RSC Advances

RSC Advances PAPER View Article Online View Journal | View Issue Experimental identification and computational characterization of a novel extracellular Cite this: RSC Adv.,2017,7,13928 metalloproteinase produced by Clostridium sordellii† Michael J. Aldape,*ab Aoxiang Tao,c Dustin D. Heeney,a Eric R. McIndoo,a John M. Frencha and Dong Xu*c Clostridium sordellii is a lethal pathogen for both animals and humans. Severe capillary leakage, toxic shock syndrome, and an extreme leukemoid reaction (LR), are hallmark features of C. sordellii infections and contribute to its high mortality rate. Here we report the discovery of a previously unknown and uncharacterized metalloproteinase of C. sordellii (referred as Mcs1) that cleaves human vascular cell adhesion molecule (VCAM)-1 in vitro, an adhesion molecule critical to hematopoietic precursor retention and leukocyte diapedesis. We successfully identified the open reading frame encoding Mcs1 within Creative Commons Attribution-NonCommercial 3.0 Unported Licence. the ATCC 9714 genome and developed an Dmcs1 mutant strain using the ClosTron mutagenesis technology. No VCAM-1 proteolysis was observed from exotoxins collected from mutant strain cultures. Using advanced protein structural modeling and molecular dynamics simulation techniques, the 3D molecular structure and conformational features of Mcs1 were also characterized. Our data demonstrates that Mcs1 proteolytic activity is controlled by the electrostatic interactions between Glu113 and Arg227 residues and the gating motions within its cleft region. This pilot interdisciplinary investigation provided crucial experimental evidence of the existence of Mcs1 in C. sordellii and Received 2nd December 2016 molecular insights into its 3D structure and proteolytic activity. These findings have the potential to help Accepted 22nd February 2017 This article is licensed under a advance new therapeutics and diagnostics against deadly C. sordellii infections. Follow-up in vitro and in DOI: 10.1039/c6ra27654g vivo work is under way to further characterize Mcs1 enzymatic kinetics and its role in C. sordellii rsc.li/rsc-advances pathogenesis. Open Access Article. Published on 02 March 2017. Downloaded 10/1/2021 9:36:05 AM. I. Introduction attributable to the expansion of the granulocytic population with a le shi to myelocytes, metamyeloctyes and band-type Clostridium sordellii is a ubiquitous Gram positive anaerobic neutrophils, serve as the sole predictor of fatal outcome for bacillus found in the soil or intestines of humans and animals C. sordellii infections.1 In addition, LR development also serves (reviewed in ref. 1). In humans, C. sordellii causes rapidly as a hallmark feature of Clostridium difficile and Clostridium progressive and lethal necrotizing so tissue infections,1–3 novyi infections.5,6 including 100% mortality in women undergoing postpartum or C. sordellii infections are manifested by the lethal and gynecological procedures and medically-induced abortions. hemorrhagic toxins (TcsL and TcsH, respectively). TcsL and TcsH C. sordellii infections are characterized by a lack of fever, a weak are members of the large clostridial cytotoxins (LCC) family, innate immune inammatory response and a marked leuke- which also consists of toxin A and toxin B from C. difficile and moid reaction (LR), where absolute circulating white blood cell alpha-toxin by C. novyi. LCCs share high homology and possess counts commonly reach 75 000 per mL whole blood.4 LR, glycosyltransferase activity, utilizing UDP-glucose or N-acetyl- glucosamine as their co-substrate to modify members of the Rho GTPase signaling superfamily. Once modied, Rho GTPases aVeterans Affairs Medical Center, Research and Development, Infectious Diseases become inoperative, causing reorganization and degradation of Section, 500 West Fort St. (Bldg 117), Boise, ID, 83702, USA. E-mail: mike.aldape@ the actin cytoskeleton, likely leading to the disruption of endo- va.gov; Fax: +1-208-422-1425; Tel: +1-208-422-1000 ext. 7659 bNorthwest Nazarene University, Nampa, ID, 83686, USA thelial barrier function and the systemic capillary leakage char- 7–10 cDepartment of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho acteristic of these clostridium infections. State University, Meridian, ID, 83642, USA. E-mail: [email protected]; Fax: +1-208- Two pairs of molecular interactions are critical to retaining 373-1834; Tel: +1-208-373-1832 polymorphonuclear leukocytes (PMNL) and PMNL precursors † Electronic supplementary information (ESI) available. See DOI: in the bone marrow during hematopoiesis: vascular cell 10.1039/c6ra27654g 13928 | RSC Adv.,2017,7,13928–13938 This journal is © The Royal Society of Chemistry 2017 View Article Online Paper RSC Advances adhesion molecule (VCAM)-1 to very late