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.

The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii.

BACKGROUND: Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. RESULTS: In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97-100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. CONCLUSIONS: The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

N-3-Hydroxy Dodecanoyl-DL-homoserine Lactone (OH-dDHL) Triggers Apoptosis of Bone Marrow-Derived Macrophages through the ER- and Mitochondria-Mediated Pathways.

Quorum sensing of Acinetobacter nosocomialis for cell-to-cell communication produces N-3-hydroxy dodecanoyl-DL-homoserine lactone (OH-dDHL) by an AnoR/I two-component system. However, OH-dDHL-driven apoptotic mechanisms in hosts have not been clearly defined. Here, we investigated the induction of apoptosis signaling pathways in bone marrow-derived macrophages treated with synthetic OH-dDHL. Moreover, the quorum-sensing system for virulence regulation was evaluated in vivo using wild-type and anoI-deletion mutant strains. OH-dDHL decreased the viability of macrophage and epithelial cells in dose- and time-dependent manners. OH-dDHL induced Ca2+ efflux and caspase-12 activation by ER stress transmembrane protein (IRE1 and ATF6a p50) aggregation and induced mitochondrial dysfunction through reactive oxygen species (ROS) production, which caused cytochrome c to leak. Pretreatment with a pan-caspase inhibitor reduced caspase-3, -8, and -9, which were activated by OH-dDHL. Pro-inflammatory cytokine and paraoxonase-2 (PON2) gene expression were increased by OH-dDHL. We showed that the anoI-deletion mutant strains have less intracellular invasion compared to the wild-type strain, and their virulence, such as colonization and dissemination, was decreased in vivo. Consequently, these findings revealed that OH-dDHL, as a virulence factor, contributes to bacterial infection and survival as well as the modification of host responses in the early stages of infection.

Therapeutic Effects of Inhibitor of ompA Expression against Carbapenem-Resistant Acinetobacter baumannii Strains.

The widespread of carbapenem-resistant Acinetobacter baumannii (CRAB) is of great concern in clinical settings worldwide. It is urgent to develop new therapeutic agents against this pathogen. This study aimed to evaluate the therapeutic potentials of compound 62520, which has been previously identified as an inhibitor of the ompA promoter activity of A. baumannii, against CRAB isolates, both in vitro and in vivo. Compound 62520 was found to inhibit the ompA expression and biofilm formation in A. baumannii ATCC 17978 at sub-inhibitory concentrations in a dose-dependent manner. These inhibitory properties were also observed in clinical CRAB isolates belonging to sequence type (ST) 191. Additionally, compound 62520 exhibited a bacteriostatic activity against clinical clonal complex (CC) 208 CRAB isolates, including ST191, and ESKAPE pathogens. This bacteriostatic activity was not different between STs of CRAB isolates. Bacterial clearance was observed in mice infected with bioimaging A. baumannii strain 24 h after treatment with compound 62520. Compound 62520 was shown to significantly increase the survival rates of both immunocompetent and neutropenic mice infected with A. baumannii ATCC 17978. This compound also increased the survival rates of mice infected with clinical CRAB isolate. These results suggest that compound 62520 is a promising scaffold to develop a novel therapeutic agent against CRAB infections.

The sensor kinase BfmS controls production of outer membrane vesicles in Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is an important opportunistic pathogen responsible for various nosocomial infections. The BfmRS two-component system plays a role in pathogenesis and antimicrobial resistance of A. baumannii via regulation of bacterial envelope structures. This study investigated the role of the sensor kinase, BfmS, in localization of outer membrane protein A (OmpA) in the outer membrane and production of outer membrane vesicles (OMVs) using wild-type A. baumannii ATCC 17978, ΔbfmS mutant, and bfmS-complemented strains. RESULTS: The ΔbfmS mutant showed hypermucoid phenotype in the culture plates, growth retardation under static culture conditions, and reduced susceptibility to aztreonam and colistin compared to the wild-type strain. The ΔbfmS mutant produced less OmpA in the outer membrane but released more OmpA via OMVs than the wild-type strain, even though expression of ompA and its protein production were not different between the two strains. The ΔbfmS mutant produced 2.35 times more OMV particles and 4.46 times more OMV proteins than the wild-type stain. The ΔbfmS mutant OMVs were more cytotoxic towards A549 cells than wild-type strain OMVs. CONCLUSIONS: The present study demonstrates that BfmS controls production of OMVs in A. baumannii. Moreover, BfmS negatively regulates antimicrobial resistance of A. baumannii and OMV-mediated host cell cytotoxicity. Our results indicate that BfmS negatively controls the pathogenic traits of A. baumannii via cell envelope structures and OMV production.

