Loratadine Combats Methicillin-Resistant Staphylococcus aureus by Modulating Virulence, Antibiotic Resistance, and Biofilm Genes

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved to become resistant to multiple classes of antibiotics. New antibiotics are costly to develop and deploy, and they have a limited effective lifespan. Antibiotic adjuvants are molecules that potentiate existing antibiotics through nontoxic mechanisms. We previously reported that loratadine, the active ingredient in Claritin, potentiates multiple cell-wall active antibiotics in vitro and disrupts biofilm formation through a hypothesized inhibition of the master regulatory kinase Stk1. Loratadine and oxacillin combined repressed the expression of key antibiotic resistance genes in the bla and mec operons. We hypothesized that additional genes involved in antibiotic resistance, biofilm formation, and other cellular pathways would be modulated when looking transcriptome-wide. To test this, we used RNA-seq to quantify transcript levels and found significant effects in gene expression, including genes controlling virulence, antibiotic resistance, metabolism, transcription (core RNA polymerase subunits and sigma factors), and translation (a plethora of genes encoding ribosomal proteins and elongation factor Tu). We further demonstrated the impacts of these transcriptional effects by investigating loratadine treatment on intracellular ATP levels, persister formation, and biofilm formation and morphology. Loratadine minimized biofilm formation in vitro and enhanced the survival of infected Caenorhabditis elegans. These pleiotropic effects and their demonstrated outcomes on MRSA virulence and survival phenotypes position loratadine as an attractive anti-infective against MRSA.

made between gene expression in cells treated with loratadine alone as compared to untreated cells.(B) In the bottom panel, comparisons were made between gene expression in cells treated with a combination of loratadine and oxacillin as compared to oxacillin alone.Solid lines represent published and verified interactions.Dashed lines represent hypothesized interactions that have not been experimentally verified.Genes that displayed increased expression as compared to the control are shaded in red.Genes that displayed decreased expression as compared to the control are shaded in green.Many genes encoding proteins that make up both the large and small subunits of the bacterial ribosome are affected by treatment with loratadine alone or in combination with oxacillin.Alternate sigma factor B (SigB) is a major transcriptional regulator controlling the expression of virulence factors and genes associated with persister formation. 13Expression of sigB is enhanced by MsaB, which also regulates expression of sarZ, whose protein SarZ is a known substrate of Stk1. 14,15 t the protein level, SigB is inhibited by RsbW, which is activated by RsbV after dephosphorylation by RsbU. 16Notably, loratadine treatment downregulated expression of sarZ, msaABCR, rsbUVW, and sigB.Treatment with loratadine alone and in combination with oxacillin lowered transcription of glpF and glpK.GlpF and GlpK have been identified as important proteins involved in glycerol uptake that are required for S. aureus to adopt the cell wall deficient L-form believed to be correlated to latent infections and persister formation. 4 Supporting Figure S10: A biphasic kill curve with gentamicin treatment is observed.
MRSA 43300 cells were treated with gentamicin at 10X MIC for 24 hours and aliquots were removed for enumeration on TSA spot plates.Results are shown as average % survival of the original time 0hr population from three biological replicates.Error bars represent the standard error of the mean.
PBP2a, a penicillin-binding protein with reduced affinity for -lactam antibiotics, to rebuild and repair the cell wall. 17The vra operon is associated with broad antibiotic resistance and tolerance activities.9][20][21] Similarly, GraRS has also been implicated in the regulation of antibiotic resistance genes and has also been shown to regulate expression of the vraRST operon to further regulate S. aureus's response to antibiotic treatment. 22Finally, cidB and lrgAB encode proteins that regulate murein hydrolase activity and whose expression correlates to reduced (cidB) or enhanced (lrgAB) tolerance to penicillin treatment. 23,24 acillin alone.Solid lines represent published and verified interactions.Dashed lines represent hypothesized interactions that have not been experimentally verified.Genes that displayed increased expression as compared to the control are shaded in red.Genes that displayed decreased expression as compared to the control are shaded in green.CcpA is a glucose responsive transcriptional regulator that controls a number of genes linked to metabolism and virulence.Phosphorylation by Stk1 reduces CcpA's DNA binding affinity.In response to elevated glucose levels, CcpA represses expression of tst, the gene that encodes the toxic shock syndrome protein TSST-1. 25SrrAB also regulates tst expression in response to oxygen levels.RNAIII and SaeRS both promote tst expression. 26Expression of hemolysins, including HlgABC and Hla, are major contributors to S. aureus virulence and promote lysis of host cells. 27xpression of hla is down-regulated by CodY, SarS, and Rot. 278][29] SaeR also regulates hlgABC expression. 30upporting Figure S15: Loratadine is not toxic to human cells in culture at concentrations used in transcriptomic analysis.The y-axis shows fluorescence measured, which reports on cell viability due to redox of alamarBlue.The x-axis shows the loratadine concentration HEK 293 cells were exposed to.

Supporting Figure S1 :
Pearson correlation between samples.A Pearson correlation coefficient was calculated for each pairwise comparison among the 12 RNA samples.Un represents untreated sample, Lor represents loratadine treated sample, Ox represents oxacillin treated sample, and Co represents cotreated sample.Each biological replicate is labelled A, B, or C. Supporting Figure S2: A Principal component analysis PCA was performed on the 12 RNA samples.Un represents untreated sample, Lor represents loratadine treated sample, Ox represents oxacillin treated sample, and Co represents cotreated sample.Each biological replicate is labelled A, B, or C. Supporting Figure S4: KEGG pathway map of the phosphotransferase system pathway showing differentially expressed genes upon cotreatment with loratadine and oxacillin compared to oxacillin only.The green to red legend represents log2 fold change in differential gene expression.Supporting Figure S6: KEGG pathway map of the terpenoid backbone biosynthesis pathway showing downregulated genes upon cotreatment of loratadine and oxacillin compared to oxacillin only.The green to red legend represents log2 fold change in differential gene expression.

Table S2 : RNA-seq reads quality control summary.
Un represents untreated sample, ox represents oxacillin treated sample, Lor represents loratadine treated sample, and co represents cotreated sample.Each biological replicate is labelled A, B, or C. Q20 and Q30 were calculated as the base number of Phred value > 20 or 30, respectively, divided by the total base value x 100%.

Table S3 :
Mapped reads summary.Un represents untreated sample, Lor represents loratadine treated sample, Ox represents oxacillin treated sample, and Co represents cotreated sample.Each biological replicate is labelled A, B, or C.

Supporting Tables S4-S9 -contain complete DEG data sets and are available in an Excel workbook Supporting Table S10: RT-qPCR Primers.
Primer sequences are written 5' to 3'.

S23 -contain completed GO and KEGG results and are available in an Excel workbook Supporting Table S24: Hub analysis of genes affected by cotreatment but not loratadine alone.
Rank order and score was generated by the cytoHubba app within Cytoscape.DGE = differential gene expression.Stk1 interaction was determined via the STRING database and published literature.

Table S26 : Loratadine enhanced C. elegans survival when infected with MRSA.
Oxacillin was used at 4g/mL and loratadine was used at a final concentration of 50M .Cotreated wells contained both oxacillin at 4g/mL and loratadine at 50M.The number of hours where at least 50% of C. elegans survived is shown.Undefined is reported when at the end of the time course experiment, over 50% of C. elegans are still surviving and cannot be scored as dead.