Introduction

Streptococcus pyogenes (Spy; Group A Streptococcus) is a human-exclusive bacterial pathogen responsible for billions of infections and more than 500,000 deaths annually, making it one of the top ten infectious causes of human mortality worldwide¹. Most infections are relatively mild, such as impetigo and pharyngitis, but serious, and often fatal diseases like bacteremia, puerperal sepsis, Streptococcal toxic shock syndrome (STSS), and necrotizing fasciitis arise when Spy becomes invasive. Virulence factors play a major role in disease progressing by facilitating tissue invasion, evading immune cell killing, and creating intracellular reservoirs⁶.

SpeB is a major virulence factor conserved amongst Spy, and is essential for colonization and proliferation within tissue in a variety of infection models⁷. Nonetheless, mutants arise during severe infections that, enigmatically, no longer express speB^8,9. These mutations are most commonly in the two-component system CovRS (CsrRS), which modulates expression of nearly 15% of Spy genes, including most virulence factors^10–12. CovRS senses the host cathelicidin peptide LL-37, produced by epithelial and immune cells as part of innate immune defense and wound repair^5,13–15. Loss-of-function covRS mutations dysregulate their virulence factors in a manner similar to constitutive LL-37 induction, decreasing production of SpeB, while increasing production of capsule (through the hasABC operon) and toxins like SLO, NADase, Mac/IdeS, and ⁴. The major inducer of speB is RopB in response to the quorum-sensing peptide SIP^16,17. In vitro, the virulence factor-related (Vfr) protein also appears to antagonize induction, on the basis that vfr mutants express greater speB^18,19. How the combination of these possible host and microbial signals are integrated to regulate SpeB during infection is unknown.

This work shows that Spy establishes a phenotypically diverse population during infection through the heterogeneous expression of speB. Using genetics, RopB, Vfr, and the CovRS system are all found to be essential for generating discrete SpeB-producing and non-producing subpopulations. We show that Vfr is a labile sensor of protease activity that is degraded by SpeB, allowing it to autoregulate it’s own expression by relieving Vfr repression. Furthermore, neutrophil serine proteases also relieve Vfr repression, allowing the bacteria to sense both influx of neutrophils and their activities, with maximal speB induction occurring in response to neutrophil extracellular traps (NETs). Despite neutrophils being the major producers of LL-37, which can lead to speB repression through CovRS, protease sensing by Vfr ensures SpeB, which is important for resisting neutrophil-produced antimicrobials including LL-37²⁰, is still expressed in its presence. Together, our results show how Spy navigates the challenge of expressing the right virulence factor at the right time by use of a circuit that integrates host and microbial cues.

Results

S. pyogenes establishes subpopulation heterogeneity in their expression of speB

To examine speB expression within Spy, we generated a transcriptional fusion of gfp with 1000 bp upstream of the speB to include P1 and P2 regions of the speB promoter¹⁶. The best-characterized regulator of speB is CovRS; speB is derepressed when CovR is phosphorylated (CovR∼P), in contrast to the capsule biosynthesis operon hasABC, which is induced when CovR is unphosphorylated¹². CovR phosphorylation depends on the histidine kinase, CovS, which is sensitive to environmental signals. To examine the contribution of speB inducers separate from this repressor, we created a tandem reporter that also has the hasABC promoter fused to rfp²¹ (Fig. 1A). To test the accuracy and precision of the reporter construct, Spy was grown in concentrations of LL-37 (300 nM) or MgCl₂ (15 mM) that did not impact bacterial growth (Fig. S1A), but which divergently regulate CovR phosphorylation of CovS¹². As expected for a quorum-sensing regulated protease¹⁷, GFP (speB) is induced as Spy approaches late log phase. The addition of LL-37 induced RFP (hasABC) and repressed both the expression of GFP (speB) (Fig. 1C) as well as production of the mature, active protease (Fig. S1B). Contrarily, GFP (speB) induction was maintained in the presence of MgCl₂, but RFP (hasABC) repressed. Examination by fluorescence microscopy recapitulated these observations, but suggested heterogeneity in these responses within the bacterial population (Fig. 1B).

