Bacteriophage defends murine gut from Escherichia coli invasion via mucosal adherence.

Bacteriophage are sophisticated cellular parasites that can not only parasitize bacteria but are increasingly recognized for their direct interactions with mammalian hosts. Phage adherence to mucus is known to mediate enhanced antimicrobial effects in vitro. However, little is known about the therapeutic efficacy of mucus-adherent phages in vivo. Here, using a combination of in vitro gastrointestinal cell lines, a gut-on-a-chip microfluidic model, and an in vivo murine gut model, we demonstrated that a E. coli phage, øPNJ-6, provided enhanced gastrointestinal persistence and antimicrobial effects. øPNJ-6 bound fucose residues, of the gut secreted glycoprotein MUC2, through domain 1 of its Hoc protein, which led to increased intestinal mucus production that was suggestive of a positive feedback loop mediated by the mucus-adherent phage. These findings extend the Bacteriophage Adherence to Mucus model into phage therapy, demonstrating that øPNJ-6 displays enhanced persistence within the murine gut, leading to targeted depletion of intestinal pathogenic bacteria.

ZMapp reduces diffusion of Ebola viral particles in fresh human cervicovaginal mucus.

Vaginal transmission from semen of male Ebola virus (EBOV) survivors has been implicated as a potential origin of Ebola virus disease (EVD) outbreaks. While EBOV in semen must traverse cervicovaginal mucus (CVM) to reach target cells, the behaviour of EBOV in CVM is poorly understood. CVM contains substantial quantities of IgG, and arrays of IgG bound to a virion can develop multiple Fc-mucin bonds, immobilizing the IgG/virion complex in mucus. Here, we measured the real-time mobility of fluorescent Ebola virus-like-particles (VLP) in 50 CVM specimens from 17 women, with and without ZMapp, a cocktail of 3 monoclonal IgGs against EBOV. ZMapp-mediated effective trapping of Ebola VLPs in CVM from a subset of women across the menstrual cycle, primarily those with Lactobacillus crispatus dominant microbiota. Our work underscores the influence of the vaginal microbiome on IgG-mucin crosslinking against EBOV and identifies bottlenecks in the sexual transmission of EBOV.

ITGB4 deficiency induces mucus hypersecretion by upregulating MUC5AC in RSV-infected airway epithelial cells.

Respiratory syncytial virus (RSV) infection is the main cause of bronchiolitis in children. Excessive mucus secretion is one of the primary symbols in RSV related lower respiratory tract infections (RSV-related LRTI), which is closely associated with the occurrence and development of asthma in later life. Integrin ß4 (ITGB4) is down-regulated in the airway epithelial cells (AECs) of asthma patients which plays a critical role in the pathogenesis of asthma. However, whether ITGB4 is involved in the pathological processes of RSV infection remains unclear. In this study, we found that decreased expression of ITGB4 was negatively correlated with the level of MUC5AC in childhood AECs following RSV infection. Moreover, ITGB4 deficiency led to mucus hypersecretion and MUC5AC overexpression in the small airway of RSV-infected mice. MUC5AC expression was upregulated by ITGB4 in HBE cells through EGFR, ERK and c-Jun pathways. EGFR inhibitors treatment inhibited mucus hypersecretion and MUC5AC overexpression in ITGB4-deficient mice after RSV infection. Together, these results demonstrated that epithelial ITGB4 deficiency induces mucus hypersecretion by upregulating the expression of MUC5AC through EGFR/ERK/c-Jun pathway, which further associated with RSV-related LRTI.

Evaluation of serum total sialic acid in moderate COVID-19 patients with and without gastrointestinal tract manifestations.

BACKGROUND: It is known that SARS-CoV-2 mostly infects the respiratory system causing pneumonia; although it can also affect the gastrointestinal tract (GIT), which covered with a bi-layer of mucus rich in glycosylated proteins that terminated by sialic acid. Therefore; this study aimed to evaluate serum total sialic acid (TSA) in moderate COVID-19 patients with and without GIT manifestations. METHODS: A total of 161 moderate COVID-19 patients without and with GIT manifestations and 50 controls were enrolled into our study. Serum electrolytes levels were measured by using colorimetric or turbidmetric commercial assay kits, while the level of serum TSA was measured by using a commercial ELISA kit. RESULTS: Our results showed that serum TSA level was highly significantly increased in moderate COVID-19 patients with GIT manifestations (81.43 ± 8.91) when compared with controls (61.24 ± 6.41) or even moderate COVID-19 patients without GIT manifestations (69.46 ± 7.03). ROC curve analysis showed that AUC for TSA is 0.84 with 76.2 % sensitivity and 73.7 % specificity in discrimination between moderate COVID-19 patients with and without GIT manifestations. Serum potassium and sodium levels were highly significantly decreased in moderate COVID-19 patients with GIT manifestations when compared with controls or even moderate COVID-19 patients without GIT manifestations; while serum calcium level was found to be significantly decreased in moderate COVID-19 patients with GIT manifestations when compared with controls. CONCLUSION: Finally, we can conclude that SA plays a crucial role in the pathogenesis of GIT complications associated with COVID-19 and could be a potential biomarker for the COVID-19 gastrointestinal complications.

