Loss of SREBP-1c ameliorates iron-induced liver fibrosis by decreasing lipocalin-2.

Sterol regulatory element-binding protein (SREBP)-1c is involved in cellular lipid homeostasis and cholesterol biosynthesis and is highly increased in nonalcoholic steatohepatitis (NASH). However, the molecular mechanism by which SREBP-1c regulates hepatic stellate cells (HSCs) activation in NASH animal models and patients have not been fully elucidated. In this study, we examined the role of SREBP-1c in NASH and the regulation of LCN2 gene expression. Wild-type and SREBP-1c knockout (1cKO) mice were fed a high-fat/high-sucrose diet, treated with carbon tetrachloride (CCl4), and subjected to lipocalin-2 (LCN2) overexpression. The role of LCN2 in NASH progression was assessed using mouse primary hepatocytes, Kupffer cells, and HSCs. LCN2 expression was examined in samples from normal patients and those with NASH. LCN2 gene expression and secretion increased in CCl4-induced liver fibrosis mice model, and SREBP-1c regulated LCN2 gene transcription. Moreover, treatment with holo-LCN2 stimulated intracellular iron accumulation and fibrosis-related gene expression in mouse primary HSCs, but these effects were not observed in 1cKO HSCs, indicating that SREBP-1c-induced LCN2 expression and secretion could stimulate HSCs activation through iron accumulation. Furthermore, LCN2 expression was strongly correlated with inflammation and fibrosis in patients with NASH. Our findings indicate that SREBP-1c regulates Lcn2 gene expression, contributing to diet-induced NASH. Reduced Lcn2 expression in 1cKO mice protects against NASH development. Therefore, the activation of Lcn2 by SREBP-1c establishes a new connection between iron and lipid metabolism, affecting inflammation and HSCs activation. These findings may lead to new therapeutic strategies for NASH.

Inhibition of Granule Cell Dispersion and Seizure Development by Astrocyte Elevated Gene-1 in a Mouse Model of Temporal Lobe Epilepsy.

Although granule cell dispersion (GCD) in the hippocampus is known to be an important feature associated with epileptic seizures in temporal lobe epilepsy (TLE), the endogenous molecules that regulate GCD are largely unknown. In the present study, we have examined whether there is any change in AEG-1 expression in the hippocampus of a kainic acid (KA)-induced mouse model of TLE. In addition, we have investigated whether the modulation of astrocyte elevated gene-1 (AEG-1) expression in the dentate gyrus (DG) by intracranial injection of adeno-associated virus 1 (AAV1) influences pathological phenotypes such as GCD formation and seizure susceptibility in a KA-treated mouse. We have identified that the protein expression of AEG-1 is upregulated in the DG of a KA-induced mouse model of TLE. We further demonstrated that AEG-1 upregulation by AAV1 delivery in the DG-induced anticonvulsant activities such as the delay of seizure onset and inhibition of spontaneous recurrent seizures (SRS) through GCD suppression in the mouse model of TLE, while the inhibition of AEG-1 expression increased susceptibility to seizures. The present observations suggest that AEG-1 is a potent regulator of GCD formation and seizure development associated with TLE, and the significant induction of AEG-1 in the DG may have therapeutic potential against epilepsy.

PmrAB controls virulence-associated traits and outer membrane vesicle biogenesis in Acinetobacter baumannii.

The PmrAB two-component system modulates colistin resistance in Acinetobacter baumannii, but its association with the virulence traits of this bacterium remains uncharacterized. This study explored the role of A. baumannii PmrAB in surface motility, biofilm formation, and outer membrane vesicle (OMV) biogenesis using wild-type (WT) A. baumannii 17978 and ΔpmrA and ΔpmrB mutant strains. The two mutant strains exhibited significantly decreased surface motility compared with that of WT strain by the low expression of abaI, abaR, A1S_0113, A1S_0115, and A1S_0116. Biofilm mass also significantly decreased in the two mutant strains at 12 h of incubation, but restored at 24 h. Under static culture conditions for 12 h, the two mutant strains showed low pgaA expression. However, the other biofilm-associated genes, such as csuC, csuE, ompA, and bap, showed different expression between the two mutant strains. Although the size of OMVs was similar among the three strains, the number of OMVs secreted from the two mutant strains slightly decreased compared with that secreted from the WT strain. Protein concentrations in the OMVs of ΔpmrA mutant significantly decreased compared with those in the OMVs of WT and ΔpmrB strains. Overall, PmrAB modulates virulence traits and OMV biogenesis in A. baumannii.

