Biology subjects

Portnoy, D. A.

Publications and source records attributed to Portnoy, D. A..

13 recordsLinked to original sources

bioRxiv2025

MAIT cells induced by engineered Listeria exhibit antibacterial and antitumor activity

Mucosal-associated invariant T (MAIT) cells are among the most conserved and abundant innate-like T cells in humans that recognize microbial-derived riboflavin precursors and elicit potent antimicrobial responses. The foodborne pathogen Listeria monocytogenes is a broad host-range facultative intracellular pathogen that lacks the riboflavin biosynthetic pathway, leading us to hypothesize that this deficiency is pathoadaptive and allows the pathogen to evade MAIT cells. Here, we show that L. monocytogenes strains engineered to produce riboflavin (L. monocytogenes-ribDEAHT) are attenuated in wild-type mice but fully virulent in MAIT cell-deficient mice. Infection with L. monocytogenes-ribDEAHT prompted rapid and robust MAIT cell expansion in multiple tissues and required the cytolytic effector perforin to eliminate infected cells in vivo and in vitro. We also assessed the therapeutic potential of L. monocytogenes-ribDEAHT-stimulated MAIT cells in both infectious disease and cancer mouse models. Therapeutic administration of L. monocytogenes-ribDEAHT provided protection against Francisella tularensis in the lungs and inhibited tumor growth even in the absence of CD8+ T cells. These findings reveal the importance of MAIT cell evasion during L. monocytogenes infection and highlight the therapeutic potential of engineered L. monocytogenes to activate and harness MAIT cells for protection against infectious disease and cancer. Significance StatementListeria monocytogenes is a bacterial pathogen that grows freely in the environment but can become intracellular following ingestion of contaminated food. Although L. monocytogenes can synthesize most metabolites required for growth, it lacks the genes necessary to produce riboflavin (vitamin B2), an essential cofactor across all domains of life. We hypothesized that lacking riboflavin biosynthesis allows L. monocytogenes to evade mucosal-associated invariant T cells (MAIT cells), which generate potent antimicrobial responses against riboflavin-producing microbes. By engineering L. monocytogenes to produce riboflavin, we show that these strains robustly activate MAIT cells and are highly attenuated in wild-type mice, but not in MAIT cell-deficient mice. Furthermore, MAIT cells activated by engineered L. monocytogenes provided therapeutic protection against other riboflavin-producing bacteria and cancer.

microbiology↗

bioRxiv2025

The mevalonate pathway of isoprenoid biosynthesis supports metabolic flexibility in Mycobacterium marinum

Isoprenoids are a diverse class of natural products that are essential in all domains of life. Most bacteria synthesize isoprenoids through either the methylerythritol phosphate (MEP) pathway or the mevalonate (MEV) pathway, while a small subset encodes both pathways, including the pathogen Mycobacterium marinum (Mm). It is unclear whether the MEV pathway is functional in Mm, or why Mm encodes seemingly redundant metabolic pathways. Here we show that the MEP pathway is essential in Mm while the MEV pathway is dispensable in culture, with the {Delta}MEV mutant having no growth defect in axenic culture but a competitive growth defect compared to WT Mm. We found that the MEV pathway does not play a role in ex vivo or in vivo infection but does play a role in survival of peroxide stress. Metabolite profiling revealed that modulation of the MEV pathway causes compensatory changes in the concentration of MEP intermediates DOXP and CDP-ME, suggesting that the MEV pathway is functional and that the pathways interact at the metabolic level. Finally, the MEV pathway is upregulated early in the shift down to hypoxia, suggesting that it may provide metabolic flexibility to this bacterium. Interestingly, we found that our complemented strains, which vary in copy number of the polyprenyl synthetase idsB2, responded differently to peroxide and UV stresses, suggesting a role for this gene as a determinant of downstream prenyl phosphate metabolism. Together, these findings suggest that MEV may serve as an anaplerotic pathway to make isoprenoids under stress conditions. ImportanceOrganisms from all domains of life utilize isoprenoids to carry out thousands of critical and auxiliary cellular processes, including signaling, membrane integrity, stress response, and host-pathogen interactions. The common precursor of all isoprenoids is synthesized via one of two biosynthetic pathways and importantly, some bacteria encode both pathways, including M. marinum. We found that only one pathway is essential in M. marinum, while the nonessential pathway may confer metabolic flexibility to help the bacterium better adapt to various environmental conditions. We also found that the polyprenyl synthetase IdsB2 plays an important role in driving such phenotypes. Further, we demonstrate metabolic interplay between both functional pathways. These insights represent the first characterization of isoprenoid biosynthesis in dual pathway-encoding mycobacteria.

