Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis

Abstract

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.

Multi-Omics Profiling Specifies Involvement of Alternative Ribosomal Proteins in Response to Zinc Limitation in Mycobacterium smegmatis

“…Transcript abundances were analyzed to determine differential expression (DE) patterns using an absolute foldchange cut-off of two (log 2 fold-change, lfc = 1), yielding 1,084 genes that are upregulated and 742 genes that are downregulated at the transcript level in the Zn 2+ -limited condition (i.e., wild type grown in ZLM vs. wild type grown in ZRM) (Figure 1A). As expected, we observed upregulation of genes in the Msm Zur regulon (Novichkov et al, 2013; Supplementary Table S1) and, as observed in Mtb (Dow et al, 2021), hundreds of genes not under control of Zur were also upregulated (or downregulated) in the Zn 2+ -limited condition (Supplementary Table S2). Transcriptional changes during gradual Zn 2+ limiting conditions when bacteria are grown in ZLM (i.e., without addition of a Zn 2+ chelator) had a strong overlap with previous reports for acute Zn 2+ depletion through addition of the Zn 2+ -specific chelator TPEN; 74% of DE genes in TPEN-treated Msm followed the same expression levels in the wild type ZLM vs. wild type ZRM comparison (Goethe et al, 2020).…”

“…The concentration of Zn 2+ available during growth has a drastic impact on the transcriptomes of mycobacteria; Msm differentially regulated 26% and Mtb regulated 17% of their respective transcriptomes during Zn 2+ -limiting conditions. The transcriptomics analysis of Zn 2+ -limited Msm yielded similar results to those described for Zn 2+ -limited Mtb, specifically regarding increased DNA repair processes (Dow et al, 2021). Over half of the genes in Mtb have orthologues in Msm; 403 of the orthologous genes were DE in Zn 2+ -limited Mtb (Dow et al, 2021).…”

“…In Mtb, analysis of the proteome supported the transcriptomic changes observed, and a conserved response to Zn 2+ -limiting conditions was described from the overlap of DE transcripts and proteins (Dow et al, 2021). Taking the same approach here, we probed the proteomes of wild type Msm grown in ZRM and ZLM using semi-quantitative spectral counting (SpC) from shotgun proteomics.…”

“…We anticipate vast and unique changes in gene and protein expression patterns in response to Zn 2+ limitation in Msm compared to Mtb, especially considering that the previously observed morphological changes (elongated cells with condensed DNA) in Zn 2+ -limited Msm do not occur in Mtb (Dow and Prisic, 2018;Dow et al, 2021). Moreover, the Zn 2+dependent morphogenesis in Msm relies on expression of Zn 2+ -independent alternative ribosomal proteins (AltRPs) (Dow and Prisic, 2018).…”

Transcriptomic Characterization of Tuberculous Sputum Reveals a Host Warburg Effect and Microbial Cholesterol Catabolism

“…Neutrophil-derived calprotectin may restrict the availability of zinc in the respiratory tract, and competition with commensals for free zinc may represent a vulnerability of M. tuberculosis in sputum. It has been proposed that zinc limitation defines a population of M. tuberculosis with anticipatory adaptations against impending immune attack, based on the evidence that zinc-limited M. tuberculosis is more resistant to oxidative stress, exhibits increased survival, and induces more severe pulmonary granulomas in mice ( 44 ). Similarly, in contrast to results in macrophage cultures ( 45 ), the M. tuberculosis sputum transcriptome is characterized by reduced activation of the PhoP regulon compared to exponential-phase cultures.…”