Biogenesis of Lipoproteins in Gram-Negative Bacteria: 50 Years of Progress

The outer membrane is the defining characteristic of Gram-negative bacteria and is crucial for the maintenance of cellular integrity. Lipoproteins are an essential component of this outer membrane and regulate broad cellular functions ranging from efflux, cellular physiology, antibiotic resistance, and pathogenicity. In the canonical model of lipoprotein biogenesis, lipoprotein precursors are first synthesized in the cytoplasm prior to extensive modifications by the consecutive action of three key enzymes: diacylglyceryl transferase (Lgt), lipoprotein signal peptidase A (LspA), and apolipoprotein N-acyltransferase (Lnt). This enzymatic process modifies lipoprotein precursors for subsequent trafficking by the Lol pathway. The function of these three enzymes were originally thought to be essential, however, in some Gram-negative bacteria, namely Acinetobacter baylyi, the third enzyme Lnt is dispensable. Here we review the function and significance of Lgt, LspA, and Lnt in outer membrane biogenesis and how non-canonical models of lipoprotein processing in Acinetobacter spp. can enhance our understanding of lipoprotein modifications and trafficking.

Download Full-text

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.

Download Full-text

Disrupting Gram-Negative Bacterial Outer Membrane Biosynthesis through Inhibition of the Lipopolysaccharide Transporter MsbA

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01142-18 ◽

2018 ◽

Vol 62 (11) ◽

Cited By ~ 10

Author(s):

Mary Kate Alexander ◽

Anh Miu ◽

Angela Oh ◽

Mike Reichelt ◽

Hoangdung Ho ◽

...

Keyword(s):

Outer Membrane ◽

Drug Targets ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Transport Pathway ◽

Gram Negative ◽

Content Type ◽

Outer Leaflet ◽

New Antibiotics ◽

Outer Membrane Biogenesis

ABSTRACTThere is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that killEscherichia colithrough inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.

Download Full-text

Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

10.26434/chemrxiv.8282204.v1 ◽

2019 ◽

Author(s):

Jiajun Wang ◽

Rémi Terrasse ◽

Jayesh Arun Bafna ◽

Lorraine Benier ◽

Mathias Winterhalter

Keyword(s):

Outer Membrane ◽

Lipid Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Multi Drug Resistance ◽

Gram Negative Bacteria ◽

Membrane Channels ◽

Gram Negative ◽

Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from Escherichia coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (kon) and lower dissociation (koff) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

Download Full-text

MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

International Journal of Molecular Sciences ◽

10.3390/ijms22105328 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5328

Author(s):

Miao Ma ◽

Margaux Lustig ◽

Michèle Salem ◽

Dominique Mengin-Lecreulx ◽

Gilles Phan ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Cell Division ◽

Membrane Proteins ◽

Outer Membrane ◽

Efflux Pump ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

Download Full-text

Shadowing the Actions of a Predator: Backlit Fluorescent Microscopy Reveals Synchronous Nonbinary Septation of Predatory Bdellovibrio inside Prey and Exit through Discrete Bdelloplast Pores

Journal of Bacteriology ◽

10.1128/jb.00914-10 ◽

2010 ◽

Vol 192 (24) ◽

pp. 6329-6335 ◽

Cited By ~ 40

Author(s):

A. K. Fenton ◽

M. Kanna ◽

R. D. Woods ◽

S.-I. Aizawa ◽

R. E. Sockett

Keyword(s):

