Characterization of Paeniclostridium sordellii Metalloproteinase-1 in vitro and in an experimental model of infection.

OBJECTIVE: Paeniclostridium sordellii is a pathogen that causes rapidly fatal infections characterized by severe edema, extreme leukemoid reaction and lack of an innate immune response. We recently identified a metalloproteinase of P. sordellii-1 (Mcs1) that cleaves human vascular cell adhesion molecule 1, an adhesion molecule important to hematopoietic precursor retention and leukocyte diapedesis. In the current study, we further characterize Mcs1 activity and investigate its role in pathogenesis. METHODS: Mcs1 peptide cleavage sequence and activity conditions were identified using a semi-quantitative fluorescence-based reporter assay. Additional host targets for Mcs1 protease activity were tested and confirmed by gel electrophoreses and western blots. Finally, Mcs1 knock out (ΔMcs1) and complemented (cMcs1) strains were developed for assessment in our animal model of myonecrosis. RESULTS: Data show that Mcs1 prefers aliphatic amino acid residues, I or L, especially when adjacent to negatively charged or noncharged-polar residues. In vitro, Mcs1 cleaved or partially cleaved human cell adhesion molecules, E-selectin and intracellular adhesion molecule-1 (ICAM-1), and mediators of innate immune infection defense, complement protein-3 and antimicrobial peptide LL-37. In vivo, infection with the ΔMcs1 P. sordellii strain had little effect on animal survival, tissue destruction or circulating white blood cell counts compared to wild type and cMcs1 strains. CONCLUSIONS: Similar to proteolytic virulence factors from other pathogens, Mcs1 is a promiscuous protease that cleaves multiple human-host factors. Despite minimal impact of Mcs1 on the murine model of P. sordellii infection, it is worth considering its role in humans and other animal models.

Staphylococcus aureus α-hemolysin promotes platelet-neutrophil aggregate formation.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes severe hemorrhagic necrotizing pneumonia associated with high mortality. Exotoxins have been implicated in the pathogenesis of this infection; however, the cellular mechanisms responsible remain largely undefined. Because platelet-neutrophil aggregates (PNAs) can dysregulate inflammatory responses and contribute to tissue destruction, we investigated whether exotoxins from MRSA could stimulate formation of PNAs in human whole blood. Strong PNA formation was stimulated by toxins from stationary phase but not log phase CA-MRSA, and α-hemolysin was singularly identified as the mediator of this activity. MRSA exotoxins also caused neutrophil (polymorphonuclear leukocyte) activation, as measured by increased CD11b expression, although platelet binding was not driven by this mechanism; rather, α-hemolysin-induced PNA formation was solely platelet P-selectin dependent. These findings suggest a role for S. aureus α-hemolysin-induced PNA formation in alveolar capillary destruction in hemorrhagic/necrotizing pneumonia caused by CA-MRSA and offer novel targets for intervention.

Characterization of Viral miRNAs during Adenovirus 14 Infection and Their Differential Expression in the Emergent Strain Adenovirus 14p1.

Human adenoviruses (HAdV) express either one or two virus-associated RNAs (VA RNAI or VA RNAII). The structure of VA RNA resembles human precursor microRNAs (pre-miRNA), and, like human pre-miRNA, VA RNA can be processed by DICER into small RNAs that resemble human miRNA. VA RNA-derived miRNA (mivaRNA) can mimic human miRNA post-transcriptional gene repression by binding to complementary sequences in the 3' UTR of host mRNA. HAdV14 is a member of the B2 subspecies of species B adenovirus, and the emergent strain HAdV14p1 is associated with severe respiratory illness that can lead to acute respiratory distress syndrome. Utilizing small RNA sequencing, we identified four main mivaRNAs generated from the HAdV14/p1 VA RNA gene, two from each of the 5' and 3' regions of the terminal stem. There were temporal expression changes in the abundance of 5' and 3' mivaRNAs, with 3' mivaRNAs more highly expressed early in infection and 5' mivaRNAs more highly expressed later in infection. In addition, there are differences in expression between the emergent and reference strains, with HAdV14 expressing more mivaRNAs early during infection and HAdV14p1 having higher expression later during infection. HAdV14/p1 mivaRNAs were also shown to repress gene expression in a luciferase gene reporter system. Our results raise the question as to whether differential expression of mivaRNAs during HAdV14p1 infection could play a role in the increased pathogenesis associated with the emergent strain.

Streptococcus pyogenes NAD+-Glycohydrolase Reduces Skeletal Muscle ßNAD+ Levels Independently of Streptolysin O.

