Rafael Rivera Lugo

All Publications

• Reprogramming <i>Listeria monocytogenes</i> flavin metabolism to improve its therapeutic safety profile and broaden innate T-cell activation MBIO Chevee, V., Lobanovska, M., Rivera-Lugo, R., Guereca, L., Feng, Y., Anaya-Sanchez, A., Garcia Castillo, J., Huckins, A. M., Lemmens, E. E., Rae, C. S., Hardy, J. W., Carrington, R., Kotula, J. W., Portnoy, D. A. 2025: e0365225

  Abstract

  Listeria monocytogenes is a facultative intracellular bacterial pathogen that is a potent inducer of cell-mediated immunity, which has led to the development of attenuated, Listeria-based cancer vaccines. L. monocytogenes strains, such as live-attenuated double-deleted Listeria (LADD), lacking two key virulence factors, ΔactA and ΔinlB, have been used safely in clinical trials and showed promising anti-tumor activity. Despite early clinical success, improving potency and safety by preventing extracellular bacterial growth is paramount for the development of further clinical applications. We describe a quadruple attenuated intracellular Listeria (QUAIL) strain that, in addition to ΔactAΔinlB, lacks ribC and ribF, which encode enzymes required for generating the essential flavin cofactors flavin mononucleotide (FMN) and flavin adenine nucleotide (FAD). QUAIL imported FMN and FAD during intracellular growth but was unable to grow extracellularly in blood or on vascular catheters in mice, which reduced its lethality. Despite its lack of extracellular growth, QUAIL maintained its immunoprotective properties, which were comparable to LADD. Furthermore, we showed that QUAIL can be engineered to synthesize riboflavin, leading to expansion and activation of mucosal-associated invariant T cells. Together, our data support the use of QUAIL as a promising therapeutic platform with an improved safety profile that is amenable to further modifications to expand its immune-activating potential.IMPORTANCEListeria-based live-attenuated cancer vaccines represent a promising therapy in many different pre-clinical tumor models and in clinical trials. Enhancing its anti-cancer immunity and increasing its safety profile will advance the clinical applications of Listeria vaccines. By manipulating Listeria monocytogenes flavin metabolism, we engineered a quadruple attenuated intracellular Listeria (QUAIL) vaccine candidate strain that has limited toxicity associated with extracellular growth in major extracellular niches in vivo, including blood and implanted catheter ports. Furthermore, we showed that QUAIL can be effectively programmed to engage innate-like T cells known as mucosal-associated invariant T cells, which could be harnessed for future cancer immunotherapies. The results presented here lay the foundation for further analysis of QUAIL as a safer, yet immunopotent L. monocytogenes vaccine or therapeutic vector.

  View details for DOI 10.1128/mbio.03652-25

  View details for Web of Science ID 001651587300001

  View details for PubMedID 41474325

• <i>Mycobacterium tuberculosis</i> triggers reduced inflammatory cytokine responses and virulence in mice lacking Tax1bp1 PLOS PATHOGENS Chin, J., Abeydeera, N., Repasy, T., Rivera-Lugo, R., Mitchell, G., Nguyen, V. Q., Zheng, W., Richards, A., Stevenson, E., Swaney, D. L., Krogan, N. J., Ernst, J. D., Cox, J. S., Budzik, J. M. 2025; 21 (10): e1012829

  Abstract

  Host responses - autophagy, cell death, and inflammation - limit the growth of bacterial pathogens while minimizing tissue damage. During the early stages of infection, Mycobacterium tuberculosis (Mtb) thwarts these and other innate immune defense mechanisms in alveolar macrophages (AMs) derived from the yolk sac; in later stages, it circumvents defenses in recruited mononuclear cells (MNCs) and survives within them despite additional cytokine stimulation from recruited T cells. The mechanisms that drive variable rates of Mtb growth in different macrophage subtypes and how Mtb manipulates inflammatory responses to grow within innate immune cells remain obscure. Here we explored the role of the host factor, Tax-1 binding protein 1 (Tax1bp1), an autophagy receptor that targets pathogens for degradation through selective autophagy and terminates pro-inflammatory cytokine responses. Unexpectedly, we found that Tax1bp1-deficient mice were less susceptible to Mtb infection, and generated reduced inflammatory cytokine responses, compared to wild-type mice; the same mutant mice exhibited decreased growth of, and inflammatory cytokine responses to, Listeria monocytogenes, suggesting that Tax1bp1 plays a role in host responses to multiple intracellular pathogens. Contrary to our previous ex vivo findings in bone marrow-derived macrophages (BMDMs), in vivo growth of Mtb in AMs and a subset of recruited MNCs was more limited in mice lacking Tax1bp1 relative to wild-type mice. To better understand these differences, we performed global protein abundance measurements in mock- and Mtb-infected AM samples ex vivo from wild-type mice. These experiments revealed that Tax1bp1 protein abundance does not significantly change early after infection in AMs but does in BMDMs; moreover, early after infection, Tax1bp1-deficiency reduced necrotic-like cell death -- an outcome that favors Mtb replication -- in AMs but not BMDMs. Together, these results show that deficiency of Tax1bp1 plays a crucial, cell type-specific role in linking the regulation of autophagy, cell death, and anti-inflammatory host responses and overall reducing bacterial growth.

