A protease-sensing circuit links neutrophil inflammation to virulence regulation in Streptococcus pyogenes

Reviewer #1 (Public review):

Summary:

This manuscript examines how Streptococcus pyogenes regulates expression of the virulence factor SpeB in response to both bacterial and host-derived cues. The authors propose that Vfr acts as a repressor of speB expression and that degradation of Vfr by SpeB or by neutrophil-derived proteases relieves this repression. This creates a model in which S. pyogenes can sense proteolytic activity during infection and use that information to tune virulence factor expression.

Strengths:

The main strength of the study is the bacterial regulatory mechanism. The dual reporter system provides a useful way to follow speB and hasABC expression, and the genetic analysis of known regulators helps validate the system. The media-swap experiments, recombinant Vfr experiments, and SpeB-mediated degradation of Vfr support the conclusion that Vfr represses speB and that proteolysis can relieve this repression. The finding that SpeB can degrade Vfr is particularly interesting because it suggests an autoregulatory mechanism that could reinforce SpeB expression once it has been initiated.

Weaknesses:

The host side of the model is less completely supported. The authors show that neutrophil lysates and protease-containing fractions can induce the speB reporter and degrade Vfr, which supports the idea that neutrophil-derived proteases can affect this circuit. However, the in vivo interpretation relies heavily on PAD4-deficient mice to implicate neutrophil extracellular traps. PAD4 deficiency is a useful perturbation, but it does not by itself distinguish loss of extracellular trap formation from changes in neutrophil recruitment, survival, degranulation, phagocytosis, oxidative killing, or other neutrophil death pathways. As a result, the current data support a role for neutrophil-associated proteolytic activity more strongly than they support a specific role for extracellular traps. This distinction is important for interpreting the central model. The bacterial circuit is well developed, but the host-derived cue remains somewhat underdefined. If the relevant signal is extracellular protease activity more broadly, then the model is still interesting, but the conclusion should be framed around neutrophil-derived proteolytic stress rather than extracellular traps specifically. If extracellular traps are the key in vivo source of protease exposure, then additional evidence would be needed to separate that mechanism from other neutrophil effector functions that remain intact in PAD4-deficient cells.

Overall:

This is a valuable study with solid evidence for a bacterial protease-sensing regulatory mechanism controlling SpeB expression. The work should be useful to investigators interested in bacterial virulence regulation, host-pathogen interactions, and how pathogens integrate immune-derived cues during infection. The impact of the study would be stronger if the host-derived signal were defined more precisely, but the bacterial Vfr-SpeB circuit provides a compelling framework for thinking about how S. pyogenes links proteolytic activity to virulence gene expression.

Reviewer #2 (Public review):

Summary:

The study examines how Streptococcus pyogenes integrates bacterial and host-derived signals to regulate SpeB, proposing that Vfr acts as a protease-sensitive repressor whose degradation relieves repression of speB. The authors further suggest that neutrophil-derived serine proteases, including those associated with inflammatory conditions, may promote this transition, and thereby counterbalance LL-37/CovRS-associated suppression of speB. The conceptual framework is interesting and potentially important for understanding how host inflammation feeds into bacterial virulence regulation.

Strengths:

The work addresses a biologically significant question and does so using a broad and generally well-integrated experimental approach, including bacterial genetics, reporter assays, recombinant protein analyses, neutrophil-derived material, human blood infection, and mouse infection models. A particular strength is the effort to connect host inflammatory processes to bacterial regulatory behavior, which gives the study conceptual reach beyond a narrow mechanistic observation. The data support the view that Vfr is relevant to speB control and that neutrophil-associated protease activity may influence this pathway.

Weaknesses:

The main limitations are mechanistic. The physiological form, localization, and abundance of Vfr are not sufficiently defined to support the proposed model at full strength, and the evidence that Vfr functions as a SpeB-labile repressor under biologically relevant conditions remains incomplete. The relationship between Vfr and the broader RopB/SIP regulatory framework is also not yet firmly established. In addition, the reporter system is not yet benchmarked closely enough against endogenous SpeB protein output, and its growth-phase dependence is insufficiently resolved, which makes it difficult in some settings to distinguish promoter activity from mature protease production. The neutrophil protease component is likewise not defined beyond a general serine protease signal, and the potentially important LL-37/CovRS/Vfr connection is underdeveloped in the main text. Overall, the conceptual advance is promising, but several of the central mechanistic claims would benefit from more direct experimental support and more cautious framing.

