COMPOSITION FOR DIAGNOSING INFECTIOUS DISEASES COMPRISING AGENT FOR MEASURING EXPRESSION LEVEL OF SREBP2

Abstract

The present invention relates to a composition for diagnosing infectious diseases, comprising an agent for measuring the expression level of sterol regulatory element-binding protein 2 (SREBP2) and, more specifically, to a composition for diagnosing infectious diseases or diagnosing the severity thereof, the composition comprising an agent for measuring the expression level of sterol regulatory element-binding protein 2 (SREBP2) or a C-terminal peptide thereof.

Claims

1. A composition for diagnosing an infectious disease comprising an agent for measuring the expression level of sterol regulatory element-binding protein 2 (SREBP2) or mRNA.

2. The composition of claim 1, wherein the SREBP2 protein consists of an amino acid sequence of SEQ ID NO: 1.

3. The composition of claim 1, wherein the SREBP2 protein is a C-terminal peptide of SREBP2.

4. The composition of claim 3, wherein the C-terminal peptide of SREBP2 consists of an amino acid sequence of SEQ ID NO: 3.

5. The composition of claim 1, wherein the infectious disease is caused by one or more infections selected from the group consisting of viruses, bacteria and fungi.

6. The composition of claim 1, wherein the infectious disease is at least one selected from the group consisting of sepsis, septic shock, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Middle East respiratory syndrome (MERS), salmonellosis, food poisoning, typhoid, paratyphoid, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), pneumonia, pulmonary tuberculosis, tuberculosis, cold, influenza, airway infection, rhinitis, nasopharyngitis, otitis media, bronchitis, lymphadenitis, parotitis, lymphadenitis, cheilitis, stomatitis, arthritis, myositis, dermatitis, vasculitis, gingivitis, periodontitis, keratitis, conjunctivitis, wound infection, peritonitis, hepatitis, osteomyelitis, cellulitis, meningitis, encephalitis, brain abscess, encephalomyelitis, meningitis, osteomyelitis, nephritis, carditis, endocarditis, enteritis, gastritis, esophagitis, duodenitis, colitis, urinary tractitis, cystitis, vaginitis, cervicitis, salpingitis, erythema infectious, dysentery, abscesses and ulcers, bacteremia, diarrhea, dysentery, enterogastritis, gastroenteritis, genitourinary abscess, open wound or injury infection, suppurative inflammation, abscess, boil, pyoderma, impetigo, folliculitis, cellulitis, postoperative wound infection, skin laceration syndrome, skin burn syndrome, thrombotic thrombocytopenia, hemolytic uremic syndrome, renal failure, pyelonephritis, glomerulonephritis, nervous system abscess, otitis media, sinusitis, pharyngitis, tonsillitis, mastoiditis, cellulitis, devotion infection, dacryocystitis, pleurisy, abdominal abscess, liver abscess, cholecystitis, spleen abscess, pericarditis, myocarditis, placenta, amniotic fluid infection, mammitis, mastitis, puerperal fever, toxic shock syndrome, lyme disease, gas gangrene, atherosclerosis, Mycobacterium avium syndrome (MAC), enterohemorrhagic Escherichia coli (EHEC) infection, enteropathogenic Escherichia coli (EPEC) infection, enterohemorrhagic Escherichia coli infection (EIEC), methicillin-resistant Staphylococcus aureus (MRSA) infection, vancomycin-resistant Staphylococcus aureus (VRSA) infection, and listerosis.

7. The composition of claim 1, wherein the composition is a composition for diagnosing the severity of the infectious disease.

8. A kit for diagnosing an infectious disease comprising the agent for measuring the expression level of sterol regulatory element-binding protein 2 (SREBP2) or mRNA according to claim 1.

9. A method for detecting SREBP2 to provide information necessary for the diagnosis of an infectious disease, comprising: (a) measuring the expression level of sterol regulatory element binding protein 2 (SREBP2) or mRNA in a biological sample provided from a patient suspected of having an infectious disease; and (b) comparing the expression level of the SREBP2 protein or mRNA with that of a normal subject and determining an infectious disease when the expression level of the SREBP2 protein or mRNA is increased compared to the normal subject.

10. The method of claim 9, wherein the biological sample is at least one selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, synovial fluid, amniotic fluid, ascites, cervical or vaginal secretions, urine and cerebrospinal fluid.

