Characterization of an Acinetobacter baumanniilptDDeletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

ABSTRACTLipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. SomeAcinetobacter baumanniistrains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such aslpxC. Here, we characterized a mutant deleted forlptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lackinglptDhad a growth defect comparable to that of anlpxCdeletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage andN-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact oflptDdeletion than oflpxCdeletion on inner and outer membrane permeability, respectively. Targeted liquid chromatography-mass spectrometry (LCMS) analysis of LPS intermediates from UDP-3-O-R-3-hydroxylauroyl-N-acetyl-α-d-glucosamine through lipid IVAshowed that the loss of LptD caused an accumulation of lipid IVA. This suggested that pathway intermediate accumulation or mislocalization caused by the blockage of later LPS pathway steps impacts envelope integrity. Supporting this notion, chemical inhibition of lipid A precursor enzymes, including LpxC and FabB/F, in thelptDdeletion strain partially rescued growth and permeability defects.IMPORTANCENew antibiotics to treat Gram-negative bacterial infections are urgently needed. Inhibition of LPS biosynthesis is attractive because this would impact viability and cell permeability. Therefore, a better understanding of this pathway is important, especially in strains such asA. baumanniiATCC 19606, where LPS biosynthesis is not essentialin vitro. We show that ATCC 19606 also survives the loss of the final translocation of LPS into the OM (lptDdeletion). Intriguingly, this impaired cell envelope integrity more than the loss of LPS biosynthesis (lpxCdeletion), presumably due to the accumulation of toxic intermediates. Supporting this, chemical inhibition of LPS biosynthesis partially reversed this permeability defect. This extends our understanding of the LPS machinery and provides insights into potential interrelationships of the target steps along this important pathway.

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LpxK Is Essential for Growth of Acinetobacter baumannii ATCC 19606: Relationship to Toxic Accumulation of Lipid A Pathway Intermediates

mSphere ◽

10.1128/msphere.00199-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 11

Author(s):

Jun-Rong Wei ◽

Daryl L. Richie ◽

Mina Mostafavi ◽

Louis E. Metzger ◽

Christopher M. Rath ◽

...

Keyword(s):

Fatty Acid ◽

Cell Growth ◽

Acinetobacter Baumannii ◽

Biosynthetic Pathway ◽

Lipid A ◽

Fatty Acid Biosynthesis ◽

Gram Negative ◽

Content Type ◽

Lipid A Biosynthesis ◽

Chemical Inhibition

ABSTRACT Acinetobacter baumannii is a Gram-negative pathogen for which new therapies are needed. The lipid A biosynthetic pathway has several potential enzyme targets for the development of anti-Gram-negative agents (e.g., LpxC). However, A. baumannii ATCC 19606 can grow in the absence of LpxC and, correspondingly, of lipid A. In contrast, we show that cellular depletion of LpxK, a kinase occurring later in the pathway, inhibits growth. Growth inhibition results from toxic accumulation of lipid A pathway intermediates, since chemical inhibition of LpxC or fatty acid biosynthesis rescues cell growth upon loss of LpxK. Overall, this suggests that targets such as LpxK can be essential for growth even in those Gram-negative bacteria that do not require lipid A biosynthesis per se. This strain provides an elegant tool to derive a better understanding of the steps in a pathway that is the focus of intense interest for the development of novel antibacterials. Acinetobacter baumannii ATCC 19606 can grow without lipid A, the major component of lipooligosaccharide. However, we previously reported that depletion of LpxH (the fourth enzyme in the lipid A biosynthetic pathway) prevented growth of this strain due to toxic accumulation of lipid A pathway intermediates. Here, we explored whether a similar phenomenon occurred with depletion of LpxK, a kinase that phosphorylates disaccharide 1-monophosphate (DSMP) at the 4′ position to yield lipid IVA. An A. baumannii ATCC 19606 derivative with LpxK expression under the control of an isopropyl β-d-1-thiogalactopyranoside (IPTG)-regulated expression system failed to grow without induction, indicating that LpxK is essential for growth. Light and electron microscopy of LpxK-depleted cells revealed morphological changes relating to the cell envelope, consistent with toxic accumulation of lipid A pathway intermediates disrupting cell membranes. Using liquid chromatography-mass spectrometry (LCMS), cellular accumulation of the detergent-like pathway intermediates DSMP and lipid X was shown. Toxic accumulation was further supported by restoration of growth upon chemical inhibition of LpxC (upstream of LpxK and the first committed step of lipid A biosynthesis) using CHIR-090. Inhibitors of fatty acid synthesis also abrogated the requirement for LpxK expression. Growth rescue with these inhibitors was possible on Mueller-Hinton agar but not on MacConkey agar. The latter contains outer membrane-impermeable bile salts, suggesting that despite growth restoration, the cell membrane permeability barrier was not restored. Therefore, LpxK is essential for growth of A. baumannii, since loss of LpxK causes accumulation of detergent-like pathway intermediates that inhibit cell growth. IMPORTANCE Acinetobacter baumannii is a Gram-negative pathogen for which new therapies are needed. The lipid A biosynthetic pathway has several potential enzyme targets for the development of anti-Gram-negative agents (e.g., LpxC). However, A. baumannii ATCC 19606 can grow in the absence of LpxC and, correspondingly, of lipid A. In contrast, we show that cellular depletion of LpxK, a kinase occurring later in the pathway, inhibits growth. Growth inhibition results from toxic accumulation of lipid A pathway intermediates, since chemical inhibition of LpxC or fatty acid biosynthesis rescues cell growth upon loss of LpxK. Overall, this suggests that targets such as LpxK can be essential for growth even in those Gram-negative bacteria that do not require lipid A biosynthesis per se. This strain provides an elegant tool to derive a better understanding of the steps in a pathway that is the focus of intense interest for the development of novel antibacterials.

