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. 2022 Jun;117(6):1447-1463.
doi: 10.1111/mmi.14919. Epub 2022 May 31.

Regulation of neutrophil myeloperoxidase inhibitor SPIN by the small RNA Teg49 in Staphylococcus aureus

Liviu Cengher  1 , Adhar C Manna  1 , Junho Cho  1 , Jomkuan Theprungsirikul  1 , Katherine Sessions  1 , William Rigby  1 , Ambrose L Cheung  1
Affiliations

Regulation of neutrophil myeloperoxidase inhibitor SPIN by the small RNA Teg49 in Staphylococcus aureus

Liviu Cengher et al. Mol Microbiol. 2022 Jun.

Abstract

Teg49 is a Staphylococcus aureus trans-acting regulatory sRNA derived from cleavage of the sarA P3 transcript. We showed by RNA-Seq here that the 5' trident-like structure in Teg49 regulates transcriptionally (direct and indirect) 22 genes distinct from sarA. Among these, Teg49 was noted to repress spn, encoding a 102 residue preprotein which yields the mature 73 residue peptide which inhibits the catalytic activity of myeloperoxidase in human neutrophils. Teg49 was found to regulate spn mRNA post-transcriptionally in strain SH1000 through 9-nt base-pairing between hairpin loop 2 of Teg49 and an exposed bulge of the spn mRNA. Mutations of the Teg49 binding site disrupted the repression of spn, leading to reduced degradation, and increased half-life of spn mRNA in the Teg49 mutant. The spn-Teg49 interaction was also confirmed with a synonymous spn mutation to yield enhanced spn expression in the mutant vs. the parent. The Teg49 mutant with increased spn expression exhibited enhanced resistance to MPO activity in vitro. Killing assays with human neutrophils showed that the Teg49 mutant was more resistant to killing after phagocytosis. Altogether, this study shows that Teg49 in S. aureus has a distinct and important regulatory profile whereby this sRNA modulates resistance to myeloperoxidase-mediated killing by human neutrophils.

Keywords: Staphylococcus aureus; spn; RNA; SPIN; gene expression; human neutrophils; myeloperoxidase; regulatory small RNA; virulence.

