Patent Application
20230357373
The present invention generally relates to a Staphylococcus pro-apoptotic peptide (herein called “corisin”) that has been found to induce acute exacerbation of pulmonary fibrosis, as well as to methods, kits and apparatus for diagnosing or evaluating fibrosis in patients and to methods and compositions for ameliorating or treating fibrosis, such as idiopathic pulmonary fibrosis.
Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease of as yet undetermined etiology; however, apoptosis of lung alveolar epithelial cells is known to play a role in disease progression. This intractable disease is associated with increased abundance of Staphylococcus and Streptococcus in the lungs, yet their roles in disease pathogenesis have remained elusive.
IPF is the most frequent form of idiopathic interstitial pneumonitis characterized by a chronic, progressive and fatal clinical outcome. See NPL1 and NPL2 (the full citations for all Non-Patent Literature Documents identified herein by the designation “NPL” are provided at the end of the present specification). The prognosis of IPF is worse than in many other types of malignancy, with a life expectancy for patients following diagnosis of the disease being only 2 to 3 years. See NPL3 and NPL4. Repetitive injury and/or apoptosis of lung epithelial cells, excessive release of profibrotic factors and enhanced lung recruitment of extracellular matrix-producing myofibroblasts play critical roles in the disease pathogenesis. See NPL2 and NPLS.
NPL6 suggests that the lung microbiome plays a causative role in IPF, with increased lung bacterial burden being associated with acute exacerbation of the disease and high mortality rate. As shown in NPL7, the relative abundance of lung microbes of the Staphylococcus and Streptococcus genera has also been associated with acceleration of the clinical progression of IPF. However, the role of these bacteria in the pathogenesis of pulmonary fibrosis has remained unclear. The capacity to culture the bacteria associated with fibrotic tissues and elucidation of their phenotypic characteristics would be ideal in clearly identifying the organisms involved in the pathogenesis of IPF; however, it is believed there has been no earlier report of bacterial isolates that are relevant to disease pathogenesis.
In NPL8 and NPL9, it was demonstrated that the lung fibrotic tissue from IPF patients and from transforming growth factor (TGF)β1 transgenic (TG) mice with lung fibrosis is characterized by an enrichment of halophilic bacteria. NPL4 substantiated this observation.
The results in NPL8 and NPL9 led us to hypothesize that the fibrotic tissue is a salty microenvironment, and that the hypersaline condition of the lung fibrotic tissue facilitates the growth of bacteria that release factors that play a role in IPF disease pathogenesis and its acute exacerbation.
In our research that led to the developments and insights described herein, we used a halophilic medium to enrich for Staphylococcus strains from lung fibrotic tissue samples originating from TGFβ1 TG mice. As a result, we found that the culture supernatants of one of the bacterial strains, namely S. nepalensis strain CNDG, contain a pro-apoptotic peptide that induces apoptosis of lung epithelial cells.
We further found that this pro-apoptotic peptide, designated herein as “corisin”, is a component of a transglycosylase conserved in diverse members of the genus Stapylococcus, and that intratrachael instillation of mice having established lung fibrosis either with corisin or the corisin-encoding S. nepalensis strain CNDG leads to acute exacerbation of the disease.
Furthermore, by performing enhanced detection of corisin in human IPF patients with acute exacerbation and comparing these results to patients without disease exacerbation, we concluded that bacteria carrying and shedding the pro-apoptotic peptide are involved in acute exacerbation of pulmonary fibrosis.
More specifically, we have found that Staphylococcus nepalensis releases corisin, a peptide conserved in diverse Staphylococci, to induce apoptosis of lung epithelial cells. The disease in mice exhibits acute exacerbation after intrapulmonary instillation of corisin or after lung infection with corisin-harboring S. nepalensis compared to untreated mice or mice infected with bacteria lacking corisin. Correspondingly, the lung corisin levels are significantly increased in human IPF patients with acute exacerbation compared to patients without disease exacerbation. This resulted in the conclusion that bacteria, which shed corisin, are involved in acute exacerbation of IPF, yielding insights to the molecular basis for the elevation of Staphylococci in pulmonary fibrosis and for the association of the Staphylococci with the worsening stage of pulmonary fibrosis.
Based on these developments and insights, we developed the following aspects of the present teachings.
In one aspect of the present teaching, methods, kits and apparatus are disclosed that comprise detecting the presence of corisin in a biological sample of the patient, preferably detection that is performed in vitro. The corisin may have, e.g., one of the amino acid sequences of SEQ ID NO: 1, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID NO: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID NO: 11, SEQ ID No: 12, or SEQ ID No: 13 disclosed herein. These methods, kits and/or apparatus may be used in the evaluation and/or diagnosis of fibrosis in the patient, such as idiopathic pulmonary fibrosis (IPF), liver cirrhosis, kidney fibrosis, cystic fibrosis, myelofibrosis, and/or mammary fibrosis. Preferably, these methods, kits and/or apparatus is (are) used in the detection and/or evaluation of idiopathic pulmonary fibrosis (IPF).
In such a method, kit or apparatus, the corisin may be detected by mass spectrometry, Western blotting, and/or enzyme-linked immunosorbent assay (ELISA) and may involve binding of the corisin to an antibody, preferably in vitro. For example, the antibody may recognize (bind to), e.g., one of the amino acid sequences of SEQ ID NO: 1, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID NO: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID NO: 11, SEQ ID No: 12, or SEQ ID No: 13 disclosed herein.
In another aspect of the present teachings, an antibody that binds to corisin is disclosed. The antibody may recognize (bind to) one of the amino acid sequences of SEQ ID NO: 1SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID NO: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID NO: 11, SEQ ID No: 12, or SEQ ID No: 13 disclosed herein and it may be a polyclonal antibody.
The antibody may be used as a medicament in preventing, ameliorating and/or treating fibrosis in a patient subject having, or suspected of having or developing, fibrosis. For example, the antibody may be provided in a pharmaceutical composition for use as a medicament to be administered to a patient in need thereof.
Such pharmaceutical compositions optionally may include one or more pharmaceutically acceptable additives, salts and/or excipients, such as preservatives, saccharides, solubilizing agents, stabilizers, carriers, diluents, bulking agents, pH buffering agents, tonicifying agents, antimicrobial agents, wetting agents, and/or emulsifying agents, preferably in an amount (e.g., a combined amount, if two or more are present) of 0.005% to 99% by weight, e.g., 0.5% to 98% by weight.
The antibody may be used in preventing, ameliorating and/or treating idiopathic pulmonary fibrosis (IPF), liver cirrhosis, kidney fibrosis, cystic fibrosis, myelofibrosis, and/or mammary fibrosis. For example, the antibody may be used in preventing, ameliorating and/or treating idiopathic pulmonary fibrosis (IPF). The antibody may be a neutralizing antibody, e.g., an antibody that blocks or inhibits negative effects of corisin in the lungs or other tissue of a patient suffering from fibrosis.
