using-mycobacterium-smegmatis-as-bioindicator-for-zinc-limited-growth

Using <i>Mycobacterium smegmatis</i> as a Bioindicator for Zinc-Limited Growth Conditions in Mycobacteria

Many bacteria build alternative ribosomes in Zn2+-limiting growth conditions by replacing Zn2+-binding ribosomal proteins with Zn2+-independent paralogs. ...

University of Hawaiʻi at Mānoa

The Mycobacterium tuberculosis genome encodes five putative 'alternative' ribosomal proteins whose expression is repressed at high Zn(2+) concentration. Each alternative protein has a primary homologue that is predicted to bind Zn(2+). We hypothesized that zinc triggers a switch between these paired homologous proteins and therefore chose one of these pairs, S18-1/S18-2, to study mechanisms of the predicted competition for their incorporation into ribosomes. Our data show that Zn(2+)-depletion causes accumulation of both S18-2 mRNA and protein. In contrast, S18-1 mRNA levels are unchanged to slightly elevated under Zn(2+)-limited conditions. However, the amount of S18-1 protein is markedly decreased. We further demonstrate that both S18 proteins interact with ribosomal protein S6, a committed step in ribosome biogenesis. Zn(2+) is absolutely required for the S18-1/S6 interaction while it is dispensable for S18-2/S6 dimer formation. These data suggest a model in which S18-1 is the dominant ribosome constituent in high zinc conditions, e.g. inside of phagosomes, but that it can be replaced by S18-2 when zinc is deficient, e.g. in the extracellular milieu. Consequently, Zn(2+)-depletion may serve as a signal for building alternative ribosomes when M. tuberculosis is released from macrophages, to allow survival in the extracellular environment.

Zinc regulates a switch between primary and alternative S18 ribosomal proteins in Mycobacterium tuberculosis.

The Mycobacterium tuberculosis Ser/Thr kinase PknB is implicated in the regulation of bacterial cell growth and cell division. The intracellular kinase function of PknB is thought to be triggered by peptidoglycan (PGN) fragments that are recognized by the extracytoplasmic domain of PknB. The PGN in the cell wall of M. tuberculosis has several unusual modifications, including the presence of N-glycolyl groups (in addition to N-acetyl groups) in the muramic acid residues and amidation of d-Glu in the peptide chains. Using synthetic PGN fragments incorporating these diverse PGN structures, we analyzed their binding characters through biolayer interferometry (BLI), NMR spectroscopy, and native mass spectrometry (nMS) techniques. The results of BLI showed that muropeptides containing 1,6-anhydro-MurNAc and longer glycan chains exhibited higher binding potency and that the fourth amino acid of the peptide stem, d-Ala, was crucial for protein recognition. Saturation transfer difference (STD) NMR spectroscopy indicated the major involvement of the stem peptide region in the PASTA-PGN fragment binding. nMS suggested that the binding stoichiometry was 1:1. The data provide the first molecular basis for the specific interaction of PGN with PknB and firmly establish PGNs as the effective ligands of PknB.

A Comprehensive Study of the Interaction between Peptidoglycan Fragments and the Extracellular Domain of Mycobacterium tuberculosis Ser/Thr Kinase PknB.

Despite increasing acknowledgment that microorganisms underpin the healthy functioning of basically all multicellular life, few cross-disciplinary teams address the diversity and function of microbiota across organisms and ecosystems. Our newly formed consortium of junior faculty spanning fields such as ecology and geoscience to mathematics and molecular biology from the University of Hawai'i at Mānoa aims to fill this gap. We are united in our mutual interest in advancing a new paradigm for biology that incorporates our modern understanding of the importance of microorganisms. As our first concerted research effort, we will assess the diversity and function of microbes across an entire watershed on the island of Oahu, Hawai'i. Due to its high ecological diversity across tractable areas of land and sea, Hawai'i provides a model system for the study of complex microbial communities and the processes they mediate. Owing to our diverse expertise, we will leverage this study system to advance the field of biology.

Synergy among Microbiota and Their Hosts: Leveraging the Hawaiian Archipelago and Local Collaborative Networks To Address Pressing Questions in Microbiome Research.

Zinc depletion does not necessarily induce ribosome hibernation in mycobacteria.

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 (Zn2+) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn2+ 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, Zn2+-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn2+-limited Mtb in vivo. Prolonged Zn2+ 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 Zn2+ 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 Zn2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn2+-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 Zn2+-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn2+ availability likely plays a key role during early interactions with host cells.

Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis.

Zinc ion (Zn) is an essential micronutrient and a potent antioxidant. However, Zn is often limited in the environment. Upon Zn limitation,  (basonym: )  undergoes a morphogenesis, which relies on alternative ribosomal proteins (AltRPs); i.e., Zn-independent paralogues of Zn-dependent ribosomal proteins. However, the underlying physiological changes triggered by Zn limitation and how AltRPs contribute to these changes were not known. In this study, we expand the knowledge of mechanisms utilized by  to endure Zn limitation, by comparing the transcriptomes and proteomes of Zn-limited and Zn-replete . We further compare, corroborate and contrast our results to those reported for the pathogenic mycobacterium, , which highlighted conservation of the upregulated oxidative stress response when Zn is limited in both mycobacteria. By comparing the multi-omics analysis of a knockout mutant lacking AltRPs (Δ) to the  wild type strain, we specify the involvement of AltRPs in the response to Zn limitation. Our results show that AltRP expression in  does not affect the conserved oxidative stress response during Zn 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-dependent paralogues; they are also involved in the transcriptomic response to the Zn-limited environment.

Article	Year
Zinc regulates a switch between primary and alternative S18 ribosomal proteins in Mycobacterium tuberculosis. Molecular microbiology\| PubMed ID: 25858183	2015
A Comprehensive Study of the Interaction between Peptidoglycan Fragments and the Extracellular Domain of Mycobacterium tuberculosis Ser/Thr Kinase PknB. Chembiochem : a European journal of chemical biology\| PubMed ID: 28851116	2017
Synergy among Microbiota and Their Hosts: Leveraging the Hawaiian Archipelago and Local Collaborative Networks To Address Pressing Questions in Microbiome Research. mSystems\| PubMed ID: 29556540	2018
Zinc depletion does not necessarily induce ribosome hibernation in mycobacteria. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30683730	2019
Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis. PLoS pathogens\| PubMed ID: 33989345	2021
Multi-Omics Profiling Specifies Involvement of Alternative Ribosomal Proteins in Response to Zinc Limitation in . Frontiers in microbiology\| PubMed ID: 35222334	2022

Sladjana Prisic

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