Simulated sunlight decreases the viability of SARS-CoV-2 in mucus.

The novel coronavirus, SARS-CoV-2, has spread into a pandemic since its emergence in Wuhan, China in December of 2019. This has been facilitated by its high transmissibility within the human population and its ability to remain viable on inanimate surfaces for an extended period. To address the latter, we examined the effect of simulated sunlight on the viability of SARS-CoV-2 spiked into tissue culture medium or mucus. The study revealed that inactivation took 37 minutes in medium and 107 minutes in mucus. These times-to-inactivation were unexpected since they are longer than have been observed in other studies. From this work, we demonstrate that sunlight represents an effective decontamination method but the speed of decontamination is variable based on the underlying matrix. This information has an important impact on the development of infection prevention and control protocols to reduce the spread of this deadly pathogen.

Gut Susceptibility to Viral Invasion: Contributing Roles of Diet, Microbiota and Enteric Nervous System to Mucosal Barrier Preservation.

The gastrointestinal lumen is a rich source of eukaryotic and prokaryotic viruses which, together with bacteria, fungi and other microorganisms comprise the gut microbiota. Pathogenic viruses inhabiting this niche have the potential to induce local as well as systemic complications; among them, the viral ability to disrupt the mucosal barrier is one mechanism associated with the promotion of diarrhea and tissue invasion. This review gathers recent evidence showing the contributing effects of diet, gut microbiota and the enteric nervous system to either support or impair the mucosal barrier in the context of viral attack.

Transmission risk of viruses in large mucosalivary droplets on the surface of objects: A time-based analysis.

The novel human coronavirus SARS-CoV-2 has been responsible for a worldwide pandemic. Although media transmission through contaminated surfaces is one of the most recognized ways of transmission, the study on the number and viability of viruses surviving on a surface after leaving the host represents a "blind spot" in current research. In this paper we have reviewed studies on the physical process of droplet evaporation on media surfaces, and analyzed the recent literature related to experiments on the decay of the viral concentration and infectious activity of SARS-CoV-2 and other viruses on those surface and in the air. The huge differences in the risk of media transmission of large saliva and sputum droplets were analyzed in terms of time elapsed. Due to the rapid decrease of water content in the evaporated droplets and the increased concentration of each component, the living environment of the virus tended to deteriorate sharply, and virus concentration plummeted within a few minutes. Although a virus can be detected in a matter of hours, tens of hours, or days, the risk of transmission is negligible compared to when it first left the host. This study suggests that the key to prevention and control is to start from the source, the earlier the better. It is extremely important to develop good public health habits, wear masks, and wash hands frequently. That said, excessive disinfection and sterilization of surfaces during a later period may have adverse effects.

Exhaled aerosol increases with COVID-19 infection, age, and obesity.

COVID-19 transmits by droplets generated from surfaces of airway mucus during processes of respiration within hosts infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. We studied respiratory droplet generation and exhalation in human and nonhuman primate subjects with and without COVID-19 infection to explore whether SARS-CoV-2 infection, and other changes in physiological state, translate into observable evolution of numbers and sizes of exhaled respiratory droplets in healthy and diseased subjects. In our observational cohort study of the exhaled breath particles of 194 healthy human subjects, and in our experimental infection study of eight nonhuman primates infected, by aerosol, with SARS-CoV-2, we found that exhaled aerosol particles vary between subjects by three orders of magnitude, with exhaled respiratory droplet number increasing with degree of COVID-19 infection and elevated BMI-years. We observed that 18% of human subjects (35) accounted for 80% of the exhaled bioaerosol of the group (194), reflecting a superspreader distribution of bioaerosol analogous to a classical 20:80 superspreader of infection distribution. These findings suggest that quantitative assessment and control of exhaled aerosol may be critical to slowing the airborne spread of COVID-19 in the absence of an effective and widely disseminated vaccine.