Acinetobacter baumannii under Acidic Conditions Induces Colistin Resistance through PmrAB Activation and Lipid A Modification.

Colistin is a last-resort antimicrobial agent for treating carbapenem-resistant Acinetobacter baumannii infections. The activation of PmrAB by several environmental signals induces colistin resistance in Gram-negative bacteria. This study investigated the molecular mechanisms of colistin resistance in A. baumannii under acidic conditions using wild-type (WT) A. baumannii 17978, ΔpmrA and ΔpmrB mutants, and pmrA-complemented strains. The pmrA or pmrB deletion did not affect the growth of A. baumannii under acidic or aerobic conditions. A. baumannii under acidic (pH 5.5) and high-iron (1 mM) conditions showed 32- and 8-fold increases in the minimum inhibitory concentrations (MICs) of colistin, respectively. The ΔpmrA and ΔpmrB mutants at pH 5.5 showed a significant decrease in colistin MICs compared to the WT strain at pH 5.5. No difference in colistin MICs was observed between WT and mutant strains under high-iron conditions. The pmrCAB expression significantly increased in the WT strain at pH 5.5 compared to the WT strain at pH 7.0. The pmrC expression significantly decreased in two mutant strains at pH 5.5 compared to the WT strain at pH 5.5. The PmrA protein was expressed in the ΔpmrA strain carrying ppmrA_FLAG plasmids at pH 5.5 but not at pH 7.0. Lipid A modification by the addition of phosphoethanolamine was observed in the WT strain at pH 5.5. In conclusion, this study demonstrated that A. baumannii under acidic conditions induces colistin resistance via the activation of pmrCAB operon and subsequent lipid A modification.

Complete genome sequence of the multidrug-resistant clinical isolate Acinetobacter baumannii KBN10P05679: Insights into antimicrobial resistance genotype and potential virulence traits.

OBJECTIVES: Acinetobacter baumannii, a nosocomial pathogen, exhibits multidrug resistance and is a major concern worldwide. We therefore aimed to evaluate the genomic features of the clinical strain A. baumannii KBN10P05679 to elucidate its antibiotic resistance mechanisms and virulence factors. METHODS: In silico multilocus sequence typing, phylogenetic identification, genome annotation, genome analysis, antibiotic susceptibility testing, and biofilm formation assay were performed, and the expression levels of antibiotic resistance- and biofilm-related genes were investigated. RESULTS: The complete genome of KBN10P05679 comprises a circular chromosome of 3 990 428 bp and two plasmids (74 294 and 8731 bp) and was assigned to the ST451 sequence type. Clusters of Orthologous Gene annotation identified 3810 genes, including those involved in amino acid transport and metabolism, transcription, inorganic ion transport, energy production and conversion, replication, recombination and repair, and carbohydrate and protein metabolism. The antibiotic resistance genes were investigated by searching the Comprehensive Antibiotic Resistance Database, and the genome was found to harbour 30 different antibiotic resistance genes. Analysis of the Virulence Factor Database revealed 86 virulence factor genes in the KBN1005679 genome. The KBN10P05679 strain was found to have a higher capacity for biofilm formation and expressed biofilm-related genes at a higher level than the other tested strains. CONCLUSIONS: The antibiotic resistance genotype and potential virulence factor-related data obtained in this study would help direct future studies for developing the control measures for this multidrug-resistant pathogen.

pKr-2 induces neurodegeneration via upregulation of microglial TLR4 in the hippocampus of AD brain.

