Functional Features of TonB Energy Transduction Systems of Acinetobacter baumannii

ABSTRACTAcinetobacter baumanniiis an opportunistic pathogen that causes severe nosocomial infections. Strain ATCC 19606Tutilizes the siderophore acinetobactin to acquire iron under iron-limiting conditions encountered in the host. Accordingly, the genome of this strain has threetonBgenes encoding proteins for energy transduction functions needed for the active transport of nutrients, including iron, through the outer membrane. Phylogenetic analysis indicates that thesetonBgenes, which are present in the genomes of all sequencedA. baumanniistrains, were acquired from different sources. Two of these genes occur as components oftonB-exbB-exbDoperons and one as a monocistronic copy; all are actively transcribed in ATCC 19606T. The abilities of components of these TonB systems to complement the growth defect ofEscherichia coliW3110 mutants KP1344 (tonB) and RA1051 (exbBD) under iron-chelated conditions further support the roles of these TonB systems in iron acquisition. Mutagenesis analysis of ATCC 19606TtonB1(subscripted numbers represent different copies of genes or proteins) andtonB2supports this hypothesis: their inactivation results in growth defects in iron-chelated media, without affecting acinetobactin biosynthesis or the production of the acinetobactin outer membrane receptor protein BauA.In vivoassays usingGalleria mellonellashow that each TonB protein is involved in, but not essential for, bacterial virulence in this infection model. Furthermore, we observed that TonB2plays a role in the ability of bacteria to bind to fibronectin and to adhere to A549 cells by uncharacterized mechanisms. Taken together, these results indicate thatA. baumanniiATCC 19606Tproduces three independent TonB proteins, which appear to provide the energy-transducing functions needed for iron acquisition and cellular processes that play a role in the virulence of this pathogen.

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Intracellular Trafficking and Persistence of Acinetobacter baumannii Requires Transcription Factor EB

mSphere ◽

10.1128/msphere.00106-18 ◽

2018 ◽

Vol 3 (2) ◽

Cited By ~ 10

Author(s):

Raquel Parra-Millán ◽

David Guerrero-Gómez ◽

Rafael Ayerbe-Algaba ◽

Maria Eugenia Pachón-Ibáñez ◽

Antonio Miranda-Vizuete ◽

...

Keyword(s):

Transcription Factor ◽

Acinetobacter Baumannii ◽

Intracellular Trafficking ◽

Nuclear Translocation ◽

A549 Cells ◽

Lung Epithelial Cells ◽

Host Cells ◽

Infection Model ◽

Content Type ◽

Transcription Factor Eb

ABSTRACT Acinetobacter baumannii is a significant human pathogen associated with hospital-acquired infections. While adhesion, an initial and important step in A. baumannii infection, is well characterized, the intracellular trafficking of this pathogen inside host cells remains poorly studied. Here, we demonstrate that transcription factor EB (TFEB) is activated after A. baumannii infection of human lung epithelial cells (A549). We also show that TFEB is required for the invasion and persistence inside A549 cells. Consequently, lysosomal biogenesis and autophagy activation were observed after TFEB activation which could increase the death of A549 cells. In addition, using the Caenorhabditis elegans infection model by A. baumannii , the TFEB orthologue HLH-30 was required for survival of the nematode to infection, although nuclear translocation of HLH-30 was not required. These results identify TFEB as a conserved key factor in the pathogenesis of A. baumannii . IMPORTANCE Adhesion is an initial and important step in Acinetobacter baumannii infections. However, the mechanism of entrance and persistence inside host cells is unclear and remains to be understood. In this study, we report that, in addition to its known role in host defense against Gram-positive bacterial infection, TFEB also plays an important role in the intracellular trafficking of A. baumannii in host cells. TFEB was activated shortly after A. baumannii infection and is required for its persistence within host cells. Additionally, using the C. elegans infection model by A. baumannii , the TFEB orthologue HLH-30 was required for survival of the nematode to infection, although nuclear translocation of HLH-30 was not required.