Imaging of bioluminescent Acinetobacter baumannii in a mouse pneumonia model.

Bioluminescence imaging is a non-invasive tool for in vivo real-time monitoring of infectious disease progression in animal models. However, no bioluminescence imaging assay has been developed to monitor Acinetobacter baumannii infections. In the current study, bioluminescent strains of A. baumannii ATCC 17978 and its isogenic ΔompA mutant were constructed by integrating the promoter of the ompA gene and the luxCDABE luciferase gene into the bacterial chromosome. In an acute murine pneumonia model, bioluminescence of the two reporter strains was clearly visible in the lungs and the bioluminescent signal increased over time. Bioluminescence was correlated with bacterial burden and histopathology in reporter strain-infected mice, suggesting that bioluminescent bacteria are useful for monitoring A. baumannii infections in animal models.

Outer membrane protein A contributes to antimicrobial resistance of Acinetobacter baumannii through the OmpA-like domain.

OBJECTIVES: Acinetobacter baumannii outer membrane protein A (AbOmpA) is involved in bacterial pathogenesis. However, the role of AbOmpA in the antimicrobial resistance of A. baumannii has not been fully elucidated. This study aimed to investigate the role of the OmpA-like domain of AbOmpA in the antimicrobial resistance of A. baumannii. METHODS: The MICs of antimicrobial agents for the WT A. baumannii ATCC 17978, ΔompA mutant, OmpA-like domain-deleted (amino acids 223-356) AbOmpA mutant and single-copy ompA-complemented strain were determined by the Etest method. The MICs of antimicrobial agents for MDR strain 1656-2 and its ΔompA mutant strains were also determined. RESULTS: The ΔompA mutant strain of ATCC 17978 was more susceptible to trimethoprim (>5.3-fold) and other antimicrobial agents tested (<2.0-fold), except tigecycline, than the WT strain. The ΔompA mutant strain of 1656-2 was more susceptible to trimethoprim (>4.0-fold), tetracycline (2.3-fold) and other antimicrobial agents (<2.0-fold), including tigecycline, colistin and imipenem, than the WT strain. The MICs of gentamicin, imipenem and nalidixic acid for the WT ATCC 17978 and ΔompA mutant strains were decreased in the presence of an efflux pump inhibitor. A mutant strain of ATCC 17978 with the OmpA-like domain of AbOmpA deleted was more susceptible (≥2.0-fold) to substrates of the resistance-nodulation-division efflux pumps, including aztreonam, gentamicin, imipenem and trimethoprim, than the WT strain. CONCLUSIONS: This study demonstrates that AbOmpA contributes to the antimicrobial resistance of A. baumannii through the OmpA-like domain.

Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells.

Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs.

Whole Genome Re-Sequencing of Three Domesticated Chicken Breeds.

Chicken is one of the most popular domesticated species worldwide, as it can serve an important role in agricultural as well as biomedical research fields. Because it inhabits almost every continent and presents diverse morphology and traits, the need of genetic markers for distinguishing each breed for various purposes has increased. The whole genome sequencing of three different breeds (White Leghorn, Korean domestic, and Araucana) that show similar coloring patterns, with the exception of the White Leghorn breed, have confirmed previously reported genomic alterations and identified many novel variants. Additionally, the Whole Genome Re-Sequencing (WGRS) approach identified an approximately 4 kb insert within SLCO1B3 responsible for blue egg shell color. Targeted investigation of pigment-related genes corroborated previously reported non-synonymous mutations, and provided deeper insight into chicken coloring, where not a single but a combination of non-synonymous mutations in the MC1R gene is likely to be responsible for altered feather coloring.