The proteomic characteristics of airway mucus from critical ill COVID-19 patients.

BACKGROUND: The pandemic of the coronavirus disease 2019 (COVID-19) has brought a global public health crisis. However, the pathogenesis underlying COVID-19 are barely understood. METHODS: In this study, we performed proteomic analyses of airway mucus obtained by bronchoscopy from severe COVID-19 patients. In total, 2351 and 2073 proteins were identified and quantified in COVID-19 patients and healthy controls, respectively. RESULTS: Among them, 92 differentiated expressed proteins (DEPs) (46 up-regulated and 46 down-regulated) were found with a fold change >1.5 or <0.67 and a p-value <0.05, and 375 proteins were uniquely present in airway mucus from COVID-19 patients. Pathway and network enrichment analyses revealed that the 92 DEPs were mostly associated with metabolic, complement and coagulation cascades, lysosome, and cholesterol metabolism pathways, and the 375 COVID-19 only proteins were mainly enriched in amino acid degradation (Valine, Leucine and Isoleucine degradation), amino acid metabolism (beta-Alanine, Tryptophan, Cysteine and Methionine metabolism), oxidative phosphorylation, phagosome, and cholesterol metabolism pathways. CONCLUSIONS: This study aims to provide fundamental data for elucidating proteomic changes of COVID-19, which may implicate further investigation of molecular targets directing at specific therapy.

Soft matter science and the COVID-19 pandemic.

Much of the science underpinning the global response to the COVID-19 pandemic lies in the soft matter domain. Coronaviruses are composite particles with a core of nucleic acids complexed to proteins surrounded by a protein-studded lipid bilayer shell. A dominant route for transmission is via air-borne aerosols and droplets. Viral interaction with polymeric body fluids, particularly mucus, and cell membranes controls their infectivity, while their interaction with skin and artificial surfaces underpins cleaning and disinfection and the efficacy of masks and other personal protective equipment. The global response to COVID-19 has highlighted gaps in the soft matter knowledge base. We survey these gaps, especially as pertaining to the transmission of the disease, and suggest questions that can (and need to) be tackled, both in response to COVID-19 and to better prepare for future viral pandemics.

Effect of Environmental Conditions on SARS-CoV-2 Stability in Human Nasal Mucus and Sputum.

We found that environmental conditions affect the stability of severe acute respiratory syndrome coronavirus 2 in nasal mucus and sputum. The virus is more stable at low-temperature and low-humidity conditions, whereas warmer temperature and higher humidity shortened half-life. Although infectious virus was undetectable after 48 hours, viral RNA remained detectable for 7 days.

Astrovirus infects actively secreting goblet cells and alters the gut mucus barrier.

Astroviruses are a global cause of pediatric diarrhea, but they are largely understudied, and it is unclear how and where they replicate in the gut. Using an in vivo model, here we report that murine astrovirus preferentially infects actively secreting small intestinal goblet cells, specialized epithelial cells that maintain the mucus barrier. Consequently, virus infection alters mucus production, leading to an increase in mucus-associated bacteria and resistance to enteropathogenic E. coli colonization. These studies establish the main target cell type and region of the gut for productive murine astrovirus infection. They further define a mechanism by which an enteric virus can regulate the mucus barrier, induce functional changes to commensal microbial communities, and alter host susceptibility to pathogenic bacteria.

The cervicovaginal mucus barrier to HIV-1 is diminished in bacterial vaginosis.