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Efflux Transporter of Siderophore Staphyloferrin A in Staphylococcus aureus Contributes to Bacterial Fitness in Abscesses and Epithelial Cells

Infection and Immunity ◽

10.1128/iai.00358-17 ◽

2017 ◽

Vol 85 (8) ◽

Cited By ~ 9

Author(s):

Hidemasa Nakaminami ◽

Chunhui Chen ◽

Que Chi Truong-Bolduc ◽

Eu Suk Kim ◽

Yin Wang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Epithelial Cells ◽

Iron Acquisition ◽

A549 Cells ◽

Growth Defect ◽

Efflux Transporter ◽

Content Type ◽

Lung Adenocarcinoma Cell Line ◽

Restricted Environment ◽

Bacterial Fitness

ABSTRACT The siderophores staphyloferrin A (SA) and staphyloferrin B (SB) of Staphylococcus aureus are essential for iron acquisition in the iron-restricted environment of the host, such as in subcutaneous abscesses. SA and SB are secreted by SfaA and SbnD transporters, respectively. To assess the further function of SfaA and SbnD in S. aureus fitness, we tested its effect on murine abscess models and intracellular replication in epithelial cells. Bacterial fitness in abscesses and in epithelial cells was studied, by comparing the parental strains RN6390 and MW2 and their ΔsfaA and ΔsbnD mutants using competition assays in a murine abscess model and invasion and replication assays with human lung adenocarcinoma cell line A549. In the murine abscess model using equal inocula of a ΔsfaA or ΔsbnD mutant and the wild-type RN6390 strain, the ΔsfaA mutant exhibited growth defects of 2.2-fold. Additionally, replication of the ΔsfaA mutant within A549 cells was decreased 3.0-fold. In complementation experiments, the ΔsfaA mutant carrying plasmid-borne sfaA restored the growth fitness in abscesses and epithelial cells. The ΔsbnD mutant, in contrast, showed no growth defect in either abscesses or epithelial cells. Our findings demonstrate that the efflux transporter of the siderophore SA contributes to the ability of S. aureus to replicate in abscesses and epithelial cells. Furthermore, fitness of S. aureus in these sites of replication is not compromised by the absence of transporter SbnD.

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Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

Infection and Immunity ◽

10.1128/iai.02925-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1354-1365 ◽

Cited By ~ 8

Author(s):

Steven E. Fiester ◽

Chika C. Nwugo ◽

William F. Penwell ◽

John M. Neary ◽

Amber C. Beckett ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Protein Transport ◽

Protein Translocation ◽

Outer Membrane Proteins ◽

Critical Role ◽

Iron Chelation ◽

Receptor Protein ◽

Outer Membrane Receptor ◽

Content Type

Acinetobacter baumanniiis a Gram-negative opportunistic nosocomial pathogen that causes pneumonia and soft tissue and systemic infections. Screening of a transposon insertion library ofA. baumanniiATCC 19606Tresulted in the identification of the 2010 derivative, which, although capable of growing well in iron-rich media, failed to prosper under iron chelation. Genetic, molecular, and functional assays showed that 2010's iron utilization-deficient phenotype is due to an insertion within the 3′ end ofsecA, which results in the production of a C-terminally truncated derivative of SecA. SecA plays a critical role in protein translocation through the SecYEG membrane channel. Accordingly, thesecAmutation resulted in undetectable amounts of the ferric acinetobactin outer membrane receptor protein BauA while not affecting the production of other acinetobactin membrane protein transport components, such as BauB and BauE, or the secretion of acinetobactin by 2010 cells cultured in the presence of subinhibitory concentrations of the synthetic iron chelator 2,2′-dipyridyl. Outer membrane proteins involved in nutrient transport, adherence, and biofilm formation were also reduced in 2010. The SecA truncation also increased production of 30 different proteins, including proteins involved in adaptation/tolerance responses. Although some of these protein changes could negatively affect the pathobiology of the 2010 derivative, its virulence defect is mainly due to its inability to acquire iron via the acinetobactin-mediated system. These results together indicate that although the C terminus of theA. baumanniiATCC 19606TSecA is not essential for viability, it plays a critical role in the production and translocation of different proteins and virulence.