In a further aspect of the present teachings, a method of treating fibrosis in a patient in need thereof may comprise administering a therapeutically effective amount of any of the above-described antibodies the patient. For example, the antibody may be administered to one or both lungs of the patient. In addition or in the alternative, the antibody may be administered intraperitoneally or by intratracheal instillation or by inhalation. Administration of the antibody preferably at least reduces the severity of the fibrosis in the subject.
It is noted that all methods of diagnosis and/or evaluation are preferably performed in vitro on a biological sample that was extracted, collected, obtained, etc. from a patient having, or suspected of having or developing, fibrosis, such as any of the types of fibrosis described above or below.
Other objects, aspects, embodiments and advantages of the present teachings will become apparent to a person skilled in the art upon reading the following detailed description in view of the Figures and appended claims.
In another aspect of the present teachings, a method for evaluating or diagnosing a subject having, or suspected of having or developing, fibrosis, may include receiving an in vitro biological sample that was collected, harvested, obtained, etc. from the subject; and detecting an amount of corisin that is present in the biological sample. Such a method may further comprise comparing the detected amount of corisin in the biological sample to one or more predetermined thresholds. The predetermined thresholds may be set, e.g., based upon levels of corisin that are typically (normally) present in healthy individuals.
The biological sample may be collected from one or both lungs of the subject.
The biological sample may be, e.g., sputum, bronchial secretion, pleural effusion, bronchoalveolar lavage fluid (BALF), and tissue collected from the bronchus or the lung.
The biological sample may be blood or bronchoalveolar lavage fluid (BALF).
In any of these methods, detection of one of the amino acid sequences of SEQ ID NO: 1, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID NO: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID NO: 11, SEQ ID No: 12, or SEQ ID No: 13 preferably serves as detection of the corisin.
In any of these methods, the patient may have, or be suspected of having or developing, idiopathic pulmonary fibrosis (IPF), liver cirrhosis, kidney fibrosis, cystic fibrosis, myelofibrosis, and/or mammary fibrosis. In particular, the present methods are advantageous for use with patients having idiopathic pulmonary fibrosis (IPF).
The corisin may be detected by mass spectrometry, Western blotting, or enzyme-linked immunosorbent assay (ELISA, e.g., by detecting corisin bound to an antibody that, e.g., recognizes one of the amino acid sequences of SEQ ID NO: 1, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID NO: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID NO: 11, SEQ ID No: 12, or SEQ ID No: 13, e.g., by binding a labeled antibody to the corisin that is bound to an antibody, which is, e.g., bound to a substrate). Kits for performing such a method may include such an antibody and one or more reagents for effecting the detection of the corisin in the biological sample.
In another aspect of the present teachings, a pharmaceutical composition for use in treating fibrosis in a patient is disclosed. The pharmaceutical composition preferably comprises a corisin-inhibitor that is capable of neutralizing corisin in a lung of the patient and/or reducing a quantity of corisin in the lung of the patient.
The corisin-inhibitor may be, e.g., a small molecule, an antagonist of corisin or an antibody to corisin. The corisin-inhibitor may act, e.g., by binding to corisin, by degrading corisin or by blocking or inhibiting the production of corisin.
The corisin-inhibitor may be used to treat patients having, or suspected of having or developing, idiopathic pulmonary fibrosis (IPF), liver cirrhosis, kidney fibrosis, cystic fibrosis, myelofibrosis, and/or mammary fibrosis, in particular idiopathic pulmonary fibrosis (IPF).
In another aspect of the present teachings, a method for identifying a corisin receptor protein may comprise searching for a corisin-binding protein present on a surface of an epithelial cell.
In another aspect of the present teachings, a method for identifying a corisin receptor protein may comprise searching for one of the amino acid sequences of SEQ ID NO: 1, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID NO: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID NO: 11, SEQ ID No: 12, or SEQ ID No: 13 in a binding protein present on a surface of an epithelial cell.
The results of the research that led to the present teachings, as well as a discussion thereof and the particular methods used in the present research are now provided in the following.
TGFβ1 (transforming growth factor) is considered to be the most important mediator of IPF. Therefore, in the experiments described below in further detail, we used transgenic (TG) mice with lung fibrosis induced by lung overexpression of human TGFβ1, as previously reported, e.g., in NPL8, NPL10, NPL11 and NPL12. Similar to the IPF disease in humans, these TGFβ1 TG mice spontaneously develop pulmonary fibrosis characterized by a predominant and progressive scarring process, fatal outcome and typical lung histopathological findings (diffuse collagen deposition, honeycomb cysts, fibroblast foci-like areas). See NPL8 and NPL11. As controls, we used a line of TGFβ1 TG mice without fibrosis that express the human transgene but not the protein. See NPL8 and NPL13.
To interrogate the hypothesis that lung fibrotic tissue is a salty microenvironment, we measured the Na+ content of lung fibrotic tissues from TGFβ1 TG mice with lung fibrosis (see NPL8), by allocating TGFβ1 TG and wild-type (WT) mice in groups according to computed tomography-based fibrosis scores (see
More specifically,
As a result of these experiments, we found there was a significantly higher concentration of Na+ in lung tissue from TGFβ1 TG mice with lung fibrosis as compared to TG mice without lung fibrosis and WT mice (see
We separated lung immune cells from each of the WT mice without fibrosis, TGFβ1 TG mice without lung fibrosis and TGFβ1 TG mice with fibrosis and compared the percentage of cells between groups. We found a significant increase in the percentage of monocyte/macrophages and regulatory (CD4+CD25+) T cells in TGFβ1 TG mice with lung fibrosis compared to WT mice and TGFβ1 TG mice without lung fibrosis (See
More specifically,
The lung tissue relative mRNA expression of fibrotic markers (connective tissue growth factor, fibronectin 1, collagen I) and of pro-fibrotic cytokines (TGFβ1, tumor necrosis factor-α, interferon-γ), chemokines (monocyte chemoattractant protein-1), vascular endothelial growth factor or inducible nitric oxide synthase were significantly increased in TGFβ1 TG mice with lung fibrosis compared to WT mice and TGFβ1 TG mice without fibrosis (see Table 2 below).
However, the lung tissue relative mRNA expression of the chloride (cystic fibrosis transmembrane conductance regulator) channels and sodium (Scnnγ, Scnnβ) channels were significantly decreased in TGFβ1 TG mice with lung fibrosis compared to WT mice and TGFβ1 TG mice without lung fibrosis (see Table 2 below). Therefore, we evaluated the correlation between variables in all WT mice and all TGFβ1 TG mice with and without fibrosis.
As a result, we found that the tissue level of sodium was inversely and significantly correlated with the mRNA expression of chloride and sodium channels and with the number of B cells. In contrast, the tissue sodium level was proportionally and significantly correlated with fibrotic markers, pro-fibrotic cytokines and with the number of monocytes/macrophages and regulatory T cells (see
More specifically, the concentration of sodium, the expression of fibrotic factors, pro-fibrotic cytokines, chemokines, angiogenic factors and the percentage of immune cells in lung tissue were assessed in lung tissue from wild-type (n=4) and TGFβ1 TG mice with (n=4) and without (n=4) lung fibrosis. Spearman correlation r values are shown in
These findings provide evidence of the detrimental role of a salty microenvironment in the process of tissue fibrosis and the implication of the tissue sodium level in the regulation of the immune response. See also NPL14.