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Interplay ofKlebsiella pneumoniaefabZandlpxCMutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis

mSphere ◽

10.1128/msphere.00508-18 ◽

2018 ◽

Vol 3 (5) ◽

Cited By ~ 9

Author(s):

Mina Mostafavi ◽

Lisha Wang ◽

Lili Xie ◽

Kenneth T. Takeoka ◽

Daryl L. Richie ◽

...

Keyword(s):

Outer Membrane ◽

Lipid A ◽

Gram Negative Bacteria ◽

Resistance Mutations ◽

Cellular Mechanisms ◽

Gram Negative ◽

Content Type ◽

Outer Leaflet ◽

Chemical Inhibition ◽

Dependent Growth

ABSTRACTTight coordination of inner and outer membrane biosynthesis is very important in Gram-negative bacteria. Biosynthesis of the lipid A moiety of lipopolysaccharide, which comprises the outer leaflet of the outer membrane has garnered interest for Gram-negative antibacterial discovery. In particular, several potent inhibitors of LpxC (the first committed step of the lipid A pathway) are described. Here we show that serial passaging ofKlebsiella pneumoniaein increasing levels of an LpxC inhibitor yielded mutants that grew only in the presence of the inhibitor. These strains had mutations infabZandlpxCoccurring together (encoding either FabZR121L/LpxCV37Gor FabZF51L/LpxCV37G).K. pneumoniaemutants having only LpxCV37Gor LpxCV37Aor various FabZ mutations alone were less susceptible to the LpxC inhibitor and did not require LpxC inhibition for growth. Western blotting revealed that LpxCV37Gaccumulated to high levels, and electron microscopy of cells harboring FabZR121L/LpxCV37Gindicated an extreme accumulation of membrane in the periplasm when cells were subcultured without LpxC inhibitor. Significant accumulation of detergent-like lipid A pathway intermediates that occur downstream of LpxC (e.g., lipid X and disaccharide monophosphate [DSMP]) was also seen. Taken together, our results suggest that redirection of lipid A pathway substrate by less active FabZ variants, combined with increased activity from LpxCV37Gwas overdriving the lipid A pathway, necessitating LpxC chemical inhibition, since native cellular maintenance of membrane homeostasis was no longer functioning.IMPORTANCEEmergence of antibiotic resistance has prompted efforts to identify and optimize novel inhibitors of antibacterial targets such as LpxC. This enzyme catalyzes the first committed step of lipid A synthesis, which is necessary to generate lipopolysaccharide and ultimately the Gram-negative protective outer membrane. Investigation of this pathway and its interrelationship with inner membrane (phospholipid) biosynthesis or other pathways is therefore highly important to the fundamental understanding of Gram-negative bacteria and by extension to antibiotic discovery. Here we exploited the availability of a novel LpxC inhibitor to engender the generation ofK. pneumoniaeresistant mutants whose growth depends on chemical inhibition of LpxC. Inhibitor dependency resulted from the interaction of different resistance mutations and was based on loss of normal cellular mechanisms required to establish membrane homeostasis. This study provides new insights into the importance of this process inK. pneumoniaeand how it may be linked to novel biosynthetic pathway inhibitors.