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Activity of Meropenem-Vaborbactam againstPseudomonas aeruginosaandAcinetobacter baumanniiin a Neutropenic Mouse Thigh Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01665-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 5

Author(s):

Mojgan Sabet ◽

Ziad Tarazi ◽

David C. Griffith

Keyword(s):

Pseudomonas Aeruginosa ◽

Acinetobacter Baumannii ◽

Clinical Isolates ◽

Infection Model ◽

Model Data ◽

Bacterial Killing ◽

Content Type

ABSTRACTWe have evaluated the activity of meropenem-vaborbactam against clinical isolates ofPseudomonas aeruginosaandAcinetobacter baumanniiin a neutropenic mouse thigh infection model. Data show that meropenem-vaborbactam regimens equivalent to 3-h infusions every 8 h with 2 g meropenem and 2 g vaborbactam produced bacterial killing against strains with MICs of 2 to 16 mg/liter and suggests that this combination may have utility in the treatment of infections caused byP. aeruginosaandA. baumannii.

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Efficacy of Humanized Cefiderocol Exposures over 72 Hours against a Diverse Group of Gram-Negative Isolates in the Neutropenic Murine Thigh Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01040-18 ◽

2018 ◽

Vol 63 (2) ◽

pp. e01040-18 ◽

Cited By ~ 3

Author(s):

Sean M. Stainton ◽

Marguerite L. Monogue ◽

Masakatsu Tsuji ◽

Yoshinori Yamano ◽

Roger Echols ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Acinetobacter Baumannii ◽

Infection Model ◽

Diverse Group ◽

Gram Negative ◽

Content Type ◽

Adaptive Resistance

ABSTRACT Herein, we evaluated sustainability of humanized exposures of cefiderocol in vivo over 72 h against pathogens with cefiderocol MICs of 0.5 to 16 μg/ml in the neutropenic murine thigh model. In Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae displaying MICs of 0.5 to 8 μg/ml (n = 11), sustained kill was observed at 72 h among 9 isolates. Postexposure MICs revealed a single 2-dilution increase in one animal compared with controls (1/54 samples, 1.8%) at 72 h. Adaptive resistance during therapy was not observed.

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Combating Carbapenem-ResistantAcinetobacter baumanniiby an Optimized Imipenem-plus-Tobramycin Dosage Regimen: Prospective Validation via Hollow-Fiber Infection and Mathematical Modeling

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02053-17 ◽

2018 ◽

Vol 62 (4) ◽

Cited By ~ 7

Author(s):

Cornelia B. Landersdorfer ◽

Rajbharan Yadav ◽

Kate E. Rogers ◽

Tae Hwan Kim ◽

Beom Soo Shin ◽

...

Keyword(s):

Mathematical Modeling ◽

Acinetobacter Baumannii ◽

Hollow Fiber ◽

Dosage Regimen ◽

Infection Model ◽

Bacterial Killing ◽

Content Type ◽

Carbapenem Resistant

ABSTRACTWe aimed to prospectively validate an optimized combination dosage regimen against a clinical carbapenem-resistantAcinetobacter baumannii(CRAB) isolate (imipenem MIC, 32 mg/liter; tobramycin MIC, 2 mg/liter). Imipenem at constant concentrations (7.6, 13.4, and 23.3 mg/liter, reflecting a range of clearances) was simulated in a 7-day hollow-fiber infection model (inoculum, ∼107.2CFU/ml) with and without tobramycin (7 mg/kg q24h, 0.5-h infusions). While monotherapies achieved no killing or failed by 24 h, this rationally optimized combination achieved >5 log10bacterial killing and suppressed resistance.