Growth of Bacteria from Fibrotic Lung Tissue
After confirming that the fibrotic tissue is a salty microenvironment, we posited that a hypersaline culture medium would best mimic the in vivo fibrotic tissue condition, and thus it would favor the growth of microbes implicated in disease pathogenesis.
Therefore, we incubated lung fibrotic tissue specimens from TGFβ1 TG and WT mice (see
Determination of the whole genome sequences, however, revealed that while one of the colonies (strain 8) corresponds to a strain of Staphylococcus nepalensis, another colony (strain 6) was a mixture of Staphylococcus spp. The whole genome sequences of the cultures designated strain 6 and strain 8 have been deposited at the Genbank database with the accession number PRJNA544423.
To further confirm the identity of strain 8, we compared its whole genome sequence with that of other Staphylococcus nepalensis strains in the Genbank database, and for strains JS9, SNUC4337, DSM15150, JS11, and JS1; the identities were 99.52%, 99.61%, 99.60%, 99.53% and 99.50%, respectively. Thus, based on the purity of strain 8 and its very high genomic homology to other Staphylococcus nepalensis strains, the bacterium of strain 8 was named Staphylococcus nepalensis with a strain designation of CNDG.
To assess the potential implication of these fibrotic tissue-derived bacterial isolates in disease pathogenesis, we cultured normal human bronchial epithelial (NHBE) cells and A549 alveolar epithelial cells in the presence of the bacterial culture supernatant and evaluated cell survival. Cells cultured in the presence of supernatants from Staphylococcus nepalensis CNDG and the mixed bacteria showed significant levels of apoptosis, caspase-3 activation and DNA fragmentation compared to cells cultured in control medium (see
Culture Supernatant with the Highest Apoptotic Activity
The culture supernatants from the mixed Staphylococcus spp. (strain 6; see
We cultured Staphylococcus nepalensis CNDG and the mixed Staphylococcus spp. in media containing 0%, 2% or 8% NaCl and used the culture supernatant to assess apoptosis by flow cytometry. We found that the apoptotic activity was significantly dependent on the salt concentration of the medium used to culture both isolates in vitro (see
The culture supernatant from bacteria was incubated at 85° C. for 15 min before assessing its pro-apoptotic activity on A549 alveolar epithelial cells at 1/10 dilution. The apoptotic activity of the culture supernatant from both Staphylococcus nepalensis CNDG and the mixed Staphylococcus spp. remained stable after heating, and the activities were significantly stronger than unheated culture supernatant (see
More specifically,
Furthermore,
These observations provided evidence that the apoptosis-inducing factor is a protein of low molecular weight, and that this soluble factor released by the bacteria enriched from the fibrotic tissue contributes to the mechanism of lung fibrosis by sealing the fate of lung epithelial cells.
We next proceeded to purify the soluble pro-apoptotic factor from the culture supernatant of Staphylococcus nepalensis strain CNDG. Successive extractions of the proteins in the supernatant were performed in n-hexane, water, ethyl acetate, ethanol and then fractionations using octadecyl-silane gel flash column chromatography and Sep-Pak followed by high-performance liquid chromatography (HPLC) (see
More specifically, fractionation of the culture supernatant was performed as described according to the methods below. The pro-apoptotic activity of the fraction on A549 alveolar epithelial cells was evaluated by flow cytometry and it is indicated in
Culture supernatant as well as ethanol, methanol or acetonitrile fractions of the culture supernatant from Staphylococcus nepalensis were then incubated in the presence of 200 μg/ml of proteinase K (PK) at 37° C. before adding to the culture medium of A549 alveolar epithelial cells at 1/10dilution. Each group had n=3.
Five micrograms of the high-performance liquid chromatography fraction (fraction 3) with biological activity was then loaded on a 15% sodium dodecyl sulfate polyacrylamide gel and silver-staining was performed using a commercial kit. Representative microphotographs out of three experiments with similar results are shown in
Subsequently, we analyzed the peptide by mass spectrometry and compared the raw data against a custom database of Staphylococcus nepalensis strain CNDG protein sequences, based on its closed genome sequence data (Genbank Accession number PRJNA544423). Mass spectrometry analysis identified a peptide of 19 amino acid residues (IVMPESSGNPNAVNPAGYR—SEQ ID NO.: 1) that corresponded to a molecular mass of 1.94 kDa, in agreement with the purified biological activity in the culture supernatant. We named this newly discovered peptide “corisin”. Homology searching revealed that the corisin sequence corresponds to a segment of transglycosylase 351 IsaA (MW: 25.6 kDa) of Staphylococcus nepalensis strain CNDG.
Structural alignment using a homology modelling server (swissmodel.expasy.org) showed that corisin shares 46.88% identity with a segment of an endo-type membrane-bound lytic murein transglycosylase A (see
Both synthetic corisin peptides recapitulated the pro-apoptotic effect of the staphylococcal isolate supernatant in a dose dependent manner (see
More specifically,
Normal human bronchial epithelial cells also showed significantly enhanced apoptosis in the presence of corisin, but not in the presence of a synthetic peptide composed of a scrambled amino acid sequence (see
More specifically,
In additional experiments using A549 alveolar epithelial cells, the pro-apoptotic activity of synthetic corisin was found to be heat-resistant (see
More specifically, the synthetic corisin (5 μM; Peptide Institute Incorporation) or scrambled peptide (5 μM; Peptide Institute Incorporation) was incubated at 85° C. for 15 min before adding to the culture medium of A549 alveolar epithelial cells for 48 h.
