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Inter-kingdom endosymbiotic interactions between bacteria and eukaryotic cells are critical to human health and disease. However, the molecular mechanisms that drive the emergence of endosymbiosis remain obscure. Here, we describe the development of a microfluidic system, named SEER (S̲ystem for the E̲volution of E̲ndosymbiotic R̲elationships), that automates the evolutionary selection of bacteria with enhanced intracellular survival and persistence within host cells, hallmarks of endosymbiosis. Using this system, we show that a laboratory strain of

Microbial interactions within a natural or engineered consortium of microbes play an important role in the functions of the consortium. Better understanding these interactions is also important for engineering microbial consortia for specific applications. As such, tools that can enable investigating microbial interactions are highly valuable. One aspect of microbial interactions that impacts community formation is how the spatial organization of individual microbes impacts interactions leading to community formation. Here, we report the development of a tool that can manipulate the spatial organization of microorganisms to investigate the role of these interactions in community formation. Our developed microfluidic platform utilizes dielectrophoretic (DEP) force to perform on-demand spatial arrangement of microorganism-encapsulated agarose gel microparticles. To demonstrate this concept, three gel microparticle manipulators composed of three independently controllable DEP electrodes were utilized for the on-demand spatial arrangement of a specific combination of microparticles, each containing

Soil microbes perform a multitude of essential biological functions, including organic matter decomposition, nutrient cycling, and soil structure preservation. Qatar is one of the most hostile and fragile arid environments on earth and is expected to face a disproportionate impact of climate change in the coming years.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, can infect animals by binding to the angiotensin-converting enzyme 2 (ACE2). Equine infection appears possible due to high homology (≈97%) between human and equine ACE2, evidence ofin vitroinfection in cell lines expressing equine ACE2, and evidence of seroconversion in horses after exposure to persons infected with SARS-CoV-2. Our objective was to examine susceptibility of cultured primary equine bronchial epithelial cells (EBECs) to a SARS-CoV-2 pseudovirus relative to human bronchial epithelial cells (HBECs; positive control). ACE2 expression in EBECs detected by immunofluorescence, western immunoblotting, and flow cytometry was lower in EBECs than in HBECs. EBECs were transduced with a lentivirus pseudotyped with the SARS-CoV-2 spike protein that binds to ACE2 and expresses the enhanced green fluorescent protein (eGFP) as a reporter. Cells were co-cultivated with the pseudovirus at a multiplicity of infection of 0.1 for 6 hours, washed, and maintained in media. After 96 hours, eGFP expression in EBECs was demonstrated by fluorescence microscopy, and mean Δ Ct values from quantitative PCR were significantly (P < 0.0001) higher in HBECs (8.78) than HBECs (3.24) indicating lower infectivity in EBECs. Equine respiratory tract cells were susceptible to infection with a SARS-CoV-2 pseudovirus. Lower replication efficiency in EBECs suggests that horses are unlikely to be an important zoonotic host of SARS-CoV-2, but viral mutations could render some strains more infectious to horses. Serological and virological monitoring of horses in contact with persons shedding SARS-CoV-2 is warranted.IMPORTANCEThis study provides the first published evidence for SARS-CoV-2 pseudovirus infection in equine airway epithelial cells, which were less susceptible to infection than cells of human origin. This was presumably due to lower ACE2 expression in equine cells, lower viral affinity for equine ACE2, or both. Our results are important considering recent evidence for asymptomatic seroconversion in horses following exposure to COVID-19 positive humans, despite this lower susceptibility, and increased affinity of viral variants of concern for equine ACE2 compared to ancestral strains. Thus, there is great need to better characterize SARS-CoV-2 susceptibility in horses for the benefit of veterinary and human health.

Changes in microbial growth kinetics and biofilm formation capacity were evaluated in bacterial cells remaining from PDT. Curcumin (CUR) used as photosensitizer, shows penetration and photodynamic effects on biofilm layers, evaluated considering parameters such as the biofilm complexity (BC), and formation time (FT) of the biofilm. Microbial growth and biofilm reformulation capacity were modified in cells remaining after partial photodynamic action. The photosensitizer (PS) permeation per bacterial biofilm layer showed to be dependent on the FT and BC in addition to the incubation time (IT) of the PS. Post-PDT effects showed (1) a delay in the onset of the exponential phase; (2) limitation of the maximum microbial amount; (3) slowdown of the growth rate. The use of PDT shows to be promising for biofilm control remnants presents slowdown and modification on the growth patterns and penetration of CUR is facilitated through the complex structure of the biofilm.

