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

2015 ◽  
Vol 83 (4) ◽  
pp. 1354-1365 ◽  
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.

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

2017 ◽  
Vol 85 (12) ◽  
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.

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

2015 ◽  
Vol 59 (6) ◽  
pp. 3246-3251 ◽  
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.

scholarly journals Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Rakesh Sikdar ◽  
Harris D. Bernstein
Keyword(s):  
Outer Membrane ◽  
Alternative Energy ◽  
Protein Translocation ◽  
Outer Membrane Proteins ◽  
Passenger Domain ◽  
Experimental Conditions ◽  
Content Type ◽  
Intrinsically Disordered ◽  
Extracellular Domains ◽  
Bacterial Outer Membrane

ABSTRACT Trimeric autotransporter adhesins (TAAs) are a family of bacterial outer membrane (OM) proteins that are comprised of three identical subunits. Each subunit contains an N-terminal extracellular (“passenger”) domain and a short C-terminal segment that contributes four β strands to a single 12-stranded β barrel. The mechanism by which the passenger domains are translocated across the OM and the energetics of the translocation reaction are poorly understood. To address these issues, we examined the secretion of modified versions of the passenger domain of UpaG, a TAA produced by Escherichia coli CFT073. Using the SpyTag-SpyCatcher system to probe passenger domain localization, we found that both intrinsically disordered polypeptides fused to the UpaG passenger domain and artificially disulfide-bonded polypeptides were secreted effectively but relatively slowly. Surprisingly, we also found that in some cases, the three nonnative passenger domain segments associated with a single trimer were secreted sequentially. Photo-cross-linking experiments indicated that incompletely assembled UpaG derivatives remained bound to the barrel assembly machinery (Bam) complex until all three passenger domains were fully secreted. Taken together, our results strongly suggest that the secretion of polypeptides through the TAA pathway is coordinated with the assembly of the β barrel domain and that the folding of passenger domains in the extracellular space maximizes the rate of secretion. Furthermore, our work provides evidence for an unprecedented sequential mode of protein translocation, at least under specific experimental conditions. IMPORTANCE Trimeric autotransporter adhesins (TAAs) are specialized bacterial outer membrane proteins consisting of three identical subunits. TAAs contain large extracellular domains that trimerize and promote virulence, but the mechanism by which they are secreted is poorly understood. We found that the extracellular domains of a native TAA were secreted rapidly but that disordered and artificially folded polypeptides fused to native passenger domains were secreted in a slow, sequential fashion. Our results strongly suggest that the efficient secretion of native extracellular domains is driven by their trimerization following export but that alternative energy sources can be harnessed to secrete nonnative polypeptides. Furthermore, we obtained evidence that TAA extracellular domains are secreted before the assembly of the linked membrane spanning domain is completed.

scholarly journals AbuO, a TolC-Like Outer Membrane Protein of Acinetobacter baumannii, Is Involved in Antimicrobial and Oxidative Stress Resistance

2014 ◽  
Vol 59 (2) ◽  
pp. 1236-1245 ◽  
Author(s):  
Vijaya Bharathi Srinivasan ◽  
Vasanth Vaidyanathan ◽  
Govindan Rajamohan
Keyword(s):  
Oxidative Stress ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Major Facilitator Superfamily ◽  
Pcr Analysis ◽  
Content Type ◽  
Major Facilitator ◽  
And Oxidative Stress

ABSTRACTAlthoughAcinetobacter baumanniiis well accepted as a nosocomial pathogen, only a few of the outer membrane proteins (OMPs) have been functionally characterized. In this study, we demonstrate the biological functions of AbuO, a homolog of TolC fromEscherichia coli. Inactivation ofabuOled to increased sensitivity to high osmolarity and oxidative stress challenge. The ΔabuOmutant displayed increased susceptibility to antibiotics, such as amikacin, carbenicillin, ceftriaxone, meropenem, streptomycin, and tigecycline, and hospital-based disinfectants, such as benzalkonium chloride and chlorhexidine. The reverse transcription (RT)-PCR analysis indicated increased expression of efflux pumps (resistance nodulation cell division [RND] efflux pumpacrD, 8-fold; SMR-typeemrEhomolog, 12-fold; and major facilitator superfamily [MFS]-typeampGhomolog, 2.7-fold) and two-component response regulators (baeR, 4.67-fold;ompR, 10.43-fold) in the ΔabuOmutant together with downregulation ofrstA(4.22-fold) and the pilin chaperone (9-fold). The isogenic mutant displayed lower virulence in a nematode model (P< 0.01). Experimental evidence for the binding of MerR-type transcriptional regulator SoxR to radiolabeledabuOpromoter suggests regulation ofabuOby SoxR inA. baumannii.