We then developed polyclonal antibody against corisin using the methods described further below. The polyclonal antibody could detect corisin in mouse lung tissue and in culture supernatant of Staphylococcus nepalensis (see
More specifically, five micrograms of lung tissue homogenate prepared from WT mice and TGFβ1 TG mice (
We then stimulated A549 alveolar epithelial cells with corisin or with culture supernatant from Staphylococcus nepalensis strain CNDG in the presence of saline, control rabbit IgG or rabbit anti-corisin IgG and assessed apoptotic cells by flow cytometry. We found significant inhibition of lung epithelial cell apoptosis induced by synthetic corisin (see
More specifically, A549 alveolar epithelial cells (2×105 cells/well) were cultured in 12-well plates and stimulated with 5 μM corisin in the presence of saline (Saline/corisin), 10 μg/ml control rabbit IgG (Control IgG/corisin) or 10 μg/ml rabbit anti-corisin IgG(Anti-corisin IgG/corisin) for 48 h. Cells cultured in the presence of saline and treated with saline (Saline/saline), control rabbit IgG (Control IgG/saline) or rabbit ant-corisin IgG (Anti-corisin IgG/saline) were used as controls. Each treatment group with n=3 (triplicates). The results are shown in
In addition, A549 alveolar epithelial cells cultured in 12-well plates were stimulated with the 1/10 dilution of the culture supernatant of Staphylococcus nepalensis strain CNDG in the presence of saline (Saline/supernatant of Staphylococcus nepalensis strain CNDG), 10 μg/ml control rabbit IgG (Control IgG/supernatant of Staphylococcus nepalensis strain CNDG) or 10 μg/ml rabbit anti-corisin IgG (Anti-corisin IgG/supernatant of Staphylococcus nepalensis strain CNDG) for 48 h. Cells cultured in medium and treated with saline (Saline/medium), control rabbit IgG (Control IgG/medium) or rabbit ant-corisin IgG (Anti-corisin IgG/medium) were used as controls. Each treatment group had n=3. Flow cytometry of A549 cells was performed after staining with propidium iodide and annexin V. The results are shown in
We prepared 6-Histidine-tagged (His-tagged) or Tag-free (the His-tag was cleaved) recombinant full-length transglycosylase 351, expressed in E. coli cells, to evaluate apoptotic activity on A549 cells. The unheated or heated recombinant His-tagged transglycosylase 351 (see
More specifically,
Corisin Exacerbates Pulmonary Fibrosis in hTGFβ1 TG mice
To investigate whether corisin can exacerbate the lung fibrotic disease in vivo, we separated TGFβ1 TG mice into three groups with matched level of lung fibrosis (see FIGS. 25A and 25B) and treated them with saline, scrambled peptide or corisin by the intratracheal route once daily for two days before euthanasia on day 3 (see
TGFβ1 TG mice receiving corisin exhibited significantly increased infiltration of macrophages, lymphocytes and neutrophils, increased collagen deposition and concentration of inflammatory cytokines and chemokines, and enhanced apoptosis of epithelial cells in the lungs compared to control mice (see
More specifically,
S. nepalensis Instillation Exacerbates Pulmonary Fibrosis
We evaluated in vivo whether bacteria that express transglycosylases containing the corisin sequence also exacerbate lung fibrosis. To this end, we intratracheally administered Staphylococcus nepalensis strain CNDG, which contains the corisin sequence, or Staphylococcus epidermidis [ATCC14990], as negative control, to germ-free TGFβ1 TG mice separated in three groups with matched lung fibrosis CT scores (see
Before this in vivo experiment, we corroborated in vitro that a synthetic peptide (IIARESNGQLHARNASGAA—SEQ. ID NO.:2) corresponding to the peptide sequence at the “corisin position” of the transglycosylase from Staphylococcus epidermidis exerts (exhibits) no pro-apoptotic effect on lung epithelial cells (see
More specifically,
We explored the presence of corisin in WT mice without fibrosis and in TGFβ1 TG mice with and without fibrosis. We found a significantly enhanced level of corisin in TGFβ1 TG mice with lung fibrosis compared to WT mice and TGFβ1 TG mice without fibrosis (see
To clarify the clinical relevance of this finding, we also evaluated corisin in human IPF patients. To this end, we collected bronchoalveolar lavage fluids from 34 IPF patients and 8 male healthy controls. The characteristics of the IPF patients are described in Table 3 below.
The level of corisin in bronchoalveolar lavage fluid (BALF) was significantly increased in IPF patients with stable disease or with acute exacerbation compared to healthy controls (see
A dramatic increase of apoptotic epithelial cells occurs in the lungs of IPF patients with acute exacerbation. See NPL15 and NPL16. The results herein provide evidence that excessive release of the bacterial-derived pro-apoptotic corisin will contribute to this fatal disease complication.
To unveil the evolutionary relationship of transglycosylases expressed by different bacteria, we constructed a phylogenetic tree based on the amino acid sequences of six transglycosylases identified in the genome of Staphylococcus nepalensis strain CNDG and their homologs in a publicly available database (www.ncbi.nlm.nih.gov/pubmed), as will be further described below.
The topology of the phylogenetic tree shows that a derivative of the transglycosylases close to the ancestral sequence splits into the two IsaA clusters (IsaA-1 and IsaA-2) and from IsaA-1 related sequences, the proteins designated SceD members likely evolved (SceD-1, SceD-2, SceD-3, SceD-4) (see
The amino acid sequence identity of corisin homologous transglycosylases from Staphylococcus xylosus, Staphylococcus cohnii and Staphylococcus nepalensis was 100%.
Furthermore, these Staphylococci shared more than 98% identity with the corresponding corisin regions of transglycosylases from other members of the IsaA-1 and IsaA-2 clusters, and 60% identity with the corresponding regions in members of the SceD clusters (see
In particular,
Sequence alignment and comparative genome analysis revealed that a pathogenic strain of Streptococcus, i.e., Streptococcus pneumoniae strain N, implicated in respiratory tract disease, contains a transglycosylase (COE35810) with a peptide sequence almost identical (a single amino acid change) to corisin.
A further examination of the genome of this bacterium unveiled a second homolog (COE67256) of the corisin-containing polypeptide (
To understand how Streptococcus pneumoniae strain N might have acquired the corisin-encoding gene, since its polypeptide sequence is highly conserved only in diverse Staphylococcus spp., we performed a search in the Genbank database and found that the polypeptide (COE35810) yields 98-100% identity with transglycosylases in different strains of Staphylococcus warneri (WP_002467055, WP_050969398, WP_126403073, and WP_107532308) (see
We further examined the genomic context of these genes in Streptococcus pneumoniae strain N in comparison with a Staphylococcus warneri strain, and found a clear conservation of synteny, despite some differences in annotation (see
We therefore hypothesized that the transglycosylase gene and other genes linked to it in Streptococcus pneumoniae strain N were acquired from a Staphylocccus warneri strain or a related species. Significantly, strains of another pathogenic bacterium are known to inhabit the human lung. For example, Mycobacterium [Mycobacteroides] abscessus harbors (contains) a variant of the transglycosylase (SKT99287). Based on a similar analysis as was described above for Streptococcus pneumoniae strain N, we inferred that the transfer was from Staphylococcus hominis or related species (see
More particularly,
From these observations, it is concluded that non-Staphylococcus organisms that have the genes encoding transglycosylases with very high homology to the Staphylococcus nepalensis transglycosylase 351 are lung-associated, thereby providing evidence of a case of horizontal gene transfer from Staphylococcus strains inhabiting the lung.
TGFβ1 (transforming growth factor) is a pleiotropic cytokine having a pivotal role in the pathogenesis of pulmonary fibrosis owing to its potent stimulatory activity on extracellular matrix synthesis, activation, differentiation and migration of myofibroblasts, epithelial-to-mesenchymal transition, and production of pro-fibrotic factors and apoptosis of alveolar epithelial cells. See NPL17 and NPL18. The development of pulmonary fibrosis in TG mice that overexpress TGFβ1 is a proof-of-concept for the critical role of this cytokine in tissue fibrosis. See NPL11. In addition, TGFβ1 may promote exacerbation of pulmonary fibrosis by directly suppressing both the innate and adaptive immune systems leading to enhanced host susceptibility to infection. See NPL19, NPL20 and NPL21.