We recently demonstrated that prothrombin kringle-2 (pKr-2) derived from blood-brain barrier (BBB) disruption could induce hippocampal neurodegeneration and object recognition impairment through neurotoxic inflammatory responses in the five familial Alzheimer's disease mutation (5XFAD) mice. In the present study, we aimed to determine whether pKr-2 induces microglial activation by stimulating toll-like receptor 4 (TLR4) upregulation and examine whether this response contributes to pKr-2-induced neuroinflammatory damage in the hippocampi of mice models. We observed that inflammatory responses induced by pKr-2 administration in the hippocampi of wild-type mice were significantly abrogated in TLR4-deficient mice (TLR4-/-), and caffeine supply or rivaroxaban treatment that inhibits the overexpression of hippocampal pKr-2 reduced TLR4 upregulation in 5XFAD mice, resulting in the inhibition of neuroinflammatory responses. Similar to the expression patterns of pKr-2, TLR4, and the TLR4 transcription factors, PU.1 and p-c-Jun, seen in the postmortem hippocampal tissues of Alzheimer's disease (AD) patients, our results additionally showed the influence of transcriptional regulation on TLR4 expression following pKr-2 expression in triggering the production of neurotoxic inflammatory mediators. Therefore, we conclude that pKr-2 may play a role in initiating upregulation of microglial TLR4, consequently inducing hippocampal neurodegeneration. Furthermore, the control of pKr-2-induced microglial TLR4 could be a useful therapeutic strategy against hippocampal neurodegeneration in AD.

ECM-targeting bacteria enhance chemotherapeutic drug efficacy by lowering IFP in tumor mouse models.

Bacterial cancer therapies aim to manipulate bacteria to effectively deploy therapeutic payloads to tumors. Attenuated bacteria alone often cannot eradicate solid tumors. Attenuated Salmonella can be engineered to deliver cytotoxic drugs to either trigger an immune response or increase antitumor efficacy when combined with chemotherapeutic drugs. However, the extracellular matrix (ECM) surrounding cancer cells forms a barrier that often limits the ability of chemotherapeutic and cytotoxic drugs to penetrate and eliminate tumors. To overcome this limitation, we developed a strategy to combine chemotherapy with an attenuated Salmonella typhimurium strain engineered to secrete HysA protein (from Staphylococcus aureus; Hyaluronidase, HAase) in tumors. The engineered Salmonella effectively degraded hyaluronan (HA), which is a major ECM constituent in tumors, and suppressed tumor growth in mouse models of pancreatic adenocarcinoma (ASPC-1) and breast cancer (4T1). Furthermore, it prolonged survival when combined with chemotherapeutic drugs (doxorubicin or gemcitabine). Upon bacterial colonization, the HAase-mediated ECM degradation decreased interstitial fluid pressure (IFP) in the tumor microenvironment. Additionally, HA degradation using HAase-expressing bacteria in vivo led to decreased binding to the receptor, CD44, expressed in tumors. This may modulate proliferation- and apoptosis-related signal pathways. Therefore, ECM-targeting bacteria can be used as a synergistic anticancer therapeutic agent to maximize chemotherapeutic drug delivery into highly invasive tumors.

Complete Genome Sequence of Acinetobacter baumannii Strain KBN10P04593, a Clinical Isolate from South Korea.

Acinetobacter baumannii is an opportunistic nosocomial pathogen that is responsible for various life-threating infections in immunocompromised hosts. We present the complete 3.93-Mb genome sequence of A. baumannii KBN10P04593, generated by combining PacBio and Illumina technologies.

Structure-based discovery of pyrazolamides as novel ERRγ inverse agonists.

