Zinc ion (Zn 2+ ) is an essential micronutrient and a potent antioxidant. However, Zn 2+ is often limited in the environment. Upon Zn 2+ limitation, Mycolicibacterium (basonym: Mycobacterium ) smegmatis (Msm) undergoes a morphogenesis, which relies on alternative ribosomal proteins (AltRPs); i.e., Zn 2+ -independent paralogues of Zn 2+ -dependent ribosomal proteins. However, the underlying physiological changes triggered by Zn 2+ limitation and how AltRPs contribute to these changes were not known. In this study, we expand the knowledge of mechanisms utilized by Msm to endure Zn 2+ limitation, by comparing the transcriptomes and proteomes of Zn 2+ -limited and Zn 2+ -replete Msm . We further compare, corroborate and contrast our results to those reported for the pathogenic mycobacterium, M. tuberculosis , which highlighted conservation of the upregulated oxidative stress response when Zn 2+ is limited in both mycobacteria. By comparing the multi-omics analysis of a knockout mutant lacking AltRPs (Δ altRP ) to the Msm wild type strain, we specify the involvement of AltRPs in the response to Zn 2+ limitation. Our results show that AltRP expression in Msm does not affect the conserved oxidative stress response during Zn 2+ limitation observed in mycobacteria, but AltRPs do significantly impact expression patterns of numerous genes that may be involved in morphogenesis or other adaptive responses. We conclude that AltRPs are not only important as functional replacements for their Zn 2+ -dependent paralogues; they are also involved in the transcriptomic response to the Zn 2+ -limited environment.
more »
« less
Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis
Mycobacterium tuberculosis ( Mtb ) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn 2+ ) availability as a likely driver of bacterial phenotypic heterogeneity in vivo . Zn 2+ sequestration is part of “nutritional immunity”, where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn 2+ -limitation is an environmental cue sensed by Mtb , as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn 2+ -limited Mtb in vivo . Prolonged Zn 2+ limitation leads to numerous physiological changes in vitro , including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn 2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn 2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn 2+ -limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn 2+ -limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn 2+ availability likely plays a key role during early interactions with host cells.
more »
« less
- Award ID(s):
- 1844854
- NSF-PAR ID:
- 10318392
- Editor(s):
- Sassetti, Christopher M.
- Date Published:
- Journal Name:
- PLOS Pathogens
- Volume:
- 17
- Issue:
- 5
- ISSN:
- 1553-7374
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
more » « less
Abstract Pathogenesis of COVID-19 by SARS-CoV-2 resulted in a global pandemic and public health emergency in 2020. Viral infection can induce oxidative stress through reactive oxygen species (ROS). Inflammation and environmental stress are major sources of oxidative stress after infection. Micronutrients such as iron, copper, zinc, and manganese play various roles in human tissues and their imbalance in blood can impact immune responses against pathogens including SARS CoV-2. We hypothesized that alteration of free metal ions during infection and metal-catalyzed oxidation plays a critical role towards pathogenesis after infection. We analyzed convalescent and hospitalized COVID-19 patient plasma using orthogonal analytical techniques to determine redox active metal concentrations, overall protein oxidation, oxidative modifications, and protein levels via proteomics to understand the consequences of metal-induced oxidative stress in COVID-19 plasma proteins. Metal analysis using ICP-MS showed significantly greater concentrations of copper in COVID-19 plasma compared to healthy controls. We demonstrate significantly greater total protein carbonylation, other oxidative modifications, and deamidation of plasma proteins in COVID-19 plasma compared to healthy controls. Proteomics analysis showed that levels of redox active proteins including hemoglobulin were elevated in COVID-19 plasma. Molecular modeling concurred with potential interactions between iron binding proteins and SARS CoV-2 surface proteins. Overall, increased levels of redox active metals and protein oxidation indicate that oxidative stress-induced protein oxidation in COVID-19 may be a consequence of the interactions of SARS-CoV-2 proteins with host cell metal binding proteins resulting in altered cellular homeostasis.