Necrotizing soft tissue infections caused by Streptococcus pyogenes (group A streptococcus [GAS]) are characterized by rapid and extensive necrosis of fascia and muscle. Molecular epidemiological studies have demonstrated a positive correlation between GAS isolates that cause invasive infections and the production of S. pyogenes NAD+-glycohydrolase (SPN), an NADase secreted by GAS, but the effect of SPN on muscle cells has not been described. Thus, using standard ßNAD+ and ATP quantification assays, we investigated the effects of SPN on cultured human skeletal muscle cell (SkMC) ßNAD+ and ATP with and without streptolysin O (SLO)-a secreted cholesterol-dependent cytolysin known to act synergistically with SPN. We found that culture supernatants from GAS strains producing SLO and SPN depleted intracellular ßNAD+ and ATP, while exotoxins from a GAS strain producing SLO and an enzymatically-inactive form of SPN had no effect on ßNAD+ or ATP. Addition of purified, enzymatically-active SPN to NADase-negative culture supernatants or sterile media reconstituted ßNAD+ depletion but had no effect ATP levels. Further, SPN-mediated ßNAD+ depletion could be augmented by SLO or the homologous cholesterol-dependent cytolysin, perfringolysin O (PFO). Remarkably, SPN-mediated ßNAD+ depletion was SkMC-specific, as purified SPN had minimal effect on epithelial cell ßNAD+. Taken together, this study identifies a previously unrecognized role for SPN as a major disruptor of skeletal muscle ßNAD+. Such activity could contribute to the rapid and widespread myonecrosis characteristic of severe GAS soft tissue infections.

The US Department of Veterans Affairs Science and Health Initiative to Combat Infectious and Emerging Life-Threatening Diseases (VA SHIELD): A Biorepository Addressing National Health Threats.

Background: The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has demonstrated the need to share data and biospecimens broadly to optimize clinical outcomes for US military Veterans. Methods: In response, the Veterans Health Administration established VA SHIELD (Science and Health Initiative to Combat Infectious and Emerging Life-threatening Diseases), a comprehensive biorepository of specimens and clinical data from affected Veterans to advance research and public health surveillance and to improve diagnostic and therapeutic capabilities. Results: VA SHIELD now comprises 12 sites collecting de-identified biospecimens from US Veterans affected by SARS-CoV-2. In addition, 2 biorepository sites, a data processing center, and a coordinating center have been established under the direction of the Veterans Affairs Office of Research and Development. Phase 1 of VA SHIELD comprises 34 157 samples. Of these, 83.8% had positive tests for SARS-CoV-2, with the remainder serving as contemporaneous controls. The samples include nasopharyngeal swabs (57.9%), plasma (27.9%), and sera (12.5%). The associated clinical and demographic information available permits the evaluation of biological data in the context of patient demographics, clinical experience and management, vaccinations, and comorbidities. Conclusions: VA SHIELD is representative of US national diversity with a significant potential to impact national healthcare. VA SHIELD will support future projects designed to better understand SARS-CoV-2 and other emergent healthcare crises. To the extent possible, VA SHIELD will facilitate the discovery of diagnostics and therapeutics intended to diminish COVID-19 morbidity and mortality and to reduce the impact of new emerging threats to the health of US Veterans and populations worldwide.

Emerging erythromycin and clindamycin resistance in group A streptococci: Efficacy of linezolid and tedizolid in experimental necrotizing infection.

OBJECTIVES: Clindamycin (CLI) and erythromycin (ERY) resistance is increasing among group A streptococci (GAS) causing invasive disease and alternative treatments are urgently required. In this study, the efficacy of the newer oxazolidinone tedizolid (TZD) was compared with the first drug in this class, linezolid (LNZ), in experimental murine myonecrosis caused by ERY-susceptible/CLI-susceptible (ERYS/CLIS) or ERY- resistant/CLI-resistant (ERYR/CLIR) GAS. METHODS: Normal adult outbred Swiss Webster female mice (10 per group) were infected intramuscularly with ERYS/CLIS (ATCC 12384) or ERYR/CLIR (15-003) GAS. Treatments began 4 h post-infection and continued for 72 h. TZD and LNZ (10, 20 and 40 mg/kg) were given intraperitoneally every 12 h. Saline, penicillin (PEN), CLI and ERY were given every 6 h. Survival and infection severity signs and symptoms were followed for 12 days. RESULTS: Both GAS strains were susceptible to LNZ, TZD and PEN; strain 15-003 was confirmed as constitutively resistant to ERY and CLI. Blood levels following a 40 mg/kg dose of LZD and TZD were 30.9 ± 4.0 µg/mL and 21.9 ± 5.3 µg/mL, respectively. Both TZD and LNZ were highly efficacious for the treatment of severe experimental myonecrosis caused by ERYS/CLIS and, importantly, ERYR/CLIR GAS. CONCLUSION: In the current era of emerging macrolide/lincosamide resistance among GAS, these data support the use of TZD and LNZ as first-line antibiotics for the treatment of life-threatening GAS infections in humans.

vfr, a novel locus affecting cysteine protease production in Streptococcus pyogenes.