Fig. 1.

To quantify this at the single-cell level, we next used flow cytometry. Size, BactoView^TM Dead 760/780, and an antibody against the Spy-specific antigen Group A Carbohydrate (anti-GAC), were used to gate on single, live cells (Fig. S1C). As expected, Spy grown with LL-37 had a population shift positive for RFP and negative for GFP (Fig. 1D). Conversely, growth with MgCl₂ resulted in a lower and rightward shift in the population. However, there was significant heterogeneity even in these induced conditions, with large populations of intermediate expression.

Genetic knockouts of known and probable SpeB regulators (Fig. 1A) were created to further validate the fluorescent reporters while defining determinants of heterogenicity. None impacted bacterial growth (Fig. S2A). A ΔcovS mutant constitutively expressed high hasABC, (RFP), while repressing speB, as expected. A ΔropB mutant did not express speB, in agreement with its reported importance for speB induction¹⁷. Interestingly, a ΔspeB mutant also showed less induction of the speB reporter, suggesting the possibility of some autoregulation (Fig. 2A). Reporter fluorescence examined by cytometry (Fig. 2B) and microscopy (Fig. S2B) were consistent with these observations. Similarly, the ΔspeB and ΔcovS mutants expressed slightly less, while ΔropB bacteria expressed little (Fig. 2B, S2B).

Fig. 2.

Vfr is a dominant repressor for speB

Since secreted bacterial factors, including the SIP quorum-sensing peptide, give the potential for bacteria to coordinate expression within the population, we next performed media swap experiments. Wild-type Spy was grown to early exponential (EE), mid-late exponential (MLE), or stationary phase (SP) and the spent media removed, filtered, then used to supplement wild-type Spy growth carrying the SpeB reporter (Fig. 3A). As expected, since the SIP inducer accumulates in stationary phase cultures where SpeB is optimally expressed, SP spent media induced GFP (speB) (Fig. 3B). Surprisingly, however, growth from earlier growth phase cultures not only lacked speB inducer activity but suppressed its expression (Fig. 3B). This suggested that these cells produced a soluble factor that dominantly repressed the ability of other cells to produce SpeB. We next repeated the media swap experiment using a Δvfr mutant, which highly expressed SpeB (Fig. S2B), to see if a soluble factor from wild-type Spy could repress this. Spent media from only early-growth phase cultures could significantly repress this (Fig. 2B).

Fig. 3.

To assess whether it was Vfr itself in the media suppressing speB, we examined the activity of recombinant Vfr (rVfr). rVfr reduced GFP (speB) expression significantly in SpyΔvfr (Fig. 3C). To assess the relative phenotypic dominance of SpeB regulation by autoregulatory mechanisms (Rop-SIP-Vfr dependent) versus environmental signaling mechanisms (CovRS-dependent), wild-type Spy containing the dual reporter was grown in the presence of CovRS inducers (LL-37 or MgCl₂) and Vfr at increasing concentrations. Both LL-37 and Vfr function synergistically in repressing speB, while Vfr antagonized MgCl₂-mediated induction of speB (Fig. 3D). Notably, speB expression in Spy Δvfr is unaffected by LL-37 or MgCl₂, further validating its dominance over CovRS regulation (Fig. S2C).

Interestingly, the Vfr structure contains several potential protease SpeB cleavage sites (Fig. 3E)²². Since degradation could explain the SpeB-dependent changes in GFP (speB) expression, rVfr was incubated in the presence and absence of purified SpeB. Vfr degradation was observed in a concentration-dependent manner (Fig. 3F). Together, these data highlight that Vfr is a SpeB-labile repressor of SpeB expression.

speB is induced in the presence of immune effectors

Neutrophils are the first line of defense during innate immune response and are quickly recruited during bacterial infections. Neutrophils highly express the immune effector LL-37⁵, which Spy recognizes through CovRS, which by current models should repress speB expression. Spy grown with neutrophil lysates induced hasABC expression, consistent with CovRS stimulation, but did not repress speB expression (Fig. 4A). Similarly, in the mouse invasive infection model and in whole human blood, Spy induced hasABC while populations of high speB expressors were maintained (Fig. 4B). Spy with regulator knockouts were also evaluated to determine consistency in mouse and blood tissue infections with in vitro regulation. Δvfr maintained high speB expression, ΔropB bacteria were negative for speB expression, and ΔcovS contained a hasABC high population (Fig. S3A).

