Phage-induced bacterial morphological changes reveal a phage-derived antimicrobial affecting cell wall integrity.

In a looming post-antibiotic era, antibiotic alternatives have become key players in the combat against pathogens. Although recent advances in genomic research allow scientists to fully explore an organism's genome in the search for novel antibacterial molecules, laborious work is still needed in order to dissect each individual gene product for its antibacterial activity. Here, we exploited phage-induced bacterial morphological changes as anchors to explore and discover a potential phage-derived antimicrobial embedded in the phage genome. We found that, upon vibriophage KVP40 infection, Vibrio parahaemolyticus exhibited morphological changes similar to those observed when treated with mecillinam, a cell wall synthesis inhibitor, suggesting the mechanism of pre-killing that KVP40 exerts inside the bacterial cell upon sieging the host. Genome analysis revealed that, of all the annotated gene products in the KVP40 genome that are involved in cell wall degradation, lytic transglycosylase (LT) is of particular interest for subsequent functional studies. A single-cell morphological analysis revealed that heterologous expression of wild-type KVP40-LT induced similar bacterial morphological changes to those treated with the whole phage or mecillinam, prior to cell burst. On the contrary, neither the morphology nor the viability of the bacteria expressing signal-peptide truncated- or catalytic mutant E80A- KVP40-LT was affected, suggesting the necessity of these domains for the antibacterial activities. Altogether, this research paves the way for the future development of the discovery of phage-derived antimicrobials that is guided through phage-induced morphological changes.

High-Resolution Bacterial Cytological Profiling Reveals Intrapopulation Morphological Variations upon Antibiotic Exposure.

Phenotypic heterogeneity is crucial to bacterial survival and could provide insights into the mechanism of action (MOA) of antibiotics, especially those with polypharmacological actions. Although phenotypic changes among individual cells could be detected by existing profiling methods, due to the data complexity, only population average data were commonly used, thereby overlooking the heterogeneity. In this study, we developed a high-resolution bacterial cytological profiling method that can capture morphological variations of bacteria upon antibiotic treatment. With an unprecedented single-cell resolution, this method classifies morphological changes of individual cells into known MOAs with an overall accuracy above 90%. We next showed that combinations of two antibiotics induce altered cell morphologies that are either unique or similar to that of an antibiotic in the combinations. With these combinatorial profiles, this method successfully revealed multiple cytological changes caused by a natural product-derived compound that, by itself, is inactive against Acinetobacter baumannii but synergistically exerts its multiple antibacterial activities in the presence of colistin. The findings have paved the way for future single-cell profiling in bacteria and have highlighted previously underappreciated intrapopulation variations caused by antibiotic perturbation.

Bacterial Cytological Profiling as a Tool To Study Mechanisms of Action of Antibiotics That Are Active against Acinetobacter baumannii.

An increasing number of multidrug-resistant Acinetobacter baumannii (MDR-AB) infections have been reported worldwide, posing a threat to public health. The establishment of methods to elucidate the mechanism of action (MOA) of A. baumannii-specific antibiotics is needed to develop novel antimicrobial therapeutics with activity against MDR-AB We previously developed bacterial cytological profiling (BCP) to understand the MOA of compounds in Escherichia coli and Bacillus subtilis Given how distantly related A. baumannii is to these species, it was unclear to what extent it could be applied. Here, we implemented BCP as an antibiotic MOA discovery platform for A. baumannii We found that the BCP platform can distinguish among six major antibiotic classes and can also subclassify antibiotics that inhibit the same cellular pathway but have different molecular targets. We used BCP to show that the compound NSC145612 inhibits the growth of A. baumannii via targeting RNA transcription. We confirmed this result by isolating and characterizing resistant mutants with mutations in the rpoB gene. Altogether, we conclude that BCP provides a useful tool for MOA studies of antibacterial compounds that are active against A. baumannii.

Melatonin stimulates the nonamyloidogenic processing of ßAPP through the positive transcriptional regulation of ADAM10 and ADAM17.

