Salmonella enterica Modulates Its Infectivity in Response to Intestinal Stimuli

ABSTRACT En route to its intestinal target cells Salmonella enterica passes different host niches and encounters various environmental cues. These are expected to promote Salmonella in the decision of changing its extracellular life style to intracellular. We find that prior incubation of bacteria in the presence of signals which are characteristic for the small intestine affects invasion in a model system: Salmonella grown at high osmotic pressure in the presence of bile or in amino acid rich medium, infect host cells most efficiently. Hence, Salmonella enterica modulates its infectivity in response to these stimuli which consequently determines the success of infection. Our results close the current gap between signal and actual behavior and may serve as a basis for further investigations for example if Salmonella has an adaptive prediction of environmental changes

A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents

SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha

Evaluation of titration method on Vero E6 and Calu-3 cells for the calibration of B.1 and VOC Alpha seeding doses and comparison of E gene copy numbers versus infectious units (determined by Q-RT-PCR and plaque assay on Vero E6 cells) in SARS-CoV-2 stocks.

(A) No significant differences in the titers were observed when titers of B.1 and VOC Alpha SARS-CoV-2 stocks were compared on Calu-3 vs. Vero E6 using TCID50 titration method. Although plaque morphology of B.1- and VOC Alpha-infected Vero E6 cells differ, Vero E6 cells are suitable to determine titers by plaque titration assay. N = 3 biologically independent experiments each conducted in triplicates. (B) Overview on virus infectivity and viral RNA concentrations, determined by plaque assay (log10 PFU/ml) and E gene assay (log10 GE/ml), of all virus stocks is shown. Direct comparison of all B.1 and VOC Alpha stocks. Statistical analysis was conducted between both viruses for genomic E gene and infectious titers, respectively. Stocks applied in gene expression analysis (triangle) and growth kinetics (square) are highlighted by symbols. GE, genome equivalents; n.s., not significant; PFU, plaque-forming units; Q-RT-PCR, quantitative real-time PCR; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VOC, variant of concern. See S1 Data. (TIF)</p

Delayed cytopathic onset of VOC Alpha SARS-CoV-2 infection.

Vero E6 cells were infected with B.1, VOC Alpha/v1, and VOC Alpha/v2 (MOI 0.01). Onset of CPE was monitored by live cell imaging until 70 hours postinfection. CPE, cytopathogenic effect; MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VOC, variant of concern. (MP4)</p

Delayed cytopathic onset of VOC Alpha SARS-CoV-2 infection.

Vero E6 cells were infected with B.1, VOC Alpha/v1, and VOC Alpha/v2 (MOI 0.001). Onset of CPE was monitored by live cell imaging until 70 hours postinfection. CPE, cytopathogenic effect; MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VOC, variant of concern. (MP4)</p

Primers used for quantitative RT-PCR.

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Similar abundance of sgN RNAs, genome replication, and low but similar expression of IFNs, proinflammatory cytokines, and ISGs in B.1 and VOC Alpha-infected H1299 cells.

NCI-H1299 cells were infected with B.1 or VOC Alpha (MOI of 2), and viral replication, viral transcription, and expression of innate immune genes were determined by Q-RT-PCR from cell lysates at 24 and 48 hours postinfection. (A) Expression of cell-associated envelope. (B) Expression of cell-associated sgN RNA. TBP was used for normalization. (C) Expression of the indicated genes was determined by specific Q-RT-PCR. TBP was used for normalization. Shown is the mean fold change +/− SD of 3 biologically independent experiments that were each conducted in quadruples. RVFV cl.13, which is devoid of its IFN antagonist NSs, was included for the expression of IFNs, ISGs, and pro-inflammatory cytokines. GE, genome equivalents; IFN, interferon; ISG, IFN-stimulated gene; Q-RT-PCR, quantitative real-time PCR; RVFV cl.13, Rift Valley Fever Virus clone 13; sgN, subgenomic nucleocapsid; TBP, TATA-binding protein. See S1 Data. (TIF)</p

Absence of detectable fitness advantages of VOC Alpha in primary human respiratory cells, organoids, and hamsters.

(A) Virus growth kinetics were performed in infected hNAECs (MOI 0.1). Samples were collected from the apical and basal side at indicated time points and titrated by plaque assay. n = 3 biological replicates. (B) Virus growth kinetics was conducted in infected bronchial AEC (MOI 0.5). Samples were collected from the apical side and titrated by plaque assay. Data are derived from 1 experiment conducted in triplicates. (C) Intestinal organoids were infected (MOI 0.05) and viral load in supernatant (left) and organoid lysates (right) was quantified at indicated time points by E-gene-specific quantitative RT-PCR. Data are derived from 4 independent experiments. (D) Virus replication was monitored in infected lung organoids (MOI 1). Samples harvested at indicated time points were titrated by plaque assay. Data are derived from 3 independent experiments. (E) Dwarf hamsters were intranasally infected (100,000 PFU) and infectious virus particles from lung homogenates were quantified using plaque assay (left). Donor hamsters were cohoused with naive animals and transmission efficiency was determined from lung homogenates at the indicated time points (right). n = 1–3 animals per experimental condition. Dotted horizontal lines indicate the lower detection limit of the plaque assays. AEC, airway epithelial culture; GE, genome equivalents; hNAEC, human nasal airway epithelial culture; MOI, multiplicity of infection; n.d., not detected; PFU, plaque-forming units; RT-PCR, real-time PCR; VOC, variant of concern. See S1 Data.</p