Fig. 4.

Immune effectors induce speB through Vfr

This high-level of expression in vivo suggested that speB could be induced, not just repressed, in the presence of immune effectors. Along with LL-37, neutrophil granules store many major inflammatory components secreted during infections^23,24. To assess whether other neutrophil derived molecules play a role in speB regulation, neutrophil lysates were fractionated and examined for their ability to induce or repress speB expression. Upon supplementation, GFP (speB) was induced by three fractions (10-12) containing active proteases (Fig. 5AB).

Fig. 5.

While Spy is resistant NET killing^25,26, the immune effectors released by neutrophils during NETosis, include not only LL-37, but proteases like neutrophil elastase (NE)²⁷. Based on the protease profile of NETs²⁸, Vfr contains several potential sites for cleavage (Fig. 5C). Neutrophils degraded rVfr (Fig. 5D), as did purified NE (Fig. S3C). The NE-specific inhibitor, BAY-678, was not sufficient to inhibit neutrophil lysate-mediated rVfr degradation, but serine inhibitors AEBSF and PMSF did (Fig. 5D, S3C), suggesting a sufficiency but not requirement for other neutrophil proteases. Moreover, Spy grown with neutrophil lysates and inhibitor AEBSF had resulted in speB inhibition expression, compared to neutrophil lysate alone (Fig. 5E). Notably, these conditions maintained hasABC expression, consistent with CovRS stimulation (Fig. S3D). Altogether, these data suggest Vfr is a substrate for both bacterial and host-mediated degradation.

SpeB and neutrophils are sufficient to induce speB expression

To examine the impact that neutrophils have on speB regulation in vivo, neutrophils were depleted by anti-Ly6G treatment, as previously²⁹, significantly decreasing their numbers (Fig. S4A). After a 24 h intradermal infection, Spy from the tissue was examined by flow cytometry for speB expression, relative to a Δvfr control to define the high-expressing population (Fig. S4B). Spy expression of speB was only moderately decreased in the absence of neutrophils (Fig. 6B). However, infection of neutropenic mice with a ΔspeB, where there is neither host or microbial proteases to cleave Vfr, prevented speB expression (Fig. 6B). Mice deficient in peptidyl-arginine deiminase 4 (PAD4) are unable to form NETs³⁰. To separately examine NET contributions PAD4^-/- mice were infected with Spy and speB expression examined. SpeB expression was reduced in PAD4^-/- mice, additively with a contribution of SpeB itself (Fig. 6C). Collectively, this data suggests that Spy integrates the sensing of both neutrophils and feedback from SpeB to titrate expression during infection.

Fig. 6.

Discussion

Since a bacterium must adapt to environmental changes, they modulate expression in response to diverse stimuli, including stress, metabolites, quorum sensing, pH, and temperature. The niche of Spy is exclusively the human, potentially limiting its exposure to the range that some other species face. Yet within this host, it is nonetheless presented with challenges. Foremost amongst these are the immune defenses, which escalate through the course of infection. Fine-tuning its regulation toward the virulence factors needed to overcome this specific challenge is key to the species. SpeB is a potent pro-inflammatory virulence factor required to establish infection and has key roles in determining the course and severity of infections⁶. Accordingly, the regulation of SpeB must be tied to circumstances where it will be advantageous. Regulation of speB is thus multifactorial, as it integrates sensors to promote colonization and survival. In this study, we highlight a novel mechanism for SpeB regulation through Vfr detection of neutrophils and their activation state. Our data shows that neutrophils, despite functioning as an LL-37 reservoir expected to repress speB expression through the CovRS system, actually induces it during invasive skin infections.

