Engineered endolysin of Klebsiella pneumoniae phage is a potent and broad-spectrum bactericidal agent against "ESKAPEE" pathogens.

The rise of antimicrobial resistance in ESKAPEE pathogens poses significant clinical challenges, especially in polymicrobial infections. Bacteriophage-derived endolysins offer promise in combating this crisis, but face practical hurdles. Our study focuses on engineering endolysins from a Klebsiella pneumoniae phage, fusing them with ApoE23 and COG133 peptides. We assessed the resulting chimeric proteins' bactericidal activity against ESKAPEE pathogens in vitro. ApoE23-Kp84B (CHU-1) reduced over 3 log units of CFU for A. baumannii, E. faecalis, K. pneumoniae within 1 h, while COG133-Kp84B (CHU-2) showed significant efficacy against S. aureus. COG133-L1-Kp84B, with a GS linker insertion in CHU-2, exhibited outstanding bactericidal activity against E. cloacae and P. aeruginosa. Scanning electron microscopy revealed alterations in bacterial morphology after treatment with engineered endolysins. Notably, CHU-1 demonstrated promising anti-biofilm and anti-persister cell activity against A. baumannii and E. faecalis but had limited efficacy in a bacteremia mouse model of their coinfection. Our findings advance the field of endolysin engineering, facilitating the customization of these proteins to target specific bacterial pathogens. This approach holds promise for the development of personalized therapies tailored to combat ESKAPEE infections effectively.

Phage therapy combats pan drug-resistant Acinetobacter baumannii infection safely and efficiently.

Phage therapy offers a promising approach to combat the growing threat of antimicrobial resistance. Yet, key questions remain regarding dosage, administration routes, combination therapy, and the causes of therapeutic failure. In this study, we focused on a novel lytic phage, ФAb4B, which specifically targeted the Acinetobacter baumannii strains with KL160 capsular polysaccharide, including the pan-drug resistant A. baumannii YQ4. ФAb4B exhibited the ability to effectively inhibit biofilm formation and eradicate mature biofilms independently of dosage. Additionally, it demonstrated a wide spectrum of antibiotic-phage synergy and did not show any cytotoxic or haemolytic effects. Continuous phage injections, both intraperitoneally and intravenously over 7 d, showed no acute toxicity in vivo. Importantly, phage therapy significantly improved neutrophil counts, outperforming ciprofloxacin. However, excessive phage injections suppressed neutrophil levels. The combinatorial treatment of phage-ciprofloxacin rescued 91% of the mice, a superior outcome compared to phage alone (67%). The efficacy of the combinatorial treatment was independent of phage dosage. Notably, prophylactic administration of the combinatorial regimen provided no protection, but even when combined with a delayed therapeutic regimen, it saved all the mice. Bacterial resistance to the phage was not a contributing factor to treatment failure. Our preclinical study systematically describes the lytic phage's effectiveness in both in vitro and in vivo settings, filling in crucial details about phage treatment against bacteriemia caused by A. baumannii, which will provide a robust foundation for the future of phage therapy.

ApoE Mimetic Peptide COG1410 Kills Mycobacterium smegmatis via Directly Interfering ClpC's ATPase Activity.

Antimicrobial peptides (AMPs) hold promise as alternatives to combat bacterial infections, addressing the urgent global threat of antibiotic resistance. COG1410, a synthetic peptide derived from apolipoprotein E, has exhibited potent antimicrobial properties against various bacterial strains, including Mycobacterium smegmatis. However, our study reveals a previously unknown resistance mechanism developed by M. smegmatis against COG1410 involving ClpC. Upon subjecting M. smegmatis to serial passages in the presence of sub-MIC COG1410, resistance emerged. The comparative genomic analysis identified a point mutation in ClpC (S437P), situated within its middle domain, which led to high resistance to COG1410 without compromising bacterial fitness. Complementation of ClpC in mutant restored bacterial sensitivity. In-depth analyses, including transcriptomic profiling and in vitro assays, uncovered that COG1410 interferes with ClpC at both transcriptional and functional levels. COG1410 not only stimulated the ATPase activity of ClpC but also enhanced the proteolytic activity of Clp protease. SPR analysis confirmed that COG1410 directly binds with ClpC. Surprisingly, the identified S437P mutation did not impact their binding affinity. This study sheds light on a unique resistance mechanism against AMPs in mycobacteria, highlighting the pivotal role of ClpC in this process. Unraveling the interplay between COG1410 and ClpC enriches our understanding of AMP-bacterial interactions, offering potential insights for developing innovative strategies to combat antibiotic resistance.