Bacterial vaginosis (BV), a condition in which the vaginal microbiota consists of community of obligate and facultative anaerobes rather than dominated by a single species of Lactobacillus, affects ~30% of women in the US. Women with BV are at 60% increased risk for HIV acquisition and are 3-times more likely to transmit HIV to an uninfected partner. As cervicovaginal mucus (CVM) is the first line of defense against mucosal pathogens and the home of the resident vaginal microbiota, we hypothesized the barrier function of CVM to HIV may be diminished in BV. Here, we characterized CVM properties including pH, lactic acid content, and Nugent score to correlate with the microbiota community composition, which was confirmed by 16S rDNA sequencing on a subset of samples. We then quantified the mobility of fluorescently-labeled HIV virions and nanoparticles to characterize the structural and adhesive barrier properties of CVM. Our analyses included women with Nugent scores categorized as intermediate (4-6) and BV (7-10), women that were either symptomatic or asymptomatic, and a small group of women before and after antibiotic treatment for symptomatic BV. Overall, we found that HIV virions had significantly increased mobility in CVM from women with BV compared to CVM from women with Lactobacillus crispatus-dominant microbiota, regardless of whether symptoms were present. We confirmed using nanoparticles and scanning electron microscopy that the impaired barrier function was due to reduced adhesive barrier properties without an obvious degradation of the physical CVM pore structure. We further confirmed a similar increase in HIV mobility in CVM from women with Lactobacillus iners-dominant microbiota, the species most associated with transitions to BV and that persists after antibiotic treatment for BV. Our findings advance the understanding of the protective role of mucus and highlight the interplay between vaginal microbiota and the innate barrier function mucus.

Bacteriophage Adherence to Mucus Mediates Preventive Protection against Pathogenic Bacteria.

Metazoans were proposed to host bacteriophages on their mucosal surfaces in a symbiotic relationship, where phages provide an external immunity against bacterial infections and the metazoans provide phages a medium for interacting with bacteria. However, scarce empirical evidence and model systems have left the phage-mucus interaction poorly understood. Here, we show that phages bind both to porcine mucus and to rainbow trout (Oncorhynchus mykiss) primary mucus, persist up to 7 days in the mucosa, and provide protection against Flavobacterium columnare Also, exposure to mucus changes the bacterial phenotype by increasing bacterial virulence and susceptibility to phage infections. This trade-off in bacterial virulence reveals ecological benefit of maintaining phages in the metazoan mucosal surfaces. Tests using other phage-bacterium pairs suggest that phage binding to mucus may be widespread in the biosphere, indicating its importance for disease, ecology, and evolution. This phenomenon may have significant potential to be exploited in preventive phage therapy.IMPORTANCE The mucosal surfaces of animals are habitat for microbes, including viruses. Bacteriophages-viruses that infect bacteria-were shown to be able to bind to mucus. This may result in a symbiotic relationship in which phages find bacterial hosts to infect, protecting the mucus-producing animal from bacterial infections in the process. Here, we studied phage binding on mucus and the effect of mucin on phage-bacterium interactions. The significance of our research is in showing that phage adhesion to mucus results in preventive protection against bacterial infections, which will serve as basis for the development of prophylactic phage therapy approaches. Besides, we also reveal that exposure to mucus upregulates bacterial virulence and that this is exploited by phages for infection, adding one additional layer to the metazoan-bacterium-phage biological interactions and ecology. This phenomenon might be widespread in the biosphere and thus crucial for understanding mucosal diseases, their outcome and treatment.

Deletion of the thymidine kinase gene attenuates Caprine alphaherpesvirus 1 in goats.

Caprine alphaherpesvirus 1 (CpHV-1) is a pathogen associated with systemic infection and respiratory disease in kids and subclinical infection or reproductive failure and abortions in adult goats. The enzyme thymidine kinase (TK) is an important viral product involved in nucleotide synthesis. This property makes the tk gene a common target for herpesvirus attenuation. Here we deleted the tk gene of a CpHV-1 isolate and characterized the recombinant CpHV-1ΔTKin vitro and in vivo. In vitro characterization revealed that the recombinant CpHV-1ΔTK replicated to similar titers and produced plaques of similar size to the parental CpHV-1 strain in BT and CRIB cell lines. Upon intranasal inoculation of young goats, the parental virus replicated more efficiently and for a longer period than the recombinant virus. In addition, infection with the parental virus resulted in mild systemic and respiratory signs whereas the kids inoculated with the recombinant CpHV-1ΔTK virus remained healthy. Goats inoculated with the parental virus also developed higher neutralizing antibody titers when compared to CpHV-1ΔTK inoculated animals. Dexamethasone (Dx) administration on days 35-39 post-inoculation did not result in virus shedding in nasal secretions, indicating lack of reactivation from latency. However, viral DNA was detected in the trigeminal ganglia of animals euthanized at 14 days post-Dx, indicating that both viruses successfully established latent infection. Our results show that the recombinant CpHV-1ΔTK presents an attenuated phenotype when compared to the parental virus, and hence may represent a promising vaccine candidate to prevent CpHV-1 disease in goats.

Situations Leading to Reduced Effectiveness of Current Hand Hygiene against Infectious Mucus from Influenza Virus-Infected Patients.