The relative contributions of these regulators could vary by disease³¹. In response to inflammation, neutrophils are recruited to infection sites to engulf and inactivate pathogens. Despite neutrophils playing a major role in combating most infections, several studies indicate they can worsen Spy infections, in particular, in the upper respiratory tract^29,32. Here, RopB can detect the SIP homologs used for quorum sensing by other Streptococci species of the upper respiratory microbiota³³. This ensures SpeB expression at low cell densities and could allow SpeB expression even before neutrophil infiltration. Importantly, the type of CovS mutants that interfere with SpeB expression do not occur during human pharyngitis or experimental mouse models of it, and ΔspeB mutants are highly attenuated, indicating its essentiality at this site²⁹. This would subsequently require SpeB expression during infections, suggesting repression by LL-37, released by the abundant neutrophils recruited during infection, is, at best, limited.

Our work suggests that Vfr functions as a biosensor of protease activity that regulates the major protease of Spy, SpeB. It is additionally degraded by neutrophil proteases, thereby relieving repression in the presence of these important immune cells. Originally identified through a transposon mutagenesis screen¹⁸, Vfr has been suspected to regulate SpeB in a RopB-dependent manner¹⁹. Our results showing that Vfr functions as a negative regulator of speB early during growth in culture is consistent with this, but the mechanism in which SIP quorum sensing relieves Vfr-mediated repression at late and stationary growth remains to be elucidated. Since the SpeB protease itself can degrade Vfr, autoregulation may play a role in this temporal regulation. However, it is not clear whether this would be relevant during infection, since the host can provide the proteases to overcome this. Similarly, CovRS mediates repression of SpeB in some in vitro conditions, such as with LL-37 alone. However, since repression is not maintained in the presence of the major LL-37-producing cells, neutrophils, the effect may be more limited during infections, either due to heterogeneity in the population or dominant effects of Vfr. Importantly, other than releasing LL-37, neutrophils undergoing NETosis or degranulating release proteases, including NE³⁴, that degrade Vfr. Together, this highlights the importance of in vivo data for examining pathways such as the regulation of SpeB, since pathways sensing multiple bacterial and host factors can intersect.

In summary, we demonstrate how Spy overcomes challenges in the temporal regulation of the virulence factor SpeB by a circuit that integrates multiple considerations in the host-pathogen interactions. Clear Vfr homologues can be found throughout the Streptococci (S. agalactiae, S. urinalis, S. pseudoporcinus, S. porcinus, S. uberis, S. iniae, S. equi, S. dysgalactiae, S. canis), despite none of these species encoding a SpeB homolog. This suggests that Vfr may be a regulator module more generally, and that through these other species senses broader factors as well. We propose a model wherein Spy, through the Vfr protein, coordinates expression of SpeB within the bacterial population. At high cell densities, quorum sensing is sufficient to induce expression. However, Vfr allows expression at lower cell densities if there is sufficient protease to degrade it. This can be accomplished if there are some SpeB-expressing cells already, thus leading to further coordination between cells independent of classic quorum-sensing, or, an override in the instance of proximate protease-producing immune cells. This further allows heterogeneity within the population, as each cell is exposed to different concentrations of SIP, LL-37, VFR, and protease. Altogether, beyond demonstrating a previously undescribed mechanism for regulating a protease through a natural biosensor for its activity, we show that this integrated regulation poises the pathogen to respond to shifts in innate immune pressure as part of the sophisticated virulence strategy of Spy.

Methods

Bacterial strains and growth conditions

All strains are described in Table 1. Spy strains were routinely grown in Todd Hewitt broth with 5% yeast (THY) at 37°C with 5% CO₂. Bacterial aliquots were washed in PBS and resuspended in PBS with 20% glycerol for storage at −80°C and grown fresh for each experiment. The bacterial mutations ΔcovS, ΔropB, and Δvfr were obtained through lambda red recombineering, as described previously³⁹. Briefly, a kanamycin resistance cassette was PCR amplified with the primer sequences outlined in Table 2, each carrying 5’ homology to the chromosomal sequence flanking the sites of the desired mutation. Spy 5448 carrying recombineering plasmid pAV488 were electroporated with the PCR product and selected for kanamycin resistance. Curing of the recombineering plasmid was achieved by selecting colonies susceptible to chloramphenicol. Gene knockout and lack of spurious secondary-site mutations were validated by whole-genome sequencing (Plasmidsaurus). Reference sequences and plasmids are detailed in Table 3.