Estrogen-related receptor-gamma (ERRγ) is an orphan nuclear receptor with high structural similarity to estrogen receptors (ERα and ß). The endogenous ligand of the receptor has yet to be identified. Only two classes of molecules-stilbene (diethylstilbestrol, 4-hydroxytamoxifen, and GSK5182) and flavonol (kaempferol) have been known to modulate the transcriptional activity of the receptor to date. Further, these agents lack selectivity to ERRγ suggesting the need for a new inverse agonist. Thus, virtual screening was used to identify pyrazolamide 7 as a novel ERRγ inverse agonist. Structure-based diversification and optimization of the compound further led to the identification of derivative 19 as a potent inverse agonist of ERRγ with selectivity over other nuclear receptors including those of ERR family. Pyrazolamide 19 exhibits strong affinity towards ERRγ and inhibits the expression of hepcidin, fibrinogen and gluconeogenic genes, which suggests that these compounds may have antimicrobial, anti-coagulant and antidiabetic activities.

Complementary Regulation of BfmRS Two-Component and AbaIR Quorum Sensing Systems to Express Virulence-Associated Genes in Acinetobacter baumannii.

Acinetobacter baumannii expresses various virulence factors to adapt to hostile environments and infect susceptible hosts. This study investigated the regulatory network of the BfmRS two-component and AbaIR quorum sensing (QS) systems in the expression of virulence-associated genes in A. baumannii ATCC 17978. The ΔbfmS mutant exhibited a significant decrease in surface motility, which presumably resulted from the low expression of pilT and A1S_0112-A1S_0119 gene cluster. The ΔbfmR mutant displayed a significant reduction in biofilm and pellicle formation due to the low expression of csu operon. The deletion of abaR did not affect the expression of bfmR or bfmS. However, the expression of abaR and abaI was upregulated in the ΔbfmR mutant. The ΔbfmR mutant also produced more autoinducers than did the wild-type strain, suggesting that BfmR negatively regulates the AbaIR QS system. The ΔbfmS mutant exhibited no autoinducer production in the bioassay system. The expression of the A1S_0112-A1S_0119 gene cluster was downregulated in the ΔabaR mutant, whereas the expression of csu operon was upregulated in this mutant with a high cell density. In conclusion, for the first time, we demonstrated that the BfmRS-AbaIR QS system axis regulated the expression of virulence-associated genes in A. baumannii. This study provides new insights into the complex network system involved in the regulation of virulence-associated genes underlying the pathogenicity of A. baumannii.

Engineering of lysin by fusion of antimicrobial peptide (cecropin A) enhances its antibacterial properties against multidrug-resistant Acinetobacter baumannii.

Most clinical isolates of Acinetobacter baumannii, a nosocomial pathogen, are multidrug-resistant (MDR), fueling the search for alternative therapies. Bacteriophage-derived endolysins have potent antibacterial activities and are considered as alternatives to antibiotics against A. baumannii infection. Gram-negative bacteria possess outer lipid membrane that prevents direct contact between the endolysins and the cell wall. We hypothesized that the fusion of antimicrobial peptide (AMP) with endolysin could help to reduce bacterial endolysin resistance and increase antimicrobial activity by membrane permeability action. Accordingly, we fused cecropin A, a commonly used AMP, with the N-terminus of AbEndolysin, which enhances the bactericidal activity of the chimeric endolysin. The bactericidal activity of cecropin A-fused AbEndolysin increased by at least 2-8 fold for various MDR A. baumannii clinical isolates. The in vitro bactericidal activity results also showed higher bacterial lysis by the chimeric endolysin than that by the parental lysin. The engineered AbEndolysin (eAbEndolysin) showed synergistic effects with the beta-lactam antibiotics cefotaxime, ceftazidime, and aztreonam, and an additive effect with meropenem and imipenem. eAbEndolysin had no cytotoxic effect on A549 cell line and rescued mice (40% survival rate) from systemic A. baumannii infection. Together, these findings suggest the potential of lysin therapy and may prompt its use as an alternative to antibiotics.

Lipocalin2 as a potential antibacterial drug against Acinetobacter baumannii infection.