microbiology↗

bioRxiv2025

TLR2 signaling uniquely destabilizes tumor Tregs to promote cancer immunotherapy

A fundamental principle of immune responses is that innate immunity promotes adaptive immunity, but in the context of cancer immunity, the importance of innate receptor signaling is poorly defined. To study how Toll-like receptor (TLR) signaling contributes to cancer immunity, we used an attenuated strain of Listeria monocytogenes (Lm) that inhibits tumor growth in mice. We found that Lm stimulation of TLR2, but not TLR5 or TLR9, was essential for tumor control. As expected, TLR2 supported the priming of Lm-specific T cells in lymphoid tissues. However, TLR2 signaling in innate immune cells within tumors also destabilized Foxp3 expression in regulatory T cells (Tregs), a function that was not mediated by other TLRs. Reduced Treg function promoted tumor antigen cross-presentation by DC1s to enhance the functionality of recalled tumor-specific CD8+ T cells directly within tumors. These findings reveal a unique capacity for TLR2 signaling to diminish immunosuppression and promote cancer immunity.

immunology↗

bioRxiv2025

Phosphorylation State Dictates Bacterial Stressosome Assembly and Function

Bacterial pathogens rely on their ability to sense and respond to environmental stressors to survive and maintain virulence. The stressosome, a 1.8-megadalton nanomachine, serves as a critical sensor and regulator of the general stress response. It is composed of multiple copies of three proteins RsbR, RsbS, and the kinase RsbT which together orchestrate activation of downstream stress adaptation pathways. Using cryo-electron microscopy, we solved the atomic structure of five Listeria monocytogenes stressosomes, capturing structural mimics of the transition between inactive and activated states using phosphomimetic and phosphodeficient mutants. Our findings reveal that phosphorylation at specific residues T175 and T209 on RsbR, and S56 on RsbS dictates stressosome assembly, stoichiometry, and activation. Specifically, phosphorylation at T175 primes the stressosome for activation, while S56 phosphorylation destabilizes the core, triggering the release of RsbT to propagate the stress response. In contrast, phosphorylation at T209 modulates stressosome composition and appears to fine-tune the intensity of the stress response. Functional analyses reveal that phosphomimetic mutants (T209E, S56D) resist oxidative stress but lose virulence in host cell model, while phosphodeficient mutants (T175A, S56A) are stress-sensitive but retain virulence. These findings establish phosphorylation as a central regulatory switch linking structural dynamics to bacterial adaptation and pathogenesis, highlighting potential targets for antimicrobial intervention.

microbiology↗

bioRxiv2024

Methylglyoxal is an antibacterial effector produced by macrophages during infection

Infected macrophages transition into aerobic glycolysis, a metabolic program crucial for control of bacterial infection. However, antimicrobial mechanisms supported by aerobic glycolysis are unclear. Methylglyoxal is a highly toxic aldehyde that modifies proteins and DNA and is produced as a side-product of glycolysis. Here we show that despite the toxicity of this aldehyde, infected macrophages generate high levels of methylglyoxal during aerobic glycolysis while downregulating the detoxification system. We use targeted mutations in mice to modulate methylglyoxal generation and show that reducing methylglyoxal production by the host promotes survival of Listeria monocytogenes and Mycobacterium tuberculosis, whereas increasing methylglyoxal levels improves control of bacterial infection. Furthermore, we show that bacteria that are unable to detoxify methylglyoxal are avirulent and experience up to 1000-fold greater genomic mutation frequency during infection. Taken together, these results suggest that methylglyoxal is an antimicrobial innate immune effector that defends the host against bacterial pathogens.

microbiology↗

bioRxiv2024

Cellular mechanisms underlying beneficial versus detrimental effects of bacterial antitumor immunotherapy