Melatonin controls many physiological functions including regulation of the circadian rhythm and clearance of free radicals and neuroprotection. Importantly, melatonin levels strongly decrease as we age and patients with Alzheimer's disease (AD) display lower melatonin than age-matched controls. Several studies have reported that melatonin can reduce aggregation and toxicity of amyloid-ß peptides that are produced from the ß-amyloid precursor protein (ßAPP). However, whether melatonin can directly regulate the ßAPP-cleaving proteases ('secretases') has not been investigated so far. In this study, we establish that melatonin stimulates the α-secretase cleavage of ßAPP in cultured neuronal and non-neuronal cells. This effect is fully reversed by ADAM10- and ADAM17-specific inhibitors and requires both plasma membrane-located melatonin receptor activation, and ERK1/2 phosphorylation. Moreover, we demonstrate that melatonin upregulates both ADAM10 and ADAM17 catalytic activities and endogenous protein levels. Importantly, genetic depletion of one or the other protease in mouse embryonic fibroblasts prevents melatonin stimulating constitutive and PKC-regulated sAPPα secretion and ADAM10/ADAM17 catalytic activities. Furthermore, we show that melatonin induces ADAM10 and ADAM17 promoter transactivation, and we identify the targeted promoter regions. Finally, we correlate melatonin-dependent sAPPα production with a protection against staurosporine-induced apoptosis. Altogether, our results provide the first demonstration that melatonin upregulates the nonamyloidogenic ADAM10 and ADAM17 proteases through melatonin receptor activation, ERK phosphorylation and the transactivation of some specific regions of their promoters and further underline the preventive rather than curative nature of melatonin regarding AD treatment.

Anti-CAMP1 IgG promotes macrophage phagocytosis of Cutibacterium acnes type II.

Among 5 types of the Christie-Atkins-Munch-Petersen factor (CAMP) of Cutibacterium acnes, CAMP1 is highly expressed in phylotype II as well as IB, and thought to be a virulence factor of opportunistic but fatal blood, soft tissue, and implant-related infections. The target of a human single-chain variable antibody fragment (scFv), recently isolated from a phage display library, has been identified as CAMP1 of phylotype II, using immunoprecipitation followed by mass spectrometry, phage display peptide biopanning, 3D-modelling, and ELISA. The IgG1 format of the antibody could enhance phagocytosis of C. acnes DMST 14916 by THP-1 human monocytes. Our results suggest that the antibody-dependent phagocytosis process is mediated by the caveolae membrane system and involves the induction of IL-1ß. This is the first report on the study of a human antibody against CAMP1 of C. acnes phylotype II, of which a potential use as therapeutic antibody against virulence C. acnes infection is postulated.

Differential gut microbiota composition in ß-Thalassemia patients and its correlation with iron overload.

Recent research highlights the significant impact of the gut microbiota on health and disease. Thalassemia, a hereditary blood disorder, requires regular blood transfusions, leading to an accumulation of iron in the body. Such changes could potentially alter the intestinal microbiota, thereby increasing the susceptibility of thalassemic patients to infection. In this study, we analyzed the fecal microbiota of 70 non-transfusion-dependent (NTDT) ß-thalassemia/HbE patients and 30 healthy controls. Our findings indicate that iron chelation intervention had no detectable effect on the microbiome profile of thalassemic patients. However, the cross-sectional analysis revealed that the bacterial diversity and community structure in patients were significantly less diverse and distinct compared to those of healthy subjects. Using reference frames, we were also able to demonstrate that bacterial taxa that are known to produce short chain fatty acids, from the genera Alistipes, Coprococcus, and Oscillospira, and those from the family Ruminococcaceae, were less prevalent in the patients. In contrast, bacterial taxa associated with an unhealthy gut, including the genus Clostridium and those from the families Fusobacteriaceae, Enterobacteriaceae, and Peptostrptococcaceae, were more prevalent in patients and found to be correlated with higher levels of ferritin. Collectively, these changes in the microbiota could be regarded as markers of raised ferritin levels, and therefore, awareness should be exercised as they could interfere, albeit indirectly, with the treatment of the co-morbidities of thalassemia.

A novel coli myophage and antibiotics synergistically inhibit the growth of the uropathogenic E. coli strain CFT073 in stoichiometric niches.

Urinary tract infections are widespread bacterial infections affecting millions of people annually, with Escherichia coli being the most prevalent. Although phage therapy has recently gained interest as a promising alternative therapy for antibiotic-resistant bacteria, several studies have raised concerns regarding the evolution of phage resistance, making the therapy ineffective. In this study, we discover a novel coli myophage designated as Killian that targets E. coli strains, including the uropathogenic E. coli (UPEC) strain CFT073. It requires at least 20 minutes for 90% of its particles to adsorb to the host cells, undergoes subcellular activities for replication for 30 minutes, and eventually lyses the cells with a burst size of about 139 particles per cell. Additionally, Killian can withstand a wide variety of temperatures (4-50°C) and pHs (4-10). Genome analysis reveals that Killian's genome consists of 169,905 base pairs with 35.5% GC content, encoding 276 open reading frames; of these, 209 are functionally annotated with no undesirable genes detected, highlighting its potential as an antibiotic alternative against UPEC. However, after an 8-hour phage treatment at high multiplicities of infection, bacterial density continuously increases, indicating an onset of bacterial growth revival. Thus, the combination study between the phage and three different antibiotics, including amikacin, ciprofloxacin, and piperacillin, was performed and showed that certain pairs of phage and antibiotics exhibited synergistic interactions in suppressing the bacterial growth revival. These findings suggest that Killian-antibiotic combinations are effective in inhibiting the growth of UPEC. IMPORTANCE Phage therapy has recently been in the spotlight as a viable alternative therapy for bacterial infections. However, several studies have raised concerns about the emergence of phage resistance that occurs during treatment, making the therapy not much effective. Here, we present the discovery of a novel E. coli myophage that, by itself, can effectively kill the uropathogenic E. coli, but the emergence of bacterial growth revival was detected during the treatment. Phage and antibiotics are then combined to improve the efficiency of the phage in suppressing the bacterial re-growth. This research would pave the way for the future development of phage-antibiotic cocktails for the sustainable use of phages for therapeutic purposes.

