsurface snow algae influence albedo and snowmelt is crucial for accurate predictions of meltwater runoff, which impacts alpine ecosystems, glacier health, and water resources. Accurate projections are essential for managing freshwater supplies for agriculture, drinking water, and other vital uses. Thus, further investigation into subsurface snow algae is necessary to improve our understanding of their role in snow albedo reduction and water resource management.

As a universal language across the bacterial kingdom, the quorum sensing signal autoinducer-2 (AI-2) can coordinate many bacterial group behaviors. However, unknown AI-2 receptors in bacteria may be more than what has been discovered so far, and there are still many unknown functions for this signal waiting to be explored. Here, we have identified a membrane-bound histidine kinase of the pathogenic bacterium Vibrio furnissii, AsrK, as a receptor that specifically detects AI-2 under low boron conditions. In contrast with another well-known AI-2 receptor LuxP that recognizes the borated form of AI-2, AsrK is found to show higher affinity with AI-2 under borate-depleted conditions, and thus boron has a negative effect on AI-2 sensing by AsrK in regulation of the biofilm and motility phenotypes. AI-2 binds to the extracytoplasmic dCache_1 domain of AsrK to inhibit its autokinase activity, thus decreasing the phosphorylation level of its cognate response regulator AsrR and activating the phosphodiesterase activity of AsrR to degrade the cellular second messenger cyclic di-GMP (c-di-GMP). AI-2 perception by the AsrK-AsrR system remarkably reduces intracellular c-di-GMP levels and enhances tolerance of V. furnissii to oxidative stress and DNA damage by upregulating the transcription of universal stress proteins including UspA1, UspA2, and UspE. Our study reveals a previously unrecognized mechanism for AI-2 detection in bacteria and also provides new insights into the important role of AI-2 in bacterial defense against oxidative stress and DNA damage.IMPORTANCEThe QS signal AI-2 is widely synthesized in bacteria and has been implicated in the regulation of numerous bacterial group behaviors. However, in contrast to the wide distribution of this signal, its receptors have only been found in a small number of bacterial species, and the underlying mechanisms for the detection of and response to AI-2 remain elusive in most bacteria. It is worth noting that the periplasmic protein LuxP is the uniquely identified receptor for AI-2 in Vibrio spp. Here, we identify a second type of AI-2 receptor, a membrane-bound histidine kinase with a periplasmic dCache_1 sensory domain, in a member of the genus Vibrio, and thus show that AI-2 enhances the defense of V. furnissii against oxidative stress and DNA damage by modulation of c-di-GMP signaling via the AsrK-AsrR two-component system. Our results reveal a previously unrecognized AI-2 sensing mechanism and expand our understanding of the physiological roles of AI-2 in bacteria.

Deoxynivalenol (DON), a mycotoxin primarily produced by Fusarium species, is commonly found in cereal grains and poses risks to human and animal health, as well as global grain trade. This study demonstrates that methyl jasmonate (MeJA), a natural plant hormone, inhibits the growth and conidiation of Fusarium graminearum. Importantly, MeJA significantly reduces DON production by suppressing TRI gene expression and toxisome formation. To explore the molecular mechanism, we identified MeJA-tolerant mutants, including a transcription factor MRT1 and cAMP-PKA pathway-related genes (FgGPA1 and FgSNT1). MeJA treatment reduced PKA activity and intracellular cAMP levels in F. graminearum, suggesting it targets the cAMP-PKA pathway. Notably, the MeJA-resistant mutant FgGPA1^R178H enhanced fungal growth, DON production, and cAMP levels in the presence of MeJA. Exogenous cAMP alleviated MeJA's inhibitory effects on DON production, further supporting this pathway's involvement. Interestingly, MeJA had no effect on all three MAP kinase pathways (Mgv1, Gpmk1, and FgHog1). Truncated and phospho-mimicking mutations in Mrt1 or FgSnt1 conferred MeJA resistance, suggesting they may act downstream of the cAMP-PKA pathway. In conclusion, MeJA presents a promising approach to control F. graminearum growth and DON production.IMPORTANCEDeoxynivalenol (DON) poses significant risks to both human and animal health and severely disrupts the global grain trade due to its prevalence as a common contaminant in wheat grains. With rising public concern over food safety, finding effective and sustainable methods to reduce DON contamination becomes increasingly urgent. In our study, we found that methyl jasmonate (MeJA), a natural plant hormone, can effectively inhibit the vegetative growth of F. graminearum and significantly reduce its DON toxin production. To explore the underlying molecular mechanism, we identified the mutations in MeJA-tolerant mutants and revealed that MeJA effectively exerts its antifungal activities by inhibiting the cAMP-PKA signaling pathway in F. graminearum. Our work provides a promising natural solution to reduce DON toxin contamination in cereal grains, enhancing food safety while decreasing the reliance on chemical fungicides and their associated environmental impact.

Sepsis-induced acute liver injury (SALI) is a prevalent and life-threatening complication associated with sepsis. The gut microbiota plays a crucial role in the maintenance of health and the development of diseases. The impact of physical exercise on gut microbiota modulation has been well-documented. However, the potential impact of gut microbiome on exercise training-induced protection against SALI remains uncertain. Here, we discovered exercise training ameliorated SALI and systemic inflammation in septic mice. Notably, gut microbiota pre-depletion abolished the protective effects of exercise training in SALI mice. Fecal microbiota transplantation treatment revealed that exercise training-associated gut microbiota contributed to the beneficial effect of exercise training on SALI. Exercise training modulated the metabolism of Ligilactobacillus and enriched betulinic acid (BA) levels in mice. Functionally, BA treatment conferred protection against SALI by inhibiting the hepatic inflammatory response in mice. BA bound and inactivated hnRNPA2B1, thus suppressing NLRP3 inflammasome activation in macrophages. Collectively, this study reveals gut microbiota is involved in the protective effects of exercise training against SALI, and gut microbiota-derived BA inhibits the hepatic inflammatory response via the hnRNPA2B1-NLRP3 axis, providing a potential therapeutic strategy for SALI.