Cell Division ◽

Outer Membrane ◽

Fluorescent Microscopy ◽

Gram Negative Bacteria ◽

Bacterial Cell Division ◽

Prey Cell ◽

Gram Negative ◽

A Genome ◽

Predatory Bacteria ◽

First Time

ABSTRACT The Bdellovibrio are miniature “living antibiotic” predatory bacteria which invade, reseal, and digest other larger Gram-negative bacteria, including pathogens. Nutrients for the replication of Bdellovibrio bacteria come entirely from the digestion of the single invaded bacterium, now called a bdelloplast, which is bound by the original prey outer membrane. Bdellovibrio bacteria are efficient digesters of prey cells, yielding on average 4 to 6 progeny from digestion of a single prey cell of a genome size similar to that of the Bdellovibrio cell itself. The developmental intrabacterial cycle of Bdellovibrio is largely unknown and has never been visualized “live.” Using the latest motorized xy stage with a very defined z-axis control and engineered periplasmically fluorescent prey allows, for the first time, accurate return and visualization without prey bleaching of developing Bdellovibrio cells using solely the inner resources of a prey cell over several hours. We show that Bdellovibrio bacteria do not follow the familiar pattern of bacterial cell division by binary fission. Instead, they septate synchronously to produce both odd and even numbers of progeny, even when two separate Bdellovibrio cells have invaded and develop within a single prey bacterium, producing two different amounts of progeny. Evolution of this novel septation pattern, allowing odd progeny yields, allows optimal use of the finite prey cell resources to produce maximal replicated, predatory bacteria. When replication is complete, Bdellovibrio cells exit the exhausted prey and are seen leaving via discrete pores rather than by breakdown of the entire outer membrane of the prey.

Download Full-text

Improved methods for producing outer membrane vesicles in Gram-negative bacteria

Research in Microbiology ◽

10.1016/j.resmic.2004.02.003 ◽

2004 ◽

Author(s):

T Henry

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Improved Methods

Download Full-text

The lipopolysaccharide transport (Lpt) machinery: A nonconventional transporter for lipopolysaccharide assembly at the outer membrane of Gram-negative bacteria

Journal of Biological Chemistry ◽

10.1074/jbc.r117.802512 ◽

2017 ◽

Vol 292 (44) ◽

pp. 17981-17990 ◽

Cited By ~ 23

Author(s):

Paola Sperandeo ◽

Alessandra M. Martorana ◽

Alessandra Polissi

Keyword(s):

Outer Membrane ◽

Gram Negative Bacteria ◽

Gram Negative

Download Full-text

Bloodstream infections in patients with hematologic malignancies and febrile neutropenia - a single center experience

Medicinski pregled ◽

10.2298/mpns2104083d ◽

2021 ◽

Vol 74 (3-4) ◽

pp. 83-89

Author(s):

Marina Dragicevic-Jojkic ◽

Ivana Urosevic ◽

Amir El Farra ◽

Borivoj Sekulic ◽

Ivanka Percic ◽

...

Keyword(s):

Febrile Neutropenia ◽

Fatal Outcome ◽

Antibiotic Sensitivity ◽

Bloodstream Infections ◽

Hematologic Malignancies ◽

High Rate ◽

Antibiotics Resistance ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Acinetobacter Spp

Introduction. Bacterial blood infections during febrile neutropenia episodes are urgent medical conditions which were and still are the main cause of morbidity and mortality among patients with hematologic malignancies. The aim of this study was to determine the incidence and clinical characteristics of bacteremia, infectious agents, presence and incidence of antibiotic resistance, as well as the treatment outcome of bloodstream infections in patients with hematologic malignancies. Material and Methods. A three-year retrospective study included 107 patients with hematologic malignancies and positive blood culture results during febrile neutropenia. Results. The most common isolates were Gram-negative bacteria (58.5%), with Escherichia coli being the most frequent pathogen. The Gram-negative microorganisms were mostly sensitive to carbapenems in 70.7%, whereas sensitivity to other antibiotics was as follows: piperacillin/ tazobactam 62%, amikacin 58.5%, and third-generation cephalosporins 50.5%. Acinetobacter spp. was sensitive only to colistin (94.1%). The antibiotic sensitivity among Gram-positive bacteria was highest to linezolid (97.1%), followed by teicoplanin (81.4%) and vancomycin (81.4%). In our patients, the mortality rate during the first 28 days from the moment of positive isolates was high (37.4%). Most patients died within the first seven days. Bacterial blood infections caused by Gram-negative bacteria were associated with significantly higher mortality (?2 = 4.92, p = 0.026). Acinetobacter spp. was isolated in almost half of the patients with fatal outcome, of whom 62.5% died in the first 24 hours. Conclusion. Bacterial bloodstream infections are severe complications with a high rate of mortality in febrile neutropenic hematological patients. Gram-negative bacteria were the most common isolates in our Clinic, with high mortality. It is of utmost importance to constantly monitor the resistance of bacteria to antibiotics, as well as to prevent and control the spread of resistant strains. Antibiotics resistance patterns should regularly be followed.

Download Full-text