scholarly journals Small-Molecule Inhibitors of Gram-Negative Lipoprotein Trafficking Discovered by Phenotypic Screening

2015 ◽  
Vol 197 (6) ◽  
pp. 1075-1082 ◽  
Author(s):  
Sarah M. McLeod ◽  
Paul R. Fleming ◽  
Kathleen MacCormack ◽  
Robert E. McLaughlin ◽  
James D. Whiteaker ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Receptor Protein ◽  
Cross Resistance ◽  
Outer Membrane Receptor ◽  
Transport Pathway ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Outer Membrane Lipoprotein ◽  
Bacterial Cytological Profiling

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process.Escherichia colimutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp fromE. colispheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.

scholarly journals Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria.

1993 ◽  
Vol 121 (6) ◽  
pp. 1233-1243 ◽  
Author(s):  
A Mayer ◽  
R Lill ◽  
W Neupert
Keyword(s):  
Outer Membrane ◽  
Protein Transport ◽  
Protein Translocation ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Surface Receptors ◽  
Outer Membranes

Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub-mitochondrial locations. While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes. The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22. Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration. The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion. Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space. In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane.

scholarly journals Functional Features of TonB Energy Transduction Systems of Acinetobacter baumannii

2013 ◽  
Vol 81 (9) ◽  
pp. 3382-3394 ◽  
Author(s):  
Daniel L. Zimbler ◽  
Brock A. Arivett ◽  
Amber C. Beckett ◽  
Sharon M. Menke ◽  
Luis A. Actis
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Iron Acquisition ◽  
A549 Cells ◽  
Energy Transduction ◽  
Receptor Protein ◽  
Growth Defect ◽  
Infection Model ◽  
Content Type ◽  
Functional Features

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.

scholarly journals Biofilm Formation Caused by Clinical Acinetobacter baumannii Isolates Is Associated with Overexpression of the AdeFGH Efflux Pump

2015 ◽  
Vol 59 (8) ◽  
pp. 4817-4825 ◽  
Author(s):  
Xinlong He ◽  
Feng Lu ◽  
Fenglai Yuan ◽  
Donglin Jiang ◽  
Peng Zhao ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Gene Transcription ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Content Type ◽  
Potential Association ◽  
Genes Encoding ◽  
Clinical Environments

ABSTRACTChronic wound infections are associated with biofilm formation, which in turn has been correlated with drug resistance. However, the mechanism by which bacteria form biofilms in clinical environments is not clearly understood. This study was designed to investigate the biofilm formation potency ofAcinetobacter baumanniiand the potential association of biofilm formation with genes encoding efflux pumps, quorum-sensing regulators, and outer membrane proteins. A total of 48 clinically isolatedA. baumanniistrains, identified by enterobacterial repetitive intergenic consensus (ERIC)-PCR as types A-II, A-III, and A-IV, were analyzed. Three representative strains, which were designatedA. baumanniiABR2, ABR11, and ABS17, were used to evaluate antimicrobial susceptibility, biofilm inducibility, and gene transcription (abaI,adeB,adeG,adeJ,carO, andompA). A significant increase in the MICs of different classes of antibiotics was observed in the biofilm cells. The formation of a biofilm was significantly induced in all the representative strains exposed to levofloxacin. The levels of gene transcription varied between bacterial genotypes, antibiotics, and antibiotic concentrations. The upregulation ofadeGcorrelated with biofilm induction. The consistent upregulation ofadeGandabaIwas detected in A-III-typeA. baumanniiin response to levofloxacin and meropenem (1/8 to 1/2× the MIC), conditions which resulted in the greatest extent of biofilm induction. This study demonstrates a potential role of the AdeFGH efflux pump in the synthesis and transport of autoinducer molecules during biofilm formation, suggesting a link between low-dose antimicrobial therapy and a high risk of biofilm infections caused byA. baumannii. This study provides useful information for the development of antibiofilm strategies.

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

2019 ◽  
Vol 201 (22) ◽  
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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