RESUMEN
PURPOSE: Cyclic guanosine monophosphate-adenosine monophosphate and other bacterial-derived cyclic di-guanosine monophosphate or cyclic di-adenosine monophosphate trigger innate immune responses through binding to stimulator of interferon genes (STING). Thus in chronic infection, such as in periodontitis, immune cells can be exposed to bacterial DNA and/or cyclic dinucleotides, potentially activating STING to cause inflammation. Thus far the cyclic GMP-AMP synthase-STING- TANK-binding kinase 1 pathway has been well characterized but a global perspective of how the presence or lack of STING affect the proteome is lacking. The aim of this study is to identify macrophage proteins that are affected by STING. EXPERIMENTAL DESIGN: Proteins are extracted from a macrophage cell line harboring STING (RAW-Blue ISG) as well as a STING knockout (STING KO) cell line (RAW-Lucia ISG-KO-STING) and global proteomics analyses are performed. RESULTS: Proteins related to kinase and phosphatase signaling, spliceosome, terpenoid backbone biosynthesis, glycosylation, ubiquitination, and phagocytosis are affected by STING knock out. CONCLUSIONS AND CLINICAL RELEVANCE: STING pathway in macrophages is related to the regulation of several proteins that are known as potent biomarkers of various cancers and autoimmune diseases. Moreover, the relation between STING and phagocytosis is demonstrated for the first time. Further validation studies will help identify molecules and pathways that may function as diagnostic or therapeutic targets.
Asunto(s)
Macrófagos/metabolismo , Proteínas de la Membrana/metabolismo , Proteómica , Animales , Biomarcadores/metabolismo , Línea Celular , Técnicas de Inactivación de Genes , Glicosilación , Macrófagos/citología , Macrófagos/inmunología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Terapia Molecular Dirigida , Fagocitosis , Transducción de Señal , UbiquitinaciónRESUMEN
Staphylococcus aureus can survive both inside and outside of phagocytic and nonphagocytic host cells. Once in the intracellular milieu, most antibiotics have reduced ability to kill S. aureus, thus resulting in relapse of infection. Consequently, there is a need for antibacterial agents that can accumulate to lethal concentrations within host cells to clear intracellular infections. We have identified tetrahydrobenzo[a or c]phenanthridine and tetrahydrobenzo[a or c]acridine compounds, synthesized via a one-flask Povarov-Doebner operation from readily available amines, aldehydes, and cyclic ketones, as potent agents against drug-resistant S. aureus. Importantly, the tetrahydrobenzo[a or c]phenanthridine and tetrahydrobenzo[a or c]acridine compounds can accumulate in macrophage cells and reduce the burden of intracellular MRSA better than the drug of choice, vancomycin. We observed that MRSA could not develop resistance (by passage 30) against tetrahydrobenzo[a or c]acridine compound 15. Moreover, tetrahydrobenzo[c]acridine compound 15 and tetrahydrobenzo[c]phenanthridine compound 16 were nontoxic to red blood cells and were nonmutagenic. Preliminary data indicated that compound 16 reduced bacterial load (MRSA USA300) in mice (thigh infection model) to the same degree as vancomycin. These observations suggest that compounds 15 and 16 and analogues thereof could become therapeutic agents for the treatment of chronic MRSA infections.
Asunto(s)
Antibacterianos/administración & dosificación , Antibacterianos/síntesis química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Infecciones Estafilocócicas/microbiología , Animales , Antibacterianos/química , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Estructura Molecular , Infecciones Estafilocócicas/tratamiento farmacológicoRESUMEN
Halogenated 4-hydroxybenzylidene indolinones have been shown to re-sensitize methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE) to methicillin and vancomycin respectively. The mechanism of antibiotic re-sensitization was however not previously studied. Here, we probe the scope of antibiotic re-sensitization and present the global proteomics analysis of S. aureus treated with GW5074, a 4-hydroxybenzylidene indolinone compound. With a minimum inhibitory concentration (MIC) of 8⯵g/mL against S. aureus, GW5074 synergized with beta-lactam antibiotics like ampicillin, carbenicillin and cloxacillin, the DNA synthesis inhibitor, ciprofloxacin, the protein synthesis inhibitor, gentamicin and the folate acid synthesis inhibitor, trimethoprim. Global proteomics analysis revealed that GW5074 treatment resulted in significant downregulation of enzymes involved in the purine biosynthesis. S. aureus proteins involved in amino acid metabolism and peptide transport were also observed to be downregulated. Interestingly, anti-virulence targets such as AgrC (a quorum sensing-related histidine kinase), AgrA (a quorum sensing-related response regulator) as well as downstream targets, such as hemolysins, lipases and proteases in S. aureus were also downregulated by GW5074. We observed that the peptidoglycan hydrolase, SceD was significantly upregulated. The activity of GW5074 on S. aureus suggests that the compound primes bacteria for the antibacterial action of ineffective antibiotics. SIGNIFICANCE: Antibiotic resistance continues to present significant challenges to the treatment of bacterial infections. Given that antibiotic resistance is a natural phenomenon and that it has become increasingly difficult to discover novel antibiotics, efforts to improve the activity of existing agents are worth pursuing. A few small molecules that re-sensitize resistant bacteria to traditional antibiotics have been described but the molecular details that underpin how these compounds work to re-sensitize bacteria remain largely unknown. In this report, global label-free quantitative proteomics was used to identify changes in the proteome that occurs when GW5074, a compound that re-sensitize MRSA to methicillin, is administered to S. aureus. The identification of pathways that are impacted by GW5074 could help identify novel targets for antibiotic re-sensitization.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/biosíntesis , Enterococcus faecalis/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Oxindoles/farmacología , ProteómicaRESUMEN
BACKGROUND/OBJECTIVES: Neonatal sepsis is an important cause of morbidity and mortality in the pediatric age group in spite of several attempts at mitigating its effects. This article determines the prevalence of neonatal sepsis and the pathogens responsible for sepsis as well as risk factors and outcome at the Babcock University Teaching Hospital. METHODS: A retrospective analysis of laboratory records of consecutive babies delivered within and outside our hospital suspected of having sepsis over a 1-year period. RESULTS: The isolation rate was 34% from 100 neonates with the predominant pathogens being coagulase-negative staphylococci (CONS), Staphylococcus aureus, and Klebsiella pneumoniae. The risk factors for sepsis were age <3 days (P = 0.03) and prematurity (P < 0.001). The mortality rate was 12% with risk factors for mortality being birth weight <2500 g (P = 0.005), prematurity (P = 0.036), premature rupture of membranes (P = 0.007), and delivery outside a tertiary hospital (P = 0.007). Meropenem, ciprofloxacin, and amikacin showed the highest rates of in vitro efficacy. CONCLUSION: We highlight the prevalent pathogens in our local facility to be a combination of CONS, S. aureus, and K. pneumoniae with susceptibility patterns showing meropenem, ciprofloxacin, and amikacin to be our most effective antimicrobials in vitro.