Both antiseptic hand rubbing (AHR) using ethanol-based disinfectants (EBDs) and antiseptic hand washing (AHW) are important means of infection control to prevent seasonal influenza A virus (IAV) outbreaks. However, previous reports suggest a reduced efficacy of ethanol disinfection against pathogens in mucus. We aimed to elucidate the situations and mechanisms underlying the reduced efficacy of EBDs against IAV in infectious mucus. We evaluated IAV inactivation and ethanol concentration change using IAV-infected patients' mucus (sputum). Additionally, AHR and AHW effectiveness against infectious mucus adhering to the hands and fingers was evaluated in 10 volunteers. Our clinical study showed that EBD effectiveness against IAV in mucus was extremely reduced compared to IAV in saline. IAV in mucus remained active despite 120 s of AHR; however, IAV in saline was completely inactivated within 30 s. Due to the low rate of diffusion/convection because of the physical properties of mucus as a hydrogel, the time required for the ethanol concentration to reach an IAV inactivation level and thus for EBDs to completely inactivate IAV was approximately eight times longer in mucus than in saline. On the other hand, AHR inactivated IAV in mucus within 30 s when the mucus dried completely because the hydrogel characteristics were lost. Additionally, AHW rapidly inactivated IAV. Until infectious mucus has completely dried, infectious IAV can remain on the hands and fingers, even after appropriate AHR using EBD, thereby increasing the risk of IAV transmission. We clarified the ineffectiveness of EBD use against IAV in infectious mucus.IMPORTANCE Antiseptic hand rubbing (AHR) and antiseptic hand washing (AHW) are important to prevent the spread of influenza A virus (IAV). This study elucidated the situations/mechanisms underlying the reduced efficacy of AHR against infectious mucus derived from IAV-infected individuals and indicated the weaknesses of the current hand hygiene regimens. Due to the low rate of diffusion/convection because of the physical properties of mucus as a hydrogel, the efficacy of AHR using ethanol-based disinfectant against mucus is greatly reduced until infectious mucus adhering to the hands/fingers has completely dried. If there is insufficient time before treating the next patient (i.e., if the infectious mucus is not completely dry), medical staff should be aware that effectiveness of AHR is reduced. Since AHW is effective against both dry and nondry infectious mucus, AHW should be adopted to compensate for these weaknesses of AHR.

Antibody-Mediated Immobilization of Virions in Mucus.

Antibodies have been shown to hinder the movement of herpes simplex virus virions in cervicovaginal mucus, as well as other viruses in other mucus secretions. However, it has not been possible to directly observe the mechanisms underlying this phenomenon, so the nature of virion-antibody-mucin interactions remain poorly understood. In this work, we analyzed thousands of virion traces from single particle tracking experiments to explicate how antibodies must cooperate to immobilize virions for relatively long time periods. First, using a clustering analysis, we observed a clear separation between two classes of virion behavior: freely diffusing and immobilized. While the proportion of freely diffusing virions decreased with antibody concentration, the magnitude of their diffusivity did not, implying an all-or-nothing dichotomy in the pathwise effect of the antibodies. Proceeding under the assumption that all binding events are reversible, we used a novel switch-point detection method to conclude that there are very few, if any, state switches on the experimental timescale of 20 s. To understand this slow state switching, we analyzed a recently proposed continuous-time Markov chain model for binding kinetics and virion movement. Model analysis implied that virion immobilization requires cooperation by multiple antibodies that are simultaneously bound to the virion and mucin matrix and that there is an entanglement phenomenon that accelerates antibody-mucin binding when a virion is immobilized. In addition to developing a widely applicable framework for analyzing multistate particle behavior, this work substantially enhances our mechanistic understanding of how antibodies can reinforce a mucus barrier against passive invasive species.

Functions of the Microbiota for the Physiology of Animal Metaorganisms.

Animals are usually regarded as independent entities within their respective environments. However, within an organism, eukaryotes and prokaryotes interact dynamically to form the so-called metaorganism or holobiont, where each partner fulfils its versatile and crucial role. This review focuses on the interplay between microorganisms and multicellular eukaryotes in the context of host physiology, in particular aging and mucus-associated crosstalk. In addition to the interactions between bacteria and the host, we highlight the importance of viruses and nonmodel organisms. Moreover, we discuss current culturing and computational methodologies that allow a deeper understanding of underlying mechanisms controlling the physiology of metaorganisms.