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The Lipid A 1-Phosphatase, LpxE, Functionally Connects Multiple Layers of Bacterial Envelope Biogenesis

mBio ◽

10.1128/mbio.00886-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 1

Author(s):

Jinshi Zhao ◽

Jinsu An ◽

Dohyeon Hwang ◽

Qinglin Wu ◽

Su Wang ◽

...

Keyword(s):

Antibiotic Resistance ◽

Outer Membrane ◽

Lipid A ◽

Host Immune System ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Phosphatase Activities ◽

O Antigen

ABSTRACT Although distinct lipid phosphatases are thought to be required for processing lipid A (component of the outer leaflet of the outer membrane), glycerophospholipid (component of the inner membrane and the inner leaflet of the outer membrane), and undecaprenyl pyrophosphate (C55-PP; precursors of peptidoglycan and O antigens of lipopolysaccharide) in Gram-negative bacteria, we report that the lipid A 1-phosphatases, LpxEs, functionally connect multiple layers of cell envelope biogenesis in Gram-negative bacteria. We found that Aquifex aeolicus LpxE structurally resembles YodM in Bacillus subtilis, a phosphatase for phosphatidylglycerol phosphate (PGP) with a weak in vitro activity on C55-PP, and rescues Escherichia coli deficient in PGP and C55-PP phosphatase activities; deletion of lpxE in Francisella novicida reduces the MIC value of bacitracin, indicating a significant contribution of LpxE to the native bacterial C55-PP phosphatase activity. Suppression of plasmid-borne lpxE in F. novicida deficient in chromosomally encoded C55-PP phosphatase activities results in cell enlargement, loss of O-antigen repeats of lipopolysaccharide, and ultimately cell death. These discoveries implicate LpxE as the first example of a multifunctional regulatory enzyme that orchestrates lipid A modification, O-antigen production, and peptidoglycan biogenesis to remodel multiple layers of the Gram-negative bacterial envelope. IMPORTANCE Dephosphorylation of the lipid A 1-phosphate by LpxE in Gram-negative bacteria plays important roles in antibiotic resistance, bacterial virulence, and modulation of the host immune system. Our results demonstrate that in addition to removing the 1-phosphate from lipid A, LpxEs also dephosphorylate undecaprenyl pyrophosphate, an important metabolite for the synthesis of the essential envelope components, peptidoglycan and O-antigen. Therefore, LpxEs participate in multiple layers of biogenesis of the Gram-negative bacterial envelope and increase antibiotic resistance. This discovery marks an important step toward understanding the regulation and biogenesis of the Gram-negative bacterial envelope.

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Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC

mBio ◽

10.1128/mbio.00674-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 13

Author(s):

Nadine Lemaître ◽

Xiaofei Liang ◽

Javaria Najeeb ◽

Chul-Jin Lee ◽

Marie Titecat ◽

...

Keyword(s):

Bacterial Infections ◽

Biosynthetic Pathway ◽

Lipid A ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Drug Resistant ◽

Bubonic Plague ◽

Gram Negative ◽

New Class

ABSTRACT The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis. Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria. IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains. IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

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Reinforcing Lipid A Acylation on the Cell Surface of Acinetobacter baumannii Promotes Cationic Antimicrobial Peptide Resistance and Desiccation Survival

mBio ◽

10.1128/mbio.00478-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 59

Author(s):

Joseph M. Boll ◽

Ashley T. Tucker ◽

Dustin R. Klein ◽

Alexander M. Beltran ◽

Jennifer S. Brodbelt ◽

...