NPL22, NPL23 and NPL24 have shown that high salt concentration impairs host defense mechanisms by suppressing the activity of antimicrobial peptides or by altering the population of immune cells. Therefore, TGFβ1 may also indirectly affect the host immune response by favoring the accumulation of salt in the extracellular space. See NPL25 and NPL26. Abnormal extracellular storage of salt may result from TGFβ1-mediated negative regulation of the surface expression of epithelial sodium and chloride channels leading to decreased transport of Na+ and Cl− ions from the alveolar airspaces across the epithelium. See also NPL27-NPL29.
Consistent with these findings, as shown in the present disclosure, we found in lung tissue a significant increase of sodium level in TGFβ1 TG mice with lung fibrosis compared to WT mice, a significant positive correlation of sodium level with fibrotic markers and pro-fibrotic cytokines, and a significant negative correlation of sodium level with lymphocyte count and sodium and chloride channels.
A recent single-cell RNA sequencing study showing that expression of several cell membrane sodium and chloride transporters is significantly altered in alveolar epithelial cells from IPF patients, thereby suggesting that ion transmembrane trafficking is disrupted in pulmonary fibrosis and favors the accumulation of salt in this fibrotic disease. See NPL30. Sodium storage appears to require the presence of fibrotic matrix, because we found no difference in the lung sodium level between TGFβ1 TG mice without fibrosis and WT mice. In this connection, previous studies have shown that sodium is stored in extracellular spaces in an osmotically inactive form by binding to negatively charged glycosaminoglycans, which are abundant in the extracellular matrix of fibrotic tissues. See NPL31-NPL35.
Overall, these observations suggest that the fibrotic tissue is a salty microenvironment (see model in
Acute exacerbation is a devastating complication of IPF. See NPL36. Nearly 50% of patients dying from IPF have a prior history of acute exacerbation and the life expectancy of patients with a previous acute exacerbation is only 3 to 4 months. See NPL37-NPL41.
There is currently no optimal therapy for acute exacerbation of IPF. See NPL36. An international working group in 2016 proposed to classify this complication into triggered (identified event: post-procedure, drug toxicity, infection, aspiration) or idiopathic (unidentified inciting event) acute exacerbation. Id. Recent data associating acute exacerbation with the lung microbiome and with the host immunosuppressive states, and retrospective studies showing the preventive effect of antibiotic therapy suggest the role of infection in the pathogenesis of acute exacerbation and progression of pulmonary fibrosis. See NPL7 and NPL42-NPL45. Further, a double-blind, randomized, placebo-controlled study showing improvement of symptoms and exercise capacity in progressive IPF patients treated with co-trimoxazole, and a subsequent double-blind follow-up and multicenter study showing significant reduction of mortality with better quality of life and less respiratory tract infections in IPF patients treated with co-trimoxazole also support the pathogenic role of bacteria in lung fibrosis. See NPL46 and NPL47.
NPL7 showed that bacteria of the Staphylococcus and Streptococcus genera worsen the clinical outcome of IPF patients, suggesting their implication in the disease progression and pathogenesis. Studies showing the relative abundance of Staphylococcus or Streptococcus genera in the fibrotic lung and its significant correlation with the host immune response in IPF patients further support the contribution of these bacteria genera in the pathogenesis of pulmonary fibrosis. See NPL6, NPL 42 and NPL48-NPL52. However, the precise mechanism remains unclear.
In the research that resulted in the present disclosure, we hypothesized that a salty culture medium would mimic the in vivo salty fibrotic tissue and thus would favor the growth of bacteria involved in the pathogenesis of lung fibrosis. We detected growth of bacteria of the genus Staphylococcus in the hypersaline media inoculated with fibrotic tissues from hTGFβ1 TG mice with advanced fibrosis, and the whole genome sequence of a pure bacterial culture revealed that it corresponds to Staphylococcus nepalensis that we categorized as “strain CNDG”. The culture supernatant of this bacterium induced apoptosis of alveolar epithelial cells, and subsequent chromatography, mass spectrometry and gene sequence analysis showed that apoptosis was induced by a peptide that we called “corisin” that corresponds to a segment of transglycosylase 351 from Staphylococcus nepalensis strain CNDG. The higher apoptotic activity of supernatants from bacteria cultured under high-salt conditions may be due to salt-dependent stimulation of bacteria growth or increased bacterial expression of the corisin-containing transglycosylase, which is a related protein that has been reported to be enhanced in expression in Staphylococcus aureus under similar conditions. See NPL53.
In additional experiments, we detected the peptide in the lung from hTGFβ1 TG mice with progressive lung fibrosis and from patients with IPF and found that intratracheal instillation of synthetic corisin or Staphylococcus nepalensis strain CNDG induces acute exacerbation of pulmonary fibrosis in association with extensive apoptosis of alveolar epithelia cells (see the model in
We found that the sequence of corisin has high homology with a region in a membrane-bound lytic transglycosylase. Lytic transglycosylases are bacterial enzymes reported to cleave the peptidoglycan component of the bacterial cell wall (see NPL55) and further perform other essential cellular functions, such as cell-wall synthesis, remodeling, resistance to antibiotics, insertion of secretion systems, flagellar assembly, release of virulence factors, sporulation and germination (Id.). Transglycosylases are ubiquitous in bacteria and an individual species may produce multiple transglycosylases with functional redundancy, to compensate in case of loss or inactivation of any member. See NPL56 and NPL57.
In the results described herein, the complete genome sequence showed that Staphylococcus nepalensis strain CNDG produces six transglycosylases, of which the transglycosylase 351, a member of the IsaA-1 cluster, harbors (contains) the corisin sequence. The full-length transglycosylase 351 did not induce apoptosis of lung epithelial cells, thereby providing evidence that the corisin peptide is active only after being released from the full-length protein. Although the mechanism of this peptide shedding is unknown, the genomic context of the Staphylococcus nepalensis CNDG strain showing the presence of peptidases surrounding the transglycosylase 351 provides evidence that they may be involved in the release of the deadly peptide.
We found that, in addition to Staphylococcus nepalensis strain CNDG, sequences similar to corisin are highly conserved in several transglycosylases from other Staphylococcus species and some members of the microbial community that inhabit the normal or fibrotic lungs, including strains of Streptococcus pneumoniae and Mycobacterium abscessus. See NPL51 and NPL58-60. This observation provides evidence that a broad range of bacteria may be the source of corisin in pulmonary fibrosis.
Although the present disclosure is believed to be a first report on the pathogenicity of a peptide derived from an IsaA homolog in a strain of Staphylococcus, it is noted that homologous proteins (i.e., IsaA and SceD) have been reported in Staphylococcus aureus to be involved in virulence. See NPL53. The Staphylococcus aureus IsaA in NPL53 corresponds to YP_501340 in the alignment shown in
Streptococcus pneumoniae and Staphylococcus species also frequently cause severe pulmonary infections with high in-hospital mortality rate in IPF patients. See NPL20, NPL58 and NPL61. Given the growing evidence that alveolar cell apoptosis plays a central role in the pathogenesis and exacerbation of IPF (see NPL62), it is reasonable to postulate that shedding of deadly peptides constitutes an important contribution to the loss of functional lung alveolar cells and to the poor clinical outcome in patients with complications of microbial infection.
