NAD kinase promotes Staphylococcus aureus pathogenesis by supporting production of virulence factors and protective enzymes

Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes the synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knock down NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus. Furthermore, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence-associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection.


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Nicotinamide adenine dinucleotide (NAD + ), its phosphorylated form (NADP + ) and their 33 reduced equivalents (NADH and NADPH) are essential cofactors shared by all living 34 organisms, including bacteria. While NAD + and NADH are important for cellular energy 35 metabolism, inflammation and senescence (Chini et al., 2021;Covarrubias et al., 2021), NADP + 36 and NADPH are key cofactors in central metabolism, being involved in tricarboxylic acid 37 (TCA) cycle, pentose phosphate pathway as well as de novo synthesis of fatty acids, cholesterol, 38 amino acids and nucleotides (Chandel, 2021). NADPH also provides the reducing power 39 necessary for the restoration of antioxidative defense systems of the cell (Chandel, 2021). Like 40 NAD(H), growing evidence suggests that NADP(H) has a broader role. In particular, the 41 NADP + derivative nicotinic acid adenine dinucleotide phosphate (NAADP), which is the major 42 intracellular calcium mobilizing molecule, links NADP(H) metabolism with calcium 43 homeostasis and signaling, development and differentiation (Galione and Chuang, 2020). 44 Whereas there are two known multi-step pathways for NAD + biosynthesis, a single 45 enzyme is responsible for the phosphorylation of NAD + /NADH into NADP + /NADPH: the 46 NAD kinase (NADK) (McGuinness and Butler, 1985; Chini et al., 2021). NADK activity was 47 first reported in the late 30's (Vestin, 1937;Von Euler and Adler, 1938), and the enzyme was 48 purified from yeast by Kornberg in 1950 (Kornberg, 1950). It is only in 2000 that the genes 49 encoding NADK were identified in Micrococcus flavus and Mycobacterium tuberculosis 50 (Kawai et al., 2000). Since then, NADK genes have been identified in all living organisms, 51 except the intracellular parasitic bacteria Chlamydia spp. on the basis of genome annotation 52 (Grose et al., 2006;Fisher et al. 2013). Given the vital role of NADPH, notably during oxidative 53 stress, NADK genes have been shown to be essential for growth of several bacteria, such as 54 Mycobacterium tuberculosis (Sassetti et al., 2003), Bacillus subtilis (Kobayashi et al., 2003), 55 Salmonella enterica (Grose et al., 2006) and Staphylococcus aureus (Chaudhuri et al., 2009;56 aureus pathogenic potential, as NADK inhibition led to an increased survival of the host in a 82 zebrafish infection model. We further demonstrate that inhibition of NADK leads to a dramatic 83 decrease of S. aureus survival in macrophages, highlighting the enzyme importance during 84 interactions with immune cells. We further show that exposures to oxidative and envelope-85 targeting antimicrobial compounds significantly decrease bacterial survival upon inhibition of 86 NADK activity. Proteomic analysis indicated that the production of protective enzymes, 87 virulence factors and the quorum-sensing response regulator AgrA relies on NADK. Thus, our 88 results reveal a key role for NADK in bacterial pathogenesis. 89

NADK contributes to S. aureus virulence in zebrafish 91
As most prokaryotes, S. aureus possesses a single NADK encoded by the highly conserved 92 ppnK gene. To investigate the role of NADK in S. aureus pathogenesis, we followed a genetic 93 approach to modulate the expression of ppnK. We used the S. aureus NADK sgRNA strain 94  (Prajsnar et al., 2008) and has been 101 previously used to study S. aureus infection dynamics (Prajsnar et al., 2012). In addition to 102 being optically transparent, zebrafish larvae innate immune system is the only one operating 103 during the first days of development, facilitating the study of interactions between bacteria and 104 neutrophils or macrophages. We first determined the stability of the pSD1 plasmid during 105 infection. Zebrafish larvae were infected at 60 hours post fertilization (hpf) by intravenous 106 injections of either S. aureus containing the empty vector pSD1 or the knockdown NADK 107 sgRNA strain. Bacteria recovered from infected larvae were plated onto BHI agar with and 108 without chloramphenicol. There was no significant difference between the number of bacteria 109 growing on BHI alone and BHI supplemented with chloramphenicol up to 48 hours post-110 infection (hpi), irrespective of the strain (Figure supplement 2A). Thus, plasmids were 111 maintained in our experimental infection conditions. We next monitored zebrafish survival 112 upon infection with 10 4 bacteria. S. aureus/pSD1 infection led to ≈ 75% fish mortality at 24 hpi 113 ( Figure 1A). Strikingly, only one larva out of 48 died when bacterial NADK was inhibited 114 ( Figure 1A). Decreased fish mortality upon S. aureus/NADK sgRNA infection was also 115 observed 48 and 72 hpi ( Figure 1A). These results indicate that NADK inhibition leads to a 116 decreased bacterial virulence. We then determined the bacterial burden of fish larvae 24 and 48 117 hpi. Interestingly, numbers of S. aureus/pSD1 and S. aureus/NADK sgRNA bacteria at both 118 time points varied depending on the viability of the fish ( Figure 1B). As observed previously 119 by Prasjnar et al. (Prajsnar et al., 2012), some fishes were able to control the infection with 10 4 120 bacteria but a high bacterial burden was associated with larval death in most cases ( Figure 1B). 121 Early mortality was observed in zebrafish in which S. aureus/pSD1 developed to high numbers 122 compared to larvae infected with the NADK knockdown strain. Together, these results suggest 123 that NADK contributes to S. aureus pathogenic potential, which depends on both bacterial and 124 host factors. To investigate the impact of NADK inhibition on phagocyte populations, we 125 infected intravenously transgenic zebrafish lines with S. aureus/pSD1 or S. aureus/NADK 126 sgRNA strains, and observed bacteria, macrophages and neutrophils at time 0, 6, 12 and 24 hpi. 127 S. aureus/pSD1 bacterial burden was higher than that of S. aureus/NADK sgRNA strain in head 128 and caudal regions observed in fish of comparable viability 12 hours ( Figure 1C  proper NADK activity. As oxidative burst is a major antimicrobial mechanism of phagocytes 166 (Fang, 2004), we next investigated the role of ROS produced by macrophages in the control of 167 the S. aureus/NADK sgRNA strain using the antioxidant agent N-acetyl cysteine (NAC). NAC 168 partially restored bacterial survival (Figure 2A), indicating that the bacterial growth defect of 169 the ppnK knockdown strain was due to its inability to cope with oxidative stress to some extent.

