Antibiotic-resistant infections are a pressing global concern, causing millions of deaths each year. Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infections in healthcare settings and is increasingly responsible for community-acquired infections that are often more difficult to treat. Antibiotic adjuvants are small molecules that potentiate antibiotics through nontoxic mechanisms and show excellent promise as novel therapeutics. Screening of low-molecular-weight compounds was employed to identify novel antibiotic adjuvant scaffolds for further elaboration. Brominated carbazoles emerged from this screening as lead compounds for further evaluation. Lead carbazoles were able to potentiate several β-lactam antibiotics in three medically relevant strains of MRSA. Gene expression studies determined that these carbazoles were dampening the transcription of key genes that modulate β-lactam resistance in MRSA. The lead brominated carbazoles represent novel scaffolds for elaboration as antibiotic adjuvants.
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.
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 non-toxic 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 differentially expressed 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 pleiotropic effects in gene expression, including genes implicated in antibiotic resistance, as well as genes critical for metabolism, transcription (RNA Polymerase subunits alpha and beta), and translation (a plethora of ribosomal protein genes and Elongation Factor Tu). Stk1 and its cognate phosphatase, stp, were also downregulated by loratadine. Together, this provides the most molecular details to date about loratadine’s function as an antibiotic adjuvant.
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. Instead of designing and deploying new antibiotics which MRSA would quickly develop resistance to, adjuvants are a key strategy to combatting 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 exerts its effects in a more comprehensive manner. We hypothesized that the expression levels of key resistance genes would decrease once cotreated with a β-lactam antibiotic (oxacillin) and the adjuvant (compound 8). 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 with compound 8 and oxacillin compared to oxacillin alone. Gene ontology categories that were significantly enriched among downregulated genes involved phosphotransferase systems. Most of the biochemical pathways enriched with significantly 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.
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