Another mechanism that may further contribute to bacterial virulence and invasiveness is horizontal transfer of bacterial genes. See NPL63. Here we found that strains of Streptococcus pneumoniae, Mycobacterium [Mycobacteroides] abscessus and several Staphylococcus species shared highly similar genome context (synteny) and sequence homology of transglycosylases containing the corisin sequence, thereby providing evidence of the involvement of horizontal gene transfer in the acquisition of this virulence factor. Staphylococcus and Streptococcus genera are common members of the human microbiota. See NPL64. Therefore, if determined that the corisin related peptides identified in the present study have similar apoptotic impact on human cells from other sites or organs, such as the kidney and liver, our view of infections by these bacteria will require re-assessment.
In light of the increasing evidence indicating the participation of the lung microbial population in the pathogenesis of IPF, the identification of corisin as a disease exacerbator substantiates the role of apoptosis in fibrotic diseases, provides a novel diagnostic marker and therapeutic target in IPF, and opens a new avenue for investigating the role of microbiomes in organ fibrosis.
The human lung epithelial cell line A549 and hypersaline media (ATCC media 1097, 2168) were obtained from the American Type Culture Collection (Manassas, VA), Dulbecco's Modified Eagle Medium (DMEM) were obtained from Sigma-Aldrich (Saint Louis, MO) and fetal bovine serum (FBS) were obtained from Bio Whittaker (Walkersville, MD). L-glutamine, penicillin and streptomycin were obtained from Invitrogen (Carlsbad, CA). Normal human bronchial epithelial (NHBE) cells were obtained from Clonetics (Walkersville, MD). Synthetic peptides were prepared and provided by Peptide Institute Incorporation (Osaka, Japan) and by ThermoFisher Scientific (Waltham, MA, USA).
The study described herein comprised 34 Japanese patients with stable idiopathic pulmonary fibrosis (IPF; mean age: 71.7-6.6 years-old, males: 29, females: 5) and eight healthy Japanese male volunteers (38.3±6.1 years old). Table 3 above describes the characteristics of the patients. Diagnosis of idiopathic pulmonary fibrosis was done following accepted international criteria according to NPL65 and NPL66. Bronchoscopy study was performed following guidelines of the American Thoracic Society and bronchoalveolar lavage fluid (BALF) samples were collected from all 34 IPF patients and 8 healthy volunteers. See NPL65. BALF samples during acute exacerbation of the disease were available in 14 out of the 34 participant IPF patients. Aliquots of unprocessed bronchoalveolar lavage fluid (BALF) collected into sterile tubes were stored at −80° C. until analysis.
We used transgenic (TG) mice in a C57BL/6J background with lung-specific overexpression of the latent form of human TGFβ1 that have been previously characterized. See NPL8 and NPL11. These TGFβ1 TG mice spontaneously develop pulmonary fibrosis from 10-weeks of age, and showed similarity to the disease in humans. Id. C57BL/6J wild-type (WT) mice were used as controls. In some of the experiments, TGFβ1 TG mice without lung fibrosis were used as controls; however, the number of mice born with the human TGFβ1 transgene positive but with no phenotype (lung fibrosis) is extremely scarce or rare and thus it was very difficult to include them in all experiments. All mice were maintained in a specific pathogen-free environment under a 12-h light/dark cycle in the facility for experimental animals of Mie University. Genotyping of TG mice were carried out using standard PCR analysis, DNA isolated from the tail of mice and primer pairs (Supplementary Table 5) as described in NPL11.
We performed radiological evaluation of the chest of the mice using a micro-CT (Latheta LCT-200, Hitachi Aloka Medical, Tokyo, Japan). Mice received isoflurane inhalation as anesthesia and were placed in a prone position for data acquisition in accordance with NPL67. Six specialists in respiratory diseases blinded to the treatment groups scored the chest CT findings based on the following criteria: score 1, normal lung findings; 2, intermediate findings; 3, slight lung fibrosis; 4, intermediate findings; 5, moderate lung fibrosis; 6, intermediate findings; and 7, advanced lung fibrosis (
Under profound anesthesia, we collected bronchoalveolar lavage fluid for biochemical analysis and cell counting. Briefly, bronchoalveolar lavage fluid was performed by cannulating the trachea with a 20-gauge needle and infusing saline solutions into the lungs in accordance with NPL68. The samples were centrifuged and the supernatants were stored at −80° C. until analysis. The cell pellets were re-suspended in physiological saline solution and the number of cells was counted. A nucleocounter from ChemoMetec (Allerød, Denmark) was used for cell counting and the cells were stained with May-Grünwald-Giemsa (Merck, Darmstadt, Germany) to count differential cells. Mice were sacrificed by anesthesia overdose, and the lungs were resected to fix in formalin, embedded in paraffin and prepared for hematoxylin and eosin staining. The severity of lung fibrosis was quantitated based on the Ashcroft criteria. See NPL67. The level of TGFβ1 was measured using a commercial enzyme immunoassay kit from BD Biosciences Pharmingen (San Diego, CA).
All subjects participating in the clinical investigation provided written informed consent and the study protocol was approved by the Ethical Committees for Clinical Investigation of Mie University (approval No: H2019064, date: 25 Apr. 2019), Matsusaka Municipal Hospital (approval date: 11 Jun. 2014), and Chuo Medical Center (approval No 2014-6, date: 2 Aug. 2014) and conducted following the Principles of the Declaration of Helsinki. The Recombinant DNA Experiment Safety Committee (approval No: 1-614 (henkol); date: 2013 15 Dec.; approval No: 1-708, date: 13 Feb. 2019) and the Committee for Animal Investigation of Mie University approved the experimental protocols (approval No: 25-20-hen1-sai1, date: 23 Jul. 2015; approval No: 29-23, date: 15 Jan. 2019) and all procedures were performed in accordance with internationally approved principles of laboratory animal care published by the U.S. National Institute of Health.
Under sterile conditions, we excised the left and right lungs after euthanasia of mice by intraperitoneal injection of an overdose of pentobarbital and placed the tissue into sterile tubes and immediately stored them at −80° C. until use.
We removed the lungs from TGFβ1 mice with or without lung fibrosis and from WT mice. The samples were sent to Shimadzu Techno-Research, Incorporation (Kyoto, Japan) for the measurement of tissue sodium content by using microwave analysis/inductively coupled plasma mass spectrometry (ICP-MS), the microwave ashing system ETHOS-TC (Milestone General) and the ICP-MS system 7700x (Agilent Technologies, Santa Clara, CA). See NPL69 and NPL70. The results are shown in
To isolate lung immune cells, after mouse sacrifice by anesthesia overdose, we incised and minced the lung tissue with scissors into 2-3 mm pieces, incubated in 0.5 mg/ml collagenase solution for 30 min at 37° C., and then filtered through a stainless steel mesh. Lung cells were separated and purified using isotonic 33% Percoll (Sigma-Aldrich, St. Louis, MO) solution. We then detected the lung immune cells by flow cytometry using the antibodies described in Table 4 below.