Available antibiotics to treat Acinetobacter baumannii infection is limited due to increasing resistance and the emergence of multiple drug-resistant strains. Hence, discovering effective agents against A. baumannii to reduce the number of infection-related deaths is imperative. In search of novel and alternative antibiotics, the antibacterial function of lipocalin2 (Lcn2) was investigated to treat systemic infections of A. baumannii using a mouse neutropenia model. We observed a significant increase in serum Lcn2 levels upon bacterial injection into the mouse, and the administration of recombinant Lcn2 (rmLcn2) extended their survival. Such protective effects were also observed in rmLcn2-pretreated macrophages, where rmLcn2 reduced the survival of the pathogen inside the macrophages. The underlying molecular mechanism of Lcn2 protection was also investigated. We observed that pretreatment of the Raw-264.7 macrophages with rmLcn2 markedly altered the expression of tonB3, which encodes a component of the transporter for ferrisiderophores in A. baumannii. However, the expression of katG, the gene encoding catalase, remained unaffected. These indicate that Lcn2-mediated defense against the pathogen is related to nutritional immunity rather than reactive oxygen species (ROS) production. Furthermore, the addition of rmLcn2 in infected mice diminished bacterial burden in multiple organs and enhanced the expression of tonB3 in the liver, spleen, and lungs of the infected mice. Increased survival rate due to rmLcn2 treatment declined when the infection model was established using lcn2-defective (lcn2-/-) mice, which indicated the necessity of endogenous Lcn2. Therefore, the antibacterial function of Lcn2 can be exploited to develop an alternative therapeutic agent against A. baumannii.

Sirtinol Supresses Trophozoites Proliferation and Encystation of Acanthamoeba via Inhibition of Sirtuin Family Protein.

The encystation of Acanthamoeba leads to the development of metabolically inactive and dormant cysts from vegetative trophozoites under unfavorable conditions. These cysts are highly resistant to anti-Acanthamoeba drugs and biocides. Therefore, the inhibition of encystation would be more effective in treating Acanthamoeba infection. In our previous study, a sirtuin family protein-Acanthamoeba silent-information regulator 2-like protein (AcSir2)-was identified, and its expression was discovered to be critical for Acanthamoeba castellanii proliferation and encystation. In this study, to develop Acanthamoeba sirtuin inhibitors, we examine the effects of sirtinol, a sirtuin inhibitor, on trophozoite growth and encystation. Sirtinol inhibited A. castellanii trophozoites proliferation (IC50=61.24 µM). The encystation rate of cells treated with sirtinol significantly decreased to 39.8% (200 µM sirtinol) after 24 hr of incubation compared to controls. In AcSir2-overexpressing cells, the transcriptional level of cyst-specific cysteine protease (CSCP), an Acanthamoeba cysteine protease involved in the encysting process, was 11.6- and 88.6-fold higher at 48 and 72 hr after induction of encystation compared to control. However, sirtinol suppresses CSCP transcription, resulting that the undegraded organelles and large molecules remained in sirtinol-treated cells during encystation. These results indicated that sirtinol sufficiently inhibited trophozoite proliferation and encystation, and can be used to treat Acanthamoeba infections.

DksA Modulates Antimicrobial Susceptibility of Acinetobacter baumannii.

The stringent response regulators, (p)ppGpp and DksA, modulate various genes involved in physiological processes, virulence, and antimicrobial resistance in pathogenic bacteria. This study investigated the role of DksA in the antimicrobial susceptibility of Acinetobacter baumannii. The ∆dksA mutant (KM0248D) of A. baumannii ATCC 17978 and its complemented strain (KM0248C) were used, in addition to the ∆dksA mutant strain (NY0298D) of clinical 1656-2 strain. The microdilution assay was used to determine the minimum inhibitory concentrations (MICs) of antimicrobial agents. Quantitative real-time PCR was performed to analyze the expression of genes associated with efflux pumps. The KM0248D strain exhibited an increase of MICs to quinolones and tetracyclines, whereas KM0248D and NY0298D strains exhibited a decrease of MICs to aminoglycosides. The expression of genes associated with efflux pumps, including adeB, adeI/J, abeM, and/or tetA, was upregulated in both ∆dksA mutant strains. The deletion of dksA altered bacterial morphology in the clinical 1656-2 strain. In conclusion, DksA modulates the antimicrobial susceptibility of A. baumannii. The ∆dksA mutant strains of A. baumannii upregulate efflux pump gene expression, whereas (p)ppGpp-deficient mutants downregulate efflux pump gene expression. (p)ppGpp and DksA conduct opposite roles in the antimicrobial susceptibility of A. baumannii via efflux pump gene regulation.