Distinct characteristics of OxyR2, a new OxyR-type regulator, ensuring expression of Peroxiredoxin 2 detoxifying low levels of hydrogen peroxide in Vibrio vulnificus.

Two peroxiredoxins, Prx1 and Prx2, were previously identified in Vibrio vulnificus. Besides OxyR1, a homologue of Escherichia coli OxyR (EcOxyR), OxyR2 that shares low homology with EcOxyR was first identified in V. vulnificus. OxyR2 activated prx2 during aerobic growth, while OxyR1 activated prx1 only when exposed to exogenous H2O2. OxyR2 was oxidized to form a reversible C206 to C215 disulphide bond by sensing low levels of H2O2, which were insufficient to oxidize OxyR1, and only the oxidized OxyR2 activated prx2. OxyR25CA, in which all cysteine residues except for C206 and C215 were replaced with alanines, and its mutants, OxyR25CA-C206S and OxyR25CA-C215S, were constructed. OxyR25CA and OxyR25CA-C215S directly bound to a specific binding sequence centred at -56.5 from the prx2 transcription start site, albeit with different binding affinities. The binding sequence consisted of four ATCGnt elements spaced by a helical turn and aligned in the twofold dyad symmetry, suggesting that OxyR2 binds DNA as a tetramer. OxyR25CA-C206S also directly bound to DNA comprising more extended sequences, indicating that oxidized and reduced OxyR2 adopt different conformational states, leading to altered DNA contacts. The oxyR2 mutation reduced cytotoxicity and growth during infection, indicating that OxyR2 is essential for the pathogenesis of V. vulnificus.

Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii.

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii.

Acinetobacter nosocomialis secretes outer membrane vesicles that induce epithelial cell death and host inflammatory responses.

Acinetobacter nosocomialis is an important nosocomial pathogen that causes a variety of opportunistic infections; however, pathogenesis of this microorganism has not yet been characterized. The aim of this study was to investigate the secretion of outer membrane vesicles (OMVs) from A. nosocomialis and to determine their cytotoxic effects and their ability to induce inflammatory responses both in vitro and in vivo by using human epithelial HEp-2 cells and a mouse model, respectively. A. nosocomialis ATCC 17903(T) secreted spherical OMVs when cultured in vitro. Proteomic analysis revealed that 147 different proteins were associated with A. nosocomialis OMVs and virulence-associated proteins, such as outer membrane protein A (OmpA), CsuA, CsuC, CsuD, PilW, hemolysin, and serine protease, were identified. A. nosocomialis OMVs were cytotoxic to HEp-2 cells. These vesicles also induced the expression of pro-inflammatory cytokine genes in the HEp-2 cells. Early inflammatory responses, such as congestion and focal neutrophilic infiltration, were observed in the lungs of mice injected with A. nosocomialis OMVs. In conclusion, A. nosocomialis OMVs are important secretory nanocomplexes that induce cytotoxicity of epithelial cells and host inflammatory responses, which may contribute to the pathogenesis of A. nosocomialis.

Identification and characterization of Acinetobacter nosocomialis BfmRS, two-component regulatory system, essential for biofilm development.

BACKGROUND: Biofilm development by bacteria is considered to be an essential stage in the bacterial infection. Acinetobacter nosocomialis is an important nosocomial pathogen causing a variety of human infections. However, characteristics and specific determinants of biofilm development have been poorly characterized in A. nosocomialis. OBJECTIVE: The aim of this study was to investigate the factors involved in the biofilm development by A. nosocomialis. METHODS: Library of random transposon mutants was constructed using the Tn5 mutagenesis. The mutant strains, in which the ability of biofilm formation was significantly impaired, were screened by gentian violet staining. The roles of BfmR and BfmS were determined by constructing a bfmR and bfmS deletion mutant and analyzing the effects of bfmR and bfmS mutation on the biofilm development and motility of A. nosocomialis. RESULTS: We identified a biofilm-defective mutant in which a transposon insertion inactivated an open reading frame encoding the BfmR in a two-component regulatory system consisting of BfmR and BfmS. The bfmR mutant revealed a significant reduction in biofilm formation and motility compared to wild-type strain. Deficiency in the biofilm formation and motility of the bfmR mutant was restored by single copy bfmR complementation. In contrast, the bfmS mutant had no effect on biofilm formation. CONCLUSION: A. nosocomialis has a two-component regulatory system, BfmRS. BfmR is a response regulator required for the initial attachment and maturation of biofilm during the biofilm development as well as the bacterial growth. BfmR could be a potential drug target for A. nosocomialis infection.

