Selection for phage resistance reduces virulence of Shigella flexneri .

There is increasing interest in phage therapy as an alternative to antibiotics for treating bacterial infections, especially using phages that select for evolutionary trade-offs between increased phage resistance and decreased fitness traits such as virulence in target bacteria. A vast repertoire of virulence factors allows the opportunistic bacterial pathogen, Shigella flexneri , to invade human gut epithelial cells, replicate intracellularly, and evade host immunity through intercellular spread. It is previously shown that OmpA is necessary for intercellular spread of S. flexneri . We hypothesized that a phage which uses OmpA as a receptor to infect S. flexneri , should select for phage-resistant mutants with attenuated intercellular spread. Here we show that phage A1-1, requires OmpA as a receptor and selects for reduced virulence in S. flexneri . We characterized five phage-resistant mutants by measuring phenotypic changes in various traits: cell-membrane permeability, total lipopolysaccharide (LPS), sensitivity to antibiotics, and susceptibility to other phages. Results separated the mutants into two groups: R1 and R2 phenotypically resembled ompA knockouts, whereas R3, R4 and R5 were similar to LPS-deficient strains. Whole genome sequencing confirmed that R1 and R2 had mutations in ompA , while R3, R4 and R5 showed mutations in LPS inner-core biosynthesis genes gmhA and gmhC . Bacterial plaque assays confirmed that all phage-resistant mutants were incapable of intercellular spread. We concluded that selection for S. flexneri resistance to phage A1-1 generally reduced virulence (i.e. intercellular spread), but this trade-off could be mediated either by mutations in ompA or in LPS-core genes that likely altered OmpA conformation. Author Summary Shigella flexneri is a facultative intracellular pathogen of humans, and a leading cause of bacillary dysentery. With few effective treatments and rising antibiotic resistance in these bacteria, there is increasing interest in alternatives to classical infection management of S. flexneri infections. Phage therapy poses an attractive alternative, particularly if a therapeutic phage can be found that results in an evolutionary trade-off between phage resistance and bacterial virulence. Here, we isolate a novel lytic phage from water collected in Cuatro Cienegas, Mexico that uses the OmpA porin of S. flexneri as a receptor. We use phenotypic assays and genome sequencing to show that phage A1-1 selects for phage-resistant mutants that can be grouped into two categories: OmpA-deficient mutants and LPS-deficient mutants. Despite these underlying mechanistic differences, we confirmed that naturally-occurring phage A1-1 selected for evolved phage resistance that coincided with impaired intercellular spread of S. flexneri in a eukaryotic infection model.

Diverse Effects of Exosomes on COVID-19: A Perspective of Progress From Transmission to Therapeutic Developments

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new strain of coronavirus and the causative agent of the current global pandemic of coronavirus disease 2019 (COVID-19). There are currently no FDA-approved antiviral drugs for COVID-19 and there is an urgent need to develop treatment strategies that can effectively suppress SARS-CoV-2 infection. Numerous approaches have been researched so far, with one of them being the emerging exosome-based therapies. Exosomes are nano-sized, lipid bilayer-enclosed structures, share structural similarities with viruses secreted from all types of cells, including those lining the respiratory tract. Importantly, the interplay between exosomes and viruses could be potentially exploited for antiviral drug and vaccine development. Exosomes are produced by virus-infected cells and play crucial roles in mediating communication between infected and uninfected cells. SARS-CoV-2 modulates the production and composition of exosomes, and can exploit exosome formation, secretion, and release pathways to promote infection, transmission, and intercellular spread. Exosomes have been exploited for therapeutic benefits in patients afflicted with various diseases including COVID-19. Furthermore, the administration of exosomes loaded with immunomodulatory cargo in combination with antiviral drugs represents a novel intervention for the treatment of diseases such as COVID-19. In particular, exosomes derived from mesenchymal stem cells (MSCs) are used as cell-free therapeutic agents. Mesenchymal stem cell derived exosomes reduces the cytokine storm and reverse the inhibition of host anti-viral defenses associated with COVID-19 and also enhances mitochondrial function repair lung injuries. We discuss the role of exosomes in relation to transmission, infection, diagnosis, treatment, therapeutics, drug delivery, and vaccines, and present some future perspectives regarding their use for combating COVID-19.

Hierarchical Cell Death Program Disrupts the Intracellular Niche Required for Burkholderia thailandensis Pathogenesis

Burkholderia infections result in a high degree of mortality when left untreated; therefore, understanding the host immune response required to control infection is critical. In this study, we uncovered a hierarchical cell death program utilized by infected cells to disrupt the intracellular niche of Burkholderia thailandensis by limiting bacterial intercellular spread, host cell-cell fusion, and bacterial replication. In macrophages, combined loss of key PANoptosis components results in extensive B. thailandensis infection-induced cell-cell fusion, bacterial replication, and increased cell death at later stages of infection compared with both wild-type (WT) and pyroptosis-deficient cells.

Investigating the roles of Listeria monocytogenes peroxidases in growth and virulence

Bacteria have necessarily evolved a protective arsenal of proteins to contend with peroxides and other reactive oxygen species generated in aerobic environments. Listeria monocytogenes encounters an onslaught of peroxide both in the environment and during infection of the mammalian host, where it is the causative agent of the foodborne illness listeriosis. Despite the importance of peroxide for the immune response to bacterial infection, the strategy by which L. monocytogenes protects against peroxide toxicity has yet to be illuminated. Here, we investigated the expression and essentiality of all the peroxidase-encoding genes during L. monocytogenes growth in vitro and during infection of murine cells in tissue culture. We found that chdC and kat were required for aerobic growth in vitro, and fri and ahpA were each required for L. monocytogenes to survive acute peroxide stress. Despite increased expression of fri, ahpA, and kat during infection of macrophages, only fri proved necessary for cytosolic growth and intercellular spread. In contrast, the proteins encoded by lmo0367, lmo0983, tpx, lmo1609, and ohrA were dispensable for aerobic growth, acute peroxide detoxification, and infection. Together, our results provide insight into the multifaceted L. monocytogenes peroxide detoxification strategy and demonstrate that L. monocytogenes encodes a functionally diverse set of peroxidase enzymes.

Extracellular Vesicles: Roles in Human Viral Infections, Immune-Diagnostic, and Therapeutic Applications

Membrane-bound vesicles that are released from cells are increasingly being studied as a medium of intercellular communication, as these act to shuttle functional proteins, such as lipids, DNA, rRNA, and miRNA, between cells during essential physiological processes. Extracellular vesicles (EVs), most commonly exosomes, are consistently produced by virus-infected cells, and they play crucial roles in mediating communication between infected and uninfected cells. Notably, pathophysiological roles for EVs have been established in various viral infections, including human immune deficiency virus (HIV), coronavirus (CoV), and human adenovirus (HAdv). Retroviruses, such as HIV, modulate the production and composition of EVs, and critically, these viruses can exploit EV formation, secretion, and release pathways to promote infection, transmission, and intercellular spread. Consequently, EV production has been investigated as a potential tool for the development of improved viral infection diagnostics and therapeutics. This review will summarize our present knowledge of EV–virus relationships, focusing on their known roles in pathophysiological pathways, immunomodulatory mechanisms, and utility for biomarker discovery. This review will also discuss the potential for EVs to be exploited as diagnostic and treatment tools for viral infection.