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Robust Suppression of Lipopolysaccharide Deficiency in Acinetobacter baumannii by Growth in Minimal Medium

Journal of Bacteriology ◽

10.1128/jb.00420-19 ◽

2019 ◽

Vol 201 (22) ◽

Cited By ~ 1

Author(s):

Emma Nagy ◽

Richard Losick ◽

Daniel Kahne

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Minimal Medium ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Growth Defects ◽

Outer Leaflet ◽

Morphological Defects ◽

Outer Membrane Biogenesis

ABSTRACT Lipopolysaccharide (LPS) is normally considered to be essential for viability in Gram-negative bacteria but can be removed in Acinetobacter baumannii. Mutant cells lacking this component of the outer membrane show growth and morphological defects. Here, we report that growth rates equivalent to the wild type can be achieved simply by propagation in minimal medium. The loss of LPS requires that cells rely on phospholipids for both leaflets of the outer membrane. We show that growth rate in the absence of LPS is not limited by nutrient availability but by the rate of outer membrane biogenesis. We hypothesize that because cells grow more slowly, outer membrane synthesis ceases to be rate limiting in minimal medium. IMPORTANCE Gram-negative bacteria are defined by their asymmetric outer membrane that consists of phospholipids on the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. LPS is essential in all but a few Gram-negative species; the reason for this differential essentiality is not well understood. One species that can survive without LPS, Acinetobacter baumannii, shows characteristic growth and morphology phenotypes. We show that these phenotypes can be suppressed under conditions of slow growth and describe how LPS loss is connected to the growth defects. In addition to better defining the challenges A. baumannii cells face in the absence of LPS, we provide a new hypothesis that may explain the species-dependent conditional essentiality.

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The Mutation of Conservative Asp268 Residue in the Peptidoglycan-Associated Domain of the OmpA Protein Affects Multiple Acinetobacter baumannii Virulence Characteristics

Molecules ◽

10.3390/molecules24101972 ◽

2019 ◽

Vol 24 (10) ◽

pp. 1972 ◽

Cited By ~ 2

Author(s):

Jūratė Skerniškytė ◽

Emilija Karazijaitė ◽

Julien Deschamps ◽

Renatas Krasauskas ◽

Romain Briandet ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Protein A ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Clinical Strain ◽

Infection Model ◽

Terminal Domain ◽

Ompa Protein

Acinetobacter baumannii is a nosocomial human pathogen of increasing concern due to its multidrug resistance profile. The outer membrane protein A (OmpA) is an abundant bacterial cell surface component involved in A. baumannii pathogenesis. It has been shown that the C-terminal domain of OmpA is located in the periplasm and non-covalently associates with the peptidoglycan layer via two conserved amino acids, thereby anchoring OmpA to the cell wall. Here, we investigated the role of one of the respective residues, D268 in OmpA of A. baumannii clinical strain Ab169, on its virulence characteristics by complementing the ΔompA mutant with the plasmid-borne ompAD268A allele. We show that while restoring the impaired biofilm formation of the ΔompA strain, the Ab169ompAD268A mutant tended to form bacterial filaments, indicating the abnormalities in cell division. Moreover, the Ab169 OmpA D268-mediated association to peptidoglycan was required for the manifestation of twitching motility, desiccation resistance, serum-induced killing, adhesion to epithelial cells and virulence in a nematode infection model, although it was dispensable for the uptake of β-lactam antibiotics by outer membrane vesicles. Overall, the results of this study demonstrate that the OmpA C-terminal domain-mediated association to peptidoglycan is critical for a number of virulent properties displayed by A. baumannii outside and within the host.