Resolution of an insidious and migratory Mycobacterium tuberculosis-associated secondary organizing pneumonia: a case report and literature review.

BACKGROUND: Organizing pneumonia (OP) is a rare interstitial lung disease. Secondary organizing pneumonia (SOP) caused by Mycobacterium tuberculosis (MTB) is extremely rare. Migratory MTB-associated SOP is more deceptive and dangerous. When insidious tuberculosis (TB) is not recognized, SOP would be misdiagnosed as cryptogenic organizing pneumonia (COP). Use of steroid hormone alone leads to the progression of TB foci or even death. Clues of distinguishing atypical TB at the background of OP is urgently needed. CASE PRESENTATION: A 56-year-old female patient was hospitalized into the local hospital because of cough and expectoration for more than half a month. Her medical history and family history showed no relation to TB or other lung diseases. Community-acquired pneumonia was diagnosed and anti-infection therapy was initialized but invalid. The patient suffered from continuous weigh loss. More puzzling, the lesions were migratory based on the chest computed tomography (CT) images. The patient was then transferred to our hospital. The immunological indexes of infection in blood and pathogenic tests in sputum and the bronchoalveolar lavage fluid were negative. The percutaneous lung puncture biopsy and pathological observation confirmed OP, but without granulomatous lesions. Additionally, pathogen detection of the punctured lung tissues by metagenomics next generation sequencing test (mNGS) were all negative. COP was highly suspected. Fortunately, the targeted next-generation sequencing (tNGS) detected MTB in the punctured lung tissues and MTB-associated SOP was definitely diagnosed. The combined therapy of anti-TB and prednisone was administrated. After treatment for 10 days, the partial lesions were significantly resorbed and the patient was discharged. In the follow-up of half a year, the patient was healthy. CONCLUSIONS: It is difficult to distinguish SOP from COP in clinical practice. Diagnosis of COP must be very cautious. Transient small nodules and cavities in the early lung image are a clue to consider TB, even though all pathogen tests are negative. tNGS is also a powerful tool to detect pathogen, ensuring prompt diagnosis of TB-related SOP. For clinicians in TB high burden countries, we encourage them to keep TB in mind before making a final diagnosis of COP.

ApoE Mimetic Peptide COG1410 Exhibits Strong Additive Interaction with Antibiotics Against Mycobacterium smegmatis.

Background: Drug-resistant tuberculosis (TB) is an emerging threat to public health worldwide. Antimicrobial peptide (AMP) is a promising solution to solve the antimicrobial resistance crisis. The apolipoprotein E mimetic peptide COG1410 has been confirmed to simultaneously have neuroprotective, anti-inflammatory, and antibacterial activity. However, whether it is effective to inhibit growth of mycobacteria has not been investigated yet. Methods: The peptide COG1410 was synthesized with conventional solid-phase peptide synthesis and qualified by HPLC and mass spectrometry. Micro-dilution method was used to determine the minimal inhibitory concentration. A time-kill assay was used to determine the bactericidal dynamics of antimicrobial peptide and relative antibiotics. Static biofilm formation was conducted in 24-well plate and the biofilm was separated from planktonic cells and collected. The mechanism of action of COG1410 was explored by TEM observation and ATP leak assay. The localization of COG1410 was observed by confocal laser scan microscopy. The drug-drug interaction was determined by a checkerboard assay. Results: COG1410 was a potent bactericidal agent against M. smegmatis in vitro and within the macrophages with MIC 16 µg/mL, but invalid against M. abscess and M. tuberculosis. A time-kill assay showed that COG1410 killed M. smegmatis as potent as clarithromycin, but faster than LL-37, another short synthetic cationic peptide. 1× MIC COG1410 almost reduced 90% biofilm formation of M. smegmatis. Additionally, COG1410 was able to penetrate the cell membrane of macrophage and inhibit intracellular M. smegmatis growth. TEM observation and ATP leak assay found that COG1410 disrupted cell membrane and caused release of cell contents. Confocal fluorescence microscopy showed that FITC-COG1410 aggregated around cell membrane instead of entering the cytoplasm. Although COG1410 had relative high cytotoxicity, it exhibited strong additive interaction with regular anti-TB antibiotics, which reduced the working concentration of COG1410 and expanding safety window. After 30 passages, there was no induced drug resistance for COG1410. Conclusion: COG1410 was a novel and potent AMP against M. smegmatis by disrupting the integrity of cell membrane.

Construction of A375·S2 Melanoma Cell Line with High Sensibility to IL-1 by Overexpressing IL-1 Receptor.