11. The method of claim 9, wherein in step (b), it is determined that the higher the expression level of SREBP2, the higher the severity of the infectious disease.

12. A composition for diagnosing an infectious disease comprising the agent for measuring the expression level of sterol regulatory element-binding protein 2 (SREBP2) or mRNA according to claim 1.

13. (canceled)

14. (canceled)

15. A method for diagnosing an infectious disease comprising the steps of: (a) measuring the expression level of sterol regulatory element binding protein 2 (SREBP2) or mRNA in a biological sample obtained from a subject suspected of having an infectious disease; (b) measuring the expression level of the SREBP2 protein or mRNA in step (a); (c) comparing the expression level of the SREBP2 protein or mRNA with that of a normal subject; and (d) diagnosing an infectious disease when the expression level of the SREBP2 protein or mRNA is increased compared to the normal subject in step (c).

16. The method of claim 15, wherein the SREBP2 protein consists of an amino acid sequence of SEQ ID NO: 1.

Description

DESCRIPTION OF DRAWINGS

[0095] FIGS. 1A to 1H illustrate results of analyzing the usefulness of SREBP2 as a marker for diagnosing the severity of SARS-CoV-2 infection (COVID-19) in the blood of patients.

[0096] FIGS. 2A to 2E illustrate results of measuring relative mRNA levels of SREBF2 (FIG. 2A), SESN1 (FIG. 2B), PCSK9 (FIG. 2C), HMGCR (FIG. 2D), and LDLR (FIG. 2E).

[0097] FIGS. 3A to 3F are results of confirming that a SREBP2 C-terminal reflects the severity of an infectious disease and may be utilized as a diagnostic marker.

[0098] FIGS. 4A to 4G are results of confirming that the activation of SREBP2 is required for vascular inflammatory responses through cholesterol release and cytokine expression.

MODES FOR THE INVENTION

[0099] Hereinafter, the present invention will be described in detail by the following Examples. However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.

[0100] Experiment Method

[0101] 1. Plasma Sample

[0102] The whole blood was collected from patients admitted to Yeungnam University Hospital who were diagnosed with SARS-CoV-2 infection (COVID-19) at the Public Health Center in Daegu. Patients with COVID-19 sepsis were defined using a criteria provided by the Sepsis Consensus Conference Committee. The whole blood of patients with pneumonia and septic shock was collected from patients admitted to Yeungnam University Hospital. Healthy volunteers were used as a control. Clinical data were collected for all patients. Plasma samples were prepared by centrifugation at 2000×g for 5 minutes within 12 hours after the whole blood collection. A human study protocol was approved by the Daegu Yeungnam University Hospital Institutional Review Board.

[0103] 2. Total Cholesterol, HDL-Cholesterol, and LDL-Cholesterol in Patient's Blood

[0104] Total cholesterol, HDL-cholesterol, and LDL-cholesterol levels in the patient's blood were analyzed using a modular DPE system.

[0105] 3. PBMC Isolation and Incubation

[0106] Samples from healthy volunteers, patients with SARS-CoV-2 pneumonia or discharged patients were obtained from the Yeungnam University Medical Center. Heparin-treated blood samples were used in a fresh state within 4 hours, and peripheral blood mononuclear cells (PBMCs) were isolated from the blood using Ficoll-Hypaquek or NycoPrepk according to the manufacturer's recommendations. Then, the purified PBMCs were obtained using an MACSprep™ PBMC isolation kit, and incubated in RPMI-1640 containing 1 mM of sodium pyruvate, 2 mM of L-glutamine, 4.5 mg/L of glucose, 10 mM of HEPES, and 2 mg/L of sodium bicarbonate.

[0107] 4. SREBP2 Transcriptional Activity Assay

[0108] The transcriptional activity of SREBP2 was determined by an ELISA method using a kit from Abcam (ab133111, Abcam) according to a manufacturer's protocol. Briefly, a nuclear extract corresponding to 30 μg of a protein content was added to each well of a 96-well plate coated with a double-stranded DNA sequence having a consensus SREBP-binding sequence (sterol regulatory element (SRE)). The nuclear extract was hybridized with the coated double-stranded DNA sequences with the consensus SRE on the plate overnight at 4° C. An activated SREBP transcription factor complex was detected at 450 nm after addition of a primary antibody specific for SREBP2 and a secondary antibody conjugated to HRP.