Bacteria engineered to express tumor antigens as a cancer vaccine have yielded mixed results. Here, we utilized an attenuated strain of Listeria monocytogenes ({Delta}actA, Lm) that does not express tumor antigen to explore the immunological response to Listeria itself in the context of intravenous (IV), intratumoral (IT), or a combination of IV+IT administration into tumor-bearing mice. Unexpectedly, we found that Lm persisted in tumors of immune competent mice, regardless of the administration route. While IT Lm alone led to the recruitment of immunosuppressive immune cells that promoted tumor growth, IV Lm followed by IT Lm controlled tumor growth. IV Lm vaccination generated a pool of anti-Lm cytotoxic CD8 T cells that killed Lm-infected non-tumor cells to control tumor growth both indirectly, by limiting cancer cell proliferation, and directly, by enhancing tumor-specific T cell responses. Our findings reveal a differential impact of IT Lm administration on tumor progression that depends on the presence of anti-Lm CD8 T cells, which alone are sufficient to promote therapeutic efficacy.

immunology↗

bioRxiv2023

Listeria monocytogenes utilizes glutathione and limited inorganic sulfur compounds as a source of essential L-cysteine

Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen that leads a biphasic lifecycle, transitioning its metabolism and selectively inducing virulence genes when it encounters mammalian hosts. Virulence gene expression is controlled by the master virulence regulator PrfA, which is allosterically activated by host- and bacterially-derived glutathione (GSH). The amino acid L-cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for L-cysteine and must import exogenous cysteine for growth and virulence. GSH is enriched in the host cytoplasm, and previous work suggests that Lm utilizes exogenous GSH for PrfA activation. Despite these observations, the import mechanism(s) for GSH remains elusive. Analysis of known GSH importers predicted a homologous importer in Lm comprised of the Ctp ABC transporter and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH/GSSG importer that is required for Lm growth at physiologically relevant concentrations. Further, we demonstrated that OppDF are required for GSH/GSSG import in an Opp-independent manner. These data support a model where Ctp and OppDF form a unique complex for GSH/GSSG import that supports growth and pathogenesis. Additionally, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H2S for growth in a CysK-dependent manner in the absence of other L-cysteine sources. These findings suggest a pathoadaptive role for partial cysteine auxotrophy in Lm, where locally high GSH/GSSG or inorganic sulfur concentrations may signal arrival to distinct host niches.

microbiology↗

bioRxiv2022

Deficiency in Galectin-3, -8, and -9 impairs immunity to chronic Mycobacterium tuberculosis infection but not acute infection with multiple intracellular pathogens

Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection remains unclear. We found that Gal-9 binds directly to Mycobacterium tuberculosis (Mtb) and Salmonella enterica serovar Typhimurium (Stm) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity in vivo, we generated Gal-3, -8, and - 9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective lysosomal trafficking but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb, Stm or Listeria monocytogenes, and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection. Author SummaryIntracellular bacterial pathogens cause many of the worlds most deadly infectious diseases. A common requirement for nearly all intracellular pathogens is the ability to damage the endomembrane compartments in which they reside, which allows pathogens access to the nutrient-rich cytosol of the host. However, membrane damage also creates a "pattern of pathogenesis" that triggers antimicrobial immune responses. Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) act as a surveillance system for membrane damage and Gal-3 and Gal-8 inhibit bacterial growth by activating autophagy, a cellular pathway that can capture cytosolic bacteria and degrade them in lysosomes. Membrane damage-sensing galectins were hypothesized to promote bacterial killing during acute infection yet their role in the immune response of an infected animal remains unclear. Here, we show that mice deficient for Gal-3, -8, and -9 had no defects in resistance to acute infection with the pathogens Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Mycobacterium tuberculosis (Mtb), and were only modestly susceptible to chronic Mtb infection. Our data suggest that Gal-3, -8 and -9 are not critical for innate immune responses during acute infection and may play a more prominent role in the adaptive immune response. These results broaden our understanding of the role of membrane damage-sensing pathways in host defense against bacterial infection.

immunology↗

bioRxiv2022

Distinct energy-coupling factor transporter subunits enable flavin acquisition and extracytosolic trafficking for extracellular electron transfer in Listeria monocytogenes

