Biology subjects

Rivera-Lugo, R.

Publications and source records attributed to Rivera-Lugo, R..

6 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↗

bioRxiv2024

Tax1bp1 enhances bacterial virulence and promotes inflammatory responses during Mycobacterium tuberculosis infection of alveolar macrophages

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. Tax1bp1s impact on host cell death is a mechanism that explains Tax1bp1s 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. Author SummaryAlthough macrophages are the first innate immune cells to encounter Mycobacterium tuberculosis during infection, M. tuberculosis has evolved the ability to persist in them. Recent studies highlight that some types of macrophages are more permissive to M. tuberculosis replication and survival than others, but the mechanisms for cell type-specific differences in M. tuberculosis growth are only beginning to be understood. We found that the host factor, Tax1bp1 (Tax-1 binding protein 1), supports M. tuberculosis growth during animal infection and in specific subsets of innate immune cells, including alveolar macrophages while restricting M. tuberculosis in bone marrow-derived macrophages. We also found that Tax1bp1 has a similar phenotype in enhancing the pathogenesis of another intracellular pathogen, Listeria monocytogenes. Compared to bone marrow-derived macrophages, in alveolar macrophages, Tax1bp1 enhances the release of inflammatory mediators and leads to necrotic-like host cell death, which is known to enhance M. tuberculosis growth. Phosphorylation of Tax1bp1 in alveolar macrophages promotes M. tuberculosis growth. Our research highlights that Tax1bp1 is a host target for host-directed therapy against M. tuberculosis and controls host responses to M. tuberculosis in a cell type-specific manner.

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↗

bioRxiv2021

Widespread bacterial protein flavinylation in functionally distinct extracytosolic redox biochemistries

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 multiheme cytochromes in facilitating longer distance electron transfer. These findings provide key mechanistic insight into an important facet of bacterial physiology and establish flavinylation as a functionally diverse mediator of extracytosolic electron transfer. Impact StatementBioinformatic and biochemical studies find that covalently bound flavins are common and participate in wide-ranging extracytosolic redox activities throughout bacterial life.

microbiology↗