LeuO, a LysR-Type Transcriptional Regulator, Is Involved in Biofilm Formation and Virulence of Acinetobacter baumannii.

Acinetobacter baumannii is an important nosocomial pathogen that can survive in different environmental conditions and poses a severe threat to public health due to its multidrug resistance properties. Research on transcriptional regulators, which play an essential role in adjusting to new environments, could provide new insights into A. baumannii pathogenesis. LysR-type transcriptional regulators (LTTRs) are structurally conserved among bacterial species and regulate virulence in many pathogens. We identified a novel LTTR, designated as LeuO encoded in the A. baumannii genome. After construction of LeuO mutant strain, transcriptome analysis showed that LeuO regulates the expression of 194 upregulated genes and 108 downregulated genes responsible for various functions and our qPCR validation of several differentially expressed genes support transcriptome data. Our results demonstrated that disruption of LeuO led to increased biofilm formation and increased pathogenicity in an animal model. However, the adherence and surface motility of the LeuO mutant were reduced compared with those of the wild-type strain. We observed some mutations on amino acids sequence of LeuO in clinical isolates. These mutations in the A. baumannii biofilm regulator LeuO may cause hyper-biofilm in the tested clinical isolates. This study is the first to demonstrate the association between the LTTR member LeuO and virulence traits of A. baumannii.

Global regulator DksA modulates virulence of Acinetobacter baumannii.

DksA with (p)ppGpp regulates a wide range of gene transcriptions during the stringent response. The aim of this study was to identify a DksA ortholog in Acinetobacter baumannii and clarify the roles of DksA in bacterial physiology and virulence. The ∆dksA mutant and its complemented strains were constructed using A. baumannii ATCC 17978. The AlS_0248 in A. baumannii ATCC 17978 was identified to dksA using sequence homology, protein structure prediction, and gene expression patterns under different culture conditions. The ∆dksA mutant strain showed a filamentous morphology compared with the wild-type (WT) strain. Bacterial growth was decreased in the ∆dksA mutant strain under static conditions. Surface motility was decreased in the ∆dksA mutant strain compared with the WT strain. In contrast, biofilm formation was increased and biofilm-associated genes, such as bfmR/S and csuC/D/E, were upregulated in the ∆dksA mutant strain. The ∆dksA mutant strain produced less autoinducers than the WT strain. The expression of abaI and abaR was significantly decreased in the ∆dksA mutant strain. Furthermore, the ∆dksA mutant strain showed less bacterial burden and milder histopathological changes in the lungs of mice than the WT strain. Mice survival was also significantly different between the ∆dksA mutant and WT strains. Conclusively, DksA is directly or indirectly involved in regulating a wide range of genes associated with bacterial physiology and virulence, which contributes to the pathogenesis of A. baumannii. Thus, DksA is a potential anti-virulence target for A. baumannii infection.

Perilipin 5 is a novel target of nuclear receptor LRH-1 to regulate hepatic triglycerides metabolism.