Roles of qseC mutation in bacterial resistance against anti-lipopolysaccharide factor isoform 3 (ALFPm3).

Propelled by global climate changes, the shrimp industry has been facing tremendous losses in production due to various disease outbreaks, particularly early mortality syndrome (EMS), a disease caused by Vibrio parahaemolyticus AHPND. Not only is the use of antibiotics as EMS control agents not yet been proven successful, but the overuse and misuse of antibiotics could also worsen one of the most challenging global health issues-antimicrobial resistance. To circumvent antibiotic usage, anti-lipopolysaccharide factor isoform 3 (ALFPm3), an antimicrobial peptide (AMP) derived from the shrimp innate immune system, was proposed as an antibiotic alternative for EMS control. However, prolonged use of AMPs could also lead to bacterial cross resistance with life-saving antibiotics used in human diseases. Here, we showed that ALFPm3-resistant strains of E. coli could be induced in vitro. Genome analysis of the resistant mutants revealed multiple mutations, with the most interesting being a qseC(L299R). A study of antibiotic susceptibility profile showed that the resistant strains harboring the qseC(L299R) not only exhibited higher degree of resistance towards polymyxin antibiotics, but also produced higher biofilm under ALFPm3 stress. Lastly, a single cell death analysis revealed that, at early-log phase when biofilm is scarce, the resistant strains were less affected by ALFPm3 treatment, suggesting additional mechanisms by which qseC orchestrates to protect the bacteria from ALFPm3. Altogether, this study uncovers involvement of qseC mutation in mechanism of resistance of the bacteria against ALFPm3 paving a way for future studies on sustainable use of ALFPm3 as an EMS control agent.

Activation of α-secretase by curcumin-aminoacid conjugates.

The extracellular senile plaques observed in Alzheimer's disease (AD) patients are mainly composed of amyloid peptides produced from the ß-amyloid precursor protein (ßAPP) by ß- and γ-secretases. A third non-amyloidogenic α-secretase activity performed by the disintegrins ADAM10 and ADAM17 occurs in the middle of the amyloid-ß peptide Aß and liberates the large sAPPα neuroprotective fragment. Since the activation of α-secretase recently emerged as a promising therapeutic approach to treat AD, the identification of natural compounds able to trigger this cleavage is highly required. Here we describe new curcumin-based modified compounds as α-secretase activators. We established that the aminoacid conjugates curcumin-isoleucine, curcumin-phenylalanine and curcumin-valine promote the constitutive α-secretase activity and increase ADAM10 immunoreactivity. Strickingly, experiments carried out under conditions mimicking the PKC/muscarinic receptor-regulated pathway display different patterns of activation by these compounds. Altogether, our data identified new lead natural compounds for the future development of powerful and stable α-secretase activators and established that some of these molecules are able to discriminate between the constitutive and regulated α-secretase pathways.

Phage-resistant Pseudomonas aeruginosa against a novel lytic phage JJ01 exhibits hypersensitivity to colistin and reduces biofilm production.

Pseudomonas aeruginosa, a major cause of nosocomial infections, has been categorized by World Health Organization as a critical pathogen urgently in need of effective therapies. Bacteriophages or phages, which are viruses that specifically kill bacteria, have been considered as alternative agents for the treatment of bacterial infections. Here, we discovered a lytic phage targeting P. aeruginosa, designated as JJ01, which was classified as a member of the Myoviridae family due to the presence of an icosahedral capsid and a contractile tail under TEM. Phage JJ01 requires at least 10 min for 90% of its particles to be adsorbed to the host cells and has a latent period of 30 min inside the host cell for its replication. JJ01 has a relatively large burst size, which releases approximately 109 particles/cell at the end of its lytic life cycle. The phage can withstand a wide range of pH values (3-10) and temperatures (4-60°C). Genome analysis showed that JJ01 possesses a complete genome of 66,346 base pairs with 55.7% of GC content, phylogenetically belonging to the genus Pbunavirus. Genome annotation further revealed that the genome encodes 92 open reading frames (ORFs) with 38 functionally predictable genes, and it contains neither tRNA nor toxin genes, such as drug-resistant or lysogenic-associated genes. Phage JJ01 is highly effective in suppressing bacterial cell growth for 12 h and eradicating biofilms established by the bacteria. Even though JJ01-resistant bacteria have emerged, the ability of phage resistance comes with the expense of the bacterial fitness cost. Some resistant strains were found to produce less biofilm and grow slower than the wild-type strain. Among the resistant isolates, the resistant strain W10 which notably loses its physiological fitness becomes eight times more susceptible to colistin and has its cell membrane compromised, compared to the wild type. Altogether, our data revealed the potential of phage JJ01 as a candidate for phage therapy against P. aeruginosa and further supports that even though the use of phages would subsequently lead to the emergence of phage-resistant bacteria, an evolutionary trade-off would make them more sensitive to antibiotics.

