Fig 1.
Impact of SARS-CoV-2 salivary gland infection on histatin gene expression and production.
(a) Parotid expression of histatin (HTN1 and HTN3) and amylase (AMY1B) genes from deceased COVID-19 subjects (n = 8) and healthy control subjects (n = 3) using bulk RNA sequencing. (b-f) Co-localization studies using in situ hybridization for SARS-CoV-2 (white dots and arrows) and immunofluorescence for histatin-3 (green) with quantitative correlation between the intensity of histatin and SARS-CoV-2 counts within infected acinar cells of parotid tissue from (b) healthy subject (white asterisk, non-specific signal) and (c, e) deceased COVID-19 patients. Acini (dotted circles) identified based on expression of AQP5 (red, apical membrane); inset (orange box) shows the presence of the viral genome in acinar cells of the parotid glands. (d, f) Per acinar cell expression of histatin and the per cell viral count are inversely proportional as shown in two representative COVID-19 cases (P7, P19).
Fig 2.
Salivary histatin-5 (Hst-5) levels and fungal colonization in (a, b) prospectively sampled hospitalized and (c, d) outpatient COVID-19 cohorts.
(a) Boxplot with Hst-5 concentrations and Candida recovery from 26 saliva samples from hospitalized COVID-19 patients and matched healthy controls. (b) Waterfall plot depicting 26 pairs of hospitalized COVID-19 patients and healthy subjects matched for race and age. Bar height represents differences among pairs in Hst-5 levels (μg/ml) between healthy controls and COVID-19 patients. (c) Bar plot depicts Hst-5 concentrations and Candida recovery of 9 healthy subjects and 23 COVID-19 outpatients’ saliva. Red marked dots indicate cultured saliva contained candidal outgrowth (d) Linear regression analysis of serially sampled COVID-19 outpatients’ saliva (n = 8) shows time-dependent restoration of Hst-5 concentration from the post-acute phase to the chronic phase, based on longitudinally collected samples from acute phase (3–15 days) to chronic phase (6–12 months). Blue line, linear fit; light blue shading, confidence interval of the linear fit.
Fig 3.
Prospective time course longitudinal analysis of saliva samples for Hst-5 and salivary α-amylase (sAA) from two subjects with moderate COVID-19.
Changes in Hst-5, sAA and cytokine concentrations in two longitudinally sampled subjects prior to and during the acute and post-acute phases of COVID-19 disease. Timeline of sampling during COVID-19 infection up to 49 and 85 days post-COVID-19 infection for subjects 1 and 2 (a and c, respectively). Measurement of Hst-5 concentrations and fungal culturing of samples from the two subjects prior to and during the acute and post-acute phases of COVID-19 disease. Line graphs in bottom rows depict similar trends for Hst-5 and Th17 associated cytokine levels for subjects 1 and 2 (b and d, respectively). Upon culturing, C. albicans was recovered from the initial 6 samples from subject 2 (c, d). Prospective time course analysis of salivary amylase (sAA) concentrations with respect to histatin-5 prior to and during the acute and post-acute phases of COVID-19 disease. Green dots denote positive C. albicans samples (e). Scatterplot depiction of correlation test of histatin-5 and amylase (sAA) levels in saliva samples from subjects 1 and 2. Triangles denote positive C. albicans samples (f). Created using Biorender.com.
Fig 4.
Ex vivo proliferation assay using pooled saliva.
(a) Workflow for histatin-5 measurement and proliferation assay using pooled saliva samples from 3 subjects under a COVID-19 infected state (PC+) and recovered state (PC-). (b) Bar plots depict Hst-5 concentrations (μg/ml) from pooled saliva samples; (c) recovered C. albicans (cells/ml) following 1 h incubation in pooled saliva samples seeded with 1x104 cells/ml of C. albicans. Created using Biorender.com.