Table 1:

Table 2:

Table 3:

Plasmids

Fluorescent reporter plasmids pDCerm-PspeB::GFP, pDCerm-PhasA::RFP, and pDCerm-PspeB::GFP-PhasA::RFP were created for this study by Polymerase Incomplete Primer Extension (PIPE) cloning technique, as previously described⁴⁰. Spy promoters were amplified from 5448, GFP from (GenBank: OM212390.1), and RFP from (GenBank: KM521211.1) for insertion into pDCerm⁴¹ using the primers in Table 2. The sequence was validated using whole plasmid sequencing (Plasmidsaurus) and constructs transformed into each Spy strain by electroporation. Expression vector pETxSUMO-Vfr was created using primers Vfr F and Vfr R to amplify vfr from GAS 5448 and the previously described PIPE primers pETxSUMO F and pETxSUMO R to amplify the expression vector⁴⁰. Reference sequences and plasmids are detailed in Table 3.

Fluorescence during growth

Spy 5448 strains grown in Todd-Hewitt Broth with 5% yeast (THY) to mid-exponential phase were used to inoculate fresh, phenol red-free RPMI supplemented with THY (5%) and erythromycin (2 µg/mL) in a 96-well black, clear-bottom plate (Costar). Cultures were grown for 10 hours at 37 °C and 5% CO₂ with measurements of absorbance (600 nm), GFP (ex. 479 nm, em. 520 nm), and RFP (ex. 579 nm, em. 616 nm) using a BioTek Synergy H1 plate reader. Expression of speB and hasABC were analyzed by fluorescence of GFP or RFP over absorbance. Supplementation with LL-37 300 nM (GeneScript) and MgCl₂ 15 mM (Sigma M9272).

Whole blood and neutrophil experiments

Whole blood was collected from healthy adult donors with informed consent and approval from the Emory University’s Children’s Clinical and Translational Discovery Core. For infections, 10⁸ CFU bacteria suspended in 100 μL of PBS and used to inoculate 400 µl of whole blood. Inoculated blood was incubated on a rotisserie mixer for 4 h. After 4 h, to lyse host cells, the inoculum was treated with Triton X 0.05% (Sigma T8787) for 15 minutes, then samples were stained and fixed for analysis. For neutrophil experiments, neutrophils were isolated from whole human blood by centrifugation in PolymorphPrep (AxisShield) as previously⁴², then diluted in RPMI. To obtain protein content from neutrophils, suspension with neutrophils in RPMI were lysed via sonication (11% amplitude for 4 minutes at 30 second intervals) and centrifuged at 6,000 x g for 5 minutes to remove cellular debris. Neutrophil lysates were used for plate reader analysis or SDS PAGE. Serine protease inhibitor AEBSF 0.6 mM (Sigma 508436) was used for plate reader analysis.

Animal experiments

All animal use and procedures were performed with approval from Emory Institutional Animal Care and Use Committee. Mice were housed in specific pathogen-free conditions with a 14 h light/10 h dark cycle in a standard ambient environment (∼20 °C and ∼50% humidity) in ABSL-2 conditions. Experiments were performed using both male and female wild-type C57Bl/6 and NET-deficient PAD4^-/-(JAX #030315)³⁰ mice of 8-12 weeks of age (Jackson Laboratories). There was no attrition or drop out of subjects. Animal were assigned to experimental groups using simple randomization. In experiments where neutrophils were depleted, 50 µg anti-Ly6G (1A8) (BioXCell) or PBS vehicle control were delivered intraperitoneally 24 h before infection. Depletion was confirmed by flow cytometry using Ly6G (Invitrogen 367-9668-82), CD11b (Invitrogen 63-0112-82), and CXCR1 (RD Systems FAB8628P) antibodies as previously²⁹. Spy 10⁸ CFU were suspended in 100 μL of PBS and injected subcutaneously, as previously³⁸. After 24 h, mice were euthanized, lesions excised and mechanically homogenized, then stained and fixed for analysis.