Distinct characteristics of two 2-Cys peroxiredoxins of Vibrio vulnificus suggesting differential roles in detoxifying oxidative stress.

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes reducing toxic peroxides. Two distinct 2-Cys Prxs, Prx1 and Prx2, were identified in Vibrio vulnificus, a facultative aerobic pathogen. Both Prxs have two conserved catalytic cysteines, C(P) and C(R), but Prx2 is more homologous in amino acid sequences to eukaryotic Prx than to Prx1. Prx2 utilized thioredoxin A as a reductant, whereas Prx1 required AhpF. Prx2 contained GGIG and FL motifs similar to the motifs conserved in sensitive Prxs and exhibited sensitivity to overoxidation. MS analysis and C(P)-SO(3)H specific immunoblotting demonstrated overoxidation of C(P) to C(P)-SO(2)H (or C(P)-SO(3)H) in vitro and in vivo, respectively. In contrast, Prx1 was robust and C(P) was not overoxidized. Discrete expression of the Prxs implied that Prx2 is induced by trace amounts of H(2)O(2) and thereby residential in cells grown aerobically. In contrast, Prx1 was occasionally expressed only in cells exposed to high levels of H(2)O(2). A mutagenesis study indicated that lack of Prx2 accumulated sufficient H(2)O(2) to induce Prx1. Kinetic properties indicated that Prx2 effectively scavenges low levels of peroxides because of its high affinity to H(2)O(2), whereas Prx1 quickly degrades higher levels of peroxides because of its high turnover rate and more efficient reactivation. This study revealed that the two Prxs are differentially optimized for detoxifying distinct ranges of H(2)O(2), and proposed that Prx2 is a residential scavenger of peroxides endogenously generated, whereas Prx1 is an occasional scavenger of peroxides exogenously encountered. Furthermore, genome sequence database search predicted widespread coexistence of the two Prxs among bacteria.

A comprehensive analysis of gorilla-specific LINE-1 retrotransposons.

BACKGROUND: Long interspersed element-1 (LINE-1 or L1) is the most abundant retrotransposons in the primate genome. They have approximately 520,000 copies and make up ~ 17% of the primate genome. Full-length L1s can mobilize to a new genomic location using their enzymatic machinery. Gorilla is the second closest species to humans after the chimpanzee, and human-gorilla split 7-12 million years ago. The gorilla genome provides an opportunity to explore primate origins and evolution. OBJECTIVE: L1s have contributed to genome diversity and variations during primate evolution. This study aimed to identify gorilla-specific L1s using a more recent version of the gorilla reference genome (Mar. 2016 GSMRT3/gorGor5). METHODS: We collected gorilla-specific L1 candidates through computational analysis and manual inspection. L1Xplorer was used to identify whether full-length gorilla-specific L1s were intact. In addition, to determine the level of sequence conservation between intact fulllength gorilla-specific L1s, two ORFs of intact L1s were aligned with the L1PA2 consensus sequence. RESULTS: 2002 gorilla-specific L1 candidates were identified through computational analysis. Among them, we manually inspected 1,883 gorilla-specific L1s, among which most of them belong to the L1PA2 subfamily and 12 were intact L1s that could influence genomic variations in the gorilla genome. Interestingly, the 12 intact full-length gorilla-specific L1s have 14 highly conserved nonsynonymous mutations, including 6 mutations and 8 mutations in ORF1 and ORF2, respectively. In comparison to the intact full-length chimpanzee-specific L1s and human-specific hot-L1s, two of these in ORF1 (L256F and E293G) were shown as gorilla-specific nonsynonymous mutations. CONCLUSION: The gorilla-specific L1s may have had significantly affected the gorilla genome to compose a genome different form that of other primates during primate evolution.

