Brominated Carbazole with Antibiotic Adjuvant Activity Displays Pleiotropic Effects in MRSA’s Transcriptome

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a major threat to human health, as the US mortality rate outweighs those from HIV, tuberculosis, and viral hepatitis combined. In the wake of the COVID-19 pandemic, antibiotic-resistant bacterial infections acquired during hospital stays have increased. Antibiotic adjuvants are a key strategy to combat these bacteria. We have evaluated several small molecule antibiotic adjuvants that have strong potentiation with β-lactam antibiotics and are likely inhibiting a master regulatory kinase, Stk1. Here, we investigated how the lead adjuvant (compound 8) exerts its effects in a more comprehensive manner. We hypothesized that the expression levels of key resistance genes would decrease once cotreated with oxacillin and the adjuvant. Furthermore, bioinformatic analyses would reveal biochemical pathways enriched in differentially expressed genes. RNA-seq analysis showed 176 and 233 genes significantly up-and downregulated, respectively, in response to cotreatment. Gene ontology categories and biochemical pathways that were significantly enriched with downregulated genes involved carbohydrate utilization, such as the citrate cycle and the phosphotransferase system. One of the most populated pathways was S. aureus infection. Results from an interaction network constructed with affected gene products supported the hypothesis that Stk1 is a target of compound 8. This study revealed a dramatic impact of our lead adjuvant on the transcriptome that is consistent with a pleiotropic effect due to Stk1 inhibition. These results point to this antibiotic adjuvant having potential broad therapeutic use in combatting MRSA.

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

“…Therefore, we treated this strain with the same conditions for RNA-seq experiments. This approach has recently been successful in uncovering transcriptome-wide alterations in gene expression due to antibiotic adjuvant compounds used alone and in combination with antibiotics . Our experimental design allowed us to measure differentially expressed genes (DEGs) that occur when S.…”

“…This approach has recently been successful in uncovering transcriptome-wide alterations in gene expression due to antibiotic adjuvant compounds used alone and in combination with antibiotics. 15 Our experimental design allowed us to measure differentially expressed genes (DEGs) that occur when S. aureus cultures were treated with loratadine, oxacillin, or both in combination ( Figure 1 A).We primarily focused on two pairwise comparisons: (1) loratadine compared to untreated and (2) cotreated compared to oxacillin treated cells. We will refer to these comparisons as Lor vs Un and Co vs Ox.…”

“…All subsequent RNA-seq steps, including additional RNA quality control ( Supporting Information Table S1 ), library construction, sequencing ( Supporting Information Table S2 ), and reference genome alignment ( Supporting Information Table S3 ), were conducted by Novogene, Inc., as reported previously. 74 Experimental details and raw and processed data were deposited to Gene Expression Omnibus (GEO) under accession number GSE227099.…”

“…Interaction networks were constructed using the STRING database in Cytoscape v 3.9.1 77 as previously reported. 74 Additionally, interaction networks were formed from each subcluster of DEGs. In all cases, individual nodes with no edges were removed from the figures.…”

Transcriptomic Analysis of Loratadine as an Antibiotic Adjuvant Against MRSA

“…This approach has recently been successful in uncovering transcriptome-wide alterations in gene expression due to antibiotic adjuvant compounds used alone and in combination with antibiotics. 11 Our experimental design allowed us to measure differentially expressed genes (DEGs) that occur due to S. aureus cultures treated with loratadine, oxacillin, or both in combination (Figure 1A). We primarily focused on two comparisons: (1) cotreated compared to oxacillin and (2) loratadine compared to untreated.…”