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The Capsular Polysaccharide of Acinetobacter baumannii Is an Obstacle for Therapeutic Passive Immunization Strategies

Infection and Immunity ◽

10.1128/iai.00591-17 ◽

2017 ◽

Vol 85 (12) ◽

Cited By ~ 14

Author(s):

Shun Xin Wang-Lin ◽

Ruth Olson ◽

Janet M. Beanan ◽

Ulrike MacDonald ◽

Joseph P. Balthasar ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Bactericidal Activity ◽

Capsular Polysaccharide ◽

Rapid Development ◽

Outer Membrane Proteins ◽

Passive Immunization ◽

Clinical Isolates ◽

Content Type ◽

Resistant Strains

ABSTRACT Acinetobacter baumannii has become an important concern for human health due to rapid development and wide spread of antimicrobial-resistant strains and high mortality associated with the infection. Passive immunizations with antisera targeting outer membrane proteins (OMPs) have shown encouraging results in protecting mice from A. baumannii infection, but monoclonal anti-OMP antibodies have not been developed, and their potential therapeutic properties have not been explored. The goal of this report is to evaluate the antibacterial activity of monoclonal antibodies (MAbs) targeting outer membrane protein A (OmpA) of A. baumannii. Five anti-OmpA MAbs were developed using hybridoma technology and showed strong binding to strain ATCC 19606. However, low antibody binding was observed when they were tested against six clinical isolates, which included extensively drug-resistant strains. In contrast, high binding to an isogenic K1 capsule-negative mutant (AB307.30) was shown, suggesting that capsular polysaccharide mediated the inhibition of MAb binding to OmpA. Anti-OmpA MAbs increased the macrophage-mediated bactericidal activity of AB307.30 but failed to increase phagocytic killing of capsule-positive strains. Capsular polysaccharide was also protective against complement-mediated bactericidal activity in human ascites in the presence and absence of opsonization. Lastly, passive immunization with anti-OmpA MAbs did not confer protection against challenge with AB307-0294, the encapsulated parent strain of AB307.30, in a mouse sepsis infection model. These results reveal the important role of capsule polysaccharide in shielding OmpA and thereby inhibiting anti-OmpA MAb binding to clinical isolates. This property of capsule hindered the therapeutic utility of anti-OmpA MAbs, and it may apply to other conserved epitopes in A. baumannii.

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N-Acetylcysteine Selectively Antagonizes the Activity of Imipenem in Pseudomonas aeruginosa by an OprD-Mediated Mechanism

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00017-15 ◽

2015 ◽

Vol 59 (6) ◽

pp. 3246-3251 ◽

Cited By ~ 10

Author(s):

Jerónimo Rodríguez-Beltrán ◽

Gabriel Cabot ◽

Estela Ynés Valencia ◽

Coloma Costas ◽

German Bou ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Acinetobacter Baumannii ◽

Outer Membrane ◽

Bacterial Species ◽

Outer Membrane Proteins ◽

Antibiotic Activity ◽

Simultaneous Treatment ◽

Content Type ◽

Sds Page

ABSTRACTThe modulating effect ofN-acetylcysteine (NAC) on the activity of different antibiotics has been studied inPseudomonas aeruginosa. Our results demonstrate that, in contrast to previous reports, only the activity of imipenem is clearly affected by NAC. MIC and checkerboard determinations indicate that the NAC-based modulation of imipenem activity is dependent mainly on OprD. SDS-PAGE of outer membrane proteins (OMPs) after NAC treatments demonstrates that NAC does not modify the expression of OprD, suggesting that NAC competitively inhibits the uptake of imipenem through OprD. Similar effects on imipenem activity were obtained withP. aeruginosaclinical isolates. Our results indicate that imipenem-susceptibleP. aeruginosastrains become resistant upon simultaneous treatment with NAC and imipenem. Moreover, the generality of the observed effects of NAC on antibiotic activity was assessed with two additional bacterial species,Escherichia coliandAcinetobacter baumannii. Caution should be taken during treatments, as the activity of imipenem may be modified by physiologically attainable concentrations of NAC, particularly during intravenous and nebulized regimes.

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