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NAD+ metabolism is a key modulator of bacterial respiratory epithelial infections

Björn Klabunde, André Wesener, Wilhelm Bertrams, Isabell Beinborn, Nicole Paczia, Kristin Surmann, Sascha Blankenburg, Jochen Wilhelm, Javier Serrania, Kèvin Knoops, Eslam M. Elsayed, Katrin Laakmann, Anna Lena Jung, Andreas Kirschbaum, Mobarak Abu Mraheil, Anke Becker, Uwe Völker, Evelyn Vollmeister, Birke J. Benedikter, Bernd Schmeck

Posted on: 17 October 2023 , updated on: 19 October 2023

Preprint posted on 14 April 2023

Article now published in Nature Communications at https://www.nature.com/articles/s41467-023-41372-w#:~:text=NAD%2B%20treatment%20of%20Spn%20dysregulated,an%20antibacterial%20mechanism%20of%20NAD%2B.

Altered NAD+ metabolism in respiratory epithelial cells by Streptococcus pneumoniae sheds light on new infection treatments.

Selected by Matheus Atella de Oliveira, Marcus Oliveira

Categories: immunology, microbiology

Updated 17 October 2023 with a postLight by Matheus Atella de Oliveira

This study was published in Nature Communications after someupdates and revisions. This includes the addition of three more authors. To address the challenge posed by NMN’s inability to enter cells, the authors have conducted further experiments involving the supplementation of both eukaryotic and bacterial cells with other NAD precursors, such as NR and NAM. The supplementation of cells with NAD precursors led to an increase in NAD/NADH levels while simultaneously reducing bacterial replication.

Interestingly, the supplementation of bacterial culture with these precursors had no discernible impact on its growth, whereas NAD treatment resulted in a reduction and delay in growth. This outcome effectively dispelled the notion that NAM, and not NAD, presented a direct antibacterial effect. This is because when NMN is degraded by eukaryotic cells, it releases NAM, which can enter the cell and serve as a precursor for NAD. However, in the context of bacterial culture alone, the precursors treatment served as a control to demonstrate that NAD is indeed responsible for inhibiting bacterial growth.

Additionally, the researchers have introduced a supplementary figure involving a Spn mutant strain that did not produce hydrogen peroxide (H2O2). This strain was used to infect BEAS-2B cells, and the subsequent qPCR analysis confirmed that the downregulation of NMNAT1 and the upregulation of NAMPT were not induced by reactive oxygen species.

In the published article, the researchers further explored the potential role of nicotinamide N-methyltransferase (NNMT) in the reduction of NAD levels within the discussion section, as this enzyme converts NAM into a non-salvageable form, competing with NAMPT, and was found to be upregulated by Spn infection.

Background

Streptococcus pneumoniae (Spn) are gram-positive encapsulated bacteria that cause upper and lower respiratory tract infections, being a major cause of death worldwide1. Upon bacterial internalization by inspiration, there is first contact between the pathogen and epithelial cells. These epithelial cells constitute the first line of defense as they can produce antibacterial agents and chemokines that recruit immune cells2. However, as most of the studies regarding Spn infection focus on immune cells, little is known about the effect of Spn infection on epithelial cells’ gene expression and metabolism.

Over the past decade, evidence has grown for a link between inflammation and NAD(H) metabolism, as the inflammatory response promotes the expression of several NAD-consuming enzymes and can also affect the expression of genes involved in NAD synthesis3. Upon NAD degradation by NAD-consuming enzymes, the reaction releases nicotinamide (NAM) as a product, which can follow different pathways: it can be recycled into NAD by the salvage pathway or it can be metabolized by the enzyme NNMT into products that are excreted in the urine4. The NAD+ Salvage pathway is responsible for synthesizing the majority of the cellular NAD4. This pathway starts with the conversion of NAM into nicotinamide mononucleotide (NMN) by NAMPT, which is then converted into NAD by NMNAT4. In this preprint, the authors aimed to understand the impact of Spn infection on epithelial cell metabolism, focusing on NAD metabolism and how alterations found could impact the outcome of the infection.

 

Key findings

Spn infection leads to a dysregulation of NAD+ metabolism and a decrease in NAD levels

Multi-omics analysis revealed dysregulation of the expression of several genes involved in NAD(H) metabolism upon Spn infection; such as the upregulation of NAMPT and downregulation of NMNAT1 in the bronchial epithelial cell line BEAS-2B, primary human bronchial epithelial cells, and lung explant. These altered expression patterns were also found in publicly available transcriptome data sets of lungs of Spn-infected mice, revealing a consistent dysregulation in the NAD salvage pathway during Spn infection. Through a metabolomic analysis, the authors also found a decrease in NAD levels in the bronchial epithelial cell line BEAS-2B upon Spn infection.

NAD has a direct antibacterial effect against Spn

The authors could show that the depletion of NMNAT or NAMPT in BEAS-2B increases bacterial burden, while decreasing NAD levels. On the other hand, NAD or NMN supplementation of Spn infected cells reduced the number of colony forming units (CFU), while only NAD treatment showed a bacteriostatic effect in Spn cultures, suggesting that the effect of NMN is due to its conversion to NAD by NMNAT.

