Biology subjects

Karakousis, P.

Publications and source records attributed to Karakousis, P..

4 recordsLinked to original sources

bioRxiv2025

The Phosphate-Specific Transport System Gene pstA1 Contributes to Rifampin Tolerance in Mycobacterium tuberculosis

Tuberculosis (TB) caused an estimated 10.8 million new cases and 1.25 million deaths in 2023. Antibiotic tolerance, the ability of bacteria to survive exposure to bactericidal antibiotics without genetic resistance mutations, contributes to the need for prolonged treatment. Targeting antibiotic tolerance mechanisms could promote accelerated clearance of Mycobacterium tuberculosis (Mtb), thereby improving medical adherence. Our previous forward genetic screen for rifampin tolerance genes identified pstA1, which is involved in phosphate-specific import. The ABC-type transporter permease PstA1 has previously been implicated in Mtb virulence, as mutants lacking this gene exhibit defective survival during phosphate limitation in vitro, in infected macrophages, and in immunocompetent mice. The minimum inhibitory concentration (MIC) of rifampin was not altered in a pstA1 deletion mutant ({Delta}pstA1), suggesting this difference in susceptibility is not due to antibiotic resistance. Consistent with a role in rifampin tolerance, time-kill assays revealed a shift in the mean duration of killing (2-log reduction) from 1.6 days in wild-type to 1.0 day in {Delta}pstA1, and complementation partially restored this phenotype. We found that pstA1 is specifically required for Mtb survival in the absence of exogenous inorganic phosphate and is important for adaptation to growth in culture without detergent, and within macrophages in an interferon-{gamma}-dependent manner. Differential expression analysis revealed that {Delta}pstA1 exhibited substantial transcriptional reprogramming with 58 differentially expressed genes, including altered expression of metabolic, DNA damage repair, and secretory pathways. PstA1 represents a novel drug target, and inhibitors could serve as adjunctive therapies to shorten treatment times, reducing opportunities for drug resistance emergence. ImportanceTuberculosis remains one of the worlds deadliest infectious diseases, causing over a million deaths annually. Current treatment requires months of antibiotic therapy, and poor adherence to these lengthy regimens contributes to the emergence of drug-resistant strains that are increasingly difficult to treat. A major barrier to shorter treatment is antibiotic tolerance, which allows bacteria to survive drug exposure without genetic resistance mutations. This study identifies the phosphate transport component PstA1 as a critical factor enabling Mycobacterium tuberculosis to tolerate the frontline antibiotic rifampin. Bacteria lacking pstA1 are eliminated more rapidly by rifampin, demonstrating that this transporter actively promotes bacterial survival during treatment. These findings suggest that drugs targeting PstA1 could be combined with standard antibiotics to accelerate bacterial clearance, potentially shortening treatment duration and improving patient adherence. Such adjunctive therapies targeting tolerance mechanisms represent a promising strategy to combat tuberculosis and reduce the global burden of drug-resistant disease.

microbiology↗

bioRxiv2022

An intranasal stringent response vaccine targeting dendritic cells as a novel adjunctive therapy against tuberculosis

Lengthy tuberculosis (TB) treatment is required to address the ability of a subpopulation of persistent Mycobacterium tuberculosis (Mtb) to remain in a non-replicating, antibiotic-tolerant state characterized by metabolic remodeling, including induction of the RelMtb-mediated stringent response. We developed a novel therapeutic DNA vaccine construct involving fusion of the relMtb gene with the immature dendritic cell-targeting gene encoding chemokine MIP-3/CCL20. To augment mucosal immune responses, intranasal delivery was also evaluated. We found that the intramuscular MIP-3/relMtb (fusion) vaccine potentiates isoniazid activity more than a similar DNA vaccine expressing relMtb alone in a chronic TB mouse model (absolute reduction of Mtb burden: 0.63 log10 colony-forming units, P=0.0001), inducing pronounced Mtb-protective immune signatures. The intranasal fusion vaccine, an approach combining relMtb fusion to MIP-3 and intranasal delivery, demonstrated the greatest therapeutic effect compared to each approach alone, as evidenced by robust Th1 and Th17 responses systemically and locally and the greatest mycobactericidal activity when combined with isoniazid (absolute reduction of Mtb burden: 1.13 log10, P<0.0001, when compared to the intramuscular vaccine targeting relMtb alone). This DNA vaccination strategy may be a promising adjunctive approach combined with standard therapy to shorten curative TB treatment, and also serve as proof-of-concept for treating other chronic infections.

immunology↗

bioRxiv2021

Progression and Resolution of SARS-1 CoV-2 Infection in Golden Syrian Hamsters

To catalyze SARS-CoV-2 research including development of novel interventive and preventive strategies, we characterized progression of disease in depth in a robust COVID-19 animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at day 2, 4, 7, 14, and 28 days post-inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal timepoints, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 post-inoculation, corresponding with widespread necrosis and inflammation. At day 7, when infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 post-inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

pathology↗

bioRxiv2020

The anti-tubercular activity of simvastatin is mediated by cholesterol-dependent regulation of autophagy via the AMPK-mTORC1-TFEB axis

Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. We investigated the mechanism of anti-tubercular action of simvastatin in Mycobacterium tuberculosis-infected human monocytic cells. We found that the anti-tubercular activity of statins was phenocopied by cholesterol-branch but not prenylation-branch inhibitors. Moreover, statin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Overall, our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, AMPK-mTORC1-TFEB axis, and intracellular infection control, and uncover new anti-tubercular therapy targets.

immunology↗