The widespread use of antibiotics drives the evolution of antimicrobial-resistant bacteria (ARB), threatening patients and healthcare professionals. Therefore, the development of novel strategies to combat resistance is recognized as a global healthcare priority. The two methods to combat ARB are development of new antibiotics or reduction in existing resistances. Development of novel antibiotics is a laborious and slow-progressing task that is no longer a safe reserve against looming risks. In this research, we suggest a method for reducing resistance to extend the efficacious lifetime of current antibiotics. Antimicrobial photodynamic therapy (aPDT) is used to generate reactive oxygen species (ROS) via the photoactivation of a photosensitizer. ROS then nonspecifically damage cellular components, leading to general impairment and cell death. Here, we test the hypothesis that concurrent treatment of bacteria with antibiotics and aPDT achieves an additive effect in the elimination of ARB. Performing aPDT with the photosensitizer methylene blue in combination with antibiotics chloramphenicol and tetracycline results in significant reductions in resistance for two methicillin-resistant

Droplet microfluidic systems have been widely deployed to interrogate biological and chemical systems. The major limitations of these systems are the relatively high error rates from critical droplet manipulation functions. To address these limitations, we describe the development of FIDELITY (Flotation and Interdigitated electrode forces on Droplets to Enable Lasting system IntegriTY), a highly sensitive and accurate size-based droplet bandpass filter that leverages the natural buoyancy of aqueous droplets and highly localized dielectrophoretic force generated by interdigitated electrode arrays. Droplet manipulation accuracies greater than 99% were achieved at a throughput of up to 100 droplets/s and separation of droplets that differed in diameter by only 6 μm was demonstrated. Last, the utility of FIDELITY was demonstrated in a droplet size quality control application and also in a droplet-based in vitro transcription/translation workflow. We anticipate FIDELITY to be integrated into a broad range of droplet microfluidic configurations to achieve exceptional operational accuracy.

We report here that nucleus accumbens–associated protein-1 (NAC1), a nuclear factor of the Broad-complex, Tramtrack, Bric-a-brac/poxvirus and zinc finger (BTB/POZ) gene family, is a negative regulator of FoxP3 in regulatory T cells (T regs ) and a critical determinant of immune tolerance. Phenotypically, NAC1 −/− mice showed substantial tolerance to the induction of autoimmunity and generated a larger amount of CD4 + T regs that exhibit a higher metabolic profile and immune-suppressive activity, increased acetylation and expression of FoxP3, and slower turnover of this transcription factor. Treatment of T regs with the proinflammatory cytokines interleukin-1β or tumor necrosis factor–α induced a robust up-regulation of NAC1 but evident down-regulation of FoxP3 as well as the acetylated FoxP3. These findings imply that NAC1 acts as a trigger of the immune response through destabilization of T regs and suppression of tolerance induction, and targeting of NAC1 warrants further exploration as a potential tolerogenic strategy for treatment of autoimmune disorders.

Bacterial pathogen identification, which is critical for human health, has historically relied on culturing organisms from clinical specimens. More recently, the application of machine learning (ML) to whole-genome sequences (WGSs) has facilitated pathogen identification. However, relying solely on genetic information to identify emerging or new pathogens is fundamentally constrained, especially if novel virulence factors exist. In addition, even WGSs with ML pipelines are unable to discern phenotypes associated with cryptic genetic loci linked to virulence. Here, we set out to determine if ML using phenotypic hallmarks of pathogenesis could assess potential pathogenic threat without using any sequence-based analysis. This approach successfully classified potential pathogenetic threat associated with previously machine-observed and unobserved bacteria with 99% and 85% accuracy, respectively. This work establishes a phenotype-based pipeline for potential pathogenic threat assessment, which we term PathEngine, and offers strategies for the identification of bacterial pathogens.