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Figures

FIGURE 1
FIGURE 1
Teg49 regulates the expression of spn, a myeloperoxidase inhibitor. (a) Nanostring nCounter data for genes in Staphylococcus aureus SH1000, Teg49 mutant, complement, and sarA mutant. This array includes two housekeeping genes HU and rpoB, spn, and several genes that are known to be regulated by SarA (i.e., aureolysin gene aur and protein a gene spa). Genes are separated with vertical dotted lines to show differentially regulated genes, housekeeping genes (HU and rpoS), one group of genes not regulated by Teg49 or SarA (groES, esxB, and pyrP) and another group detected by RNA-seq but not confirmed by Nanostring (purH and ribD). Data were analyzed using a two-way ANOVA using Dunnett’s test for multiple comparisons. p values are * = .05, ** = .01, *** = .001, **** ≤ .001. (b) qRT-PCR validation of spn expression in SH1000, Teg49 mutant, Teg49 complement, and sarA mutant at 4th h of growth. Fold change was calculated by 2(−ΔΔCt). Significant differences were calculated using a one-way ANOVA followed by Tukey’s multiple comparison test compared to the Teg49 mutant. p-values indicated are * = .05, ** = .01, *** = .001.
FIGURE 2
FIGURE 2
Characterization of spn expression during growth. (a) Growth of strain SH1000 as determined by OD600 in a spectrophotometer with a 1 cm cuvette. (b) The left Y-axis represents a time-course of spn expression by qRT-PCR at OD corresponding to growth in (a). Increasing levels of the spn transcript were detected as cells reached the stationary phase and then tapered. The Y axis on the right is a densitometric analysis of a northern blot of Teg49 during 8 h. growth after dilution from an overnight culture. The timepoints for the densitometry are approximate but are not identical to those in (a). (c) OD600 growth curve for wild type SH100 and ΔTeg49 with or without the pALC1484:Spn promoter GFP fusion plasmid and the control empty vector in SH1000. (d) Fluorescence of the same strains (as in (c)) was measured in AU over OD600. OD600 was measured using a Tecan infinity 1000 with a 100 μl volume in a standard 96 well plate and reached a plateau at OD600 = 0.29/0.30. There was no significant difference in fluorescence between the SH1000 and ΔTeg49 strain containing the spn promoter fusion plasmid.
FIGURE 3
FIGURE 3
Predicted interaction between Teg49 and spn RNAs. (a) The predicted minimum free energy (MFE) RNA structure of spn mRNA from RNAfold. The spn mRNA is labeled with 5′ and 3′ UTR in green, and the IntaRNA predicted Teg49 target region is labeled in red. The region is within the ORF of spn and is predicted to be exposed. (b) The Teg49 sRNA is labeled with each of its hairpins, and the predicted spn binding region on hairpin 2 is shown in red. This binding region is located on the 5′ side of the hairpin 2 loop. (c) A closeup of the predicted interaction between spn mRNA and Teg49. In yellow are the residues in the predicted region for spn binding in Teg49, in orange is the predicted target region for binding to Teg49 in the spn coding sequence. IntaRNA prediction matches nucleotide 242 in spn mRNA to nucleotide 69 in Teg49 (U/a) and continues to nucleotide 250 in spn mRNA and nucleotide 61 in Teg49 (a/U).
FIGURE 4
FIGURE 4
The interaction of Teg49 and spn mRNA for proper spn regulation. (a) qRT-PCR looking at spn expression in Teg49 loop mutants at 4th h. of growth. Hairpin loop 1, 2, and 3 replacement mutants that retained the predicted structure of Teg49 were evaluated. Significance is measured using a one-way ANOVA using Dunnett’s multiple tests. p-values are * = .05, ** = .01, *** = .001, **** ≤ .001. (b) qRT-PCR looking at spn expression in spn mutants with synonymous (retaining the same amino acid -see text) and non-synonymous mutations (3 altered amino acids) in the predicted site for Teg49 interaction at 4th h. of growth. Significance is measured using a one-way ANOVA using Dunnett’s multiple tests. p-values are * = .05, ** = .01, *** = .001, **** ≤ .001. (c) Northern blots showing hybridizing spn band at serial time points in Teg49 mutant and SH1000 after addition of rifampin at time 0. (d) qRT-PCR for spn expression in SH1000 wild type, Teg49 mutant, and complement, as well as saeRS transposon mutant (sae mutant), RNase III deletion mutant (Δrnc), and complement at 4th h. of growth. Significance is measured using a one-way ANOVA using Dunnett’s multiple tests. p-values are * = .05, ** = .01, *** = .001, **** ≤ .001.
FIGURE 5
FIGURE 5
Teg49 mutants have enhanced neutrophil survival mediated through increased spn expression. (a) Diagrams of the enzymatic pathways that generate HOCl in in vitro MPO assay alongside the physiological enzymatic pathway in neutrophils that generates HOCl. In both cases, Teg49 regulation of spn affects the last step, MPO activity. In the in vitro assay, glucose oxidase is used to generate hydrogen peroxide, as compared to in vivo in neutrophils that require NADPH oxidase in the presence of oxygen to generate oxygen radicals (O*2−), and then superoxide dismutase (SOD) to yield H2O2. In both cases, MPO uses H2O2 and chloride to generate hypochlorous acid (HOCl), a powerful antimicrobial antioxidant. (b) Myeloperoxidase assay looking at cell survival in an MPO assay developed in vitro. Wild type SH1000, Teg49 mutant, complement, spn deletion mutant, complement, and double mutant were compared. A sarA mutant was added as a control. Significance is measured using a one-way ANOVA using Dunnett’s multiple tests. p-values are * = .05, ** = .01, *** = .001, **** ≤ .001. (c) Bacterial survival in human PMNs which have phagocytosed Staphylococcus aureus for a 30-min interval at an initial MOI of 8. The strains used are similar to those in (b). Significance is measured using a one-way ANOVA using Dunnett’s multiple tests. p-values are * = .05, ** = .01, *** = .001, **** ≤ .001.
FIGURE 6
FIGURE 6
Model for Teg49 function in neutrophil myeloperoxidase tolerance. Model of how regulation of spn mRNA by Teg49 in S. aureus increases survival in PMN phagolysosomes. In this representative image, myeloperoxidase activity within the phagolysosome of phagocytes (PMNs in this case) generates hypochlorous acid to kill bacterial cells. Myeloperoxidase inhibitor (encoded by spn) inhibits MPO activity to reduce HOCl production. In this study, we provide an extra layer of regulation by showing that sRNA Teg49 can repress spn expression post-transcriptionally by base-pairing with spn mRNA to facilitate degradation and hence reduce translation.
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