Three groups of TGFβ1 TG mice (each n=5 or n=4) with matched grade (level) of lung fibrosis as assessed by CT score underwent intratracheal instillation of corisin or scrambled peptide or 0.9% NaCl solution on days 1 and 2 and sacrificed on day 3 to evaluate changes in lung inflammation and fibrosis. WT mice (n=3) without lung fibrosis treated with 0.9% NaCl solution were used as controls.
Intratracheal Instillation of Staphylococcus nepalensis
We administered by oral gavage 200 μl of a solution containing a cocktail of antibiotics including vancomycin (0.5 mg/ml), neomycin (1 mg/ml), ampicillin (1 mg/ml), metronidazole (1 mg/ml) and gentamycin (1 mg/ml) once a day for 4 days to three groups of TGFβ1 TG mice. All mice had a matched grade of lung fibrosis as assessed by CT score. On the 5th day, one group of mice received intra-tracheal instillation of 1×108 colony forming units (75 μl) of Staphylococcus nepalensis strain CNDG or Staphylococcus epidermidis ATCC14990 and sacrificed after 2 days. Germ-free TGFβ1 TG mice treated with 0.9% NaCl solution were used as controls.
Lungs from TGFβ1 TG mice with lung fibrosis and from WT mice were used for in vitro microbial culture. The lung tissue specimens were washed with PBS and inoculated into ATCC medium 1097 (8% NaCl) and cultured at 37° C. with shaking at 220 rpm until growth was visible. Bacterial colonies were isolated by plating the liquid medium-cultured organisms on an ATCC medium 1097 agar plates. Each single colony was inoculated into liquid ATCC medium 1097 (8% NaCl) and cultured at 37° C. at 220 rpm for 24 h. The cultures were centrifuged for 5 min at 4,000 rpm at 4° C. to pellet the cells, and the resulting supernatant was filtered through a MILLEXGP filter unit (0.22 um, Millipore) to remove any remaining cells and used as the spent bacterial medium.
We harvested bacterial cells from a single colony in exponential phase growth, immersed in a fixative overnight at 4° C. and collected microphotographs using phase contrast microscopy (Frederick Seitz Materials Research Lab, UIUC) in accordance with NPL71.
Genome sequencing was carried out with a combination of Oxford Nanopore Sequencing and Illumina Miseq nano sequencing that produced 6.3 Gbases and 1.6 million (2×250) nucleotides with perfect Qscores. Briefly, genomic DNA from the bacterial strain (400 ng) was converted into a Nanopore library with the Rapid Barcoding library kit SQK-RAD004. The library was sequenced on a SpotON R9.4.1 FLO-MIN106 flowcell for 48 h on a GridION sequencer. Base-calling was performed with Guppy 1.4.3, and demultiplexing was done with Porechops 0.2.3. The majority of the reads were 6 kb to 30 kb in length, although reads as long as 94 kb were also obtained. The Illumina Miseq sequencing was carried out by preparing shotgun genomic libraries with the Hyper Library construction kit from Kapa Biosystems (Roche). The library was quantitated by qPCR and sequenced on one MiSeq Nano flowcell for 251 cycles from each end of the fragments using a MiSeq 500-cycle sequencing kit version 2. Fastq files were generated and demultiplexed with the bcl2fastq v2.20 Conversion Software (Illumina).
A workflow was developed to perform four assemblies as follows, primarily to assess quality using different assembly strategies to find the best overall assembly. Initial assembly of the Oxford Nanopore data was carried out using Canu (NPL72), followed by polishing using Nanopolish (NPL73) and Pilon (utilizing the Illumina MiSeq reads—NPL74), and finally the genome was re-oriented using Circlator (NPL75). Another hybrid genome assembly was carried out using SPAdes (NPL76), followed by reorienting the genome using Circlator. A hybrid genome assembly was also carried out using Unicycler (NPL77). The final hybrid genome assembly was generated using Unicycler, with the Canu assembly above as the assembly backbone.
All assemblies were quality-assessed using BUSCO (NPL78) and QUAST (NPL79) and compared to a relevant reference genome using MUMmer. See NPL80. Assemblies were then followed by an annotation run using the tool Prokka (NPL81). After evaluation, the best overall assembly was determined using the best overall BUSCO scores in combination with overall assembly metrics.
Bacterial culture supernatants were prepared from cultures grown in Halomonas medium (8% NaCl, 0.75% casamino acids, 0.5% proteose peptone, 0.1% yeast extract, 0.3% sodium citrate, 2% magnesium sulfate heptahydrate, 0.05% potassium phosphate dibasic, 0.05% ammonium iron (II) sulfate hexahydrate) with shaking at 37° C. Bacterial cells were removed by centrifugation (17,000 x g, for 10 min at 4° C.) and filtration through 0.2 μm filters (Corning). Supernatants were size fractionated into high molecular weight (HMW) and low molecular weight (LMW) fractions by ultrafiltration with Ultracel-10K filters (Amicon), separated into aliquots and frozen at −20° C. In some experiments, bacterial culture supernatants were heat-treated (85° C., 15 min) before size fractionation. Equal volumes of supernatants were separated by 17.5% Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver-stained using the Daiichi 2-D Silver Staining Kit (Daiichi, Tokyo, Japan).
The A549 and NHBE cells were cultured in DMEM supplemented with 10% fetal calf serum, 0.03% (w/v) L-glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin in a humidified, 5% CO2 atmosphere at 37° C. We used A549 cell lines in most experiments because they have higher potential growth and mimic the phenotype of alveolar type II cells more than primary NHBE cells (NPL82, NPL83); and in addition, these primary cells usually easily change phenotype or become senescent after a short period of culture.
The bacterial culture supernatant (2 liters) was successively partitioned between n-hexane and water, and then ethyl acetate and water (2 L each, two times) (
Dried samples were suspended in 0.1% formic acid (FA) in 5% acetonitrile (ACN), and 2 μg of peptides were injected into a Thermo UltiMate 3000 UHPLC system. Reversed phase separation of sample peptides was accomplished using a 15 cm Acclaim PepMap 100 C18 column with mobile phases of 0.1% FA in water (A) and 0.1% FA in ACN (B). Peptides were eluted using a gradient of 2% B to 35% B over 60 minutes followed by 35% to 50% B over 5 minutes at a flow rate of 300 μl/min. The UHPLC system was coupled online to a Thermo Orbitrap Q-Exactive HFX (Biopharma Option) mass spectrometer operated in the data dependent mode. Precursor scans from 300 to 1,500 m/z (120,000 resolution) were followed by collision induced dissociation (CID) of the most abundant precursors over a maximum cycle time of 3 s (3e4 AGC, 35% NCE, 1.6 m/z isolation window, 60 s dynamic exclusion window).
The raw data were analyzed using Mascot 1.6 against a custom database containing the protein library of the Staphylococcus nepalensis CNDG genomic DNA, and the large and small plasmids encoded polypeptides (total of 3,541 protein sequences). No enzyme was specified. Peptide mass tolerance and fragment mass tolerances were set to 10 ppm and 0.1 Da, respectively. Variable modifications included oxidation of methionine residues (see mass spectrophotometry data in Supplementary Information).