  View details for DOI 10.1371/journal.ppat.1012829

  View details for Web of Science ID 001606537600001

  View details for PubMedID 41171885

  View details for PubMedCentralID PMC12588459

• Tax1bp1 enhances bacterial virulence and promotes inflammatory responses during Mycobacterium tuberculosis infection of alveolar macrophages. bioRxiv : the preprint server for biology Chin, J., Abeydeera, N., Repasy, T., Rivera-Lugo, R., Mitchell, G., Nguyen, V. Q., Zheng, W., Richards, A., Swaney, D. L., Krogan, N. J., Ernst, J. D., Cox, J. S., Budzik, J. M. 2024

  Abstract

  Crosstalk between autophagy, host cell death, and inflammatory host responses to bacterial pathogens enables effective innate immune responses that limit bacterial growth while minimizing coincidental host damage. Mycobacterium tuberculosis (Mtb) thwarts innate immune defense mechanisms in alveolar macrophages (AMs) during the initial stages of infection and in recruited bone marrow-derived cells during later stages of infection. However, how protective inflammatory responses are achieved during Mtb infection and the variation of the response in different macrophage subtypes remain obscure. Here, we show that the autophagy receptor Tax1bp1 plays a critical role in enhancing inflammatory cytokine production and increasing the susceptibility of mice to Mtb infection. Surprisingly, although Tax1bp1 restricts Mtb growth during infection of bone marrow-derived macrophages (BMDMs) (Budzik et al. 2020) and terminates cytokine production in response to cytokine stimulation or viral infection, Tax1bp1 instead promotes Mtb growth in AMs, neutrophils, and a subset of recruited monocyte-derived cells from the bone marrow. Tax1bp1 also leads to increases in bacterial growth and inflammatory responses during infection of mice with Listeria monocytogenes, an intracellular pathogen that is not effectively targeted to canonical autophagy. In Mtb-infected AMs but not BMDMs, Tax1bp1 enhances necrotic-like cell death early after infection, reprogramming the mode of host cell death to favor Mtb replication in AMs. Tax1bp1's impact on host cell death is a mechanism that explains Tax1bp1's cell type-specific role in the control of Mtb growth. Similar to Tax1bp1-deficiency in AMs, the expression of phosphosite-deficient Tax1bp1 restricts Mtb growth. Together, these results show that Tax1bp1 plays a crucial role in linking the regulation of autophagy, cell death, and pro-inflammatory host responses and enhancing susceptibility to bacterial infection.

  View details for DOI 10.1101/2024.12.16.628616

  View details for PubMedID 39763950

  View details for PubMedCentralID PMC11702572

• Deficiency in Galectin-3, -8, and -9 impairs immunity to chronic Mycobacterium tuberculosis infection but not acute infection with multiple intracellular pathogens. PLoS pathogens Morrison, H. M., Craft, J., Rivera-Lugo, R., Johnson, J. R., Golovkine, G. R., Bell, S. L., Dodd, C. E., Van Dis, E., Beatty, W. L., Margolis, S. R., Repasy, T., Shaker, I., Lee, A. Y., Vance, R. E., Stanley, S. A., Watson, R. O., Krogan, N. J., Portnoy, D. A., Penn, B. H., Cox, J. S. 2023; 19 (6): e1011088

  Abstract

  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 in vivo 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, we generated Gal-3, -8, and -9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective autophagic flux 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.