A variety of electron transfer mechanisms link bacterial cytosolic electron pools with functionally diverse redox activities in the cell envelope and extracellular space. In Listeria monocytogenes, the ApbE-like enzyme FmnB catalyzes extracytosolic protein flavinylation, covalently linking a flavin cofactor to proteins that transfer electrons to extracellular acceptors. L. monocytogenes uses an energy-coupling factor (ECF) transporter complex that contains distinct substrate-binding, transmembrane, ATPase A, and ATPase A subunits (RibU, EcfT, EcfA, and EcfA) to import environmental flavins, but the basis of extracytosolic flavin trafficking for FmnB flavinylation remains poorly defined. In this study, we show that the proteins EetB and FmnA are related to ECF transporter substrate-binding and transmembrane subunits, respectively, and are essential for exporting flavins from the cytosol for flavinylation. Comparisons of the flavin import versus export capabilities of L. monocytogenes strains lacking different ECF transporter subunits demonstrates a strict directionality of substrate-binding subunit transport but partial functional redundancy of transmembrane and ATPase subunits. Based on these results, we propose that ECF transporter complexes with different subunit compositions execute directional flavin import/export through a broadly conserved mechanism. Finally, we present genomic context analyses which show that related ECF exporter genes are distributed across the Firmicutes phylum and frequently co-localize with genes encoding flavinylated extracytosolic proteins. These findings clarify the basis of ECF transporter export and extracytosolic flavin cofactor trafficking in Firmicutes.

microbiology↗

bioRxiv2021

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1,000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX rescued this phenotype. These studies show that NAD+ regeneration, rather than oxidative phosphorylation, represents the primary role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

microbiology↗

bioRxiv2020

Comparative analysis of macrophage post-translational modifications during intracellular bacterial pathogen infection

Macrophages activate robust antimicrobial functions upon engulfing virulent bacteria, yet a wide array of pathogens paradoxically thrive within these innate immune cells. To probe the pathogen-macrophage interface, we used proteomics to comprehensively quantify changes in post-translational modifications (PTMs) of host proteins during infection with three evolutionarily diverse intracellular pathogens: Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes. Comparing global phosphorylation and ubiquitylation patterns identified extensive reprogramming of cellular pathways during infection, with ubiquitylation patterns revealing unique pathogen-specific molecular response signatures undetectable by transcriptional profiling. Differential PTM changes during infection with attenuated M. tuberculosis cells lacking the ESX-1 virulence determinant revealed extensive modification of phagosome dynamics and antiviral type I interferon activation. We found that M. tuberculosis-mediated activation of the antiviral OASL1-IRF7 pathway promotes bacterial replication, uncovering a new mechanism of virus-bacterial synergy. Our data reveals remarkable specificity in innate cellular responses to complex stimuli and provides a resource for deeper understanding of host-pathogen interactions.

microbiology↗

bioRxiv2020

Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons

Type I interferons (IFNs) are essential for anti-viral immunity, but often impair protective immune responses during bacterial infections. An important question is how type I IFNs are strongly induced during viral infections, and yet are appropriately restrained during bacterial infections. The Super susceptibility to tuberculosis 1 (Sst1) locus in mice confers resistance to diverse bacterial infections. Here we provide evidence that Sp140 is a gene encoded within the Sst1 locus that represses type I IFN transcription during bacterial infections. We generated Sp140-/- mice and find they are susceptible to infection by Legionella pneumophila and Mycobacterium tuberculosis. Susceptibility of Sp140-/- mice to bacterial infection was rescued by crosses to mice lacking the type I IFN receptor (Ifnar-/-). Our results implicate Sp140 as an important negative regulator of type I IFNs that is essential for resistance to bacterial infections. Impact StatementRepression of type I interferons by SP140 is essential for resistance to Legionella pneumophila and Mycobacterium tuberculosis.

immunology↗

bioRxiv2017

An Mtb-Human Protein-Protein Interaction Map Reveals that Bacterial LpqN Antagonizes CBL, a Host Ubiquitin Ligase that Regulates the Balance Between Anti-Viral and Anti-Bacterial Responses

Although macrophages are armed with potent anti-bacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage-intrinsic bacterial control, and the Mtb strategies to overcome them are poorly understood. To further study these processes, we used a systematic affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two new factors involved in Mtb pathogenesis - the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl-/- macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between anti-bacterial and anti-viral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map as a resource for developing a deeper understanding of the intricate interactions between Mtb and its host.

microbiology↗