A gene unique to Streptococcus pyogenes, called vfr, that negatively regulates speB, an important extracellular proteinase, has been identified. Disruption of vfr markedly increased SpeB production in a clinical strain of S. pyogenes and relieved its growth phase dependency. These findings may provide important insights into the pathogenesis of invasive S. pyogenes infections.

Enrichment of Antigen-Specific Class-Switched B Cells from Individuals Naturally Immunized by Infection with Group A Streptococcus.

The low frequency of circulating antigen-specific memory B cells is a considerable obstacle in the discovery and development of human monoclonal antibodies for therapeutic application. Here, we evaluate two solid-phase isolation methods to enrich the number of antigen-specific B cells from individuals naturally immunized against streptolysin O (SLO), a key virulence factor and known immunogen of group A streptococcus (GAS). Class-switched B cells obtained from individuals with a history of GAS infection were separated from peripheral blood mononuclear cells (PBMCs) by immunomagnetic methods. SLO-specific B cells were further enriched directly by binding to SLO monomers and captured by streptavidin-coated magnetic microbeads or indirectly by binding a fluorescently labeled SLO-streptavidin tetramer and captured by anti-fluorophore immunomagnetic microbeads. SLO-bound B cells were quantitated by flow cytometry and/or expanded in batch culture to determine IgG specificity. From individuals who have suffered a GAS infection ≥2 years prior, only the direct method enriched SLO-specific B cells, as determined by flow cytometry. Likewise, in batch culture, B cells isolated by the direct method resulted in an average of 375-fold enrichment in anti-SLO IgG, while no enrichment was observed for B cells isolated by the indirect method. The direct method established here provides a simple approach to increase low-frequency antigen-specific B cell populations supporting many downstream applications, such as immortalization of B cells, cloning of immunoglobulin genes, or purification of antibodies from supernatant for future study. Overall, this process is efficient, is inexpensive, and can be applied to many naturally immunogenic antigens.IMPORTANCE Bacteria called group A streptococci can cause a variety of skin and soft tissue infections ranging from mild pharyngitis ("strep throat") to deadly necrotizing fasciitis (sometimes called "flesh-eating" disease). In each case, the development of disease and the degree of tissue damage are mediated by toxins released from the bacteria during infection. Consequently, novel therapies aimed at clearing bacterial toxins are greatly needed. One promising new treatment is the utilization of monoclonal antibodies delivered as an immunotherapeutic for toxin neutralization. However, current methods of antibody development are laborious and costly. Here, we report a method to enrich and increase the detection of highly desirable antigen-specific memory B cells from individuals previously exposed to GAS using a cost-effective and less-time-intensive strategy. We envision that this method will be incorporated into many applications supporting the development of immunotherapeutics.

In vitro production of panton-valentine leukocidin among strains of methicillin-resistant Staphylococcus aureus causing diverse infections.

BACKGROUND: Community-acquired methicillin-resistant Staphylococcus aureus strains have recently been associated with severe necrotizing infections. Greater than 75% of these strains carry the genes for Panton-Valentine leukocidin (PVL), suggesting that this toxin may mediate these severe infections. However, to date, studies have not provided evidence of toxin production. METHODS: Twenty-nine community-acquired methicillin-resistant Staphylococcus aureus and 2 community-acquired methicillin-susceptible S. aureus strains were collected from patients with infections of varying severity. Strains were analyzed for the presence of lukF-PV and SCCmecA type. PVL production in lukF-PV gene-positive strains was measured by ELISA, and the amount produced was analyzed relative to severity of infection. RESULTS: Only 2 of the 31 strains tested, 1 methicillin-resistant Staphylococcus aureus abscess isolate and 1 nasal carriage methicillin-susceptible S. aureus isolate, were lukF-PV negative. All methicillin-resistant Staphylococcus aureus strains were SCCmec type IV. PVL was produced by all strains harboring lukF-PV, although a marked strain-to-strain variation was observed. Twenty-six (90%) of 29 strains produced 50-350 ng/mL of PVL; the remaining strains produced PVL in excess of 500 ng/mL. The quantity of PVL produced in vitro did not correlate with severity of infection. CONCLUSIONS: Although PVL likely plays an important role in the pathogenesis of these infections, its mere presence is not solely responsible for the increased severity. Factors that up-regulate toxin synthesis in vivo could contribute to more-severe disease and worse outcomes in patients with community-acquired methicillin-resistant Staphylococcus aureus infection.