  View details for DOI 10.1371/journal.ppat.1011088

  View details for PubMedID 37352334

  View details for PubMedCentralID PMC10325092

• Autophagy restricts Mycobacterium tuberculosis during acute infection in mice. Nature microbiology Golovkine, G. R., Roberts, A. W., Morrison, H. M., Rivera-Lugo, R., McCall, R. M., Nilsson, H., Garelis, N. E., Repasy, T., Cronce, M., Budzik, J., Van Dis, E., Popov, L. M., Mitchell, G., Zalpuri, R., Jorgens, D., Cox, J. S. 2023; 8 (5): 819-832

  Abstract

  Whether or not autophagy has a role in defence against Mycobacterium tuberculosis infection remains unresolved. Previously, conditional knockdown of the core autophagy component ATG5 in myeloid cells was reported to confer extreme susceptibility to M. tuberculosis in mice, whereas depletion of other autophagy factors had no effect on infection. We show that doubling cre gene dosage to more robustly deplete ATG16L1 or ATG7 resulted in increased M. tuberculosis growth and host susceptibility in mice, although ATG5-depleted mice are more sensitive than ATG16L1- or ATG7-depleted mice. We imaged individual macrophages infected with M. tuberculosis and identified a shift from apoptosis to rapid necrosis in autophagy-depleted cells. This effect was dependent on phagosome permeabilization by M. tuberculosis. We monitored infected cells by electron microscopy, showing that autophagy protects the host macrophage by partially reducing mycobacterial access to the cytosol. We conclude that autophagy has an important role in defence against M. tuberculosis in mammals.

  View details for DOI 10.1038/s41564-023-01354-6

  View details for PubMedID 37037941

  View details for PubMedCentralID PMC11027733

• Distinct Energy-Coupling Factor Transporter Subunits Enable Flavin Acquisition and Extracytosolic Trafficking for Extracellular Electron Transfer in Listeria monocytogenes. mBio Rivera-Lugo, R., Huang, S., Lee, F., Méheust, R., Iavarone, A. T., Sidebottom, A. M., Oldfield, E., Portnoy, D. A., Light, S. H. 2023; 14 (1): e0308522

  Abstract

  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 EetB and FmnA proteins 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 demonstrate 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 that show that related ECF exporter genes are distributed across members of the phylum Firmicutes and frequently colocalize with genes encoding flavinylated extracytosolic proteins. These findings clarify the basis of ECF transporter export and extracytosolic flavin cofactor trafficking in Firmicutes. IMPORTANCE Bacteria import vitamins and other essential compounds from their surroundings but also traffic related compounds from the cytosol to the cell envelope where they serve various functions. Studying the foodborne pathogen Listeria monocytogenes, we find that the modular use of subunits from a prominent class of bacterial transporters enables the import of environmental vitamin B2 cofactors and the extracytosolic trafficking of a vitamin B2-derived cofactor that facilitates redox reactions in the cell envelope. These studies clarify the basis of bidirectional small-molecule transport across the cytoplasmic membrane and the assembly of redox-active proteins within the cell envelope and extracellular space.

  View details for DOI 10.1128/mbio.03085-22

  View details for PubMedID 36744898

  View details for PubMedCentralID PMC9973259

• Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis. eLife Rivera-Lugo, R., Deng, D., Anaya-Sanchez, A., Tejedor-Sanz, S., Tang, E., Reyes Ruiz, V. M., Smith, H. B., Titov, D. V., Sauer, J. D., Skaar, E. P., Ajo-Franklin, C. M., Portnoy, D. A., Light, S. H. 2022; 11

  Abstract

  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 >1000-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 expression rescued this phenotype. These studies show that NAD+ regeneration represents a major role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

  View details for DOI 10.7554/eLife.75424

  View details for PubMedID 35380108

  View details for PubMedCentralID PMC9094743

• RibU is an essential determinant of Listeria pathogenesis that mediates acquisition of FMN and FAD during intracellular growth. Proceedings of the National Academy of Sciences of the United States of America Rivera-Lugo, R., Light, S. H., Garelis, N. E., Portnoy, D. A. 2022; 119 (13): e2122173119

  Abstract

  Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential riboflavin-derived cofactors involved in a myriad of redox reactions across all forms of life. Nevertheless, the basis of flavin acquisition strategies by riboflavin auxotrophic pathogens remains poorly defined. In this study, we examined how the facultative intracellular pathogen Listeria monocytogenes, a riboflavin auxotroph, acquires flavins during infection. A L. monocytogenes mutant lacking the putative riboflavin transporter (RibU) was completely avirulent in mice but had no detectable growth defect in nutrient-rich media. However, unlike wild type, the RibU mutant was unable to grow in defined media supplemented with FMN or FAD or to replicate in macrophages starved for riboflavin. Consistent with RibU functioning to scavenge FMN and FAD inside host cells, a mutant unable to convert riboflavin to FMN or FAD retained virulence and grew in cultured macrophages and in spleens and livers of infected mice. However, this FMN- and FAD-requiring strain was unable to grow in the gallbladder or intestines, where L. monocytogenes normally grows extracellularly, suggesting that these sites do not contain sufficient flavin cofactors to promote replication. Thus, by deleting genes required to synthesize FMN and FAD, we converted L. monocytogenes from a facultative to an obligate intracellular pathogen. Collectively, these data indicate that L. monocytogenes requires riboflavin to grow extracellularly in vivo but scavenges FMN and FAD to grow in host cells.