Keyword(s):

Health Care ◽

Acinetobacter Baumannii ◽

Outer Membrane ◽

Lipid A ◽

Treatment Options ◽

Cationic Antimicrobial Peptide ◽

Gram Negative ◽

Content Type ◽

Hospital Environments ◽

Lipid A Biosynthesis

ABSTRACTAcinetobacter baumanniiis an emerging Gram-negative pathogen found in hospitals and intensive care units. In order to persist in hospital environments,A. baumanniiwithstands desiccative conditions and can rapidly develop multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the conserved lipid A component of the Gram-negative outer membrane to lyse the bacterial cell. However, many Gram-negative pathogenic bacteria, includingA. baumannii, fortify their outer membrane with hepta-acylated lipid A to protect the cell from CAMP-dependent cell lysis. Whereas inEscherichia coliandSalmonella, increased production of the outer membrane acyltransferase PagP results in formation of protective hepta-acylated lipid A, which reinforces the lipopolysaccharide portion of the outer membrane barrier,A. baumanniidoes not carry a gene that encodes a PagP homolog. Instead,A. baumanniihas evolved a PagP-independent mechanism to synthesize protective hepta-acylated lipid A. Taking advantage of a recently adaptedA. baumanniigenetic recombineering system, we characterized two putative acyltransferases inA. baumanniidesignated LpxLAb(A. baumanniiLpxL) and LpxMAb(A. baumanniiLpxM), which transfer one and two lauroyl (C12:0) acyl chains, respectively, during lipid A biosynthesis. Hepta-acylation ofA. baumanniilipid A promoted resistance to vertebrate and polymyxin CAMPs, which are prescribed as last-resort treatment options. Intriguingly, our analysis also showed that LpxMAb-dependent acylation of lipid A is essential forA. baumanniidesiccation survival, a key resistance mechanism for survival in hospital environments. Compounds that inhibit LpxMAb-dependent hepta-acylation of lipid A could act synergistically with CAMPs to provide innovative transmission prevention strategies and treat multidrug-resistant infections.IMPORTANCEAcinetobacter baumanniiinfections can be life threatening, and disease can progress in a variety of host tissues. Current antibiotic regimen and disinfectant strategies have failed to limit nosocomialA. baumanniiinfections. Instead, the rate ofA. baumanniiinfection among health care communities has skyrocketed due to the bacterium's adaptability. Its aptitude for survival over extended periods on inanimate objects, such as catheters, respirators, and surfaces in intensive care units, or on the hands of health care workers and its ability to rapidly develop antibiotic resistance makeA. baumanniia threat to health care communities. Emergence of multidrug- and extremely drug-resistantA. baumanniiillustrates the ineffectiveness of current prevention and treatment options. Our analysis to understand howA. baumanniiresists cationic antimicrobial peptide (CAMP)-mediated and desiccative killing revealed two lipid A acyltransferases that produce protective hepta-acylated lipid A. Our work suggests that inhibiting lipid A biosynthesis by targeting the acyltransferase LpxMAb(A. baumanniiLpxM) could provide a novel target to combat this pathogen.

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Characterization of Two Novel Lipopolysaccharide Phosphoethanolamine Transferases in Pasteurella multocida and Their Role in Resistance to Cathelicidin-2

Infection and Immunity ◽

10.1128/iai.00557-17 ◽

2017 ◽

Vol 85 (11) ◽

Cited By ~ 6

Author(s):

Marina Harper ◽

Amy Wright ◽

Frank St. Michael ◽

Jianjun Li ◽

Deanna Deveson Lucas ◽

...

Keyword(s):

Pasteurella Multocida ◽

Lipid A ◽

Bacterial Pathogen ◽

Cationic Antimicrobial Peptide ◽

Gram Negative ◽

Content Type ◽

New Family ◽

Maximum Protection ◽

Phosphoethanolamine Transferases

ABSTRACT The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen Pasteurella multocida has phosphoethanolamine (PEtn) residues attached to lipid A, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and galactose. In this report, we show that PEtn is transferred to lipid A by the P. multocida EptA homologue, PetL, and is transferred to galactose by a novel PEtn transferase that is unique to P. multocida called PetG. Transcriptomic analyses indicated that petL expression was positively regulated by the global regulator Fis and negatively regulated by an Hfq-dependent small RNA. Importantly, we have identified a novel PEtn transferase called PetK that is responsible for PEtn addition to the single Kdo molecule (Kdo1), directly linked to lipid A in the P. multocida glycoform A LPS. In vitro assays showed that the presence of a functional petL and petK, and therefore the presence of PEtn on lipid A and Kdo1, was essential for resistance to the cationic, antimicrobial peptide cathelicidin-2. The importance of PEtn on Kdo1 and the identification of the transferase responsible for this addition have not previously been shown. Phylogenetic analysis revealed that PetK is the first representative of a new family of predicted PEtn transferases. The PetK family consists of uncharacterized proteins from a range of Gram-negative bacteria that produce LPS glycoforms with only one Kdo molecule, including pathogenic species within the genera Vibrio, Bordetella, and Haemophilus. We predict that many of these bacteria will require the addition of PEtn to Kdo for maximum protection against host antimicrobial peptides.