Reviewer #3 (Public review):

Summary:

SpeB is a cysteine protease secreted during infection by Streptococcus pyogenes (Spy). SpeB has been extensively investigated for its role in pathogenesis, which involves proteolytic processing of both Spy virulence factors and host proteins. Regulation of speB expression is complex and includes growth phase regulation, a quorum-sensing system, the transcription factor RopB, and the global regulatory system CovRS (CsrRS). Guerra et al now attempt to refine the current model of regulation of SpeB expression, focusing on the Spy protein Vfr, which has been suggested previously to act as a negative regulator of SpeB expression. In the current study, neutrophil lysates (representing proteases released during NETosis) are shown to degrade Vfr and to relieve repression of SpeB. At high cell density, SpeB itself also degrades Vfr, which may allow autoregulation of SpeB expression. These observations are unsurprising as the broad protease activities of both neutrophil proteases and SpeB are well known. Nonetheless, the data presented fill in additional details in our understanding of the complex regulation of an important Spy virulence factor.

Strengths:

(1) Construction of a GFP reporter strain provided a facile methodology for tracking speB promoter activity in a variety of experimental setups.

(2) A Vfr deletion mutant was a useful tool to investigate the role of Vfr in SpeB regulation, and mutants in speB and ropB were important controls.

(3) Experiments using neutrophil lysates in vitro, as well as in vivo studies of mice depleted of neutrophils with anti-Ly6G or in PAD4-/- mice (that cannot form NETs) support the hypothesis that neutrophil proteases derepress speB expression by degrading Vfr.

Weaknesses:

(1) The introduction and all the experiments in Figure 1 focus on CovRS, which turns out to be largely tangential to the overall story developed by the rest of the study. On the other hand, the complex and well-studied regulation of speB expression by RopB and the SIP quorum-sensing system is only minimally described. A better framing would be a more detailed introduction to the current model of speB/RopB/SIP/quorum sensing/growth phase regulation. CovRS could be introduced later as its relevance is really just to show that neutrophil lysates or NETs do more than simply providing LL-37, which signals through CsrS, as another regulator of speB expression.

(2) Vfr, as the central focus of the paper, also deserves a more thorough introduction to provide context for the study. For example, reference 19 (Shelburne et al, 2011) showed reduced transcription of speB in a vfr mutant, an effect that could be complemented by expressing vfr or a 39-aa N-terminal fragment in trans. That study presented evidence that the N-terminal peptide binds to RopB, which may prevent RopB from upregulating SpeB expression. Do the authors concur with that model? As it stands, the discussion and model in Figure 1A imply a direct regulatory effect of Vfr on speB expression rather than an indirect one through regulation of RopB. If direct regulation of speB by Vfr is a consideration, it should be investigated more thoroughly, e.g., by promoter-binding assays, CHIP-seq, etc.

(3) Use of single-cell flow cytometry generally confirmed results observed in batch culture. The authors also comment repeatedly on the heterogeneity of individual cell fluorescence representing both speB and has operon expression. However, the reason(s) for heterogeneity in gene expression are not explored, e.g., differences in individual cell growth rate in batch culture, variable loss of reporter plasmid during infection experiments, etc).

(4) Lines 116-118 and Figure 3C: Incubation of recombinant Vfr with Spy Dvfr reduced SpeB expression, but the degree of suppression is modest compared to that seen in wild-type Spy. How does the concentration of rVfr added compare to that present in the culture fluid of wild-type Spy? (Also, the concentration of rVfr used is unclear: the figure says 3 µg/ml and the legend says 0.3 mg/ml, i.e., 300 µg/ml).

(5) Lines 125-126: "...the Vfr structure contains several potential protease SpeB cleavage sites..." The role of Vfr in degrading SpeB could be clarified by identifying the predicted cleavage products, e.g., by mass spec, after co-incubation of the two recombinant proteins.

(6) Lines 122-124: "Notably, speB expression in Spy Dvfr is unaffected by LL-37 or MgCl2, further validating its [Vfr's?] dominance over CovRS regulation." This statement is an oversimplification and is potentially misleading: LL-37 is degraded by SpeB (Nyberg et al, JBC 2004), which likely explains why the addition of LL-37 fails to signal through CovRS to repress SpeB in Spy Dvfr since SpeB is produced continuously in that strain. By contrast, SpeB is only produced during the stationary phase in the wild type, so LL-37 remains active throughout the exponential phase and represses SpeB expression. The response to the CovRS ligand MgCl2 is similar (or greater) in Spy Dvfr compared to wild type (Figure S2C).

(7) Lines 153-154 and Figure 6E: Growing wild type Spy in the presence of neutrophil lysates with or without a protease inhibitor stimulated or repressed speB expression in a manner consistent with degradation (or not) of Vfr. It would be confirmatory and informative to do the same experiment with the Spy Dvfr strain.

(8) Clarity of writing could be improved, particularly by eliminating pronouns of indefinite reference (it, its, this) in contexts in which the subject is ambiguous (examples at lines 62, 89, 111, 114, 115, 123, 183, 190, 193, 204, 205, 210, 217, 221, 222, 224).