Identifying antibodies (Abs) that neutralize infectious agents is the first step for developing therapeutics, vaccines, and diagnostic tools for those infectious agents. However, current approaches for identifying neutralizing Abs (nAbs) typically rely on dilution-based assays that are costly, inefficient, and only survey a small subset of the entire repertoire. There are also intrinsic biases in many steps of conventional nAb identification processes. More importantly, conventional assays rely on simply Ab-antigen binding assays, which may not result in identifying the most potent nAbs, as the strongest binder may not be the most potent nAb. Droplet microfluidic systems have the capability to overcome such limitations by conducting complex multi-step assays with high reliability, resolution, and throughput in a pico-liter volume water-in-oil emulsion droplet format. Here, we describe the development of PRESCIENT (Platform for the Rapid Evaluation of antibody SucCess using Integrated microfluidics ENabled Technology), a droplet microfluidic system that can enable high-throughput single-cell resolution identification of nAb repertoires elicited in response to a viral infection. We demonstrated PRESCIENT’s ability to identify Abs that neutralize a model viral agent, Murine coronavirus (Murine Hepatitis Virus), which causes high mortality rates in experimentally infected mice. In-droplet infection of host cells by the virus was first demonstrated, followed by demonstration of in-droplet neutralization by nAbs produced from a single Ab-producing hybridoma cell. Finally, fluorescent intensity analyses of two populations of hybridoma cell lines, (nAb-producing and non-nAb-producing hybridoma cell lines) successfully discriminated between the two populations. The presented strategy and platform have the potential to identify and investigate neutralizing activities against a broad range of potential infectious agents for which nAbs have yet to be discovered, significantly advancing the nAb identification process, as well as reinvigorating the field of Ab discovery, characterization, and development.

Immunotherapy has led to impressive advances in the treatment of autoimmune and pro-inflammatory disorders; yet, its clinical outcomes remain limited by a variety of factors including the pro-inflammatory microenvironment (IME). Discovering effective immunomodulatory agents, and the mechanisms by which they control disease, will lead to innovative strategies for enhancing the effectiveness of current immunotherapeutic approaches. We have metabolically engineered an attenuated bacterial strain (i.e., Brucella melitensis 16M ΔvjbR, BmΔvjbR) to produce indole, a tryptophan metabolite that controls the fate and function of regulatory T cells (Tregs). We demonstrated that treatment with this strain polarized M2 macrophages (Mφ) which produced anti-inflammatory cytokines (e.g., IL-10) and promoted Treg function; moreover, when combined with adoptive cell transfer (ACT) of Tregs, a single treatment with our engineered bacterial strain dramatically reduced the incidence and score of autoimmune arthritis and decreased joint damage. These findings show how a metabolically engineered bacterium can constitute a powerful vehicle for improving the efficacy of immunotherapy, defeating autoimmunity and reducing inflammation by remodeling the IME and augmenting Treg function.

eEF-2K has important roles in stress responses and cellular metabolism. We report here a previously unappreciated but critical role of eEF-2K in regulating the fate and cytocidal activity of CD8 + T cells. CD8 + T cells from eEF-2K KO mice were more proliferative but had lower survival than their wild-type counterparts after their activation, followed by occurrence of premature senescence and exhaustion. eEF-2K KO CD8 + T cells were more metabolically active and showed hyperactivation of the Akt-mTOR-S6K pathway. Loss of eEF-2K substantially impaired the activity of CD8 + T cells. Furthermore, the antitumor efficacy and tumor infiltration of the CAR-CD8 + T cells lacking eEF-2K were notably reduced as compared to the control CAR-CD8 + T cells. Thus, eEF-2K is critically required for sustaining the viability and function of cytotoxic CD8 + T cells, and therapeutic augmentation of this kinase may be exploited as a novel approach to reinforcing CAR-T therapy against cancer.

The MHC class I-mediated antigen presentation pathway plays a critical role in antiviral immunity. Here we show that the MHC class I pathway is targeted by SARS-CoV-2. Analysis of the gene expression profile from COVID-19 patients as well as SARS-CoV-2 infected epithelial cell lines reveals that the induction of the MHC class I pathway is inhibited by SARS-CoV-2 infection. We show that NLRC5, an MHC class I transactivator, is suppressed both transcriptionally and functionally by the SARS-CoV-2 ORF6 protein, providing a mechanistic link. SARS-CoV-2 ORF6 hampers type II interferon-mediated STAT1 signaling, resulting in diminished upregulation of NLRC5 and IRF1 gene expression. Moreover, SARS-CoV-2 ORF6 inhibits NLRC5 function via blocking karyopherin complex-dependent nuclear import of NLRC5. Collectively, our study uncovers an immune evasion mechanism of SARS-CoV-2 that targets the function of key MHC class I transcriptional regulators, STAT1-IRF1-NLRC5., The presentation of viral antigens to T cells via the MHC molecules is a critical component of the host response to viral infection. Here the authors suggest SARS-CoV-2 possesses the immune evasion strategy against the MHC class I pathway by targeting key transcriptional regulators.

Our understanding of how the host immune system thwarts bacterial evasive mechanisms remains incomplete. Here, we show that host protease neutrophil elastase acts on Acinetobacter baumannii and Pseudomonas aeruginosa to destroy factors that prevent serum-associated, complement-directed killing. The protease activity also enhances bacterial susceptibility to antibiotics in sera. These findings implicate a new paradigm where host protease activity on bacteria acts combinatorially with the host complement system and antibiotics to defeat bacterial pathogens.