Flow Cytometry

All samples were pelleted at 12,000 x g and washed with PBS with 1 mM EDTA. For viability, samples were stained using BactoView^TM Dead 760/780 (Biotium cat. 40113) as per manufacturer protocol, then strained through a 100 µm filter (Avantor). Samples were fixed using 4% paraformaldehyde for 30 minutes. To stain for Group A Carbohydrate (GAC), unique to Spy, samples were incubated with goat anti-GAC antibody (Fitzgerald 70-XG70_R) for 1 hour, then rabbit anti-goat APC (Invitrogen A56570) for 1 hour. Samples were analyzed using BD FACSymphony A3 with excitation lasers: FITC, PE-594-A, APC, APC-Cy7. Appropriate single-color controls were used as compensation controls for these experiments. Data was analyzed using FlowJo, RRID:SCR_008520. For analysis, speB expressors were determined based on fluorescent intensity. Non-expressors were determined by background fluorescence from the Spy empty vector negative control, below 10². Low expressors were identified between the ranges of 10² and 10³, whereas high expressors were identified above 10³.

Protein expression and purification

Expression plasmid pETxSUMO-Vfr was introduced to Escherichia coli Rosetta (DE3) and induced at 37°C for 3 hour with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) when O.D. 600 reached 0.4-0.6. Cells were pelleted resuspended in buffer (20 mM Tris-HCl pH 8.0) and disrupted with sonication. Lysates were centrifuged at 20,000 x g 10 minutes at 4°C. Inclusion bodies were washed twice with wash buffer 1 (20 mM Tris-HCl, 2 M urea, 0.3 M NaCl, 2% TritonX-100 pH 8.0) and dissolved in binding buffer (20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol pH 8.0). Sample was mixed at low speed for 40 minutes at room temperature and then filter-sterilized through 0.22 µm filter (Avantor). Filtered sample was loaded to a washed and equilibrated HisTrap HP column (Cytivia) with 0.5 ml NiSO₄. Sample within the column was washed with binding buffer and wash buffer 2 (20 mM Tris-HCl, 0.5 M NaCl, 6 M urea, 1 mM 2-mercaptoethanol pH 8.0). Refolding was performed with FPLC (AKTA start) using a urea linear gradient up to 6 M urea. Gradient volume was 30 mL and flow rate was 1 mL/minute. Sample was eluted using an imidazole linear gradient up to 400 mM imidazole in elution buffer (20 mM Tris-HCl, 0.5 M NaCl, 400 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0). Gradient volume was 10 mL and flow rate was 0.5 mL/minute. Fractions containing eluted protein were pooled and quantified. SpeB was purified as previously described ^38,43.

Vfr Cleavage Assay

For in vitro cleavage experiments, recombinant Vfr (rVfr) (0.3 mg/mL) was incubated with titrations of purified SpeB (0.1 - 25 µg/mL) or recombinant Neutrophil Elastase (1 µg/mL; Sigma 324681) in assay buffer (Tris 25 mM, 300 mM NaCl, 2 mM dithiothreitol) at 37°C for 2 hours. Proteins and their cleavage products were then separated by SDS-PAGE and visualized by AcquaStain (Bulldog Bio). For neutrophil protein cleavage, purified rVfr (0.3 mg/mL) was incubated with neutrophil soluble protein (as described above) from 10⁶ cells/mL in RPMI supplemented with 2 mM dithiothreitol for 12 hours. Protease inhibitors AEBSF, Chymostatin, and PMSF were used as per manufacture protocol (G Biosciences 786-207). Neutrophil Elastase selective protease inhibitor BAY-687 (Cayman 18615) was used in titrating quantities (0.1 - 20 µg/mL).

Protein Modeling and Cleavage Prediction

The structure of Vfr was modeled as a dimer in AlphaFold Server v3 and visualized in PyMOL, RRID:SCR_000305. Sites of potential SpeB cleavage were predicted as previously described based on known targets⁴⁴ using ScanProsite (Expasy) with a search for the motif [IVFYM]-[ADEGKSTN] and the included condition for a net negative charge sidechain charge in the P1’-P5’ region. Sites of NET cleavage are from the motif previously described²⁸.