A549 and NBHE cells (4×105 cells/well) were seeded into 12-well plates, cultured to sub-confluency, washed and then cultured in serum free medium containing 10% of each bacterial supernatant for 48 h. Non-inoculated hypersaline medium was used as control. The cells were analyzed for apoptosis by flow cytometry (FACScan, BD Biosciences, Oxford, UK) after staining with fluorescein-labelled annexin V and propidium iodide (FITC Annexin V Apoptosis Detection Kit with PI, Biolegend, San Diego, CA). Flow cytometry gating strategy used in the experiments is described in
The cells for Western blot analysis were washed twice with ice-cold phosphate-buffered saline and then lysed in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease/phosphatase inhibitors (1 mM orthovandate, 50 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 5 μg/mL leupeptin, 2 μg/mL aprotinin, 5 mM sodium fluoride). The suspensions were centrifuged (17,000 x g, 10 min at 4° C.), and the protein content was determined using Pierce BCA protein assay kit (Thermo Fisher Scientific Incorporation, Waltham, MA). Equal amounts of cellular lysate protein were mixed with Laemmli sample buffer and separated by SDS-PAGE. Western blotting was then performed after electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose membranes and using anti-phospho-Akt, anti-Akt, anti-cleaved caspase-3 or anti-β-actin antibody (Cell Signaling, Danvers, MA). See NPL67. The intensity of the bands was quantified by densitometry using the public domain NIH imageJ program (Wayne Rasband, NIH, Research Service Branch).
Immunohistochemistry
Staining of terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) was performed at the Biopathology Institute Corporation (Kunisaki, Oita, Japan) by using Alexa Fluor 594 goat anti-rabbit IgG and slow-fade gold-antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) or by using ApopTag terminal deoxynucleotidyl transferase (Merck Millipore, Burlington, MA), anti-digoxigenin-peroxidase and 3,3′-diaminobenzidine. Quantification of apoptotic areas was performed using the WinROOF software (Mitani Corporation, Tokyo, Japan) and the values were averaged for each individual mouse.
We extracted total RNA from cells or lung tissue using Sepasol RNA-I Super G reagent (Nacalai Tesque Inc., Kyoto, Japan), synthesized cDNA from 2 μg of total RNA with oligo-dT primer and ReverTra Ace Reverse Transcriptase (Toyobo Life Science Department, Osaka, Japan) and then performed standard PCR using primers described in Table 5 below.
indicates data missing or illegible when filed
PCR was performed with 26 to 35 cycles depending on the gene, denaturation at 94° C. for 30 s, annealing at 65° C. for 30 s, elongation at 72° C. for 1 min followed by a further extension at 72° C. for 5 min. See NPL67. The expression of mRNA was normalized against the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression.
A549 cells (10×104 cells/ml) were plated on a collagen-coated 8-well chamber slides (BD Bioscience, San Jose, CA) and cultured until semi-confluent. Cells were serum-starved for 6 h and stimulated with the pro-apoptotic peptide (5 μM) for 16 h. Cells were fixed with 2% fresh formaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at room temperature. After washing with 0.1 M cacodylate buffer (pH 7.4), they were postfixed with 1% OsO4 in the same buffer for 2 h at 4° C. The samples were rinsed with distilled water, stained with 1% aqueous uranyl acetate for 2 h or overnight at room temperature, dehydrated with ethanol and propylene oxide, and embedded in epon (Epon 812 resin, Nakalai). After removal of the cells from the glass, ultra-thin sections (94 nm) were cut, stained with uranyl acetate and Reynolds's lead citrate, and viewed with a transmission electron microscope (JEM-1010, JEOL, Tokyo, Japan).
We performed DNA content/cell cycle analysis by flow cytometry after culturing the cells for 48 h in the presence or absence of the bacterial supernatant fraction. Cell cycle distribution was evaluated after treating the cells with propidium iodide. Cell viability was performed using a commercial cell counting kit (Dojindo, Tokyo, Japan). The samples used in the assays were fractionated after gel filtration using a Sephadex G25 column.
Expression of S. nepalensis IsaA Transglycosylases
The genes encoding Staphylococcus nepalensis strain CNDG transglycosylase 351 and transglycosylase 531 were synthesized with E. coli optimized codons, amplified to add terminal A and cloned into the TA-cloning vector pGEM-T Easy (Promega, Madison, WI). The genes were then excised and cloned into a modified pET28a vector and transformed into E. coli BL21 DE3 cells and expressed and purified as 6-Histidine tagged (His-tag) proteins. See NPL86.
Protein A purified rabbit polyclonal antibody against the pro-apoptotic peptide (corisin) was developed by Eurofins Genomics (Tokyo, Japan) using the sequence NH2-C+IVMPESSGNPNAVNPAGYR-COOH (SEQ ID NO:1).
A band at the corresponding molecular weight for the target peptide can be observed in Western blotting of mouse lung tissue samples and culture supernatant of Staphylococcus nepalensis strain CNDG (
The purified anti-corisin IgG antibody was used at 1/1000 dilution for Western blotting in lung tissue. We measured the concentration of corisin in body fluids using a competitive enzyme immune assay. Briefly, the purified corisin from transglycosylase 351 was coated on a 96-well plate at a final concentration of 2 μg/ml in phosphate-buffered saline at 4° C. overnight. After blocking and appropriate washing, the standards, samples and 5 ng/ml of anti-corisin were added to the wells and incubated at 4° C. overnight. The wells were then washed before adding horseradish peroxidase-conjugated goat anti-rabbit IgG (R&D System), as the secondary antibody, in a phosphate-buffered saline solution containing 5 μg/mL human IgG. After appropriate washing and incubation, substrate solution was added for color development and absorbance read at 450 nm. Values were extrapolated from a standard curve prepared using several concentrations of the peptide.
The five transglycosylase polypeptides (CNDG_8p_00351, CNDG_8p_00513, CNDG_8p_00157, CNDG_8p_00159, and CNDG_8p_00845) were used to search the Genbank protein database (ncbi.nlm.nih.gov/protein/) to retrieve homologous proteins. The protein sequences were aligned with the MUltiple Sequence Comparison by Log-Expectation (MUSCLE) program and the alignment was used in generating a phylogenetic tree based on the neighbor joining method with bootstrap value of 1,000 replicates. All of these programs are available in Geneious Prime 2016 version (www.geneious.com).
More specifically, the phylogenetic tree shown in
Data are described as the mean ±standard deviation of the means (S.D.) unless otherwise specified. The statistical difference between two variables was assessed by Mann-Whitney U test and the difference between three or more variables by analysis of variance using Tukey's test for post-hoc analysis. P value <0.05 was considered statistically significant. We performed the statistical analysis using GraphPad Prism vs 7 (GraphPad Software, Inc., San Diego, CA).
Additional embodiments of the present disclosure include, but are not limited to:
This application claims priority to U.S. Patent Application No. 62/948,983 filed on Dec. 17, 2019, the contents of which are fully incorporated herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/065280 | 12/16/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62948983 | Dec 2019 | US |