Experimental identification and computational characterization of a novel extracellular metalloproteinase produced by Clostridium sordellii.

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 the ATCC 9714 genome and developed an Δmcs1 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 molecular insights into its 3D structure and proteolytic activity. These findings have the potential to help advance new therapeutics and diagnostics against deadly C. sordellii infections. Follow-up in vitro and in vivo work is under way to further characterize Mcs1 enzymatic kinetics and its role in C. sordellii pathogenesis.

Cardiac myocyte dysfunction induced by streptolysin O is membrane pore and calcium dependent.

Septic cardiomyopathy is a severe complication among some patients who develop group A streptococcal toxic shock syndrome. Despite the importance of cardiac dysfunction in determining prognosis, very little is known about mechanisms that reduce cardiac output in association with streptococcal infection. Here, we investigated the effects of streptococcal extracellular toxins on mechanical contractility of electrically paced primary murine cardiomyocytes. Our data demonstrate that streptolysin O (SLO) is the major streptococcal toxin responsible for cardiomyocyte contractile dysfunction. Streptolysin O dose-dependently affected cardiac myocyte function in discrete stages. Exposure to SLO caused a failure of cardiac cells to respond to electrical pacing, followed by spontaneous dysregulated contractions and augmented strength of contraction. Central to these SLO-mediated effects is a marked influx of calcium into the cytosol through SLO-mediated pores in the cytoplasmic membrane. Such calcium mobilization in response to SLO correlated temporally with hypercontractility and unpaced contractions. During continued exposure to SLO, cardiomyocytes exhibited periods of reversion to normal electrical pacing suggestive of membrane lesion repair and restoration of calcium handling. Together, these observations are consistent with the clinical observation that septic cardiomyopathy is a reversible condition in patients who survive streptococcal toxic shock syndrome. These data provide strong evidence that streptococcal exotoxins, specifically SLO, can directly impact cardiac mechanical function.

Impact of antibiotics on expression of virulence-associated exotoxin genes in methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

Extracellular protein toxins contribute to the pathogenesis of a wide variety of Staphylococcus aureus infections. The present study investigated the effects that cell-wall active antibiotics and protein-synthesis inhibitors have on transcription and translation of genes for Panton-Valentine leukocidin, alpha-hemolysin, and toxic-shock syndrome toxin 1, in both methicillin-sensitive and methicillin-resistant S. aureus. Subinhibitory concentrations of nafcillin induced and prolonged mRNA for Panton-Valentine leukocidin, alpha-toxin, and toxic-shock syndrome toxin 1 and increased toxin production. In contrast, clindamycin and linezolid markedly suppressed translation, but not transcription, of toxin genes. These results suggest (1) that protein-synthesis inhibition is an important consideration in the selection of antimicrobial agents to treat serious infections caused by toxin-producing gram-positive pathogens and (2) that, by inducing and enhancing toxin production, inadvertent use of beta-lactam antibiotics to treat methicillin-resistant S. aureus infections may contribute to worse outcomes.

Identification and characterization of bicistronic speB and prsA gene expression in the group A Streptococcus.

Severe, invasive group A streptococcal infections have reemerged worldwide, and extracellular toxins, including streptococcal pyrogenic exotoxin B (SpeB), have been implicated in pathogenesis. The genetic regulation of SpeB is not fully understood, and the mechanisms involved in the processing of the protoxin to its enzymatically active form have not been definitively established. The present work demonstrated that the genes encoding SpeB (speB) and a peptidyl-prolyl isomerase (prsA) constitute an operon with transcription initiated from two promoters upstream of speB. Further, the speB-prsA operon was transcribed as a bicistronic mRNA. This finding is in contrast to the generally accepted notion that speB is transcribed only as a monocistronic gene. In addition, prsA has its own promoter, and transcription from this promoter starts in early log phase, prior to the transcription of speB. Genomic disruption of prsA decreased the production of enzymatically active SpeB but not the level of the pro-SpeB zymogen. Taken together, these results demonstrate that prsA is required for production of fully mature, enzymatically active SpeB.