Spn promotes the downregulation of NMNAT gene expression through its virulence factor pneumolysin

The authors could show that the BEAS-2B infection with a pneumolysin (Ply)-deficient mutant of Spn does not affect NMNAT1 gene expression, and that treatment of the cells with isolated Ply promotes a downregulation of NMNAT1, suggesting that this is the mechanism by which Spn downregulates NMNAT1 expression.

NAD displays direct antibacterial effects through disturbing Spn energy metabolism

As a strategy to investigate the mechanisms by which NAD was restricting Spn growth, the authors cultivated Spn in a culture medium with high NAD concentration in order to generate NAD-resistant strains. The three strains generated hereby presented a mutation in a gene involved in the encapsulation of the bacteria, a process that involves a high expenditure of ATP. Therefore, the resistant strains presented loss of the capsule and higher ATP amounts when compared to wild-type. NAD treatment of wild-type Spn promoted a reduction in bacterial replication and in the ATP levels, and ATP boosting through pyruvate supplementation abolished the antibacterial effects of the NAD treatment, revealing that the antibacterial effects of NAD are due to its implications on energy metabolism.

 

Why I think this preprint is important

This preprint is important because it addresses the need for a deeper understanding of lower respiratory infections, which are a leading cause of global mortality. By focusing on the respiratory epithelium, this study reveals the dysregulation of key enzymes and metabolites involved in NAD+ metabolism during Spn infection. The authors demonstrate that manipulating NAD+ metabolism can inhibit Spn growth, suggesting it as a potential therapeutic target to treat Spn infection. Overall, this work contributes to our understanding of host-pathogen interactions, identifying the NAD+ metabolism as a potential avenue for developing treatments against bacterial infections.

 

Questions and suggestions for the authors

1- There are genes represented in Figure 1D (for the purpose of summarizing NAD metabolism dysregulation) that are not represented either on the proteomic analysis or on the transcriptome heatmap. The authors could consider representing NAD metabolism-related genes that are differentially expressed in the graphs mentioned above to clarify the fold change.

2- Regarding the statistical analysis, as the authors performed a paired-t test, the normalization should be made upon the average values of the control samples so the standard deviation and the distribution of the values should be taken into consideration. Given that paired tests assume a Gaussian distribution of the data sets, did you perform any statistical analysis to verify this issue? A more detailed explanation of the criteria for selecting specific statistical tests would be beneficial.

3- If the authors want to investigate whether the downregulation of NMNAT1 is the mechanism by which the NAD levels are found to decrease during Spn infection, they could perform a NAD(H) quantification assay in the BEAS-2B cells infected with the ply-deficient Spn, as this mutant did not cause any alterations in the expression of NMNAT1. The authors could also review the axis titles of Figure 5. D-F and standardize it.

4- The rate-limiting step of the salvage pathway is the reaction catalyzed by NAMPT. The changes in NMNAT1 expression found are very mild, and therefore it is hard to conclude that this is the mechanism by which NAD levels are decreased upon Spn infection. Did you find any differential expression of the other NMNAT isoforms, such as NMNAT2 and NMNAT3?  You could consider evaluating NNMT expression in primary cell culture models and in lung explant during Spn infection and performing a NAD(H) quantification assay on these models. This would provide a more comprehensive insight into whether NNMT upregulation could be contributing to the decrease in NAD(H) levels.

5- As the ATP quantitation was made using a luminescent based assay, the loss of capsule could affect the luminescence signal and therefore it would be appropriate to measure the ATP levels through other techniques as well.

6- Considering Spn is predominantly an extracellular pathogen5 and NAD is mainly a cytosolic compound, do you think that the direct antibacterial effect of NAD would be relevant under physiological conditions? If you quantified the NAD extracellular levels in BEAS-2B cultures under Spn infection, we suggest that they represent the NAD concentration in the graph and not only the control relative values. As part of the discussion section, it would be worth considering that NAD could be displaying both direct and indirect antibacterial effects, as it has already been demonstrated that NAD consuming enzymes such as PARPs can display antibacterial effects6.

 

References

  1. Chen H, Matsumoto H, Horita N, Hara Y, Kobayashi N, Kaneko T. Prognostic factors for mortality in invasive pneumococcal disease in adult: a system review and meta-analysis. Sci Rep 2021; 11: 11865.
  2. Kuek LE, Lee RJ. First contact: the role of respiratory cilia in host-pathogen interactions in the airways. Am J Physiol-Lung Cell Mol Physiol 2020; 319: L603–L619.
  3. Nacarelli T, Zhang R. NAD+ metabolism controls inflammation during senescence. Mol Cell Oncol 2019; 6: 1605819.
  4. Xie N, Zhang L, Gao W, Huang C, Huber PE, Zhou X, Li C, Shen G, Zou B. NAD+ metabolism: pathophysiologic mechanisms and therapeutic potential. Signal Transduct Target Ther 2020; 5: 1–37.
  5. Lemon JK, Weiser JN. Degradation products of the extracellular pathogen Streptococcus pneumoniae access the cytosol via its pore-forming toxin. mBio 2015; 6: e02110-14.
  6. Miettinen M, Vedantham M, Pulliainen AT. Host poly(ADP-ribose) polymerases (PARPs) in acute and chronic bacterial infections. Microbes Infect 2019; 21: 423–431.

 

doi: https://doi.org/10.1242/prelights.35768

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