Mansonone G and its derivatives exhibit membrane permeabilizing activities against bacteria.

In an era where the rate of bacteria evolving to be resistant to clinically-used antibiotics far exceeds that of antibiotic discovery, the search for new sources of antibacterial agents has expanded tremendously. In recent years, interest in plant-based natural products as promising sources of antibacterial agents has taken an upward trend. Mansonones, botanically-derived naphthoqionones, having many uses in Asian traditional medicine-including anti-infective roles-have sparked interest as a possible source of antibacterial agents. Here, we show that mansonone G, extracted from Mansonia gagei Drumm. heartwoods, possessed antibacterial activities towards Bacillus subtilis, Staphylococcus aureus and Escherichia coli lptD4213, inhibiting the growth of the bacteria at 15.6 µM, 62.5 µM and 125 µM, respectively. Fourteen derivatives of mansonone G were synthesized successfully and were found to have a similar antibacterial spectrum to that of the parent compound, with some derivatives possessing improved antibacterial activities. Bacterial cytological profiling analysis showed that mansonone G harbors membrane permeabilizing activities against B. subtilis and E. coli lptD4213. Temporal analysis of SYTOX Green staining among individual cells showed that mansonone G rapidly permeabilized bacterial membrane within 10 min, with SYTOX Green intensity reaching 13-fold above that of the control. Collectively, these findings highlight the importance of mansonone G and its derivatives as potential antibacterial agents, paving the way for further modifications in order to improve their antibacterial spectrum.

A novel vibriophage exhibits inhibitory activity against host protein synthesis machinery.

Since the emergence of deadly pathogens and multidrug-resistant bacteria at an alarmingly increased rate, bacteriophages have been developed as a controlling bioagent to prevent the spread of pathogenic bacteria. One of these pathogens, disease-causing Vibrio parahaemolyticus (VPAHPND) which induces acute hepatopancreatic necrosis, is considered one of the deadliest shrimp pathogens, and has recently become resistant to various classes of antibiotics. Here, we discovered a novel vibriophage that specifically targets the vibrio host, VPAHPND. The vibriophage, designated Seahorse, was classified in the family Siphoviridae because of its icosahedral capsid surrounded by head fibers and a non-contractile long tail. Phage Seahorse was able to infect the host in a broad range of pH and temperatures, and it had a relatively short latent period (nearly 30 minutes) in which it produced progeny at 72 particles per cell at the end of its lytic cycle. Upon phage infection, the host nucleoid condensed and became toroidal, similar to the bacterial DNA morphology seen during tetracycline treatment, suggesting that phage Seahorse hijacked host biosynthesis pathways through protein translation. As phage Seahorse genome encodes 48 open reading frames with many hypothetical proteins, this genome could be a potential untapped resource for the discovery of phage-derived therapeutic proteins.

The Polyherbal Wattana Formula Displays Anti-Amyloidogenic Properties by Increasing α-Secretase Activities.

Alzheimer's disease is characterized by the deposition of insoluble amyloid-ß peptides produced from the ß-amyloid precursor protein (ßAPP). Because α-secretase cleavage by ADAM10 and ADAM17 takes place in the middle of Aß, its activation is considered as a promising anti-AD therapeutic track. Here we establish that the polyherbal Wattana formula (WNF) stimulates sAPPα production in cells of neuronal and non-neuronal origins through an increase of both ADAM10 and ADAM17 catalytic activities with no modification of BACE1 activity and expression. This effect is blocked by specific inhibition or genetic depletion of these disintegrins and we show that WNF up-regulates ADAM10 transcription and ADAM17 maturation. In addition, WNF reduces Aß40 and Aß42 generation in human cell lines. Altogether, WNF presents all the characteristics of a potent preventive anti-Alzheimer formula. Importantly, this natural recipe, currently prescribed to patients for the treatment of other symptoms without any secondary effect, can be tested immediately for further clinical studies.