Importance: Sepsis is characterized by a dysregulated immune response to an infection that leads to multiple organ dysfunction. The occurrence of acute liver injury is frequently observed during the initial stage of sepsis and is directly linked to mortality in the intensive care unit. The preventive effect of physical exercise on SALI is well recognized, yet the underlying mechanism remains poorly elucidated. Exercise training alters the gut microbiome in mice, increasing the abundance of Ligilactobacillus and promoting the generation of BA. Additionally, BA supplementation can suppress the NLRP3 inflammasome activation in macrophages by directly binding to hnRNPA2B1, thereby mitigating SALI. These results highlight the beneficial role of gut microbiota-derived BA in inhibiting the hepatic inflammatory response, which represents a crucial stride toward implementing microbiome-based therapeutic strategies for the clinical management of sepsis.

Mutations affecting Clostridioides difficile flagellin (FliC) have been shown to be hypervirulent in animal models and display increased toxin production and alterations in central metabolism. The regulation of flagellin levels in bacteria is governed by a tripartite regulatory network involving fliC, fliW, and csrA, which creates a feedback system to regulate flagella production. Through genomic analysis of C. difficile clade 5 strains (non-motile), we identified they have jettisoned many of the genes required for flagellum biosynthesis yet retain the major flagellin gene fliC and regulatory gene fliW. We therefore investigated the roles of fliC, fliW, and csrA in the clade 5 ribotype 078 strain C. difficile 1015, which lacks flagella and is non-motile. Analysis of mutations in fliC, fliW, and csrA (and all combinations) on C. difficile pathogenesis indicated that FliW plays a central role in C. difficile virulence as animals infected with strains carrying a deletion of fliW showed decreased survival and increased disease severity. These in vivo findings were supported by in vitro studies showing that mutations impacting the activity of FliW showed increased toxin production. We further identified that FliW can interact with the toxin-positive regulator TcdR, indicating that modulation of toxin production via FliW occurs by sequestering TcdR from activating toxin transcription. Furthermore, disruption of the fliC-fliW-csrA network results in significant changes in carbon source utilization and sporulation. This work highlights that key proteins involved in flagellar biosynthesis retain their regulatory roles in C. difficile pathogenesis and physiology independent of their functions in motility.

Importance: Clostridioides difficile is a leading cause of nosocomial antibiotic-associated diarrhea in developed countries with many known virulence factors. In several pathogens, motility and virulence are intimately linked by regulatory networks that allow coordination of these processes in pathogenesis and physiology. Regulation of C. difficile toxin production by FliC has been demonstrated in vitro and in vivo and has been proposed to link motility and virulence. Here, we show that clinically important, non-motile C. difficile strains have conserved FliC and regulatory partners FliW and CsrA, despite lacking the rest of the machinery to produce functional flagella. Our work highlights a novel role for flagellin outside of its role in motility and FliW in the pathogenesis and physiology of C. difficile.

Haemophilus ducreyi causes the genital ulcer disease chancroid and cutaneous ulcers in children. To study its pathogenesis, we developed a human challenge model in which we infect the skin on the upper arm of human volunteers with H. ducreyi to the pustular stage of disease. The model has been used to define lesional architecture, describe the immune infiltrate into the infected sites using flow cytometry, and explore the molecular basis of the immune response using bulk RNA-seq. Here, we used single cell RNA-seq (scRNA-seq) and spatial transcriptomics to simultaneously characterize multiple cell types within infected human skin and determine the cellular origin of differentially expressed transcripts that we had previously identified by bulk RNA-seq. We obtained paired biopsies of pustules and wounded (mock infected) sites from five volunteers for scRNA-seq. We identified 13 major cell types, including T- and NK-like cells, macrophages, dendritic cells, as well as other cell types typically found in the skin. Immune cell types were enriched in pustules, and some subtypes within the major cell types were exclusive to pustules. Sufficient tissue specimens for spatial transcriptomics were available from four of the volunteers. T- and NK-like cells were highly associated with multiple antigen presentation cell types. In pustules, type I interferon stimulation was high in areas that were high in antigen presentation-especially in macrophages near the abscess-compared to wounds. Together, our data provide a high-resolution view of the cellular immune response to the infection of the skin with a human pathogen.IMPORTANCEA high-resolution view of the immune infiltrate due to infection with an extracellular bacterial pathogen in human skin has not yet been defined. Here, we used the human skin pathogen Haemophilus ducreyi in a human challenge model to identify on a single cell level the types of cells that are present in volunteers who fail to spontaneously clear infection and form pustules. We identified 13 major cell types. Immune cells and immune-activated stromal cells were enriched in pustules compared to wounded (mock infected) sites. Pustules formed despite the expression of multiple pro-inflammatory cytokines, such as IL-1β and type I interferon. Interferon stimulation was most evident in macrophages, which were proximal to the abscess. The pro-inflammatory response within the pustule may be tempered by regulatory T cells and cells that express indoleamine 2,3-dioxygenase, leading to failure of the immune system to clear H. ducreyi.