Liver receptor homolog-1 (LRH-1) has emerged as a regulator of hepatic glucose, bile acid, and mitochondrial metabolism. However, the functional mechanism underlying the effect of LRH-1 on lipid mobilization has not been addressed. This study investigated the regulatory function of LRH-1 in lipid metabolism in maintaining a normal liver physiological state during fasting. The Lrh-1f/f and LRH-1 liver-specific knockout (Lrh-1LKO) mice were either fed or fasted for 24 h, and the liver and serum were isolated. The livers were used for qPCR, western blot, and histological analysis. Primary hepatocytes were isolated for immunocytochemistry assessments of lipids. During fasting, the Lrh-1LKO mice showed increased accumulation of triglycerides in the liver compared to that in Lrh-1f/f mice. Interestingly, in the Lrh-1LKO liver, decreases in perilipin 5 (PLIN5) expression and genes involved in ß-oxidation were observed. In addition, the LRH-1 agonist dialauroylphosphatidylcholine also enhanced PLIN5 expression in human cultured HepG2 cells. To identify new target genes of LRH-1, these findings directed us to analyze the Plin5 promoter sequence, which revealed -1620/-1614 to be a putative binding site for LRH-1. This was confirmed by promoter activity and chromatin immunoprecipitation assays. Additionally, fasted Lrh-1f/f primary hepatocytes showed increased co-localization of PLIN5 in lipid droplets (LDs) compared to that in fasted Lrh-1LKO primary hepatocytes. Overall, these findings suggest that PLIN5 might be a novel target of LRH-1 to mobilize LDs, protect the liver from lipid overload, and manage the cellular needs during fasting. [BMB Reports 2021; 54(9): 476-481].

Characterization of a Novel Phage ΦAb1656-2 and Its Endolysin with Higher Antimicrobial Activity against Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen, which is a problem worldwide due to the emergence of a difficult-to-treat multidrug-resistant A. baumannii (MDRAB). Endolysins are hydrolytic enzymes produced by a bacteriophage that can be used as a potential therapeutic agent for multidrug-resistant bacterial infection in replacing antibiotics. Here, we isolated a novel bacteriophage through prophage induction using mitomycin C from clinical A. baumannii 1656-2. Morphologically, ΦAb1656-2 was identified as a Siphoviridae family bacteriophage, which can infect MDRAB. The whole genome of ΦAb1656-2 was sequenced, and it showed that it is 50.9 kb with a G + C content of 38.6% and 68 putative open reading frames (ORFs). A novel endolysin named AbEndolysin with an N-acetylmuramidase-containing catalytic domain was identified, expressed, and purified from ΦAb1656-2. Recombinant AbEndolysin showed significant antibacterial activity against MDRAB clinical strains without any outer membrane permeabilizer. These results suggest that AbEndolysin could represent a potential antimicrobial agent for treating MDRAB clinical isolates.

ppGpp signaling plays a critical role in virulence of Acinetobacter baumannii.

Acinetobacter baumannii, a major nosocomial pathogen, survives in diverse hospital environments, and its multidrug resistance is a major concern. The ppGpp-dependent stringent response mediates the reprogramming of genes with diverse functions in several bacteria. We investigated whether ppGpp is involved in A. baumannii's pathogenesis by examining biofilm formation, surface motility, adhesion, invasion, and mouse infection studies. Transcriptome analysis of early stationary phase cultures revealed 498 differentially-expressed genes (≥ 2-fold change) in a ppGpp-deficient A. baumannii strain; 220 and 278 genes were up and downregulated, respectively. Csu operon expression, important in pilus biosynthesis during early biofilm formation, was significantly reduced in the ppGpp-deficient strain. Our findings suggest that ppGpp signaling influences A. baumannii biofilm formation, surface motility, adherence, and virulence. We showed the association between ppGpp and pathogenicity in A. baumannii for the first time; ppGpp may be a novel antivirulence target in A. baumannii.

The mechanism of gap creation by a multifunctional nuclease during base excision repair.

During base excision repair, a transient single-stranded DNA (ssDNA) gap is produced at the apurinic/apyrimidinic (AP) site. Exonuclease III, capable of performing both AP endonuclease and exonuclease activity, are responsible for gap creation in bacteria. We used single-molecule fluorescence resonance energy transfer to examine the mechanism of gap creation. We found an AP site anchor-based mechanism by which the intrinsically distributive enzyme binds strongly to the AP site and becomes a processive enzyme, rapidly creating a gap and an associated transient ssDNA loop. The gap size is determined by the rigidity of the ssDNA loop and the duplex stability of the DNA and is limited to a few nucleotides to maintain genomic stability. When the 3' end is released from the AP endonuclease, polymerase I quickly initiates DNA synthesis and fills the gap. Our work provides previously unidentified insights into how a signal of DNA damage changes the enzymatic functions.