Limited proteolysis of neutrophil granule proteins by the bacterial protease RgpB depletes neutrophil antimicrobial capacity.

Neutrophils are highly abundant in the gingival tissues where they play an essential role in immune homeostasis by preventing microbial invasion. Here, we show that the oral periodontal pathogen Porphyromonas gingivalis utilizes its cysteine proteases (gingipains) to disengage phagosomal antimicrobial capacity. Arginine gingipains are a sub-family of trypsin-like proteases produced by P. gingivalis that cleave several host proteins at arginine residues. We find that RgpB-mediated proteolysis of host proteins is not limited to the extracellular or plasma membrane-associated host proteins, but it can also degrade several intracellular proteins in neutrophils. Using 2D-DIGE coupled with mass spectrometry, we identified several cytoskeletal and cytoplasmic proteins, including metabolic enzymes and antimicrobial proteins such as neutrophil elastase, myeloperoxidase, and proteinase 3 within neutrophil granules that were cleaved by RgpB. Strikingly, despite the breakdown of multiple proteins, RgpB-treated neutrophils did not undergo apoptosis but instead increased integrin expression and underwent broad transcriptional changes consistent with proinflammatory programming. However, despite their primed status and augmented inflammatory capacity, RgpB-treated neutrophils were conducive to intracellular bacterial survival due to the reduced activity of granule proteins and oxidative burst. Thus, our data show a previously unknown role for P. gingivalis proteases in the attenuation of neutrophil microbicidal capacity via proteolysis of intracellular proteins.

The TOG protein Stu2 is regulated by acetylation.

Stu2 in S. cerevisiae is a member of the XMAP215/Dis1/CKAP5/ch-TOG family of MAPs and has multiple functions in controlling microtubules, including microtubule polymerization, microtubule depolymerization, linking chromosomes to the kinetochore, and assembly of γ-TuSCs at the SPB. Whereas phosphorylation has been shown to be critical for Stu2 localization at the kinetochore, other regulatory mechanisms that control Stu2 function are still poorly understood. Here, we show that a novel form of Stu2 regulation occurs through the acetylation of three lysine residues at K252, K469, and K870, which are located in three distinct domains of Stu2. Alteration of acetylation through acetyl-mimetic and acetyl-blocking mutations did not impact the essential function of Stu2. Instead, these mutations lead to a decrease in chromosome stability, as well as changes in resistance to the microtubule depolymerization drug, benomyl. In agreement with our in silico modeling, several acetylation-mimetic mutants displayed increased interactions with γ-tubulin. Taken together, these data suggest that Stu2 acetylation can govern multiple Stu2 functions, including chromosome stability and interactions at the SPB.

Response Regulator CD1688 Is a Negative Modulator of Sporulation in Clostridioides difficile.

Two-component signal transduction systems (TCSs), consisting of a sensor histidine kinase (HK) and a response regulator (RR), sense environmental stimuli and then modulate cellular responses, typically through changes in gene expression. Our previous work identified the DNA binding motif of CD1586, an RR implicated in Clostridioides difficile strain R20291 sporulation. To determine the role of this RR in the sporulation pathway in C. difficile, we generated a deletion strain of cd1688 in the historical 630 strain, the homolog of cd1586. The C. difficile Δcd1688 strain exhibited a hypersporulation phenotype, suggesting that CD1688 negatively regulates sporulation. Complementation of the C. difficile Δcd1688 strain restored sporulation. In contrast, a nonphosphorylatable copy of cd1688 did not restore sporulation to wild-type (WT) levels, indicating that CD1688 must be phosphorylated to properly modulate sporulation. Expression of the master regulator spo0A, the sporulation-specific sigma factors sigF, sigE, sigG, and sigK, and a signaling protein encoded by spoIIR was increased in the C. difficile Δcd1688 strain compared to WT. In line with the increased spoIIR expression, we detected an increase in mature SigE at an earlier time point, which arises from SpoIIR-mediated processing of pro-SigE. Taken together, our data suggest that CD1688 is a novel negative modulator of sporulation in C. difficile and contributes to mediating progression through the spore developmental pathway. These results add to our growing understanding of the complex regulatory events involved in C. difficile sporulation, insight that could be exploited for novel therapeutic development. IMPORTANCE Clostridioides difficile causes severe gastrointestinal illness and is a leading cause of nosocomial infections in the United States. This pathogen produces metabolically dormant spores that are the major vehicle of transmission between hosts. The sporulation pathway involves an intricate regulatory network that controls a succession of morphological changes necessary to produce spores. The environmental signals inducing the sporulation pathway are not well understood in C. difficile. This work identified a response regulator, CD1688, that, when deleted, led to a hypersporulation phenotype, indicating that it typically acts to repress sporulation. Improving our understanding of the regulatory mechanisms modulating sporulation in C. difficile could provide novel strategies to eliminate or reduce spore production, thus decreasing transmission and disease relapse.