[0109] 5. NF-kB Transcriptional Activity Assay

[0110] As known in the related art, the preparation of the nuclear extract and TransAM analysis were performed. The activity of individual NF-κB subunits was determined using an ELISA-based NF-κB family transcription factor assay kit. Briefly, the nuclear extract (2 μg) was added and incubated in a 96-well plate coated with NF-κB consensus oligonucleotides. The captured complex was incubated with a specific NF-κB primary Ab and then detected using an HRP-conjugated secondary antibody included in the kit. Finally, OD values were measured at 450 nm.

[0111] 6. SREBP2 C-Terminal ELISA

[0112] A competitive ELISA was performed using an antibody recognizing an SREBP2 C-terminal. An SREBP2 C-terminal (639-1031aa) protein was diluted to 2 μg/100 μl, coated on a Nunc-Immuno™ MicroWell™ 96 well plate, and incubated overnight at 4° C. Before using, the plate was washed 3 times with PBST and blocked with 3% BSA at 37° C. for 30 minutes. The primary antibody (1:2000 dilution) and plasma samples (20 μg) were pre-incubated at 37° C. for 1 hour, then the pre-incubated samples were transferred to a peptide-coated plate and incubated at 37° C. for 1 hour. The plate was washed 5 times with PBST. The secondary antibody (1:5000 dilution) was incubated at 37° C. for 30 minutes, and then the plate was washed 5 times with PBST. The washed plate was treated with 100 μl/well of a TMB ELISA substrate at 37° C. for 10 minutes, and then 100 μl/well of a stop solution was added. The detection was performed at 450 nm with a microplate reader.

[0113] Experimental Results

[0114] 1. SREBP2 was Highly Activated in PBMCs of Patients with SARS-CoV-2 Infection, and Subsequently had a Cytotoxic Effect on PBMCs

[0115] In the blood of patients with SARS-CoV-2 infection (COVID-19), the levels of total cholesterol (Ch), high-density lipoprotein (HDL-Ch), and low-density lipoprotein (LDL-Ch) were lower than those of a normal subject, and were lower in intensive care unit (ICU) patients than in non-ICU patients (results not shown). There were no significant associated diseases in each group. According to the analysis, the SREBP2 activity increased as the severity of SARS-CoV-2 infection increased from non-ICU to ICU (FIG. 1A), which was inversely proportional to the tendency in cholesterol levels. The activation level of SREBP2 was increased in dead patients than in living patients (FIG. 1B), and accordingly, SREBP2 may be suggested as an indicator of the severity of SARS-CoV-2 infection.

[0116] In addition, NF-κB, known as a crosstalk molecule of SREBP2, showed a similar increasing tendency as the severity of SARS-CoV-2 infection increased (FIGS. 1C and 1D). As the severity of SARS-CoV-2 infection increased, the production of inflammatory cytokines such as IL-1β and TNF-α by SREBP2 or NF-κB also increased (FIGS. 1E and 1F).

[0117] When PBMCs from patients infected with SARS-CoV-2 were incubated in vitro, the viability of PBMCs isolated from the blood of ICU patients with SARS-CoV-2 infection and the blood of patients with acute respiratory syndrome (ARDS) decreased more rapidly over time than that of a normal subject (FIG. 1G). In addition, the activation level of SREBP2 increased in incubated PBMCs over time (FIG. 1H).

[0118] According to the qRT-PCR result, SREBF2 mRNA was increased in a severity-dependent manner in patients with SARS-CoV-2 infection, and the levels of sestrin 1 (SESN1) and proprotein convertase subtilisin/kexin type 9 (PCSK9), which were known to regulate lipids, also showed a similar increasing trend as the severity of SARS-CoV-2 infection increased (FIG. 2). On the other hand, the mRNA level of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), an enzyme that acts upstream of cholesterol synthesis, and a low-density lipoprotein receptor (LDLR) did not change regardless of the severity of SARS-CoV-2 infection (FIG. 2). These results suggest that SARS-CoV-2 infection increases the activity of SREBP2 as an inflammatory transcription factor, while a direct synthesis pathway of cholesterol by SREBP2 is inhibited.

[0119] 2. Expression Level of SREBP2 C-Terminal Reflects Severity of SARS-CoV-2 Infection.