Screening of small molecules attenuating biofilm formation of Acinetobacter baumannii by inhibition of ompA promoter activity.

Anti-virulence therapeutic strategies are promising alternatives against drug-resistant pathogens. Outer membrane protein A (OmpA) plays a versatile role in the pathogenesis and antimicrobial resistance of Acinetobacter baumannii. Therefore, OmpA is an innovative target for anti-virulence therapy against A. baumannii. This study aimed to develop a high-throughput screening (HTS) system to discover small molecules inhibiting the ompA promoter activity of A. baumannii and screen chemical compounds using the bacterial growth-based HTS system. The ompA promoter and open reading frame of nptI fusion plasmids that controlled the expression of nptI encoding resistance to kanamycin by the ompA promoter were constructed and then transformed into A. baumannii ATCC 17978. This reporter strain was applied to screen small molecules inhibiting the ompA promoter activity in a chemical library. Of the 7,520 chemical compounds, 15 exhibited ≥ 70% growth inhibition of the report strain cultured in media containing kanamycin. Three compounds inhibited the expression of ompA and OmpA in the outer membrane of A. baumannii ATCC 17978, which subsequently reduced biofilm formation. In conclusion, our reporter strain is useful for large-scale screening of small molecules inhibiting the ompA expression in A. baumannii. Hit compounds identified by the HTS system are promising scaffolds to develop novel therapeutics against A. baumannii.

470 nm LED Irradiation Inhibits the Invasiveness of CD133-positive Human Colorectal Cancer Stem Cells by Suppressing the Cyclooxygenase-2/prostaglandin E2 Pathway.

BACKGROUND/AIM: Recurrence and metastasis of cancer caused by cancer stem cells (CSCs) is a challenge to overcome. Low level laser therapy is a new treatment strategy to suppress their invasiveness. We have assessed the inhibitory effects of 470 nm blue LED on the invasiveness of them to determine the molecular mechanisms of anti-invasiveness. MATERIALS AND METHODS: The effects of blue LEDs on their viability, proliferation and invasion were analyzed using MTT and transwell methods. In addition, the anti-invasiveness effect of blue LED on them was evaluated by zymography, semi-quantitative RT-PCR and western blot analysis. RESULTS: Irradiation with blue LED at 3 J/cm2 resulted in inhibition of their viability, proliferation and invasiveness. Their matrix metalloproteinase 2 (MMP-2) and MMP-9 activities were reduced by blue LED irradiation. Semi-quantitative RT-PCR also showed similar results. In addition, western blotting analyses showed that cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) synthesis were significantly inhibited by LED irradiation in CD133+ colorectal CSCs. CONCLUSION: Down-regulation of the COX-2/PGE2 signaling pathway by blue LED irradiation led to reduce expression of MMP-2 and MMP-9, inhibiting the invasiveness of CD133+ colorectal CSC.

Distinctive Roles of Two Acinetobactin Isomers in Challenging Host Nutritional Immunity.