antigen (VLA)-4, and In support of this hypothesis, we report here the identica- the chemokine/chemokine receptor pair of stromal cell derived tion of an uncharacterized metalloproteinase produced by factor (SDF)-1 (aka CXCL12) to its receptor CXCR4. VCAM-1 and C. sordellii, which we refer to as Mcs1 (metalloproteinase of SDF-1 are constitutively expressed by both bone marrow stromal C. sordellii – 1). We show that Mcs1 specically cleaves human cells and the sinusoidal venous endothelium of the hemato- VCAM-1, an adhesion molecule critical to the retention of poietic compartment. Granulocytic precursors express high hematopoietic precursors to the bone marrow environment and levels of CXCR4 and VLA-4, and during cell maturation these leukocyte diapedesis. We successfully identied the Mcs1 open expression levels progressively decrease, thus facilitating an reading frame (ORF) in the C. sordellii ATCC 9714 strain orderly release of mature granulocytes into the peripheral genome. Inactivating this gene utilizing the ClosTron tech- circulation. Several studies have demonstrated that targeted nology resulted in a loss of VCAM-1 cleaving ability. Using disruption of the VCAM-1/VLA-4 or CXCR4/SDF-1 axes results in advanced computational modeling methods, we determined a rapid, unregulated mobilization of hematopoietic precursors the three-dimensional atomic structure of Mcs1 and charac- into the peripheral circulation.11–14 terized the molecular features that are important to Mcs1 For unknown reasons, the C. sordellii LR drastically exceeds proteolytic activity. those observed during C. difficile and C. novyi infection, suggesting that exotoxins unique to C. sordellii contribute to this II. Results profound LR phenomenon. Our group recently demonstrated that the C. sordellii neuraminidase (NanS) both directly induces Mcs1 was originally identied following observations that promyelocytic proliferation and modi es human VCAM-1 in crude, log phase C. sordellii exotoxin preparations grown in 15 D vitro. However, animals infected with nans-mutant C. sor- brain–heart infusion broth possessed proteolytic activity that dellii knockouts still developed LR, suggesting other virulence factors were responsible for augmenting the dramatic C. sor- Creative Commons Attribution-NonCommercial 3.0 Unported Licence. dellii-associated LR (manuscript in progress). This article is licensed under a Open Access Article. Published on 02 March 2017. Downloaded 10/1/2021 9:36:05 AM. Fig. 1 The C. sordellii metalloproteinase Mcs1 cleaves human VCAM- 1. (A) Recombinant human VCAM-1 was incubated with either uninoculated culture media (lane 1), C. sordellii exotoxins (lane 2), or C. sordellii exotoxins pre-treated with protease inhibitor cocktail specific Fig. 2 Confirmation of mcs1 mutant strain in C. sordellii ATCC 9714. for serine, cysteine, aspartic, and aminopeptidase activity (lane 3). (B) Western blot analysis demonstrating a loss of VCAM-1 cleaving activity Recombinant human VCAM-1 was incubated with either uninoculated by the mcs1 mutant strain. Recombinant human VCAM-1 was incu- culture media (lane 1), C. sordellii exotoxins (lane 2), C. sordellii bated with either uninoculated culture media (lane 1), wild-type C. exotoxins pre-treated with EDTA (lane 3), or heat inactivated C. sor- sordellii exotoxins (lane 2), or Dmcs1 mutant C. sordellii exotoxins (lane dellii exotoxins (lane 4). Western blots were developed with mono- 3). Western blots were developed with monoclonal anti-human clonal anti-human VCAM-1 antibody. VCAM-1 antibody. This journal is © The Royal Society of Chemistry 2017 RSC Adv.,2017,7, 13928–13938 | 13929 View Article Online RSC Advances Paper cleaved human vascular cell adhesion molecule (VCAM)-1 in 00259 (ORF_00259; extracellular neutral metalloproteinase) was vitro (Fig. 1A, lane 2). Proteolytic cleavage of VCAM-1 by the the single best potential Mcs1 candidate based on the predicted unidentied protease generated a single, visibly truncated function and physical characteristics of the gene product VCAM-1 species with a molecular mass of 42 kDa (Fig. 1A, lane (molecular weight of 56.9 kDa and isoelectric focusing point 2), corresponding to about 47% of its complete form. Pre- 4.79), and clear biochemical parallels to a partially puried treatment of the toxin preparation with protease inhibitors form of the protease from crude exotoxin preparations specic for serine, cysteine, aspartic, and aminopeptidase (approximate molecular weight: 50.0 kDa, approximate activities did not prevent VCAM-1 cleavage (Fig. 1A, lane 3). isoelectric point: 4.2–5.3). The Mcs1 ORF was also identied in 5 However, pre-treatment of the crude C. sordellii exotoxin prep- out of 5 additional C. sordellii strains using PCR analysis

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