NADK protects S. aureus from antimicrobial defense compounds 204
Hydrogen peroxide is one of the reactive oxygen species generated upon activation of 205 macrophages (Fang, 2004). We therefore compared growth of the S. aureus strain containing 206 the empty vector to that of the NADK knockdown strain, upon exposure to increasing 207 concentrations of hydrogen peroxide (H2O2). While the S. aureus/pSD1 strain resisted to high 208 doses of H2O2, the S. aureus/NADK sgRNA strain showed increasing growth deficiency when 209 exposed to H2O2 concentrations ranging from 50 to 500 µM H2O2 ( Figure 3A). Expectedly, 210 treatment of the S. aureus/pSD1 strain with catalase alone or catalase and H2O2 did not affect 211 growth ( Figure 3B). In contrast, addition of catalase, which catalyzes hydrogen peroxide 212 dismutation into water and oxygen, rescued the growth defect of the S. aureus/NADK sgRNA 213 strain upon H2O2 treatment ( Figure 3B). Taken together, our results show that NADK protects We thus investigated the effects of NADK knockdown on S. aureus sensitivity to lysozyme. 218 Since S. aureus is highly resistant to lysozyme, we treated bacteria with lysozyme and the 219 bacteriocin lysostaphin, a combination which potentiates their antibacterial activity against S. 220 aureus (Cisani et al., 1982). At subinhibitory concentrations for the S. aureus/pSD1 strain, the 221 S. aureus/NADK sgRNA strain showed decreased viability when exposed to lysozyme and 222 lysostaphin ( Figure 3C

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Data are representative of at least three independent experiments.

NADK supports S. aureus protective enzymes 242
To elucidate mechanisms of action of NADK, unbiased mass spectrometry-based proteomics 243 was used to identify proteins differentially expressed upon NADK knockdown. Relative protein 244 abundance was compared in the S. aureus strain containing the empty vector and the NADK 245 knockdown strain grown in BHI. Interestingly, lower levels of major antioxidant enzymes were 246 detected in the NADK sgRNA strain (Table 1 and table supplement 1 aureus/pSD1 strain than in the NADK deficient strain (Table supplement 2 and table  256 supplement 3), as it had been observed when strains were grown in BHI. To determine the 257 effect of NADK knockdown on S. aureus proteome in vivo, relative protein abundance was 258 analyzed in zebrafish infected with the S. aureus strain containing the empty vector or the 259 NADK knockdown strain. The D-alanyl carrier protein DltC, which mediates CAMP 260 resistance, and antioxidant enzymes, such as SodA, KatA, thiol peroxidase Tpx and alkyl 261 hydroperoxide reductase subunit AhpF, were detected in zebrafish infected with S. 262 aureus/pSD1 (Tables S4 and S5). In contrast, these proteins were undetectable in S.

Christie-Atkins-Munch-Peterson test 464
S. aureus hemolytic activity was performed as described previously (Adhikari et al. 2007). 465 Briefly, S. aureus RN4220 was streaked vertically at the center of a sheep blood agar plate. S. 466 aureus/pSD1 and S. aureus/NADK sgRNA strains were then streaked perpendicularly to the 467 RN4220 strain. The plate was incubated at 37°C for 18 hours. Blood agar plates were then 468 photographed with a Scan500 imager (Interscience). The assay was repeated three times. Trypsin was selected as protease allowing for up to two missed cleavages. The minimum 673 peptide length was set to 5 amino acids and the peptide mass was limited to a maximum of 674 8,000 Da. The false discovery rate (FDR) for peptide and protein identification was set to 0.01. 675 The main search peptide tolerance was set to 4.5 ppm and to 20 ppm for the MS/MS match 676 tolerance. Second peptides were enabled to identify co-fragmentation events. One unique 677 peptide to the protein group was required for the protein identification. A false discovery rate 678 cut-off of 1 % was applied at the peptide and protein levels. The mass spectrometry proteomics 679 data have been deposited to the ProteomeXchange Consortium via the PRIDE partner 680 repository with the dataset identifier PXD032968. 681 The statistical analysis of the proteomics data was performed as follows: Five biological 682 replicates were acquired per condition. To highlight significantly differentially abundant 683 proteins between two conditions, differential analyses were conducted through the following 684 Results are expressed as means ± SEM of at least 3 replicates. Statistical analysis was performed 704 using GraphPad Prism (GraphPad Prism Ò 9.1.2. Software). One-way, two-ways ANOVA or t-705 test were used to compare data. Differences between groups were considered significant when 706 the p value was lower than 0.05. Survival data were plotted using the Kaplan-Meier estimator 707 and log-rank (Mantel-Cox) tests were performed to assess differences between groups.