Three distinct proteases are responsible for overall cell surface proteolysis in Streptococcus thermophilus .

The lactic acid bacterium Streptococcus thermophilus was believed to display only two distinct proteases at the cell surface, namely the cell-envelope protease PrtS and the house-keeping protease HtrA. Using peptidomics, we demonstrate here the existence of an additional active cell-surface protease, which shares significant homology with the SepM protease of Streptococcus mutans . Although all three proteases—PrtS, HtrA, and SepM—are involved in the turnover of surface proteins, they demonstrate distinct substrate specificities. In particular, SepM cleaves proteins involved in cell wall metabolism and cell elongation, and its inactivation has consequences for cell morphology. When all three proteases are inactivated, the residual cell-surface proteolysis of S. thermophilus is approximately 5% of that of the wild-type strain. Importance Streptococcus thermophilus is a lactic acid bacterium widely used as a starter in the dairy industry. Due to its "generally recognized as safe" status and its weak cell-surface proteolytic activity, it is also considered to be a potential bacterial vector for heterologous protein production. Our identification of a new cell surface protease made it possible to construct a mutant strain with a 95% reduction in surface proteolysis, which could be useful in numerous biotechnological applications.

Hormonal intervention for the treatment of veterans with COVID-19 requiring hospitalization (HITCH): a multicenter, phase 2 randomized controlled trial of best supportive care vs best supportive care plus degarelix: study protocol for a randomized controlled trial

Background Therapeutic targeting of host-cell factors required for SARS-CoV-2 entry is an alternative strategy to ameliorate COVID-19 severity. SARS-CoV-2 entry into lung epithelium requires the TMPRSS2 cell surface protease. Pre-clinical and correlative data in humans suggest that anti-androgenic therapies can reduce the expression of TMPRSS2 on lung epithelium. Accordingly, we hypothesize that therapeutic targeting of androgen receptor signaling via degarelix, a luteinizing hormone-releasing hormone (LHRH) antagonist, will suppress COVID-19 infection and ameliorate symptom severity. Methods This is a randomized phase 2, placebo-controlled, double-blind clinical trial in 198 patients to compare efficacy of degarelix plus best supportive care versus placebo plus best supportive care on improving the clinical outcomes of male Veterans who have been hospitalized due to COVID-19. Enrolled patients must have documented infection with SARS-CoV-2 based on a positive reverse transcriptase polymerase chain reaction result performed on a nasopharyngeal swab and have a severity of illness of level 3–5 (hospitalized but not requiring invasive mechanical ventilation). Patients stratified by age, history of hypertension, and severity are centrally randomized 2:1 (degarelix: placebo). The composite primary endpoint is mortality, ongoing need for hospitalization, or requirement for mechanical ventilation at 15 after randomization. Important secondary endpoints include time to clinical improvement, inpatient mortality, length of hospitalization, duration of mechanical ventilation, time to achieve a normal temperature, and the maximum severity of COVID-19 illness. Exploratory analyses aim to assess the association of cytokines, viral load, and various comorbidities with outcome. In addition, TMPRSS2 expression in target tissue and development of anti-viral antibodies will also be investigated. Discussion In this trial, we repurpose the FDA approved LHRH antagonist degarelix, commonly used for prostate cancer, to suppress TMPRSS2, a host cell surface protease required for SARS-CoV-2 cell entry. The objective is to determine if temporary androgen suppression with a single dose of degarelix improves the clinical outcomes of patients hospitalized due to COVID-19. Trial registration ClinicalTrials.gov NCT04397718. Registered on May 21, 2020

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells

SummarySARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2’ cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.

Hepatocyte growth factor activator inhibitor type 2 (HAI-2) modulates hepcidin expression by inhibiting the cell surface protease matriptase-2

Matriptase-2, a recently identified cell surface protease, is the key enzyme of iron homoeostasis modulating the expression of the liver peptide hormone hepcidin. HAI (hepatocyte growth factor activator inhibitor) types 1 and 2 (HAI-1 and HAI-2 respectively) have been shown to inhibit the close homologue, i.e. matriptase. By co-expressing matriptase-2 and the inhibitor HAI-2 we have identified HAI-2 displaying high inhibitory potential against matriptase-2 at the cell surface as well as in conditioned medium. Accordingly, complex formation between matriptase-2 and HAI-2 was demonstrated by isolation of the complex via immobilizing either HAI-2 or matriptase-2 from lysates and conditioned medium of co-expressing cells. Furthermore, HAI-2 indirectly influences the expression of the hepcidin-encoding gene HAMP. The inhibitor abrogates the matriptase-2-mediated suppression of HAMP expression, presumably by inhibiting the supposed potential of matriptase-2 to cleave membrane-bound HJV (haemojuvelin). Taken together, the results of the present study have characterized HAI-2 as an inhibitor of matriptase-2 that modulates the synthesis of hepcidin and provides new insights into the regulatory mechanism of iron homoeostasis, with clinical importance for a treatment of iron overload diseases.