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CD137 Expressed on Neutrophils Plays Dual Roles in Antibacterial Responses against Gram-Positive and Gram-Negative Bacterial Infections

Infection and Immunity ◽

10.1128/iai.00115-13 ◽

2013 ◽

Vol 81 (6) ◽

pp. 2168-2177 ◽

Cited By ~ 8

Author(s):

Quang-Tam Nguyen ◽

Thu-Ha T. Nguyen ◽

Seong-A. Ju ◽

Yea-Sol Lee ◽

Seung Hyun Han ◽

...

Keyword(s):

Bacterial Infections ◽

Costimulatory Molecule ◽

Host Responses ◽

Gram Positive ◽

Necrosis Factor Alpha ◽

Gram Negative ◽

Content Type ◽

Stimulation Of

ABSTRACTSevere sepsis and septic shock caused mainly by bacterial infections are life-threatening conditions that urge the development of novel therapies. However, host responses to and pathophysiology of sepsis have not been clearly understood, which remains a major obstacle for the development of effective therapeutics. Recently, we have shown that stimulation of a costimulatory molecule, CD137, enhanced survival of mice infected with the Gram-positive (G+) intracellular bacteriumListeria monocytogenesbut decreased survival in a polymicrobial sepsis model. Herein, we report that CD137 deficiency or blocking of CD137 signaling decreased antibacterial responses of mice infected with G+bacteria (Staphylococcus aureus,Streptococcus pneumoniae, andEnterococcus faecalis) but increased these responses in mice infected with Gram-negative (G−) bacteria (Escherichia coli,Pseudomonas aeruginosa, andSalmonella entericaserovar Typhimurium). Consistent with these findings, stimulation of CD137 by administration of agonistic antibody enhanced responses against G+bacteria, whereas it decreased these responses against G−bacteria. Neutrophils were responsible for CD137-mediated opposite roles in control of G+and G−bacterial infections. Stimulation of CD137 enhanced activities of neutrophils againstS. aureusbut decreased these activities againstE. coli, while CD137 blocking produced opposite results with the stimulation of CD137in vivoandin vitro. Furthermore, we found that combined signaling of CD137 and Toll-like receptor 2 (TLR2) induced synergistic production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) by neutrophils, but combined signaling of CD137 and TLR4 did not. Our data strongly suggest that CD137 may play a dual role in sepsis in association with TLRs.

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Isolation of Phage Lysins That Effectively KillPseudomonas aeruginosain Mouse Models of Lung and Skin Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00024-19 ◽

2019 ◽

Vol 63 (7) ◽

Cited By ~ 13

Author(s):

Assaf Raz ◽

Anna Serrano ◽

Anaise Hernandez ◽

Chad W. Euler ◽

Vincent A. Fischetti

Keyword(s):

Outer Membrane ◽

Bacterial Infections ◽

Skin Infection ◽

Therapeutic Potential ◽

Treatment Options ◽

Opportunistic Pathogen ◽

Infection Model ◽

Gram Negative ◽

Content Type ◽

Phage Lysins

ABSTRACTMultidrug resistance (MDR) is rapidly increasing in prevalence among isolates of the opportunistic pathogenPseudomonas aeruginosa, leaving few treatment options. Phage lysins are cell wall hydrolases that have a demonstrated therapeutic potential against Gram-positive pathogens; however, the outer membrane of Gram-negative bacteria prevents most lysins from reaching the peptidoglycan, making them less effective as therapeutics. Nevertheless, a few lysins from Gram-negative bacterial phage can penetrate the bacterial outer membrane with the aid of an amphipathic tail found in the molecule’s termini. In this work, we took a phylogenetic approach to systematically identify those lysins fromP. aeruginosaphage that would be most effective therapeutically. We isolated and performed preliminary characterization of 16 lysins and chose 2 lysins, PlyPa03 and PlyPa91, which exhibited >5-log killing activity againstP. aeruginosaand other Gram-negative pathogens (particularlyKlebsiellaandEnterobacter). These lysins showed rapid killing kinetics and were active in the presence of high concentrations of salt and urea and under pH conditions ranging from 5.0 to 10.0. Activity was not inhibited in the presence of the pulmonary surfactant beractant (Survanta). While neither enzyme was active in 100% human serum, PlyPa91 retained activity in low serum concentrations. The lysins were effective in the treatment of aP. aeruginosaskin infection in a mouse model, and PlyPa91 protected mice in a lung infection model, making these lysins potential drug candidates for Gram-negative bacterial infections of the skin or respiratory mucosa.