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Heat treatment denatures viral proteins that comprise the virion, making the virus incapable of infecting a host. Coronavirus (CoV) virions contain single‐stranded RNA genomes with a lipid envelope and four proteins, three of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant‐type chemicals. Prior studies have shown that a temperature as low as 75°C with a treatment duration of 15 min can effectively inactivate CoV. The degree of CoV heat inactivation greatly depends on the length of heat treatment time and the temperature applied. With the goal of finding whether sub‐second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple fluidic system that can measure sub‐second heat inactivation of CoV. The system is composed of a stainless‐steel capillary immersed in a temperature‐controlled oil bath followed by an ice bath, through which virus solution can flow at various speeds. Flowing virus solution at different speeds, along with temperature control and monitoring system, allows the virus to be exposed to the desired temperature and treatment durations with high accuracy. Using mouse hepatitis virus, a betacoronavirus, as a model CoV system, we identified that 71.8°C for 0.51 s exposure is sufficient to obtain >5 Log10 reduction in viral titer (starting titer: 5 × 107 PFU/ml), and that when exposed to 83.4°C for 1.03 s, the virus was completely inactivated (>6 Log10 reduction)., A simple system was developed to investigate sub‐second heat inactivation of coronaviruses. Using mouse hepatitis virus, a betacoronavirus, as a model system, the authors identified that 71.8°C for 0.51 s exposure is sufficient to obtain >5 Log10 reduction in viral titer, and that when exposed to 83.4°C for 1.03 s, the virus was completely inactivated. Validated by a COMSOL simulation on temperature distribution, these findings are essential for developing sub‐second heat disinfection applications.

Summary Phagocytosis and autophagy play critical roles in immune defense. The human fungal pathogen Cryptococcus neoformans (Cn) subverts host autophagy-initiation complex (AIC)-related proteins, to promote its phagocytosis and intracellular parasitism of host cells. The mechanisms by which the pathogen engages host AIC-related proteins remain obscure. Here, we show that the recruitment of host AIC proteins to forming phagosomes is dependent upon the activity of CD44, a host cell surface receptor that engages fungal hyaluronic acid (HA). This interaction elevates intracellular Ca2+ concentrations and activates CaMKKβ and its downstream target AMPKα, which results in activation of ULK1 and the recruitment of AIC components. Moreover, we demonstrate that HA-coated beads efficiently recruit AIC components to phagosomes and CD44 interacts with AIC components. Taken together, these findings show that fungal HA plays a critical role in directing the internalization and productive intracellular membrane trafficking of a fungal pathogen of global importance., Graphical abstract, Highlights • Fungal HA drives non-canonical and ligand-induced autophagy in phagocytic cells • Cn recruits host CD44 to forming phagocytic cups to initiate fungal internalization • Fungal HA-CD44 interactions elevate intracellular Ca2+ levels and activate CaMKKβ • A Ca2+-CaMKKβ-AMPK-ULK1 signaling axis is involved in HA-CD44 induced autophagy, Immunology; Mycology

Heat treatment denatures viral proteins that comprise the virion, making virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA genomes with a lipid envelope and 4 proteins, 3 of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a temperature of as low as 75 °C and treatment duration of 15 min can effectively inactivate CoV. The applicability of a CoV heat inactivation method greatly depends on the length of time of a heat treatment and the temperature needed to inactivate the virus. With the goal of finding conditions where sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple system that can measure sub-second heat inactivation of CoV. The system is composed of capillary stainless-steel tubing immersed in a temperature-controlled oil bath followed by an ice bath, through which virus solution can be flowed at various speeds. Flowing virus solution at different speeds, along with a real-time temperature monitoring system, allows the virus to be accurately exposed to a desired temperature for various durations of time. Using mouse hepatitis virus (MHV), a beta-coronavirus, as a model system, we identified that 85.2 °C for 0.48 s exposure is sufficient to obtain > 5 Log10 reduction in viral titer (starting titer: 5 × 107 PFU/mL), and that when exposed to 83.4 °C for 0.95 s, the virus was completely inactivated (zero titer, > 6 Log10 reduction).IMPORTANCEThree coronaviruses (CoVs) have now caused global outbreaks within the past 20 years, with the COVID19 pandemic caused by SARS-CoV-2 still ongoing. Methods that can rapidly inactivate viruses, especially CoVs, can play critical roles in ensuring public safety and safeguarding personal health. Heat treatment of viruses to inactive them can be an efficient and inexpensive method, with the potential to be incorporated into various human-occupied spaces. In this work, a simple system that can heat-treat viruses for extremely short period was developed and utilized to show that sub-second exposure of CoV to heat is sufficient to inactivate CoV. This opens up the possibility of developing instruments and methods of disinfecting CoV in diverse settings, including rapid liquid disinfection and airborne virus disinfection. The developed method can also be broadly utilized to assess heat sensitivity of viruses other viral pathogens of interest and develop sub-second rapid heat inactivation approaches.