  View details for DOI 10.1073/pnas.2122173119

  View details for PubMedID 35316134

  View details for PubMedCentralID PMC9060500

• Post-translational flavinylation is associated with diverse extracytosolic redox functionalities throughout bacterial life. eLife Méheust, R., Huang, S., Rivera-Lugo, R., Banfield, J. F., Light, S. H. 2021; 10

  Abstract

  Disparate redox activities that take place beyond the bounds of the prokaryotic cell cytosol must connect to membrane or cytosolic electron pools. Proteins post-translationally flavinylated by the enzyme ApbE mediate electron transfer in several characterized extracytosolic redox systems but the breadth of functions of this modification remains unknown. Here, we present a comprehensive bioinformatic analysis of 31,910 prokaryotic genomes that provides evidence of extracytosolic ApbEs within ~50% of bacteria and the involvement of flavinylation in numerous uncharacterized biochemical processes. By mining flavinylation-associated gene clusters, we identify five protein classes responsible for transmembrane electron transfer and two domains of unknown function (DUF2271 and DUF3570) that are flavinylated by ApbE. We observe flavinylation/iron transporter gene colocalization patterns that implicate functions in iron reduction and assimilation. We find associations with characterized and uncharacterized respiratory oxidoreductases that highlight roles of flavinylation in respiratory electron transport chains. Finally, we identify interspecies gene cluster variability consistent with flavinylation/cytochrome functional redundancies and discover a class of 'multi-flavinylated proteins' that may resemble multi-heme cytochromes in facilitating longer distance electron transfer. These findings provide mechanistic insight into an important facet of bacterial physiology and establish flavinylation as a functionally diverse mediator of extracytosolic electron transfer.

  View details for DOI 10.7554/eLife.66878

  View details for PubMedID 34032212

  View details for PubMedCentralID PMC8238504

• A flavin-based extracellular electron transfer mechanism in diverse Gram-positive bacteria. Nature Light, S. H., Su, L., Rivera-Lugo, R., Cornejo, J. A., Louie, A., Iavarone, A. T., Ajo-Franklin, C. M., Portnoy, D. A. 2018; 562 (7725): 140-144

  Abstract

  Extracellular electron transfer (EET) describes microbial bioelectrochemical processes in which electrons are transferred from the cytosol to the exterior of the cell1. Mineral-respiring bacteria use elaborate haem-based electron transfer mechanisms2-4 but the existence and mechanistic basis of other EETs remain largely unknown. Here we show that the food-borne pathogen Listeria monocytogenes uses a distinctive flavin-based EET mechanism to deliver electrons to iron or an electrode. By performing a forward genetic screen to identify L. monocytogenes mutants with diminished extracellular ferric iron reductase activity, we identified an eight-gene locus that is responsible for EET. This locus encodes a specialized NADH dehydrogenase that segregates EET from aerobic respiration by channelling electrons to a discrete membrane-localized quinone pool. Other proteins facilitate the assembly of an abundant extracellular flavoprotein that, in conjunction with free-molecule flavin shuttles, mediates electron transfer to extracellular acceptors. This system thus establishes a simple electron conduit that is compatible with the single-membrane structure of the Gram-positive cell. Activation of EET supports growth on non-fermentable carbon sources, and an EET mutant exhibited a competitive defect within the mouse gastrointestinal tract. Orthologues of the genes responsible for EET are present in hundreds of species across the Firmicutes phylum, including multiple pathogens and commensal members of the intestinal microbiota, and correlate with EET activity in assayed strains. These findings suggest a greater prevalence of EET-based growth capabilities and establish a previously underappreciated relevance for electrogenic bacteria across diverse environments, including host-associated microbial communities and infectious disease.

  View details for DOI 10.1038/s41586-018-0498-z

  View details for PubMedID 30209391

  View details for PubMedCentralID PMC6221200