The human pathogen Acinetobacter baumannii produces and utilizes acinetobactin for iron assimilation. Although two isomeric structures of acinetobactin, one featuring an oxazoline (Oxa) and the other with an isoxazolidinone (Isox) at the core, have been identified, their differential roles as virulence factors for successful infection have yet to be established. This study provides direct evidence that Oxa supplies iron more efficiently than Isox, primarily owing to its specific recognition by the cognate outer membrane receptor, BauA. The other components in the acinetobactin uptake machinery appear not to discriminate these isomers. Interestingly, Oxa was found to form a stable iron complex that is resistant to release of the chelated iron upon competition by Isox, despite their comparable apparent affinities to Fe(III). In addition, both Oxa and Isox were found to be competent iron chelators successfully scavenging iron from host metal sequestering proteins responsible for nutritional immunity. These observations collectively led us to propose a new model for acinetobactin-based iron assimilation at infection sites. Namely, Oxa is the principal siderophore mediating the core Fe(III) supply chain for A. baumannii, whereas Isox plays a minor role in the iron delivery and, alternatively, functions as an auxiliary iron collector that channels the iron pool toward Oxa. The unique siderophore utilization mechanism proposed here represents an intriguing strategy for pathogen adaptation under the various nutritional stresses encountered at infection sites. IMPORTANCE Acinetobacter baumannii has acquired antibiotic resistance at an alarming rate, and it is becoming a serious threat to society, particularly due to the paucity of effective treatment options. Acinetobactin is a siderophore of Acinetobacter baumannii, responsible for active iron supply, and it serves as a key virulence factor to counter host nutritional immunity during infection. While two acinetobactin isomers were identified, their distinctive roles for successful infection of Acinetobacter baumannii remained unsettled. This study clearly identified the isomer containing an oxazoline core as the principal siderophore based on comparative analysis of the specificity of the acinetobactin uptake machinery, the stability of the corresponding iron complexes, and the iron scavenging activity against the host iron sequestering proteins. Our findings are anticipated to stimulate efforts to discover a potent antivirulence agent against Acinetobacter baumannii that exploits the acinetobactin-based iron assimilation mechanism.

AbaR is a LuxR type regulator essential for motility and the formation of biofilm and pellicle in Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is a major opportunistic pathogen causing nosocomial infections. Acinetobacter baumannii possesses a quorum sensing system consisting of abaI, encoding an autoinducer synthase, and abaR, encoding a putative LuxR type regulator. AbaI is required for motility and biofilm formation in A. baumannii. However, the functions of AbaR on the expression of abaI, motility, and the formation of biofilm and pellicle have not yet been explored. OBJECTIVE: The aim of this study was to investigate the effects of abaR mutation on the expression of abaI, motility, and the formation of biofilm and pellicle. METHODS: Functions of AbaR were assessed by the construction of an isogenic mutant and by evaluating the effects of abaR mutation on the expression of abaI, motility, and the formation of biofilm and pellicle. RESULTS: The abaR mutant revealed a significant decrease in the expression of abaI. The disruption of abaR resulted in substantial defects in motility and the formation of biofilm and pellicle. Introduction of abaR in trans complemented the defects. CONCLUSIONS: AbaR of A. baumannii is required for the expression of abaI and plays important roles in motility and the formation of biofilm and pellicle. AbaR may be considered to be a target of anti-biofilm agents.

Zur-regulated lipoprotein A contributes to the fitness of Acinetobacter baumannii.

Acinetobacter baumannii is a notorious nosocomial pathogen that commonly infects severely ill patients. Zinc (Zn) is essential to survive and adapt to different environment and host niches in A. baumannii. Of the Zinc uptake regulator (Zur)-regulated genes in A. baumannii, the A1S_3412 gene encoding a Zur-regulated lipoprotein A (ZrlA) is critical for cell envelope integrity and overcoming antibiotic exposure. This study investigated whether ZrlA contributes to the fitness of A. baumannii in vitro and in vivo using the wildtype A. baumannii ATCC 17978, ΔzrlA mutant, and zrlAcomplemented strains. The ΔzrlA mutant showed reduced biofilm formation, surface motility, and adherence to and invasion of epithelial cells compared to the wild-type strain. In a mouse pneumonia model, the ?zrlA mutant showed significantly lower bacterial numbers in the blood than the wildtype strain. These virulence traits were restored in the zrlAcomplemented strain. Under static conditions, the expression of csuCDE, which are involved in the chaperone-usher pili assembly system, was significantly lower in the ΔzrlA mutant than in the wild-type strain. Moreover, the expression of the bfmR/S genes, which regulate the CsuA/BABCDE system, was significantly lower in the ΔzrlA mutant under static conditions than in the wild-type strain. Our results indicate that the zrlA gene plays a role in the fitness of A. baumannii by regulating the BfmR/S two-component system and subsequently the CsuA/BABCDE chaperone-usher pili assembly system, suggesting it as a potential target for anti-virulence strategies against A. baumannii.