-
Hatfull, Graham F. (Ed.)ABSTRACT The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune, and antibiotic insults. There is considerable evidence of mycomembrane plasticity during infection and in response to host-mimicking stresses. Since mycobacteria are resource and energy limited under these conditions, it is likely that remodeling has distinct requirements from those of the well-characterized biosynthetic program that operates during unrestricted growth. Unexpectedly, we found that mycomembrane remodeling in nutrient-starved, nonreplicating mycobacteria includes synthesis in addition to turnover. Mycomembrane synthesis under these conditions occurs along the cell periphery, in contrast to the polar assembly of actively growing cells, and both liberates and relies on the nonmammalian disaccharide trehalose. In the absence of trehalose recycling, de novo trehalose synthesis fuels mycomembrane remodeling. However, mycobacteria experience ATP depletion, enhanced respiration, and redox stress, hallmarks of futile cycling and the collateral dysfunction elicited by some bactericidal antibiotics. Inefficient energy metabolism compromises the survival of trehalose recycling mutants in macrophages. Our data suggest that trehalose recycling alleviates the energetic burden of mycomembrane remodeling under stress. Cell envelope recycling pathways are emerging targets for sensitizing resource-limited bacterial pathogens to host and antibiotic pressure. IMPORTANCE The glucose-based disaccharide trehalose is a stress protectant and carbon source in many nonmammalian cells. Mycobacteria are relatively unique in that they use trehalose for an additional, extracytoplasmic purpose: to build their outer “myco” membrane. In these organisms, trehalose connects mycomembrane biosynthesis and turnover to central carbon metabolism. Key to this connection is the retrograde transporter LpqY-SugABC. Unexpectedly, we found that nongrowing mycobacteria synthesize mycomembrane under carbon limitation but do not require LpqY-SugABC. In the absence of trehalose recycling, compensatory anabolism allows mycomembrane biosynthesis to continue. However, this workaround comes at a cost, namely, ATP consumption, increased respiration, and oxidative stress. Strikingly, these phenotypes resemble those elicited by futile cycles and some bactericidal antibiotics. We demonstrate that inefficient energy metabolism attenuates trehalose recycling mutant Mycobacterium tuberculosis in macrophages. Energy-expensive macromolecule biosynthesis triggered in the absence of recycling may be a new paradigm for boosting host activity against bacterial pathogens.more » « less
-
Abstract Background Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia. Methods scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo. Results Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat. Conclusion Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo.more » « less
-
Bonomo, Robert A. (Ed.)ABSTRACT Streptococcus agalactiae , also known as group B Streptococcus (GBS), is a Gram-positive encapsulated bacterium that colonizes the gastrointestinal tract of 30 to 50% of humans. GBS causes invasive infection during pregnancy that can lead to chorioamnionitis, funisitis, preterm prelabor rupture of membranes (PPROM), preterm birth, neonatal sepsis, and maternal and fetal demise. Upon infecting the host, GBS encounters sentinel innate immune cells, such as macrophages, within reproductive tissues. Once phagocytosed by macrophages, GBS upregulates the expression of the gene npx , which encodes an NADH peroxidase. GBS mutants with an npx deletion (Δ npx ) are exquisitely sensitive to reactive oxygen stress. Furthermore, we have shown that npx is required for GBS survival in both THP-1 and placental macrophages. In an in vivo murine model of ascending GBS vaginal infection during pregnancy, npx is required for invading reproductive tissues and is critical for inducing disease progression, including PPROM and preterm birth. Reproductive tissue cytokine production was also significantly diminished in Δ npx mutant-infected animals compared to that in animals infected with wild-type (WT) GBS. Complementation in trans reversed this phenotype, indicating that npx is critical for GBS survival and the initiation of proinflammatory signaling in the gravid host. IMPORTANCE This study sheds new light on the way that group B Streptococcus (GBS) defends itself against oxidative stress in the infected host. The enzyme encoded by the GBS gene npx is an NADH peroxidase that, our study reveals, provides defense against macrophage-derived reactive oxygen stress and facilitates infections of the uterus during pregnancy. This enzyme could represent a tractable target for future treatment strategies against invasive GBS infections.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.ppat.1009570