We described an operation that co-overexpress interleukin receptor 1 (IL-1R1) and its co-receptor (IL-1R1AcP) genes in wild-type A375·S2 cells in order to increase their sensibility to IL-1. Firstly, laser scanning confocal microscope observed that IL-1R1 could be expressed on the surface of A375·S2 cells. qPCR was performed to estimate the ratio of two genes and result showed the ratio was almost 4.57:1. Then two genes were linked to vectors and co-transfected into A375·S2 cells. qPCR and Western blotting showed the protein content improved markedly. Finally, MTS assay was executed and the sensitivity of A375·S2 cells that co-transfected receptors to IL-1ß increased significantly. Another MTS assay showed the cell activity variation changed significantly (P < 0.05) and the reliability of the experiment was high, indicating that cell line established in this study could be further used for the activity test of IL-1Ra. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01027-8.

Apolipoprotein E mimetic peptide COG1410 combats pandrug-resistant Acinetobacter baumannii.

The emergence of pandrug-resistant bacteria breaks through the last line of defense and raises fear among people of incurable infections. In the post-antibiotic era, the pharmaceutical field turns to seek non-conventional anti-infective agents. Antimicrobial peptides are considered a prospective solution to the crisis of antimicrobial resistance. In this study, we evaluated the antimicrobial efficiency of an ApoE mimetic peptide, COG1410, which has been confirmed to exhibit strong neural protective activity and immunomodulatory function. COG1410 showed potent antimicrobial activity against pandrug-resistant Acinetobacter baumannii, even eliminating large inocula (108 CFU/ml) within 30 min. LC99.9 in PBS and 50% pooled human plasma was 2 µg/ml (1.4 µM) and 8 µg/ml (5.6 µM), respectively. Moreover, COG1410 exhibited biofilm inhibition and eradication activity, excellent stability in human plasma, and a low propensity to induce resistance. Although COG1410 easily entered bacterial cytoplasm and bound to DNA nonspecifically, the major mechanism of COG1410 killing was to disrupt the integrity of cell membrane and lead to leakage of cytoplasmic contents, without causing obvious pores on the cell surface or cell lysis. Additionally, transcriptome analysis showed that treatment with COG1410-enriched genes involved a series of oxidation-reduction processes. DCFH-DA probe detected an increased ROS level in the presence of COG1410, indicating ROS was another hit of this AMP. Furthermore, the action of COG1410 did not depend on the electronic interaction with the LPS layer, in contrast to polymyxin B. The strong synergistic interaction between COG1410 and polymyxin B dramatically reduced the working concentration of COG1410, expanding the safety window of the application. C. elegans infection model showed that combined therapy of COG1410 and polymyxin B was capable of significantly rescuing the infected nematodes. Taken together, our study demonstrates that COG1410 is a promising drug candidate in the battle against pandrug-resistant A. baumannii.

miR-615 facilitates porcine epidemic diarrhea virus replication by targeting IRAK1 to inhibit type III interferon expression.

Porcine epidemic diarrhea virus (PEDV) in the Coronavirus family is a highly contagious enteric pathogen in the swine industry, which has evolved mechanisms to evade host innate immune responses. The PEDV-mediated inhibition of interferons (IFNs) has been linked to the nuclear factor-kappa B (NF-κB) pathway. MicroRNAs (miRNAs) are involved in virus-host interactions and IFN-I regulation. However, the mechanism by which the PEDV regulates IFN during PEDV infection has not yet been investigated in its natural target cells. We here report a novel mechanism of viral immune escape involving miR-615, which was screened from a high-throughput sequencing library of porcine intestinal epithelial cells (IECs) infected with PEDV. PEDV infection altered the profiles of miRNAs and the activities of several pathways involved in innate immunity. Overexpression of miR-615 increased PEDV replication, inhibited IFN expression, downregulated the NF-κB pathway, and blocked p65 nuclear translocation. In contrast, knockdown of miR-615 enhanced IFN expression, suppressed PEDV replication, and activated the NF-κB pathway. We further determined that IRAK1 is the target gene of miR-615 in IECs. Our findings show that miR-615 suppresses activation of the NF-κB pathway by suppressing the IRAK1 protein and reducing the generation of IFN-IIIs, which in turn facilitates PEDV infection in IECs. Moreover, miR-615 inhibited PEDV replication and NF-κB pathway activation in both IECs and MARC-145 cells. These findings support an important role for miR-615 in the innate immune regulation of PEDV infections and provide a novel perspective for developing new treatments.