[0120] The role of the SREBP2 N-terminal has been confirmed in many studies. When the SREBP2 N-terminal and C-terminal are cleaved by S113 and S2P, the N-terminal move to the nucleus to ultimately regulate the synthesis of cholesterol. However, the role of the SREBP2 C-terminal has not yet been reported.

[0121] The present inventors hypothesized that the SREBP2 C-terminal should be released from the blood of SARS-CoV-2 infected patients in response to the activation degree of SREBP2. In particular, the level of the SREBP2 C-terminal increased rapidly in patients with severe SARS-CoV-2 infection, including ICU patients and dead patients (FIGS. 3A and 3B). The SREBP2 C-terminal was also released in severe sepsis (septic shock) (FIG. 3C), which suggested the usability of the SREBP2 C-terminal as a general marker for infectious diseases.

[0122] This usability is supported by increased levels of lactate dehydrogenase (LDH) and C-reactive protein (CRP) in the blood of SARS-CoV-2 infected patients (FIGS. 3D and 3E). Such a high level of the SREBP2 C-terminal was closely associated with hyperinflammation in lung tissues of patients with SARS-CoV-2 infection. Computed tomography (CT) images of ICU patients with high SREBP2 C-terminal levels in the plasma (right panel of FIG. 3G) had more severe lung inflammation than non-ICU patients with low SREBP2 C-terminal levels (left panel of FIG. 3F).

[0123] 3. SREBP2 C-Terminal is an Indicator of an Uncontrolled Vasculature and May be Protected by Pharmacological Inhibition or Knockdown of SREBP2.

[0124] Western blot assay was performed to demonstrate different translocation targets of the N-terminal and C-terminal of SREBP2. Upon exposure to LPS, the expression of the N-terminal of SREBP2 transiently increased with time in a whole cell lysate (WCL) of HUVECs (FIG. 4A). On the other hand, the SREBP2 C-terminal was highly expressed in a supernatant at the end of LPS stimulation (24 hr) (FIG. 4A). Similarly, in other cell types such as HEK293 and HUVEC, the SREBP2 N-terminal was detected in the cell lysate, but was not observed in a culture medium (results not shown). Unlike SREBP2 as a late mediator, the NF-κB activation by LPS was increased at an early age. This is interpreted as mediating severe lung damage by a crosstalk between NF-κB and SREBP2. The SREBP2 C-terminal was detectable in both the WCL and the culture supernatant, but the level was higher in the supernatant (FIG. 4A).

[0125] A notable difference between the N-terminal and the C-terminal of SREBP2 was an increasing ratio with simulation time. This was consistent with time-dependent NF-κB activation upon LPS treatment (FIG. 4B). In contrast to the monotonic increase in SREBP2 N-terminal, the SREBP2 C-terminal increased dramatically at 24 hr after LPS stimulation (FIG. 4C).

[0126] The duration of LPS stimulation induced different results in cholesterol metabolism. Through Filipin staining for visualizing intracellular cholesterol, it was confirmed that cholesterol was accumulated in HUVECs after 12 hours of LPS stimulation (FIG. 4D). However, the cholesterol level was decreased after 24 hr of LPS stimulation (FIG. 4D). As a result of western blot assay of an ATP-binding cassette transporter (ABCA1), also known as a cholesterol efflux regulatory protein (CERP), a more reduced expression was confirmed after 24 hours than after 12 hours (FIG. 4E).

[0127] In HUVECs, LPS stimulation induces upregulated release of inflammatory cytokines (top panel of FIG. 4F). However, genetic knockout of SREBP2 was able to suppress the cytokine storm even after LPS stimulation (lower panel of FIG. 4F). Pharmacological inhibition of NF-κB, SREBP2 and S113 (PF-429242) and shRNA of SREBP2 inhibited vascular bather destruction even under LPS stimulation (FIG. 4G). SREBP2-overexpressing (SREBP2 O/E) HUVECs were more severely damaged by LPS (FIG. 4G).

INDUSTRIAL APPLICABILITY

[0128] According to the present invention, since the expression level of SREBP2 or the C-terminal peptide thereof increases in proportion to the severity of the disease in infectious diseases, these markers can be very useful for diagnosing these infectious diseases and predicting the severity of these infectious diseases and thus, the industrial applicability is very high.