Protein Fractionation

Neutrophils isolated from whole blood (as above) were pelleted at 2,000 x g for 5 minutes and washed in Tris 20 mM, 1 M NaCl pH 8.0, then disrupted with sonication on ice. Neutrophil lysate was centrifuged at 20,000 x g at 4°C and filter sterilized through 0.22 µm filter (Avantor) then separated by anionic exchange by FPLC (Cytiva). Neutrophil protein was fractioned with a linear NaCl gradient up to 1 M NaCl in 20 mM of Tris. Fractions were assessed by SDS-PAGE and assayed with Spy containing GFP (PspeB::gfp) fluorescent reporter.

Protease activity

Internally-quenched FRET peptides IFFDTWDNE, TWDNEAYVH, EAYVHDAPV, and HDAPVRSLN that detect neutrophil proteases⁴⁵ were pooled (2.5 µM each) in PBS, 1 mM CaCl2, 0.01% Tween-20 (Sigma P7949) and unincubated with each column fraction. After 1 hour incubation at 37°C, the proteolysis was measured using an Nivo plate reader (PerkinElmer) with fluorophore excitation at 323 nm and emission at 398 nm as previously⁴⁵. Measurements of SpeB activity within bacterial supernatants was performed as previously with the fluorescent peptide sub103, internally quenched with an N-terminal Mca and the C-terminal Lys-Dnp (CPC Scientific)⁴³. In triplicate, 10 µM of peptide was incubated in assay buffer (PBS with 2 mM dithiothreitol) with 10 µL of supernatant at 37°C for 30 minutes. Kinetic fluorescence was measured every 30 sec (ex. 323 nm, em. 398 nm) using a Victor Nivo plate reader (PerkinElmer).

Statistics

GraphPad Prism, RRID:SCR_002798, was used to evaluate statistical significance. Unless otherwise stated, one-way ANOVA with Dunnett’s multiple comparisons test was used for the statistical analysis of experiments and P values < 0.05 were considered significant.

Data availability

All supporting data have been supplied with the manuscript.

Supplementary Figures

Fig. S1.

Fig. S2.

Fig. S3.

Fig. S4.

Acknowledgements

We thank Victor Nizet and Andrew Varble for strains, the anonymous blood donors, and members of LaRock lab for helpful discussions. This work was supported in part by the Emory University Integrated Cellular Imaging Core Facility (RRID:SCR_023534), the Emory Flow Cytometry Core (EFCC) Facility (RRID:SCR_023536), and the Children’s Healthcare of Atlanta and Emory University’s Children’s Clinical and Translational Discovery Core. Additional support was provided by the National Center for Georgia Clinical & Translational Science Alliance of the National Institutes of Health under Award Number UL1TR002378. This work was supported by the National Institute of Allergy and Infectious Diseases of the NIH under award numbers AI153071 (C.N.L.) and AI180089 (C.N.L.), training grants AI106699 (S.G.), AI179103 (S.G.), an American Heart Association Predoctoral Fellowship 25PRE1372818 (A.D.), and a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease award (C.N.L.). The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institutes of Health.

Additional information

Author Contributions

Conceptualization, C.N.L; investigation and analysis, S.G., A.D., D.L.L, C.N.L.; writing of the original draft, S.G. and C.N.L.; reviewing and editing, S.G. and C.N.L.; funding, S.G., A.D., C.N.L.; supervision, C.N.L.

Funding

HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (AI153071)

• Christopher LaRock

HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (AI180089)

• Christopher LaRock

HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (AI106699)

• Stephanie Guerra

HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (AI179103)

• Stephanie Guerra

American Heart Association (AHA)

https://doi.org/10.58275/aha.25pre1372818.pc.gr.227140

• Ananya Dash

Burroughs Wellcome Fund (BWF) (Investigator in the Pathogenesis of Infectious Disease)

• Christopher LaRock

Additional files

Supplementary file 1. Fill size gels from primary figures.

Supplementary file 2. Raw data for Figures 1-6.

Supplementary file 3. Raw data for Supplemental Figures.