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John J Mekalanos

Publications and source records attributed to John J Mekalanos.

2 recordsLinked to original sources
PubMed2026

Deletion of the Salmonella pathogenicity island 2 gene, spiC, in attenuated Salmonella Typhimurium VNP20009 optimizes its potential for bacterial schwannoma therapy.

UNLABELLED: Recent advances in systems biology and immunotherapy have spurred the investigation of bacteria as therapeutic vehicles for cancer treatment. Currently, Bacillus Calmette-Guérin remains the only FDA-approved bacterial cancer therapy; it is a live attenuated mycobacterium that is indicated for the treatment and prophylaxis of carcinoma in situ of the urinary bladder and for the prophylaxis of primary or recurrent papillary tumors following transurethral resection. Although safety concerns have been raised, attenuated Salmonella Typhimurium strains such as VNP20009 have advanced to clinical trials targeting fast-growing human tumors. Notably, this strain induces robust immunological control of slow-growing tumors such as NF2-related schwannomatosis (NF2-SWN) in preclinical murine models. Here, we genetically characterize VNP20009 with the goal of constructing genetically defined attenuated strains that retain its promising therapeutic features while improving safety. Specifically, we investigated the contribution of the Salmonella pathogenicity island I (SPI-1) and SPI-2 type III secretion systems to antitumor efficacy and biosafety. Mutation of the SPI-1 gene sipB, a key structural component required for SPI-1 type III secretion system function, partially reduced tumor control in NF2-SWN murine schwannoma models, suggesting that bacterial invasion alone does not fully account for antitumor activity. In contrast, deletion of the SPI-2 gene spiC, a key effector required for intracellular survival, preserved robust tumor regression in NF2-SWN murine schwannoma models while improving safety and reducing systemic toxicity. To create a genetically defined and tractable platform, we generated two attenuated strains-AST101 and AST101-ΔspiC-which retain key mutations present in VNP20009 but lack ill-characterized background mutations. In the syngeneic NF2-SWN mouse schwannoma model, both strains significantly suppressed tumor growth compared to PBS. Collectively, these findings support the development of rationally engineered Salmonella Typhimurium strains with enhanced safety and preserved antitumor efficacy. IMPORTANCE: Given long-standing safety concerns surrounding the therapeutic use of live bacteria, we constructed a ΔspiC mutant of VNP20009 and demonstrated that it provides a markedly improved safety profile while retaining antitumor efficacy in NF2-related schwannomatosis mouse schwannoma models. In addition, we created two genetically defined Salmonella Typhimurium strains, AST01 and AST01-ΔspiC, which incorporate the key-targeted mutations found in VNP20009 and VNP20009-ΔspiC, respectively. These engineered strains offer a well-defined genetic background, enabling precise investigation of the bacterial traits responsible for Salmonella Typhimurium-mediated tumor control and thus further improvement of attenuated strains optimized for bacteriotherapy of neoplasms.

Salmonella typhimurium↗
PubMed2026

Actuation of CRP activating region 3 by acetylation modulates V. cholerae sugar utilization and virulence.

UNLABELLED: The cyclic AMP receptor protein or CRP is a global regulator of bacterial metabolism that activates transcription of genes required for utilization of alternative carbon sources in response to the second messenger cAMP, which is synthesized in the setting of glucose scarcity. CRP activates transcription through contact with RNA polymerase at three sites termed activating regions (ARs) 1-3. AR3 was previously reported to be functional only when CRP K52 was mutated to a neutral residue and to be essential for transcription only in the absence of AR1 and AR2. Multiple proteomic studies have reported acetylation of CRP K52. This post-translational modification is predicted to activate AR3. To probe the role of K52 acetylation (K52QAc) and AR3 at the genome level, we used ChIP-seq and RNA-seq analysis to compare WT CRP with a CRP K52Q mutant that mimics CRP K52Ac. We report that CRP K52Q binds to hundreds of new sites on the chromosome, resulting in increased abundance of known as well as previously unknown transcripts. These transcripts increase uptake and metabolism of dietary sugars such as maltose and galactose, repress acetate consumption, and augment virulence gene expression. We attribute the repression of acetate consumption to a novel small RNA, CrbZ, which is positively regulated by CRP K52Q in LB broth and by WT CRP specifically in minimal medium containing maltose. This study highlights the role of post-translational modifications in molding the CRP regulon to optimize pathogen metabolism and virulence gene expression in the human intestine in response to nutritional cues. SIGNIFICANCE STATEMENT: As a model in the field of bacterial transcription, the structure and function of the cAMP receptor protein (CRP), a global transcription regulator, has been exhaustively investigated. These studies have established three activating regions (ARs) where CRP contacts RNA polymerase, of which only two were thought to participate in transcription activation by native CRP. Here we provide evidence that post-translational acetylation of V. cholerae CRP lysine 52 actuates AR3, enabling occupancy of hundreds of novel CRP binding sites and the transcription of genes encoding novel small RNAs. These changes alter virulence gene expression, promote utilization of dietary carbon sources, and delay acetate uptake. We propose that acetylation of CRP K52 engages AR3, thus optimizing V. cholerae fitness in the human intestine.

Journal Article↗