Summary The viral antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress hepatitis B virus (HBV) infection. After adoptive transfer, PSC-CTLs can infiltrate into the liver to suppress HBV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs provoke the antiviral response remain to be fully elucidated. In this study, we generated the functional HBV surface Ag-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the underlying mechanisms of the CTL-mediated antiviral replication in a murine model. We show that adoptive transfer of HBV surface Ag-specific iPSC-CTLs greatly suppressed HBV replication and prevented HBV surface Ag expression. We further demonstrate that the adoptive transfer significantly increased T cell accumulation and production of antiviral cytokines. These results indicate that stem cell-derived viral Ag-specific CTLs can robustly accumulate in the liver and suppress HBV replication through producing antiviral cytokines., Graphical Abstract, Highlights • Generation of functional viral Ag-specific CTLs from iPSCs, i.e., iPSC-CTLs • Viral Ag-specific iPSC-CTLs suppress HBV replication in a murine model • Adoptive transfer of viral Ag-specific iPSC-CTLs generates persistent α-HBV T cells • Adoptive transfer of viral Ag-specific iPSC-CTLs produces antiviral IFN-γ and TNF-⍺, Immunology; Functional Aspects of Cell Biology

SummaryPhagocytosis and autophagy play critical roles in immune defense. Cryptococcus neoformans (Cn), a fungal pathogen that causes fatal infection, subverts the host autophagy initiation complex (AIC) and its upstream regulatory proteins, to promote its phagocytosis and intracellular parasitism of host cells. The mechanisms by which the pathogen engages host AIC proteins remain obscure. Here, we show that the recruitment of host AIC proteins to forming phagosomes is dependent upon the activity of CD44, a host cell surface receptor that engages fungal hyaluronic acid (HA). This interaction elevates intracellular Ca2+ concentrations and activates CaMKKβ and its downstream target AMPKα, which results in activation of ULK1 and the recruitment of AIC components. Moreover, we demonstrate that HA-coated beads efficiently recruit AIC components to phagosomes. Taken together, these findings show that fungal HA plays a critical role in directing the internalization and productive intracellular membrane trafficking of a fungal pathogen of global importance.Graphical AbstractIn BriefDing et al. reveal that interactions between fungal hyaluronic acid (HA) and host CD44 activate a Ca2+ - CaMKKβ-AMPK-ULK1 signaling pathway that recruits autophagy initiation complex components to forming phagosomes to drive fungal internalization.HighlightsFungal HA interactions with host cells drive a novel non-canonical, ligand-induced, autophagy pathway in phagocytic cellsCryptococcus neoformans recruits host CD44, together with AIC components and regulatory proteins, to forming phagocytic cups to initiate host cell internalizationFungal HA interactions with CD44 on host cell surfaces elevate intracellular Ca2+ concentrations, leading to activation of CaMKKβA Ca2+-CaMKKβ-AMPK-ULK1 signaling axis is involved in HA and CD44 induced autophagy protein recruitment during Cn internalization

Acinetobacter baumannii is an important causative agent of nosocomial infections worldwide. The pathogen also readily acquires resistance to antibiotics, and pan-resistant strains have been reported. A. baumannii is widely regarded as an extracellular bacterial pathogen. However, accumulating evidence demonstrates that the pathogen can invade, survive or persist in infected mammalian cells. Unfortunately, the molecular mechanisms controlling these processes remain poorly understood. Here, we show that Drosophila S2 cells provide several attractive advantages as a model system for investigating the intracellular lifestyle of the pathogen, including susceptibility to bacterial intracellular replication and limited infection-induced host cell death. We also show that the Drosophila system can be used to rapidly identify host factors, including MAP kinase proteins, which confer susceptibility to intracellular parasitism. Finally, analysis of the Drosophila system suggested that host proteins that regulate organelle biogenesis and membrane trafficking contribute to regulating the intracellular lifestyle of the pathogen. Taken together, these findings establish a novel model system for elucidating interactions between A. baumannii and host cells, define new factors that regulate bacterial invasion or intracellular persistence, and identify subcellular compartments in host cells that interact with the pathogen.