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Phosphate Groups of Lipid A Are Essential for Salmonella enterica Serovar Typhimurium Virulence and Affect Innate and Adaptive Immunity

Infection and Immunity ◽

10.1128/iai.00123-12 ◽

2012 ◽

Vol 80 (9) ◽

pp. 3215-3224 ◽

Cited By ~ 46

Author(s):

Qingke Kong ◽

David A. Six ◽

Qing Liu ◽

Lillian Gu ◽

Shifeng Wang ◽

...

Keyword(s):

Outer Membrane ◽

Adaptive Immunity ◽

Salmonella Enterica ◽

Lipid A ◽

Outer Membrane Proteins ◽

Polymyxin B ◽

Content Type ◽

Serovar Typhimurium ◽

Phosphate Groups

ABSTRACTLipid A is a key component of the outer membrane of Gram-negative bacteria and stimulates proinflammatory responses via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. Its endotoxic activity depends on the number and length of acyl chains and its phosphorylation state. InSalmonella entericaserovar Typhimurium, removal of the secondary laurate or myristate chain in lipid A results in bacterial attenuation and growth defectsin vitro. However, the roles of the two lipid A phosphate groups in bacterial virulence and immunogenicity remain unknown. Here, we used anS. TyphimuriummsbB pagL pagP lpxRmutant, carrying penta-acylated lipid A, as the parent strain to construct a series of mutants synthesizing 1-dephosphorylated, 4′-dephosphorylated, or nonphosphorylated penta-acylated lipid A. Dephosphorylated mutants exhibited increased sensitivity to deoxycholate and showed increased resistance to polymyxin B. Removal of both phosphate groups severely attenuated the mutants when administered orally to BALB/c mice, but the mutants colonized the lymphatic tissues and were sufficiently immunogenic to protect the host from challenge with wild-typeS. Typhimurium. Mice receivingS. Typhimurium with 1-dephosphorylated or nonphosphorylated penta-acylated lipid A exhibited reduced levels of cytokines. Attenuated and dephosphorylatedSalmonellavaccines were able to induce adaptive immunity against heterologous (PspA ofStreptococcus pneumoniae) and homologous antigens (lipopolysaccharide [LPS] and outer membrane proteins [OMPs]).

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Outer Membrane Targeting of Pseudomonas aeruginosa Proteins Shows Variable Dependence on the Components of Bam and Lol Machineries

mBio ◽

10.1128/mbio.00246-11 ◽

2011 ◽

Vol 2 (6) ◽

Cited By ~ 31

Author(s):

Hanh H. Hoang ◽

Nicholas N. Nickerson ◽

Vincent T. Lee ◽

Anastasia Kazimirova ◽

Mohamed Chami ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Protein Translocation ◽

Lipid Vesicles ◽

Structural Features ◽

Gram Negative Bacteria ◽

Variable Effect ◽

Gram Negative ◽

Content Type

ABSTRACT In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. IMPORTANCE Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect, but the severity (and therefore, Lol and Bam dependence), varies with individual proteins. XcpQ, the secretin component of the type II secretion apparatus, is translocated into the OM without the assistance of Bam or Lol machineries. The hypothesis that XcpQ, after secretion across the cytoplasmic membrane, does not utilize the OM targeting machineries was supported by demonstrating that in vitro-synthesized XcpQ (but not the other P. aeruginosa secretins) can spontaneously incorporate into lipid vesicles. Therefore, the requirement for ancillary factors appears to be, in certain instances, dictated by the intrinsic properties of individual OM proteins, conceivably reflecting their propensities to misfold during periplasmic transit.

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