Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231’s early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231’s efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models. Importance: The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19), a pandemic that has claimed over 700,000 human lives. The only SARS-CoV-2 antiviral, for emergency use, is remdesivir, targeting the viral polymerase complex. PF-00835231 is a pre-clinical lead compound with an alternate target, the main SARS-CoV-2 protease 3CLpro (Mpro). Here, we perform a comparative analysis of PF-00835231 and remdesivir in A549+ACE2 cells, using isolates of two major SARS-CoV-2 clades. PF-00835231 is antiviral for both clades, and, in this assay, statistically more potent than remdesivir. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps and validates PF-00835231’s time of action. Both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 in human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective antiviral for SARS-CoV-2, addresses concerns from non-human in vitro models, and supports further studies with this compound.

Staphylococcus aureus have evolved into diverse lineages, known as clonal complexes (“CC”), which exhibit differences in the coding sequences of core virulence factors. Whether these alterations impact functionality is poorly understood. Here, we studied the highly polymorphic pore-forming toxin LukAB. We discovered that the LukAB toxin variants produced by S. aureus CC30 and CC45 kill human phagocytes regardless of whether CD11b, the previously established LukAB receptor, is present, and instead target the human hydrogen voltage-gated channel 1 (HVCN1). Biochemical studies identified the domain within human HVCN1 that drives LukAB species specificity, enabling the generation of humanized HVCN1 mice with enhanced susceptibility to CC30 LukAB and to bloodstream infection caused by CC30 S. aureus strains. Altogether, this work advances our understanding of an important S. aureus toxin and underscores the importance of considering genetic variation to characterizing virulence factors and understand the tug of war between pathogens and the host.

Salmonella enterica serovar Typhi is the etiologic agent of typhoid fever, a major public health problem in the developing world. Moving toward and adhering to the intestinal epithelium represents key initial steps of infection by S. Typhi. We examined the role of the S. Typhi yrbE gene, which encodes an inner membrane phospholipid transporter, in these interactions with epithelial cells. Disruption of yrbE resulted in elevated expression of flagellin and a hypermotile phenotype. It also significantly reduced the ability of S. Typhi to adhere to the HeLa epithelial cell line and to polarized primary epithelial cells derived from human ileal organoids. Interestingly, the yrbE-deficient strain of S. Typhi induced higher production of interleukin-8 from the primary human ileal epithelial cell monolayers compared to the wild-type bacteria. Deletion of the flagellin gene (fliC) in the yrbE-deficient S. Typhi inhibited motility and attenuated interleukin-8 production, but it did not correct the defect in adhesion. We also disrupted yrbE in S. Typhimurium. In contrast to the results in S. Typhi, the deficiency of yrbE in S. Typhimurium had no significant effect on flagellin expression, motility or adhesion to HeLa cells. Correspondingly, the lack of yrbE also had no effect on association with the intestine or the severity of intestinal inflammation in the mouse model of S. Typhimurium infection. Thus, our results point to an important and serovar-specific role played by yrbE in the early stages of intestinal infection by S. Typhi.

The intestinal microbiota has several effects on host physiology. Previous work from our laboratory demonstrated that the microbiota influences systemic iron homeostasis in mouse colitis models by altering inflammation‐induced expression of the iron‐regulating hormone hepcidin. In the present study, we examined the impact of the gut commensal bacterium Bacteroides fragilis on the expression of the iron exporter ferroportin, the target of hepcidin action, in macrophages, the cell type that plays a pivotal role in iron recycling. Mouse bone marrow‐derived macrophages were exposed to B. fragilis and were analyzed by quantitative real‐time polymerase chain reaction and Western blotting. We found that B. fragilis down‐regulated ferroportin transcription independently of bacterial viability. Medium conditioned by the bacteria also reduced ferroportin expression, indicating the involvement of soluble factors, possibly Toll‐like receptor ligands. Consistent with this idea, several of these ligands were able to down‐regulate ferroportin. The B. fragilis‐induced decrease in ferroportin was functionally important since it produced a significant increase in intracellular iron concentrations that prevented the effects of the iron chelator deferoxamine on Salmonella‐induced IL‐6 and IL‐1β production. Our results thus reveal that B. fragilis can influence macrophage iron handling and inflammatory responses by modulating ferroportin expression.

The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial cultures at air-liquid interface (HAE) are a physiologically relevant in vitro model of this heterogeneous tissue, enabling numerous studies of airway disease. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining capacity for differentiation to HAE. However, gene expression and innate immune function in HAE derived from BCi-NS1.1 versus primary cells have not been fully characterized. Here, combining single cell RNA-Seq (scRNA-Seq), immunohistochemistry, and functional experimentation, we confirm at high resolution that BCi-NS1.1 and primary HAE cultures are largely similar in morphology, cell type composition, and overall transcriptional patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1 HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus. Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.

Proteases play key roles in viral replication cycles. They can provide cleavage maturation of viral glycoproteins, processing of viral polyproteins, or disassembly of viral capsids. Thus, proteases constitute ideal targets for antiviral intervention: pharmaceutically, by small molecule inhibitors, or naturally, by host immune responses. Indeed, we and others have shown that individual members of the Serine protease inhibitor (SERPIN) family have specific antiviral function by blocking proteolytic steps inherent to viral replication cycles. Whether additional members of the large SERPIN family possess antiviral activity and whether SERPINs function as part of the antiviral cell-intrinsic immune response, is currently unknown. Here, we found that specific SERPINs are produced upon infection with clinically relevant respiratory viruses in vitro and in vivo, and in concert with classical interferon-stimulated genes. We next developed a structure-based in silico screen to uncover non-canonical SERPIN-protease pairs. We identified several SERPINs with potential antiviral function, including: SERPINE1 targeting cathepsin L, required for SARS-CoV-2 entry; SERPINB8 targeting furin, required for glycoprotein maturation cleavage of numerous viruses; and SERPINB2 targeting adenovirus protease, which suggests the first direct-acting antiviral SERPIN. Our study demonstrates how proteolysis is modulated for antiviral defense and how this process could inform antiviral targets against clinically relevant respiratory pathogens.