Targeting Cysteine Metabolism Reveals New Weaknesses in Acinetobacter baumannii
Research Summary: Cysteine homeostasis in A. baumannii relies on a finely balanced network of biosynthetic and uptake pathways that supports metabolism, antibiotic stress survival, and in vivo fitness.
Researcher Spotlight
Avik Pathak is a PhD candidate in Prof. Ranjana Pathania’s laboratory in the Department of BSBE, IIT Roorkee. His work focuses on understanding pathophysiologically important pathways and their regulation in Acinetobacter baumannii, a critical nosocomial pathogen.
Linkedin: https://in.linkedin.com/in/avik-pathak-a568181b7
Instagram: https://www.instagram.com/avik_pathak._/
Lab: Prof. Ranjana Pathania, Indian Institute of Technology Roorkee
Lab website: https://www.thepathanialab.org/
What was the core problem you aimed to solve with this research?
Acinetobacter baumannii is a nosocomial pathogen that causes infections at diverse sites of the body, including the meninges, respiratory tract, bloodstream, and urinary tract. The challenge posed by this pathogen is further compounded by its ability to acquire resistance determinants and develop resistance to clinically relevant antibiotics. Considering the contemporary landscape of antibiotic resistance, there is a pressing need to identify novel targets for therapeutic interventions.
The ability of A. baumannii to thrive and establish infection in diverse host niches can largely be attributed to its robust and adaptable metabolic circuitry, which represents an underexplored pool of plausible drug targets. However, metabolic pathways are inherently complex, and pathogens often possess alternative pathways to ensure the supply of critical metabolites.
With the goal of identifying metabolic vulnerabilities in this pathogen, we focused on delineating the pathways that sustain intracellular cysteine homeostasis. Cysteine is a sulfur-containing amino acid that is critical for the functionality of several enzymes involved in diverse physiological processes. We sought to understand how A. baumannii maintains intracellular cysteine balance and to determine the consequences of destabilizing this homeostasis. Our work provides insights into metabolic resilience and exposed vulnerabilities that can be explored further for the development of novel therapeutic strategies.
How did you go about solving this problem?
To address this question, we systematically disrupted the two major routes that contribute to intracellular cysteine homeostasis in A. baumannii, cysteine biosynthesis and cystine uptake, using a chromosomal recombineering approach. To understand the consequences of perturbing cysteine homeostasis, we employed an integrated transcriptomic, metabolomic, microbiological, and imaging-based approach. RNA-seq and untargeted metabolomics were used to delineate global alterations in gene expression and metabolic networks. Antibiotic uptake and killing assays were performed to evaluate the impact of disrupting cysteine homeostasis on antibiotic susceptibility, while scanning electron microscopy was used to assess changes in cellular morphology. Finally, the pathophysiological importance of cysteine homeostasis was investigated by evaluating the fitness of the mutants in a murine pneumonia infection model.
“Our work on cysteine metabolism reveals metabolic resilience and vulnerabilities in A. baumannii, paving the way for future therapeutic development.” – Prof. Ranjana Pathania
How would you explain your research outcomes (Key findings) to the non-scientific community?
Acinetobacter baumannii is a dangerous pathogen that causes serious hospital-acquired infections and has a high potential to develop resistance to different antibiotics. As antibiotic resistance continues to make infections harder to treat, developing new drugs that target previously unexplored pathways has become an urgent priority. Towards that end, we focused on cysteine, an amino acid that is required for the proper functioning of many important proteins and enzymes in the bacterial cell. We found that disrupting cysteine metabolism severely impaired the pathogen’s growth and led to a significant loss of fitness in a mouse infection model. These findings demonstrate that cysteine metabolism plays a critical role in the survival and infection-causing ability of A. baumannii and may represent a promising target for the development of future therapies.
What are the potential implications of your findings for the field and society?
Our work shows that disruption of cysteine biosynthesis leads to widespread metabolic dysregulation, leading to an increase in antibiotic susceptibility. Our in vivo murine pneumonia infection model showed that cysteine biosynthesis mutants are more prone to clearance upon antibiotic treatment. Considering this, combination therapies combining cysteine biosynthesis inhibitors and conventional antibiotics, represents an effective strategy to treat infections caused by A. baumannii. Furthermore, we found that simultaneous disruption of cysteine biosynthesis and cystine transport results in synthetic lethality and markedly compromises the pathogen’s ability to establish infection. Therefore, simultaneous inhibition of cysteine biosynthesis and cystine uptake may provide an effective therapeutic approach for combating infections caused by A. baumannii.
What was the exciting moment during your research?
An exciting moment during our research was when we were able to validate our hypothesis on synthetic lethality. We were studying a cysteine biosynthesis mutant in which both serine acetyltransferase genes had been disrupted. Although the mutant showed defects in metabolic homeostasis, it was still able to grow in a complex medium. This led us to hypothesize that the bacterium might be scavenging cystine from the environment through a dedicated transporter.
Transcriptomic analysis revealed a cystine transporter that was upregulated in the mutant. However, repeated attempts to generate a triple mutant lacking both biosynthetic genes and the transporter were unsuccessful, suggesting a possible synthetic lethal interaction. We could make a double mutant of the transporter and the predominant serine acetyltransferase. Interestingly, the strain exhibited improved growth when supplied with very high concentrations of cysteine, indicating that in the absence of the cystine transporter, the cells could still acquire cysteine through an unknown low-affinity uptake system. Based on this observation, we retried making the triple deletion mutant and screened them in a medium containing very high concentration of cysteine, and we could make the triple deletion mutant. The mutant could not grow in standard LB medium but regained growth when cysteine was added, providing direct evidence that loss of both cysteine biosynthesis and cystine uptake is synthetically lethal.
This resolved a long-standing experimental challenge, and revealed the critical interplay between cysteine biosynthesis and cystine acquisition in maintaining cellular survival.
Paper reference: Pathak A, Saini S, and Pathania R. Disruption of cysteine metabolism leads to synthetic lethality and in vivo fitness impairment in Acinetobacter baumannii. mBio 0:e00842-26. https://doi.org/10.1128/mbio.00842-26
